Water Analysis Handbook - Hach

WATER ANALYSIS
HANDBOOK
7th Edition
Photometric Procedures
Titration Procedures
Ion-Selective Electrode Procedures
Microbiological Procedures
Chemical Procedures Explained
Digesting Liquids, Oils and Solids
HACH COMPANY
(970) 669-3050 or (800) 227-4224
Loveland, Colorado, U.S.A.
Hach Company, 2008, 2012.

All rights reserved. Printed in the U.S.A.
Table of Contents
Applications Guide ........................................................................................................................... 11
Section 1 Abbreviations and Conversions .................................................................................. 15
1.1 Procedure abbreviations ............................................................................................................. 15
1.2 Conversions ................................................................................................................................ 16
Section 2 Laboratory Practices ...................................................................................................... 19

2.1 Temperature ............................................................................................................................... 19
2.2 Mixing ......................................................................................................................................... 19
2.3 Digestion ..................................................................................................................................... 20
2.4 Distillation ................................................................................................................................... 20
2.5 Filtration ...................................................................................................................................... 21
2.6 Reagents .................................................................................................................................... 24
2.7 Sample dilution ........................................................................................................................... 24
2.8 AccuVac Ampuls ...................................................................................................................... 26
2.9 PermaChem pillows ................................................................................................................. 27
2.10 Sample cells ............................................................................................................................. 28
2.11 Other apparatus ........................................................................................................................ 29
2.12 Achieve accuracy in measurement ........................................................................................... 29
Section 3 Chemical Analysis........................................................................................................... 31

3.1 Sample collection, preservation and storage .............................................................................. 31
3.2 Interferences ............................................................................................................................... 39
3.3 Method performance................................................................................................................... 40
3.4 Prepare a calibration curve ......................................................................................................... 43
3.5 Adapt procedures to other spectrophotometers ......................................................................... 43
Section 4 Sample Pretreatment by Digestion ............................................................................. 47

4.1 USEPA-approved digestions ...................................................................................................... 47
4.2 General Digesdahl digestion....................................................................................................... 48
Section 5 Water Management and Safety .................................................................................... 55

5.1 Waste minimization..................................................................................................................... 55
5.2 Regulatory overview ................................................................................................................... 55
5.3 Hazardous waste ........................................................................................................................ 55
5.4 Management of specific wastes.................................................................................................. 58
5.5 Resources................................................................................................................................... 58
5.6 Safety.......................................................................................................................................... 59
5.7 Material safety data sheets ......................................................................................................... 60
Section 6 International Guideline Comparison ........................................................................... 63

Section 7 Definitions of USEPA Approved and Accepted........................................................ 65
7.1 USEPA approved........................................................................................................................ 65
7.2 USEPA accepted ........................................................................................................................ 65
Sample cells and apparatus
Procedures for Analysis................................................................................................................... 69

Acid-Base, Acid Determination and Base Determination ....................................................................... 71
Acid-Base, Sodium Hydroxide for meq/L of Acid Sulfuric Acid for meq/L of Base ................................. 77
Acidity, Methyl Orange, Sodium Hydroxide with a Buret ........................................................................ 83
Acidity, Phenolphthalein, Sodium Hydroxide with a Buret ..................................................................... 87
Acidity, Methyl Orange and Phenolphthalein (Total) Acidity ................................................................... 91
Alachlor, Immunoassay Method.............................................................................................................. 97
Alkalinity, Phenolphthalein and Total Alkalinity..................................................................................... 105
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Alkalinity, USEPA Buret Titration Method ............................................................................................. 111
Aluminum, Aluminon Method.............................................................................................................................................. 117
Aluminum, Eriochrome Cyanine R Method........................................................................................... 125
Arsenic, Silver Diethyldithiocarbamate Method .................................................................................... 133
Atrazine, Immunoassay ........................................................................................................................ 141
Barium, Turbidimetric Method............................................................................................................... 151
Benzotriazole/Tolyltriazole, UV Photolysis Method............................................................................... 157
Boron, Carmine Method........................................................................................................................ 163
Boron, Azomethine-H Method .............................................................................................................. 169
Scope and Application: Test preparation ........................................................................................ 169
Bromine, DPD Method.......................................................................................................................... 177
Cadmium, Dithizone Method ................................................................................................................ 185
Carbon Dioxide, Digital Titrator Method Using Sodium Hydroxide ....................................................... 193
Carbon Dioxide, Buret Titration Method ............................................................................................... 197
Chelant, Free, Digital Titrator Using Magnesium Chloride ................................................................... 201
Chelant, Total, Bismuth Nitrate Method ................................................................................................ 205
Chloramine (Mono);
Nitrogen, Free Ammonia, Indophenol Method...................................................................................... 209
Chloramine (Mono), Indophenol Method .............................................................................................. 219
Chloramine (Mono), Indophenol Method .............................................................................................. 225
Chloride, Mercuric Thiocyanate Method ............................................................................................... 231
Chloride, Mercuric Nitrate Method ........................................................................................................ 237
Chloride, Silver Nitrate Method............................................................................................................. 243
Chloride, USEPA Silver Nitrate Buret Titration Method ........................................................................ 249
Chlorine Dioxide, DPD Method............................................................................................................. 255
Chlorine Dioxide, Direct Reading Method............................................................................................. 263
Chlorine Dioxide, Chlorophenol Red Method ....................................................................................... 267
Chlorine Dioxide, Amaranth Method..................................................................................................... 273
Chlorine Dioxide, Direct Reading Method............................................................................................. 277
Chlorine Demand/Requirement, .......................................................................................................... 281
Chlorine, Free, DPD Rapid Liquid Method ........................................................................................... 289
Chlorine, Free, DPD Method ................................................................................................................ 295
Chlorine, Free, USEPA DPD Method ................................................................................................... 301
Chlorine, Free, USEPA Amperometric Buret Titration Method............................................................. 309
Chlorine, Free, DPD Method ................................................................................................................ 313
Chlorine, Free, Indophenol ................................................................................................................... 319
Chlorine, Total, USEPA DPD Method ................................................................................................... 327
Chlorine, Total, DPD Rapid Liquid Method ........................................................................................... 335
Chlorine, Total, DPD Method ................................................................................................................ 341
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Chlorine, Free and Total, DPD-FEAS Method ...................................................................................... 373
Chlorine, Free, Amperometric Forward Titration using 0.00564 N PAO............................................... 379
Chlorine, Hypochlorite, Iodometric Method........................................................................................... 385
Chlorine, Total, Amperometric Back Titration ....................................................................................... 389
Chlorine, Total, Amperometric Forward Titration .................................................................................. 399
Chlorine, Total, Iodometric Method using Sodium Thiosulfate.............................................................. 405
Chlorine, Total, USEPA Amperometric Buret Titration Method............................................................. 409
Chlorine, Total, Iodometric Method Using Sodium Thiosulfate ............................................................. 413
Chromate, Titration Method using Sodium Thiosulfate......................................................................... 421
Chromium, Hexavalent, USEPA 1,5-Diphenylcarbohydrazide Method ................................................ 427
Chromium, Total, Alkaline Hypobromite Oxidation Method, ................................................................. 433
Cobalt, 1-(2-Pyridylazo)-2-Naphthol (PAN) Method ............................................................................. 439
Color, True and Apparent, Platinum-Cobalt Standard Method, , ......................................................... 445
Color, ADMI, ADMI Weighted Ordinate Method ................................................................................... 449
Copper, USEPA Bicinchoninate Method............................................................................................... 455
Copper, Porphyrin Method.................................................................................................................... 463
Cyanide, Pyridine-Pyrazalone Method ................................................................................................. 469
Cyanuric Acid, Turbidimetric Method.................................................................................................... 479
Fluoride, USEPA SPADNS Method...................................................................................................... 483
Fluoride, USEPA SPADNS 2................................................................................................................ 491
Formaldehyde, MBTH Method.............................................................................................................. 499
Hardness, Calcium and Magnesium;
Calmagite Colorimetric Method....................................................................................................... 505
Hardness, Calcium and Magnesium; Chlorophosphonazo Colorimetric Method ................................. 511
Hardness, Total, Calcium and Magnesium; Chlorophosphonazo Rapid Liquid Method ....................... 517
Hardness, Calcium, Titration Method using EDTA ............................................................................... 523
Hardness, Calcium, USEPA Buret Titration Method............................................................................. 529
Hardness, Total, Sequential, Titration Method using EDTA.................................................................. 537
Hardness, Total, Titration Method using EDTA..................................................................................... 545
Hardness, Total, USEPA ManVer 2 Buret Titration Method ................................................................. 553
Hardness, Total, Sequential, Buret Titration Method ............................................................................ 561
Hydrazine, p-Dimethylaminobenzaldehyde Method ............................................................................. 569
Iodine, DPD Method ............................................................................................................................ 575
Iron, Ferrozine Method ....................................................................................................................... 583
Iron, Ferrous, 1-10 Phenanthroline Method.......................................................................................... 589
Iron, Total, USEPA FerroVer Method.................................................................................................. 595
Iron, Total, TPTZ Method ...................................................................................................................... 603
Iron, FerroZine Rapid Liquid Method .................................................................................................. 611
Iron, Total, FerroMo Method ................................................................................................................. 619
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Iron, TitraVer Titration Method.............................................................................................................. 625
Lead, USEPA Dithizone Method........................................................................................................... 629
Lead, LeadTrak Fast Column Extraction Method .............................................................................. 637
Manganese, USEPA Periodate Oxidation Method ............................................................................... 645
Manganese, 1-(2-Pyridylazo)-2-Naphthol PAN Method ....................................................................... 651
Mercury, Cold Vapor Mercury Concentration Method........................................................................... 657
Molybdenum, Mercaptoacetic Acid Method.......................................................................................... 669
Molybdenum, Ternary Complex Method............................................................................................... 675
Nickel, USEPA Heptoxime Method....................................................................................................... 681
Nickel, 1-(2 Pyridylazo)-2-Napthol (PAN) Method ................................................................................ 687
Nitrate, Chromotropic Acid Method....................................................................................................... 693
Nitrate, Cadmium Reduction Method.................................................................................................... 699
Nitrate, UV Screening Method .............................................................................................................. 725
Nitrite, Ferrous Sulfate Method............................................................................................................. 729
Nitrite, Diazotization Method................................................................................................................. 733
Nitrite, USEPA Diazotization................................................................................................................. 737
Nitrite, Ceric Acid Titration Method ....................................................................................................... 743
Nitrogen, Ammonia, USEPA Nessler Method....................................................................................... 749
Nitrogen, Ammonia, Salicylate Method................................................................................................. 755
Nitrogen, Free Ammonia, Indophenol Method...................................................................................... 773
Nitrogen, Total, Persulfate Digestion Method ....................................................................................... 781
Nitrogen, Total Inorganic, Titanium Trichloride Reduction Method ....................................................... 789
Nitrogen, Total Kjeldahl, Nessler Method (Digestion Required)............................................................ 797
Nitrogen, Total, Persulfate Digestion Method ....................................................................................... 805
Oil and Grease, USEPA Hexane Extractable Gravimetric Method....................................................... 813
Oil and Grease, USEPA Solid Phase Extraction Method ..................................................................... 829
Organic Carbon, Total, Direct Method .................................................................................................. 841
Organic Constituents
UV Absorbing (UV-254), Direct Reading Method ................................................................................. 865
Oxygen Demand, Biochemical, Dilution Method .................................................................................. 871
Oxygen Demand, Chemical, Manganese III Reactor Digestion Method (with optional chloride removal)..
885
Oxygen Demand, Chemical, (Manganese III Reactor Digestion Method) ........................................... 895
Manganese III Reactor Digestion Method (without chloride removal) .................................................. 895
Oxygen Demand, Chemical, USEPA Reactor Digestion Method ......................................................... 901
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Oxygen, Dissolved, HRDO Method ...................................................................................................... 913
Oxygen, Dissolved, Indigo Carmine Method ........................................................................................ 917
Oxygen, Dissolved, Ultra High Range Method ..................................................................................... 921
Oxygen, Dissolved, Azide Modification of Winkler Method................................................................... 925
Oxygen, Dissolved, USEPA Azide Modification of Winkler Method ..................................................... 933
Oxygen Scavengers, Iron Reduction Method for Oxygen Scavengers ................................................ 939
Ozone, Indigo Method .......................................................................................................................... 945
Polychlorinated Biphenyls (PCB) in Soil, Immunoassay Method.......................................................... 949
Phenols, USEPA 4-Aminoantipyrine Method........................................................................................ 959
Phosphonates, Persulfate UV Oxidation Method ................................................................................. 967
Phosphorus, Acid Hydrolyzable Digestion, USEPA Acid Digestion Method......................................... 975
Phosphorus, Acid Hydrolyzable, PhosVer 3 with Acid Hydrolysis Method ....................................... 979
Phosphorus, Reactive (Orthophosphate), Molybdovanadate Method .................................................. 987
Phosphorus, Reactive (Orthophosphate), Amino Acid Method ............................................................ 995
Phosphorus, Reactive, Molybdovanadate Rapid Liquid Method ........................................................ 1001
Phosphorus, Reactive, Ascorbic Acid Rapid Liquid Method............................................................... 1009
Phosphorus, Reactive (Orthophosphate), USEPA PhosVer 3 (Ascorbic Acid) Method ..................... 1017
Phosphorus, Reactive (Orthophosphate), USEPA PhosVer 3 Method ............................................ 1025
Phosphorus, Reactive
(Orthophosphate), Molybdovanadate Method .................................................................................... 1031
Phosphorus, Total, USEPA
PhosVer 3 with Acid Persulfate Digestion Method ..................................................................... 1039
Phosphorus, Total, Digestion, USEPA Acid Persulfate Digestion Method.......................................... 1047
Phosphorus, Total, Molybdovanadate Method with Acid Persulfate Digestion ................................... 1051
Potassium, Tetraphenylborate Method............................................................................................... 1059
Quaternary Ammonium Compounds, Direct Binary Complex Method ............................................... 1065
Salinity, Mercuric Nitrate Method ........................................................................................................ 1071
Selenium, Diaminobenzidine Method ................................................................................................. 1075
Silica, Silicomolybdate Method ........................................................................................................... 1085
Silica, Heteropoly Blue Method........................................................................................................... 1091
Silica, Heteropoly Blue Rapid Liquid Method...................................................................................... 1097
Silica, Heteropoly Blue Method........................................................................................................... 1105
Silver, Colorimetric Method................................................................................................................. 1113
Solids, Settleable Matter, Direct Measurement................................................................................... 1121
Solids, Nonfilterable Suspended Solids; Total and Volatile, USEPA Gravimetric Method .................. 1123
Solids, Total Filterable
(Total Dissolved Solids), USEPA Gravimetric Method ........................................................................ 1129
Solids, Volatile Dissolved and Fixed Dissolved, Gravimetric Method ................................................. 1133
Solids, Total Volatile and Fixed, Gravimetric Method.......................................................................... 1139
Solids, Total, USEPA Gravimetric Method .......................................................................................... 1143
Suspended Solids, Photometric Method............................................................................................. 1147
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Table of Contents
Sulfate, USEPA SulfaVer 4 Method.................................................................................................... 1151
Sulfide, USEPA Methylene Blue Method............................................................................................ 1159
Sulfite, Iodate-Iodide Buret Titration Method ...................................................................................... 1163
Sulfite, Iodate-Iodide Method.............................................................................................................. 1167
Sulfite, Colorimetric Method................................................................................................................ 1171
Surfactants, Anionic (Detergents), Crystal Violet Method................................................................... 1175
Tannin and Lignin, Tyrosine Method................................................................................................... 1181
Toxicity, ToxTrak Method , ............................................................................................................................................ 1185
TPH (Total Petroleum Hydrocarbons), Immunoassay ........................................................................ 1191
Trihalomethanes, THM Plus Method............................................................................................... 1203
Trihalomethane Formation Potential (THMFP), ................................................................................. 1213
Volatile Acids, Esterification Method................................................................................................... 1221
Volatile Acids, Sodium Hydroxide Method .......................................................................................... 1227
Volatile Acids, Buret Titration Method................................................................................................. 1231
Zinc, USEPA Zincon Method .............................................................................................................. 1235
Microbiology .................................................................................................................................. 1241

Bacteria Test Guidelines, ................................................................................................................... 1243
Membrane Filtration Guidelines, ........................................................................................................ 1247
Bacteria, Membrane Filtration Method................................................................................................ 1255
ColiformsTotal, Fecal and E. coli, USEPA Membrane Filtration Method......................................... 1261
ColiformsFecal, USEPA Membrane Filtration Method .................................................................... 1275
ColiformsE. coli, USEPA Membrane Filtration Method ................................................................... 1283
ColiformsE. coli, USEPA Membrane Filtration Method ................................................................... 1289
ColiformsTotal and E. coli, USEPA Membrane Filtration Method ................................................... 1295
Enterococci, Membrane Filtration Method .......................................................................................... 1303
Heterotrophic Bacteria, Membrane Filtration Method ......................................................................... 1309
Pseudomonas, Membrane Filtration Method...................................................................................... 1333
MPN Dilution Guidelines, ColiformsFecal, USEPA A-1 Medium..................................................... 1343
ColiformsTotal and E. Coli, Lauryl Tryptose with MUG Broth.......................................................... 1349
ColiformsTotal, Fecal and E. Coli, USEPA Lauryl Tryptose Broth presumptive test with BGB, EC Medium and EC/MUG confirmation ...................................................................................................... 1359
ColiformsTotal, Fecal and E. Coli, USEPA Lauryl Tryptose Broth presumptive test with BGB, EC Medium and EC/MUG confirmation ...................................................................................................... 1369
Bacteria, Hydrogen Sulfide Producing, Most Probable Number (MPN) Method ................................ 1377
Coliforms, Presence/Absence ............................................................................................................ 1381
Bacteria, Hydrogen Sulfide Producing, Presence/Absence (P/A) Method ........................................ 1387
Heterotrophic Bacteria, Pour Plate Method ........................................................................................ 1391
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Table of Contents
Total Aerobic Bacteria, Yeasts and Molds, Total Aerobic Bacteria (Amber)/Yeast and Mold (Red)
Total Aerobic Bacteria (Amber)/Total Coliform (Red)
Total Aerobic Bacteria (Amber)/Disinfection Control (Purple)....................................................... 1425
Electrochemistry ........................................................................................................................... 1431

Conductivity, USEPA Direct Measurement Method ............................................................................ 1433
Fluoride in Acid Solutions, Direct Measurement ISE Method ............................................................. 1439
Fluoride in Drinking Water, USEPA Direct Measurement ISE Method ............................................... 1447
Nitrate, Direct Measurement ISE Method ........................................................................................... 1457
Nitrate, Direct Measurement ISE Method ........................................................................................... 1469
Nitrogen, Ammonia, USEPA Direct Measurement ISE Method.......................................................... 1479
Nitrogen, Ammonia, USEPA Known Addition ISE Method ................................................................. 1489
Sodium, Direct Measurement ISE Method ......................................................................................... 1497
Oxygen, Dissolved, Direct Measurement Method .............................................................................. 1507
Oxygen Demand, Biochemical, Dilution Method ................................................................................ 1515
Oxygen, Dissolved, Direct Measurement Method .............................................................................. 1529
Oxidation Reduction Potential
(ORP), Direct Measurement Method .................................................................................................. 1533
pH, USEPA Electrode Method............................................................................................................ 1539
Chemical Procedures Explained ................................................................................................ 1545

Acidity, For water, wastewater and seawater ..................................................................................... 1547
Alkalinity, For water, wastewater and seawater .................................................................................. 1549
Aluminum, For water........................................................................................................................... 1552
Barium, For water, wastewater, oil-field water and seawater ............................................................. 1553
Boron, For water and wastewater....................................................................................................... 1554
Benzotriazole and Tolyltriazole, For water .......................................................................................... 1556
Carbon Dioxide, For water and seawater ........................................................................................... 1557
Chloramine (Mono), For water and wastewater.................................................................................. 1558
Chloramine (Mono);
Nitrogen, Free Ammonia, For determining free ammonia and monochloramine simultaneously in finished
chloraminated water ..................................................................................................................... 1559
Chloride, For water and wastewater ................................................................................................... 1561
Chlorine Dioxide, For water and wastewater ...................................................................................... 1563
Chlorine, Free and Total, For water, wastewater and seawater ......................................................... 1565
Chromium, For water and wastewater................................................................................................ 1567
Cobalt, For water ................................................................................................................................ 1568
Copper, For water, wastewater and seawater .................................................................................... 1570
Cyanide, For water, wastewater and seawater................................................................................... 1573
Fluoride, For water and seawater ....................................................................................................... 1575
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Table of Contents
Formaldehyde, For water.................................................................................................................... 1577
Hardness, For water, wastewater and seawater ................................................................................ 1579
Hydrazine, For water and boiler water................................................................................................ 1583
Iron, For water and seawater.............................................................................................................. 1584
Langelier and Aggressive Indices, Method 8073................................................................................ 1588
Lead, For water and wastewater ........................................................................................................ 1593
Manganese, For water and wastewater.............................................................................................. 1594
Mercury, Cold Vapor, Molybdenum, Molybdate, For water ................................................................. 1597
Nickel, For water................................................................................................................................. 1599
Nitrogen, Ammonia, For water, wastewater and seawater ................................................................. 1600
Nitrogen, Kjeldahl, For water and wastewater .................................................................................... 1601
Nitrogen, Nitrate, For water and wastewater ...................................................................................... 1602
Nitrogen, Nitrite, For water and wastewater ....................................................................................... 1604
Nitrogen, Total, For water, wastewater and seawater......................................................................... 1606
Organic Carbon, Total, For water and wastewater ............................................................................. 1607
Oxygen Demand, Chemical, Mn III, For water and wastewater ......................................................... 1608
Oxygen Demand, Chemical, For wastewater ..................................................................................... 1610
Oxygen, Dissolved, For water, wastewater, and seawater................................................................. 1612
Oxygen Scavengers, For water .......................................................................................................... 1615
Ozone, For water ................................................................................................................................ 1616
pH, pH Indicators, For water and wastewater..................................................................................... 1621
Phenols, For water, wastewater and seawater................................................................................... 1626
Phosphonates, For water.................................................................................................................... 1627
Phosphorous, For water, wastewater and seawater........................................................................... 1628
Potassium, For water and wastewater................................................................................................ 1630
Selenium, For water and wastewater ................................................................................................. 1631
Silica, For water and seawater ........................................................................................................... 1632
Sulfate, For water, seawater and oil-field water.................................................................................. 1633
Sulfide, For water, wastewater and seawater..................................................................................... 1634
Sulfite, For water, wastewater and seawater...................................................................................... 1635
Turbidity, Zinc, For water and wastewater .......................................................................................... 1639
Technical Support ......................................................................................................................... 1641
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Acid/Base
Acidity
Alkalinity
Aluminum
Arsenic
Ascorbic Acid
Bacteria
Boron
Bromine
Cadmium
Calcium
Carbon Dioxide
Chelants
Chloride
Chlorine
Chlorine Dioxide
Chromate
Chromium
(Hexavalent)
Chromium (Total)
Cobalt
COD
Color
Conductivity
Copper
Cyanide
Cyanuric Acid
Detergents
Water Conditioning
Wastewater, Municipal
Wastewater, Industrial
Ultrapure Water
Textile Industry
Solid Waste/Sludge
Semiconductor Manufacture
Pulp & Paper Mills
Power Plant Utilities
Pools & Spas
Pharmaceutical Manufacture
Petroleum Industry
Metals/Mining, Mfg & Finishing
Food/Feed Industry
Environmental Testing
Education
Drinking Water
Commercial Laundries
Barium
BOD
Chlorine Production
Chemical Manufacture
Boiler/Cooling Water
Beverages/Bottled Water
Aquarium Testing
Aquaculture
Agriculture
Applications Guide
Applications Guide
Page 11
Hydrazine
Hydrogen
Peroxide
Dissolved Oxygen
Erythorbic Acid
Fluoride
Formaldehyde
Gluteraldehyde
Glycols
Hardness
Hydrogen Sulfide
Iodide
Iodine
Iron (Ferrous)
Iron (Total)
Lead
Manganese
Mercury
Molybdenum
Nickel
Nitrogen
Ammonia
Nitrogen (Total)
Nitrogen (Nitrite)
Oil and Grease
Oxygen Scavenger
Applications Guide
Page 12
Water Conditioning
Ultrapure Water
Textile Industry
Pulp & Paper Mills
Solid Waste/Sludge
Pools & Spas
Petroleum Industry
Food/Feed Industry
Education
Drinking Water
Nitrogen
(Monochloramine)
Nitrogen (TKN)
Nitrogen
(Inorganic)
Nitrogen (Nitrate)
Chlorine Production
Aquarium Testing
Aquaculture
Agriculture
Applications Guide
Ozone
PCB
Permanganate
pH
Phenols
Phosphate
Phosphonates
Phosphorus
Potassium
QAC
Salinity
Selenium
Silica
Silver
Sodium
Sodium Chromate
Tannin
Toxicity
TPH
Triazole
Volatile Acids
Water in Oil
Zinc
Water Conditioning
Ultrapure Water
Textile Industry
Solid Waste/Sludge
Pulp & Paper Mills
Pools & Spas
Petroleum Industry
Sulfite
Turbidity
Sulfide
TDS
Sodium
Hydroxide
Sulfate
Food/Feed Industry
Education
Drinking Water
Chlorine Production
Aquarium Testing
Aquaculture
Agriculture
Applications Guide
Applications Guide
Page 13
Applications Guide
Page 14
Section 1
Abbreviations and Conversions
1.1 Procedure abbreviations

The abbreviations in Abbreviations table are common in written chemical procedures:
Table 1 Abbreviations
Abbreviation Definition
Abbreviation Definition
degree(s) Celsius (Centigrade)
litervolume equal to one cubic decimeter

(dm3)
degree(s) Fahrenheit
LR
low range
ACS
American Chemical Society reagent grade

purity
MDL
method detection limit
MDB
marked dropping bottle
mg/L
milligrams per liter (ppm)
g/L
micrograms per liter (ppb)
APHA
Standard
Methods
Standard Methods for the Examination of

Water and Wastewater, published jointly by
the American Public Health Association
(APHA), the American Water Works
Association (AWWA) and the Water
Environment Federation (WEF), is the
standard reference work for water analysis.
Order from Hach Company, requesting
Catalog No. 2270800 or from the Publication
Office of the APHA. Many procedures
contained in this manual are based on
Standard Methods.
mL
milliliter1/1000 of a liter. It is approximately

the same as a cubic centimeter (and is
sometimes called a cc).
MR
medium range
NIPDWR
National Interim Primary Drinking Water

Regulations
AV
AccuVac
NPDES
National Pollutant Discharge Elimination

System
Bicn
bicinchoninate
phosphorus
conc
concentrated
PCB
poly chlorinated biphenyl
DB
dropping bottle
ppb
parts per billion
DBP
disinfection by-products
ppm
parts per million
CFR
Code of Federal Regulations
RL
Rapid Liquid
EDL
Estimated detection limit
SCDB
self-contained dropping bottle
EPA
Environmental Protection Agency
THM
trihalomethane
F&T
free and total
TNT
Test N Tube
FM
FerroMo
TOC
total organic carbon
FV
FerroVer
TPH
total petroleum hydrocarbons
FZ
FerroZine
TPTZ
2,4,6-Tri-(2-Pyridyl)-1,3,5-Triazine
grams
USEPA
United States Environmental

Protection Agency
gr/gal
grains per gallon (1 gr/gal = 17.12 mg/L)
ULR
ultra low range
HR
high range
Page 15
1.2 Conversions
1.2.1 Chemical species
Species conversion factors for many commonly used chemicals are listed in the
Conversion factors table.
Table 2 Conversion factors
Page 16
To Convert From...
To...
Multiply By...
mg/L Al
mg/L Al2O3
1.8895
mg/L B
mg/L H3BO3
5.7
mg/L Ca-CaCO3
mg/L Ca2+
0.4004
mg/L CaCO3
mg/L Ca2+
0.4004
mg/L CaCO3
mg/L Mg2+
0.2428
g/L Carbo.
g/L Hydro.
1.92
g/L Carbo.
g/L ISA
2.69
g/L Carbo.
g/L MEKO
3.15
mg/L Cr6+
mg/L CrO42
2.231
mg/L Cr6+
mg/L Na2CrO4
3.115
mg/L Cr6+
mg/L Cr2O72
2.077
mg/L MgCaCO3
mg/L Mg2+
0.2428
mg/L Mn
mg/L KMnO4
2.876
mg/L Mn
mg/L MnO4
2.165
mg/L Mo6+
mg/L MoO42
1.667
mg/L Mo6+
mg/L Na2MoO4
2.146
mg/L N
mg/L NH3
1.216
mg/L N
mg/L NO3
4.427
mg/L Cl2
mg/L NH2Cl
0.726
mg/L Cl2
mg/L N
0.197
mg/L NH3N
mg/L NH3
1.216
mg/L NH3N
mg/L NH4+
1.288
mg/L NO2
mg/L NaNO2
1.5
mg/L NO2
mg/L NO2N
0.3045
mg/L NO2N
mg/L NaNO2
4.926
g/L NO2N
g/L NaNO2
4.926
mg/L NO2N
mg/L NO2
3.284
g/L NO2N
g/L NO2
3.284
mg/L NO3N
mg/L NO3
4.427
mg/L PO43
mg/L P
0.3261
g/L PO43
g/L P
0.3261
mg/L PO43
mg/L P2O5
0.7473
g/L PO43
g/L P2O5
0.7473
mg/L SiO2
mg/L Si
0.4674
g/L SiO2
g/L Si
0.4674

1.2.2 Hardness conversion
See the Hardness conversion factors table for the factors to convert hardness from one
unit of measure to another. For example, to convert mg/L CaCO3 to German
parts/100,000 CaO, multiply the value in mg/L x 0.056.
Table 3 Hardness conversion factors
mg/L
CaCO3
British
gr/gal
(Imperial)
CaCO3
American
gr/gal (US)
CaCO3
French
Parts/
100,000 CaCO3
German
Parts/
100,000 CaO
meq/L1
g/L CaO
lb/cu ft
CaCO3
mg/L
CaCO3
1.0
0.07
0.058
0.1
0.056
0.02
5.6x104
6.23x105
English
gr/gal
CaCO3
14.3
1.0
0.83
1.43
0.83
0.286
8.0x103
8.9x104
US gr/gal
CaCO3
17.1
1.2
1.0
1.72
0.96
0.343
9.66x103
1.07x103
Fr. p/
100,000
CaCO3
10.0
0.7
0.58
1.0
0.56
0.2
5.6x103
6.23x104
Ger.
p/100,000
CaO
17.9
1.25
1.04
1.79
1.0
0.358
1x102
1.12x103
Units of
Measure
50.0
3.5
2.9
5.0
2.8
1.0
2.8x102
3.11x102
g/L CaO
1790.0
125.0
104.2
179.0
100.0
35.8
1.0
0.112
lb/cu ft
CaCO3
16,100.0
1,123.0
935.0
1,610.0
900.0
321.0
9.0
1.0
meq/L
1 epm/L
or mval/L
meq
= N 1000
L
Note: -----------
Page 17
Page 18
Section 2
Laboratory Practices
2.1 Temperature
Most methods perform accurately when the sample temperature is between 20 and 25 C
(68 to 77 F). A note in the individual procedure will state any special temperature
requirements.
2.2 Mixing
Swirling is recommended when mixing samples in a graduated cylinder or a titration
flask. Swirling is the gentlest method of mixing and offers the least chance for
atmospheric contamination when testing for carbon dioxide and other gases.
1. Grip the cylinder (or flask) firmly with the tips of the thumb and first two fingers (see
the Swirl a cylinder and invert a sample cell figure).
2. Hold the cylinder at a 45-degree angle and make a circling motion from the wrist.
3. Move the cylinder in an approximately 12-inch circle, creating enough rotation to
complete the mixing in a few turns.
Mixing sample in a square sample cell:
1. Grasp the neck of the cell with the thumb and index finger of one hand. Rest the
concave bottom of the cell on the tip of the index finger of the other hand.
2. Mix by rotating the cell quickly one way and then in the reverse direction.
Figure 1 Rotate a sample cell
Page 19
Inverting allows for thorough mixing in a capped sample cell or a mixing cylinder.
1. Hold the cell or cylinder, in a vertical position with the cap on top.
2. Invert so that the cap is on the bottom. Return the cell to its original position (see
Swirl a cylinder and invert a sample cell figure.) Repeat as needed.
Swirl a cylinder and invert a sample cell
Figure 2 Swirl a cylinder and invert a sample cell
2.3 Digestion
Several procedures require sample digestion. Digestion uses chemicals and heat to
break down a substance into components that can be analyzed. This section briefly
describes three different digestion procedures.
The Digesdahl system is a process that yields a digest suitable for the determination of
metals, total phosphorus and total Kjeldahl nitrogen (TKN). It is rapid, convenient and is
very effective at destroying interfering organic materials.
For USEPA reporting purposes, USEPA-approved digestions are required. USEPA
presents two digestions (mild and vigorous) for metals analysis. Other digestion
procedures are required for mercury, arsenic, phosphorus and TKN.
See Sample Pretreatment by Digestion for more information on sample digestion.
2.4 Distillation
Distillation is an effective, easy and safe method of separating some chemical
components for analysis. The following equipment is recommended for distillation:
Page 20
General Purpose Distillation Apparatus (Catalog No. 2265300), shown in the General
purpose distillation apparatus figure
Arsenic Distillation Apparatus Set (Catalog No. 2265400)
Cyanide Distillation Apparatus Set (Catalog No. 2265800)
General Purpose Heater and Support Apparatus (Catalog No. 2274400, 115 VAC,
60 Hz)
General Purpose Heater and Support Apparatus (Catalog No. 2274402, 230 VAC,
50 Hz)
Note: When ordering the Cyanide or Arsenic Distillation Apparatus, always order in conjunction with
the General Purpose Distillation Apparatus and the General Heater and Support Apparatus.
The Distillation Apparatus is suitable for water and wastewater that requires sample
pretreatment by distillation. Applications for the General Purpose Apparatus include:
fluoride, albuminoid nitrogen, ammonia nitrogen, phenols, selenium and volatile acids.
The General Purpose Heater and Support Apparatus provides efficient heating and
anchoring of the glassware.
General purpose distillation apparatus
Figure 3 General purpose distillation apparatus
2.5 Filtration
Filtration separates particulates from an aqueous sample. It uses a porous medium that
retains particulates but allows liquids to pass through. It is useful for removing turbidity
(which may interfere in colorimetric analyses) from water samples.
The two methods most frequently used filtration methods are vacuum and gravity
filtration.
Page 21
2.5.1 Vacuum filtration
Vacuum filtration uses both suction and gravity to draw the liquid through the filter. An
aspirator or vacuum pump is used to create suction (see the Vacuum filtration figure).
Vacuum filtration is faster than gravity filtration alone.
To filter using a vacuum:
1. Use tweezers to place a filter paper into the filter holder.
2. Place the filter holder assembly in the filtering flask. Dampen the filter with deionized
water to make sure adhesion to the holder.
3. Position the funnel housing on the filter holder assembly.
4. While applying a vacuum to the filtering flask, transfer the sample to the filtering
apparatus.
5. When the filtration is complete, slowly release the vacuum from the filtering flask and
transfer the solution from the filter flask to another container.
Vacuum filtration
Figure 4 Vacuum filtration
2.5.2 Required apparatus for vacuum filtration

Table 4 Required apparatus for vacuum filtration
Description
Filter Discs, glass fiber, 47-mm
Unit
Catalog. No.
100/pkg
253000
Filter Holder, membrane, 47-mm
each
1352900
Flask, filtering, 500-mL
each
54649
Select one of the following:

Pump, vacuum, hand operated
each
2824800
Pump, vacuum, portable, 115 VAC
each
1469700
Pump, vacuum, portable, 230 VAC
each
2824801
2074145
each
1428200
Tubing, vacuum
Tweezers
Page 22
2.5.3 Gravity filtration
Many chemical procedures use gravity filtration. This requires filter paper, a conical funnel
and a receiving flask (see the Gravity filtration figure). Gravity filtration is better for
retaining fine particles. The rate of filtration increases as the volume increases in the filter
cone, but never fill the cone more than three-quarters full.
Note: Pretreatment using acid and heat is often necessary for metals testing. Filter paper will not
withstand such an environment; therefore, vacuum filtration with glass fiber filter discs is
recommended. Also, glass filter discs do not retain colored species like the paper filters do.
To filter using gravity:

1. Place a folded filter paper into the funnel.
2. Dampen the filter paper with deionized water so it adheres to the funnel.
3. Place the funnel into an Erlenmeyer flask or graduated cylinder.
4. Pour the sample into the funnel.
Gravity filtration
Figure 5 Gravity filtration
Table 5 Required apparatus for gravity filtration

Description
Unit
Cylinder, graduated, 100-mL
each
50842
Funnel, poly, 65-mm
each
108367
100/pkg
189457
each
50543
Filter Paper, 12.5-cm, pleated

Flask, Erlenmeyer, 125-mL
Catalog. No.
Page 23
2.6 Reagents
2.6.1 Reagent and standard stability
In general, reagents and standards have the maximum shelf life when stored in a location
that is cool, dark, and dry. The product label will specify any special storage needs.
It is always good laboratory practice to date chemicals upon receipt and to rotate supplies
so the older supplies are used first. When the reagent shelf life is unknown or in doubt,
run a standard to check reagent effectiveness.
Absorption of moisture, carbon dioxide or other gases from the atmosphere, bacterial
action, high temperatures or light (with photosensitive compounds) may affect the reagent
shelf life. In some cases, reaction with the storage container or interaction of reagent
components may occur.
2.6.2 Reagent blank

In several tests, the contribution of the reagent(s) to the final reading is of such a
magnitude that it must be compensated for whenever the test is performed. Reagent
blank refers to that portion of the test result contributed solely by the reagent. This
produces a positive error in the test results.
Every effort is made to produce reagents with the lowest possible blank; and, for most
reagents, it is less than 0.009 absorbance units. However, it is sometimes impossible or
impractical to produce reagents with such a low blank. When using such reagents, it is
best to determine the reagent blank by performing the procedure using high-quality water
(deionized, distilled, etc.) in place of sample to zero the instrument. The resulting value
is then expressed in the concentration units of the test and is subtracted from each
sample determination that uses the same reagent lot. Spectrophotometer software allows
the reagent blank value to be stored and subtracted automatically from each sample
value. The reagent blank needs to be determined only at first use, when a new lot of
reagent has been opened or if contamination is suspected.
In most tests, the reagent blank is so small the instrument may be zeroed on either an
untreated portion of the original water sample or on deionized water. This will not result in
a significant loss of accuracy unless the test is for very low levels of the species of
interest. It is then best to use a reagent blank prepared as above.
2.7 Sample dilution

Most colorimetric tests use volumes of 10 and 25 mL. However, in some tests, the color
developed in the sample may be too intense to be measured due to high levels of
analyteor or unexpected colors may develop due to an interference. In either case, dilute
the sample to produce a measurable endpoint or to determine if interfering substances
are present.
To dilute the sample:
1. Pipet the chosen sample portion into a clean graduated cylinder (or volumetric flask
for more accurate work).
2. Fill the cylinder (or flask) to the desired volume with deionized water.
3. Mix well. Use the diluted sample when running the test.
The Sample dilution volumes table shows relative quantities and the multiplication factors
to use with a 25 mL graduated cylinder. The concentration of the sample is equal to the
diluted sample result multiplied by the multiplication factor.
Page 24
Table 6 Sample dilution volumes
mL Deionized Water
used to bring the volume
to 25 mL
Sample
volume (mL)
1 For
Multiplication factor
25.0
0.0
12.5
12.5
10.01
15.0
2.5
5.01
20.0
2.51
22.5
10
1.01
24.0
25
0.2501
24.75
100
sample sizes of 10 mL or less, use a pipet to measure the sample into the graduated cylinder or volumetric flask.
More accurate dilutions can be done with a pipet and a 100-mL volumetric flask
(See table for Multiplication factors for diluting to 100 mL).
1. Pipet the sample and dilute to volume with deionized water.
2. Stopper and invert to mix.stopper and invert to mix.
Table 7 Multiplication factors for diluting to 100 mL
Sample volume (mL)
Multiplication factor
100
50
20
10
10
25
50
2.7.1 Sample dilution with interfering substances

Sample dilution may influence the level at which a substance interferes. The effect of the
interferences decreases as the dilution increases. In other words, higher levels of an
interfering substance can be tolerated in the original sample if it is diluted before analysis.
Example:
Copper does not interfere at or below 100 mg/L for a 25 mL sample in a procedure. If the
sample volume is diluted with an equal volume of water, what is the level at which copper
will not interfere?
Total volume
----------------------------------------- = Dilution factor
Sample volume
25 ---------= 2
12.5
Interference Level Dilution Factor = Interference level in sample
100 2 = 200
The level at which copper will not interfere in the diluted sample is at or below 200 mg/L.
Page 25
2.8 AccuVac Ampuls

AccuVac Ampuls contain pre-measured powder or liquid vacuum-packed in optical-quality
glass ampules.
To use AccuVac Ampuls:
1. Collect the sample in a beaker or other open container.
2. Use one of the following methods to break the tip off the ampule:
Use the optional AccuVac Snapper (Catalog. No. 2405200). See How to use the
AccuVac snapper figure for instructions or;
Place the ampule tip well below the sample surface and break the tip off (see How
to use AccuVac Ampuls figure) against the beaker wall. The break must be far
enough below the surface to prevent air from being drawn in as the level of the
sample drops.
3. Secure an ampule cap over the tip of the ampule. Invert the ampule several times to
dissolve the reagent. The cap protects from broken glass and provides a grip for
inserting and removing the ampul from the cell holder. Wipe the ampule with a
lint-free cloth to remove fingerprints.
Note: Without the cap, the liquid will stay in the ampule when inverted. DO NOT place fingers
over broken glass!
4. Insert the ampule into the sample cell holder and read the results directly.
How to use AccuVac Ampuls
Figure 6 How to use AccuVac Ampuls
2.8.1 Using the AccuVac Snapper

To use the AccuVac Snapper:
1. Hold the Snapper with the open end up.
2. Gently slip the ampule into the Snapper, point first, until the tip touches the ramp at
the bottom of the Snapper.
3. Hold the Snapper between the index and middle finger (like a syringe). With the
ampule tip down, lower the Snapper into the sample until the ampule shoulder is wet.
4. Press on the flat end of ampule with the thumb (as if depressing the plunger on a
syringe) until the tip snaps. Allow the ampule to fill before removing from sample.
5. Rinse the wet end of the Snapper and ampule with clean water, if desired. Remove
the ampule from Snapper.
6. Dispose of the ampule tip (retained in the Snapper) in a suitable waste receptacle.
Page 26
How to use the AccuVac snapper
Figure 7 How to use the AccuVac snapper
2.9 PermaChem pillows

Permachem pillows utilize powdered reagents to minimize deterioration and the risk of
reagent spills.
To use PermaChem pillows (see the How to open PermaChem pillows figure):
1. Tap the pillow on a hard surface to collect the powdered reagent in the bottom.
2. Locate the tear notch and tear (or cut) across, holding the pillow away from the face.
3. Using two hands, push both sides toward each other to form a spout.
4. Pour the pillow contents into the sample cell and continue the procedure according to
the instructions.
How to open PermaChem pillows
Figure 8 How to open PermaChem pillows
Page 27
2.10 Sample cells

A set of sample cells are shipped with each photometric instrument. The same solution in
both cells will give the same absorbance (within 0.002 Abs for properly matched cells).
For more information, see the section on Match sample cells.
For accurate results, use only the sample cells specified in each procedure. Due to
differences in cell pathlengths, sample cell substitution will introduce bias in test results.
For example, 1-inch square cells have a pathlength approximately 8% longer than 1-inch
round cells; substitution of round cells for square cells will introduce a bias in the reading.
2.10.1 Orientation of sample cells

To minimize measurement variability when using a particular cell, always orient the cell in
the same manner before placing it into the cell holder. The fill marks on the cells can be
used as orientation guides for positioning the cells.
2.10.2 Maintain sample cells

When not in use, store the sample cells in the supplied boxes to protect from scratches
and breakage. After use, empty and clean the cells; avoid leaving colored solutions in the
cells for extended periods of time.
2.10.3 Clean sample cells

Most laboratory detergents can be used at recommended concentrations. Neutral
detergents, such as Liquinox, are safer when regular cleaning is required. Cleaning times
are reduced by increasing the temperature or by using an ultrasonic bath. Finish by
rinsing a few times with deionized water and allow to air dry.
Cells may also be cleaned with acid, followed by rinsing thoroughly with deionized water.
Note: Always use acid to clean cells used for low-level metal tests.
Individual procedures may require special cleaning methods. When using a brush to
clean cells, take extra care to avoid scratching the inner surfaces of the cells.
2.10.4 Match sample cells

Although sample cells shipped with the spectrophotometer instrument are distortion-free,
nicks and scratches from handling may cause an optical mismatch between two sample
cells and introduce error into the test results. This type of error may be avoided by
optically matching the sample cells as follows:
Note: Refer to the spectrophotometer user manual for specific steps required to select wavelengths
and zero the instrument.
1. Power on the instrument. Make sure Display Lock is off or the Reading mode is set to
Continuous.
2. Select a wavelength of 510 nm or the wavelength to be used for the test.
3. Pour at least 10 mL (25 mL for 25-mL cells) of deionized water into each of two
sample cells.
4. Place one sample cell into the cell holder with the fill mark facing the user.
5. Zero the instrument.
6. Place the other sample cell into the cell holder with the fill line facing the user.
7. Wait for the value to stabilize and read the absorbance. Record the resulting
absorbance.
Page 28
8. Turn the cell 180 and repeat step 6. Try to achieve an absorbance value within
0.002 Abs of the first cell. Note the orientation of the cell.
If the sample cells cannot be matched within 0.002 Abs, they may still be used by
compensating for the difference. For example, if the second cell reads 0.003 absorbance
units higher than the first cell, correct future readings (when using these two cells) by
subtracting 0.003 absorbance units (or the equivalent concentration) from the reading.
Likewise, if the second cell reads 0.003 absorbance units, add 0.003 absorbance units
to the reading.
2.11 Other apparatus

2.11.1 Boiling aids
Boiling is required in some procedures. Under some conditions bumping may occur
causing sample loss or injury. Bumping is caused by the sudden, almost explosive,
conversion of water to steam as it is heated. Use of a boiling aid, such as Boiling Chips
(Catalog. No. 1483531), reduces bumping.
Make sure the boiling aids will not contaminate the sample. Do not use boiling aids
(except glass beads, Catalog. No. 259600) more than once. Use a large enough flask or
beaker to allow significant headspace above the solution. Loosely covering the sample
during boiling will prevent splashing, reduce the chances of contamination and minimize
sample loss.
Individual procedures will recommend the specific boiling aid to use.
2.12 Achieve accuracy in measurement

2.12.1 Pipets and graduated cylinders
When smaller sample quantities are used, the accuracy of measurements becomes
increasingly important. The Reading the meniscus figure illustrates the proper way to
read the sample level using the meniscus formed when the liquid wets the graduated
cylinder or pipet walls.
Reading the meniscus
50
45
40
35
Figure 9 Reading the meniscus

Before filling, rinse the pipet or cylinder two or three times with the sample to be tested.
Use a pipet filler or pipet bulb to draw the sample into the pipet.
CAUTION
Never pipet chemical reagent solutions or samples by mouth.
When filling a pipet, keep the tip of the pipet below the surface of the sample as the
sample is drawn into the pipet.
Page 29
Serological pipets have marks that indicate the volume of liquid delivered by the pipet.
The marks may extend to the tip of the pipet or may be only on the straight portion of the
tube. If the marks are only on the straight part of the tube, fill serological pipets to the
zero mark and discharge the sample by draining the sample until the meniscus is level
with the desired mark. If the serological pipet has marks extending to the tip of the pipet,
fill the pipet to the desired volume and drain all the sample from the pipet. Then use a
pipet filler to blow the sample out of the pipet tip with the pipet filler for accurate
measurements.
Volumetric (transfer) pipets have a bulb in the middle and a single ring above the bulb to
indicate the volume of liquid when it is filled to the mark. To discharge a volumetric pipet,
hold the tip of the pipet at a slight angle against the container wall and drain. Do not
discharge the solution remaining in the tip of the pipet after draining. Volumetric pipets
are designed to retain a small amount of sample in the pipet tip.
If droplets of sample cling to the walls of the pipet, the pipet is dirty and is not delivering
the correct amount of sample. Wash the pipet thoroughly with a laboratory detergent or
cleaning solution and rinse several times with deionized water.
2.12.2 The Pour-Thru cell

The Pour-Thru Cell is an optional accessory that improves accuracy and makes
measuring more convenient for rapid liquid methods. Methods that use 25-mL samples
and sample cells can use the Pour-Thru Cell if specified in the procedure. The Pour-Thru
Cell cannot be used with 10-mL sample sizes and reagents.The Cell cannot be used
directly with a method unless it is specified in the procedure. For more information, see
the photometer user manual.
See the photometer user manual for installation and operation instructions.
Pour the solution into the funnel of the installed Pour-Thru Cell Module. Avoid spilling
solution onto the instrument.
The funnel height and orientation may be adjusted. The funnel height determines the
speed of sample flow through the cell. The higher the funnel, the faster the flow.
To minimize air bubbles, adjust the funnel so that it drains completely with the final
level of liquid in the tube about 5 cm (2 inches) below the tip of the funnel.
Take instrument readings after the solution has stopped flowing through the cell.
Always rinse the cell thoroughly with deionized water after each series of tests or as
often as specified in the procedure.
Occasionally, remove the Pour-Thru Cell to check for any accumulation of film on the
windows. If the windows appear hazy, soak the cell in a detergent bath and rinse
thoroughly with deionized water.
Page 30
Section 3
Chemical Analysis
3.1 Sample collection, preservation and storage

Correct sampling and storage are critical for accurate testing. Sampling devices and
containers must be thoroughly clean to prevent carryover from previous samples.
Preserve the sample according to test-specific information about sample preservation.
3.1.1 Collecting water samples

Use a clean container. Rinse the container several times with the water to be sampled
before taking the sample. Document the location and procedure used for each sample
taken. For example:
From a tapTake samples as close as possible to the source of the supply. This
decreases the influence of the distribution system on the sample. Let the water run long
enough to flush the system. Fill sample containers slowly with a gentle stream to avoid
turbulence and air bubbles.
From a wellLet the pump run long enough to draw fresh groundwater into the system.
Collect a sample from a tap near the well.
From open watersSample as near the middle of the body of water as is practical; at
least several feet from the shore or edge of the tank. Take the sample under the surface
of the water. When using a capped container, submerge it before removin
3.1.1.1 Types of containers
The Example on page 23 lists recommended containers for specific parameters.
Polypropylene and Polyethylene
Quartz or TFE (tetrafluoroethylene, Teflon) higher quality and price
GlassGlass provides a good general-purpose container. Avoid using soft-glass

containers to collect samples to be tested for metals in the microgram-per-liter range.
When determining silver, store samples in dark containers such as amber or

brown glass.
Acid washing will thoroughly clean sample containers before use.
3.1.1.2 Acid washing

If a procedure suggests acid washing, follow these steps:
1. Clean the glassware or plasticware with laboratory detergent. Phosphate-free
detergent is best. (When determining phosphates, phosphate-free detergent must
be used.)
2. Rinse well with tap water.
3. Rinse with a 1:1 hydrochloric acid solution or a 1:1 nitric acid solution. (Nitric acid is
best when testing for lead or other metals.)
4. Rinse well with deionized water. For chromium, 1215 rinses may be necessary.
When determining ammonia and Kjeldahl nitrogen, the rinse water must be
ammonia-free.
5. Air dry. Protect the glassware from fumes and other sources of contamination during
storage.
Use chromic acid or chromium-free substitutes to remove organic deposits from glass
containers. Afterward, rinse thoroughly with water to remove all traces of chromium.
Avoid introducing metal contaminants from containers, distilled water or membrane filters.
Page 31
Chemical Analysis
3.1.1.3 Sample splits
Samples must often be split or divided into separate containers for intra- or
inter-laboratory use in studies, confirmation, alternative techniques or maintaining
additional sample for reference and stability studies.
It is very important that sample splits be done correctly:
Collect a large volume of sample in a single container and transfer to smaller

containers; do not fill the smaller containers individually from the water source.
Thoroughly mix samples containing particulates or solids before splitting so that all the
splits are homogeneous.
If the sample requires filtering before analysis or storage, filter the entire sample
before splitting.
Use the same kind of container for all the splits.
Analyze biologically active splits on the same day or as close to the same day as is
possible.
Preserve all splits in the same way; if this is not done, the differing methods must be
fully documented.
When testing for volatile contaminants, fill containers samples to overflowing and cap
carefully. Do not leave any headspace or air in the container.
3.1.2 Storage and preservation

Because chemical and biological processes continue after collection, analyze the sample
as soon as possible. This also reduces the chance for error and minimizes labor. When
immediate analysis is not possible, the sample must be preserved. Preservation methods
include pH control, chemical addition, refrigeration and freezing.
Comparison of international drinking water and FDA bottled water guidelines presents an
overview of preservation methods and holding times for specific procedures.
Preserve aluminum, cadmium, chromium, cobalt, copper, iron, lead, nickel, potassium,
silver and zinc samples for at least 24 hours as follows:
1. Add one Nitric Acid Solution Pillow 1:1 (Catalog No. 254098) per liter of sample.
2. Check the pH with pH indicator paper or a pH meter to assure the pH is 2 or less.
Add additional pillows if necessary.
3. Adjust the sample pH prior to analysis by raising the pH to 4.5 with Sodium
Hydroxide Standard Solution, 1 N or 5 N.
3.1.3 Correcting for volume additions

When using a large volume of preservative or neutralizer, account for dilution by the acid
added to preserve the sample and/or the base used to adjust the pH to the range of the
procedure. Make this correction as follows:
1. Determine the volume of initial sample, the volume of acid and base added and the
total final volume of the sample.
2. Divide the total volume by the initial volume.
3. Multiply the test result by this factor.
Page 32
Chemical Analysis
Example:
A one-liter sample was preserved with 2 mL of nitric acid. It was neutralized with 5 mL of
5 N sodium hydroxide. The result of the analysis procedure was 10.00 mg/L. What is the
volume correction factor and correct result?
1.
2.
Total Volume = 1000 mL + 2 mL + 5 mL = 1007 mL

1007
------------- = 1.007 = volume correction factor
1000
10.0 mg/L 1.007 = 10.07 mg/L = correct result
Table 8 Required containers, preservation techniques and holding times 1

Container2
Preservation3,4
Maximum holding time5
Coliform, fecal and total
P, G
Cool, 4 C, 0.008%
Na2S2O3
6 hours
Fecal streptococci
P, G
Cool, 4 C, 0.008%
Na2S2O3
6 hours
P, G
Cool, 4 C
36 hours
Parameter name
Bacterial tests
Aquatic toxicity tests

Toxicity, acute and chronic
Chemical tests
Acidity
P, G
Cool, 4 C
14 days
Alkalinity
P, G
Cool, 4 C
14 days
Ammonia
P, G
Cool, 4 C, H2SO4 to pH<2
28 days
Biochemical oxygen demand

(BOD)
P, G
Cool, 4 C
48 hours
Boron
P, PFTE or quartz
HNO3 to pH<2
6 months
Bromide
P, G
None required
28 days
Biochemical oxygen demand,

carbonaceous
P, G
Cool, 4 C
48 hours
Chemical oxygen demand
P, G
28 days
Chloride
P, G
None required
28 days
Chlorine, total residual
P, G
None required
Analyze immediately
Color
P, G
Cool, 4 C
48 hours
Cyanide, total and amenable

to chlorination
P, G
Cool, 4 C, NaOH to pH>12,

0.6 g ascorbic acid6
14 days7
Fluoride
None required
28 days
Hardness
P, G
HNO3 to pH<2, H2SO4 to

pH<2
6 months
Hydrogen ion (pH)
P, G
None required
Analyze immediately
Kjeldahl and organic nitrogen
P, G
Cool 4 C, H2SO4 to pH<2
28 days
Chromium VI
P, G
Cool, 4 C
24 hours
Mercury
P, G
HNO3 to pH<2
28 days
Metals, except boron,

chromium VI and mercury
P, G
HNO3 to pH<2
6 months
Nitrate
P, G
Cool, 4 C
48 hours
Nitrate-nitrite
P, G
28 days
Nitrite
P, G
Cool, 4 C
48 hours
Metals8
Page 33
Chemical Analysis
Table 8 Required containers, preservation techniques and holding times (continued)1
Oil and grease
Cool, 4 C, HCl or H2SO4 to

pH<2
28 days
Organic Carbon
P, G
Cool, 4 C, HCl or H2SO4 or

H3PO4 to pH<2
28 days
Orthophosphate
P, G
Filter immediately; Cool, 4 C
48 hours
Oxygen, dissolved probe
G Bottle and top
None required
Analyze immediately
Winkler
G Bottle and top
Fix on site and store in dark
8 hours
48. Phenols
G only
28 days
Phosphorus, elemental
Cool, 4 C
48 hours
Phosphorus, total
P, G
28 days
Residue, Total
P, G
Cool, 4 C
7 days
Residue, Filterable
P, G
Cool, 4 C
7 days
Residue, Nonfilterable (TSS)
P, G
Cool, 4 C
7 days
Residue, Settleable
P, G
Cool, 4 C
48 hours
Residue, Volatile
P, G
Cool, 4 C
7 days
Silica
P, PFTE or quartz
Cool, 4 C
28 days
Specific Conductance
P, G
Cool, 4 C
28 days
Sulfate
P, G
Cool, 4 C
28 days
P, G
Cool 4 C, add zinc acetate

plus sodium hydroxide to
pH>9
7 days
Analyze immediately
Sulfide
Sulfite
P, G
none required
Surfactants
P, G
Cool, 4 C
48 hours
Temperature
P, G
None required
Analyze immediately
Turbidity
P, G
Cool, 4 C
48 hours
1 This
table was adapted from Table II in the Code of Federal Regulations, July 1, 2000, Title 40, Part 136.3 (40 CFR 136.3), pages 2325. Most organic
tests are not included.
2 Polyethylene
(P) or glass (G) or PTFE Teflon
3 Sample
preservation should be performed immediately upon sample collection. For composite chemical samples each portion should be preserved at
the time of collection. When use of an automated sampler makes it impossible to preserve each portion, then chemical samples may be preserved by
maintaining at 4 C until compositing and sample splitting is completed.
4 When
any sample is to be shipped by common carrier or sent through United States Mails, it must comply with the Department of Transportation
Hazardous Material Regulations (49 CFR Part 172). The person offering such material for transportation is responsible for ensuring such compliance.
For the preservation requirements of Table II, the Office of Hazardous Materials, Materials Transportation Bureau, Department of Transportation has
determined that the Hazardous Materials Regulations do not apply to the following materials: Hydrochloric acid (HCl) in water solutions at
concentrations of 0.04% by weight or less (pH about 1.96 or greater); Nitric acid (HNO3) in water solutions at concentrations of 0.15% by weight or less
(pH about 1.62 or greater); Sulfuric acid (H2SO4) in water solutions at concentrations of 0.35% by weight or less (pH about 1.15 or greater); and
Sodium hydroxide (NaOH) in water solutions at concentrations of 0.080% by weight or less (pH about 12.30 or less).
5 Samples
should be analyzed as soon as possible after collection. The times listed are the maximum times that samples may be held before analysis
and still be considered valid. Samples may be held for longer periods only if the permittee or monitoring laboratory has data on file to show that the
specific types of samples under study are stable for the longer time and has received a variance from the Regional Administrator under 136.3(e).
Some samples may not be stable for the maximum time period given in the table. A permittee or monitoring laboratory is obligated to hold the sample
for a shorter time if knowledge exists to show that this is necessary to maintain sample stability. See 136.3(e) for details. The term analyze
immediately usually means within 15 minutes or less after sample collection.
6 Should
only be used in the presence of residual chlorine.
7 Maximum
holding time is 24 hours when sulfide is present. Optionally all samples may be tested with lead acetate paper before pH adjustments in order
to determine if sulfide is present. If sulfide is present, it can be removed by the addition of cadmium nitrate powder until a negative spot test is obtained.
The sample is filtered and then NaOH is added to pH 12.
8 Samples
should be filtered immediately on-site before adding preservative for dissolved metals.
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Chemical Analysis
3.1.4 Checking for accuracy and precision
Accuracy defines the closeness of a test result to the true value. Precision defines the
closeness of repeated measurements to each other. Although precise results suggest
accuracy, they can be inaccurate. Both the accuracy and the precision of test results can
be evaluated by using standard additions or standard solutions.
Accurate,
not precise
Not accurate,
not precise
Precise,
not accurate
Accurate and
precise
Figure 10 Precision and accuracy illustrated
3.1.5 Standard solutions

A standard solution may be ordered as a prepared reagent or it may be made in the
laboratory. It is a solution of a known composition and concentration. The accuracy of the
analysis system may be checked by using a standard solution in place of the sample
water in a procedure.
3.1.6 Standard additions

Standard Additions is a common technique for checking test results. Other names are
spiking and known additions. The technique can test for interferences, bad reagents,
faulty instruments and incorrect procedures.
Perform Standard Additions by adding a measured small amount of a standard solution to
the sample and repeating the test. Use the same reagents, equipment and technique.
The result should be about 100% recovery. If not, there is an identifiable problem.
If Standard Additions works for the test, a Standard Additions Method section will be in
the procedure under Accuracy Check. Follow the detailed instructions given.
If the result is about 100% recovery for each addition, everything is working properly and
the results are correct.
If the result is not approximately 100% recovery for each addition, a problem exists. To
determine if the cause is an interference, repeat the Standard Additions using deionized
water as the sample. If the result is approximately 100% recovery for each addition, an
interference exists.
If results did show good recoveries with the deionized water, use the following checklist to
find the problem:
1. Check that the procedure is followed exactly:
a. Are the correct reagents used in the correct order?
b. Is the correct time allowed for color to develop?
c. Is the correct glassware being used?
d. Is the glassware clean?
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Chemical Analysis
e. Does the test need a specific sample temperature?
f.
Is the sample pH in the correct range?
Refer to the written procedure to answer these questions.

2. Check the performance of the instrument per instructions in the user manual.
3. Check the reagents. Repeat the Standard Additions using new, fresh reagents.
If results are good, the original reagents were faulty.
4. If nothing else is wrong, the standard is almost certainly defective. Repeat the
Standard Additions with a new standard.
If the problem is still uncertain, call Technical Customer Support at 800-227-4224 (U.S.A.)
or 303-669-3050 for assistance.
3.1.7 Troubleshoot a test when results are in doubt

If the results from any chemistry are in doubt, troubleshoot as follows:
1. Run a proof-of-accuracy check. Take a standard solution, which has a known
concentration, through the same steps as the original sample. Include sampling and
storage, digestion and colorimetric determination, if applicable. If the results of the
standard solution check are correct, skip to step 4 below. If there is a variation in the
expected results, go to step 2.
2. If the standard solutions check does not match the expected results, check the
instrument set-up and method procedure as follows:
a. Verify that the correct program number for the test being performed is selected.
b. Verify that the units of concentration of the standard match the displayed units.
(One of the alternative forms of the analyte may be in the display.) For example:
Molybdenum may be shown as Mo instead of MoO4.
c. Verify that the sample cells specified in the procedures are used.
d. Verify that the reagents are correct for the sample size being analyzed.
e. Verify that the reagent blank value stored is for the current procedure. It may be
from a previous lot of reagents and therefore not representative of the current
reagent lot.
f.
Verify the calibration curve adjustment (Standard Adjust) currently in use. The
factory-stored default calibration should be used initially to check the standard.
g. Verify that the dilution factor option is correct.

If the instrument setup is correct and the method procedure specifics are being followed
correctly, go to step 3.
3. If the standard solution check does not match the expected results, check the
reagents used in the test and the analytical technique as follows:
a. Determine the age of the reagents used in the test. Many factors affect reagent
shelf life (i.e., storage temperature, storage conditions, microbial contamination).
Replace suspect reagents and run the standards check again.
b. Run a deionized or distilled water blank through the entire process; include
sampling and storage, digestion and colorimetric determination. Some chemicals
will add a small amount of color to a test; this is not considered unusual. However,
color development in excess of 10% of the range of the test may indicate a
problem with one of the reagents or the dilution water.
c. To troubleshoot the procedure, delete the parts one by one. First, using the
standard solution, omit preservation and storage, doing only digestion and
colorimetry. If this analysis is correct, examine the procedure used to store the
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Chemical Analysis
sample. Make sure that it is the procedure prescribed for the chosen parameter.
If the sample is acidified for storage, be sure the correct acid is used and the
sample is adjusted to the proper pH level before testing.
If the standards check is still incorrect, run the standard on just the colorimetry. If the
results are correct, examine the digestion procedure. Make sure that the amount of
reagents used and the pH after the digestion are correct for the procedure. (See the
procedure for the parameter in question.)
4. If the standard solution gives a correct value, but the results of the sample
measurement are questionable, there may be an interference in the sample. To
check for an interference:
a. Spike the sample. Use a standard addition test instead of a standard solution test
to include any possible interferences.
b. To two cells containing fresh sample water, add an amount of standard equal to
two times the concentration of the sample.
c. Process both samples using the same reagents, instruments and technique. The
spiked sample should show an increase equal to the amount of standard added.
d. Calculate percent recovery as shown below. Ideally, the results should be 100%,
with results from 90 to 110% considered acceptable. Refer to the procedure notes
for possible interferences and ways to eliminate them.
e. Run a series of dilutions on the sample. Make sure the sample is within the range
of the test. A sample out of range for the method may give erroneous results
because of under- or over-development of the color, excess turbidity or even
sample bleaching. Run a series of dilutions to check for this possibility.
f.
Because it may not be feasible to determine the cause of the interference, diluting
the sample past the point of interference is often the most economical and
efficient means of getting the correct result. If it is not possible to dilute out an
interference without diluting out the parameter to be measured, use a different
method, such as a different chemistry or an ion-selective electrode to measure the
parameter.
Calculating percent recovery:

1. Measure the unknown sample concentration.
2. Calculate the theoretical concentration of the spiked sample using the following
formula:
( Cu Vu ) + ( Cs Vs )
Theoretical Concentration = ------------------------------------------------------Vu + Vs
Where:
Cu = measured concentration of the unknown sample
Vu = volume of the unknown sample
Cs = concentration of the standard
Vs = volume of the standard
3. Measure the spiked sample concentration.

4. Divide the spiked sample concentration by the theoretical concentration and multiply
by 100.
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Chemical Analysis
For example:
A sample was tested for manganese and the result was 4.5 mg/L. A separate 97-mL
portion of the same sample was spiked with 3 mL of a 100 mg/L standard solution of
manganese. This spiked solution was tested again for manganese using the same
method. The result was 7.1 mg/L.
The theoretical concentration of the spiked sample is:
( 4.5 mg/L 97 mL ) + ( 100 mg/L 3 mL )
------------------------------------------------------------------------------------------------------------ = 7.4 mg/L
97 mL + 3 mL
The percent spike recovery is:

7.1 mg/L
----------------------- 100 = 96%
7.4 mg/L
USEPA calculation
The USEPA requires a more stringent calculation for percent recovery. This formula
calculates the percent recovery only for the standard added to the spiked sample and
yields a lower value than the above calculation. A complete explanation for the USEPA
formula is in USEPA Publication SW-846. The USEPA percent recovery formula is:
100 ( X s X u )
%R = ---------------------------------K
Where:
Xs = measured value of the spiked sample
Xu = measured value for the unspiked sample, adjusted for the dilution of the spike volume
K = known value of the spike in the sample
Example:
A sample measures 10 mg/L. A separate 100-mL portion of the sample was spiked with
5 mL of a 100-mg/L standard solution. The spiked solution was measured by the same
method as the original sample. The result was 13.7 mg/L.
X s = 13.7 mg/L
10 mg/L 100 mL
X u = ------------------------------------------------ = 9.5 mg/L
105 mL
5 mL 100 mg/L
K = -------------------------------------------- = 4.8 mg/L
105 mL
100 ( 13.7 mg/L 9.5 mg/L )
%R = ----------------------------------------------------------------------------- = 88%
4.8 mg/L
Acceptable percent recovery values are 80120%.
3.1.8 Adjusting the standard curve

Spectrophotometers typically have many programs permanently installed in memory.
Many programs include a pre-programmed calibration curve. Each curve is the result of
an extensive calibration performed under ideal conditions and is normally adequate for
most testing. Deviations from the curve can occur from using compromised testing
reagents, defective sample cells, incorrect test procedure, incorrect technique or other
correctable causes. Interfering substances or other causes may be beyond the analysts
control.
In some situations, using the pre-programmed curve may not be convenient:
Page 38
Running tests where the reagents are highly variable from lot to lot.
Running tests where frequent calibration curve checks are required.
Testing samples which give a consistent test interference.
Consider the following before adjusting the calibration curve:
Chemical Analysis
Will future test results be improved by adjusting the curve?
Are interfering substances consistent in all the samples tested?
Estimated detection limit, sensitivity, precision and test range information provided
with the procedure may not apply to an adjusted curve calibration.
The calibration curves can be adjusted by following the steps found in the test procedure.
Generally, add test reagents to a blank and standard solution. Working carefully is
important. After the adjustment, it is wise to run standard solutions of several
concentrations to make sure the adjusted curve is satisfactory. Performing standard
additions on typical samples may also help determine if the adjusted curve is acceptable.
Adjusting a measurement is a two-step process. First, the instrument measures the
sample using the pre-programmed calibration. Second, it multiplies this measurement by
an adjustment factor. The factor is the same for all concentrations. The instrument will
remember the factor until the program is exited and will display the standard adjustment
icon when it is used. Return to the pre-programmed curve any time by selecting the
original stored program from the main menu.
3.2 Interferences
Interferences are contaminants in a sample that are capable of causing changes in color
development, turbidity or unusual colors and odors, thereby creating errors in the results.
A list of common interferences is included in each procedure. Reagents are formulated to
eliminate many interferences; remove others by pretreating the sample as instructed in
the procedure.
Test strips are available for many of the common interferences. These can be
conveniently used to screen samples for the presence of interferences.
When test results appear inaccurate, develop an unexpected color or develop an unusual
odor or turbidity, repeat the test on a sample diluted with deionized water. (See Sample
dilution.) Correct the results for the dilution and compare them with those from the original
test. If they differ significantly, make a second dilution and check it against the first.
Repeat the dilutions until the same result (after volume corrections) is achieved twice in
succession.
For more information on interferences, see Standard additions. The APHA Standard
Methods book, an excellent reference for water analysis, also covers interferences in the
General Introduction.
pH interference
Chemical reactions are often pH dependent. Reagents contain buffers to adjust the pH of
the sample to the correct range. However, the reagent buffer may not be strong enough
for samples that are highly buffered or have an extreme pH.
The Sampling and Storage section of each procedure gives the pH range for that test.
Before testing, adjust the sample to the proper pH as instructed in the procedure or by
following these steps:
1. Measure the pH of the analyzed sample with a pH meter.
Note: Use pH paper when testing for chloride, potassium or silver to avoid contamination.
2. Prepare a reagent blank using deionized water as the sample. Add all reagents
called for in the procedure. Timer sequences, etc., may be ignored. Mix well.
3. Measure the pH of the reagent blank with a pH meter.
4. Compare the pH values of the analyzed sample with the reagent blank.
Page 39
Chemical Analysis
5. If there is little difference in the values of the analyzed sample and the reagent blank,
then pH interference is not the problem. Follow the Accuracy Check for the specific
procedure to more clearly identify the problem.
6. If there is a large difference between the value of the analyzed sample and the
reagent blank, adjust the sample pH to the value of the reagent blank. Adjust the
sample pH to this same pH for all future samples before analysis. Use the
appropriate acid, usually nitric acid, to lower the pH. Use the appropriate base,
usually sodium hydroxide, to raise the pH. Adjust the final result for any dilution
caused by adding acid or base; see Correcting for volume additions.
7. Analyze the sample as before.
8. Some purchased standards may be very acidic and will not work directly with test
procedures. Adjust the pH of these standards as described above. Adjust the final
concentration of the standard for the dilution. The standard solutions suggested in the
procedures are formulated so that no pH adjustment is necessary.
3.3 Method performance

3.3.1 Estimated detection limit (EDL)
Ranges for chemical measurements have limits. The lower limit is important because it
determines whether a measurement is different from zero. Many experts disagree about
the definition of this detection limit and determining it can be difficult. The Code of Federal
Regulations (40 CFR, Part 136, Appendix B) provides a procedure to determine the
Method Detection Limit or MDL. The MDL is the lowest concentration that is different
from zero with a 99% level of confidence. A measurement below this MDL is highly
suspect.
The MDL is not fixed; it varies for each reagent lot, instrument, analyst, sample type, etc.
Therefore, a published MDL may be a useful guide, but is only accurate for a specific set
of circumstances. Each analyst should determine a more accurate MDL for each specific
sample matrix using the same equipment, reagents and standards that will routinely be
used for measurements.
A sensitivity value (concentration change equivalent to an absorbance change of 0.010
abs) is provided as an estimate of the lower detection limit of each test. The sensitivity
value may be treated as an EDL for the purposes of MDL determination. It can be
considered a good starting concentration when determining a MDL. Do not use the EDL
as the MDL. The conditions for MDL determination must be exactly the same as the
conditions used for analysis. The EDL may be useful to the analyst as a starting point in
determining a MDL or as a way to compare methods. Measurements below the EDL may
also be valuable because they can show a trend, indicate the presence of analyte and/or
provide statistical data. However, these values have a large uncertainty.
3.3.2 Method detection limit (MDL)

This method is in accordance with the USEPA definition in 40 CFR, Part 136, Appendix B
in the 7-1-94 edition. The USEPA defines the method detection limit (MDL) as the
minimum concentration that can be determined with a 99% level of confidence that the
true concentration is greater than zero. Since the MDL will vary from analyst to analyst, it
is important that the MDL be determined under actual operating conditions.
Page 40
Chemical Analysis
The procedure for determining MDL is based on replicate analyses at a concentration
1 to 5 times the estimated detection limit. The MDL value is calculated from the standard
deviation of the replicate study results multiplied by the appropriate t value for a 99%
confidence interval. For this definition, the MDL does not account for variation in sample
composition and can only be achieved under ideal conditions.
1. Estimate the detection limit. Use the sensitivity value stated in the Method
Performance section of the analysis procedure.
2. Prepare a laboratory standard of the analyte, 1 to 5 times the estimated detection
limit, in deionized water that is free of the analyte.
3. Analyze at least seven portions of the laboratory standard and record each result.
4. Calculate the average and the standard deviation (s) of the results.
5. Compute the MDL using the appropriate t value (see table below) and the standard
deviation value:
MDL = t x s
Number of test portions
t Value
3.143
2.998
2.896
10
2.821
For example:
The EDL for measuring iron using an iron test is 0.003 mg/L. An analyst accurately
prepared 1 liter of a 0.010 mg/L (about 3x the EDL) laboratory standard by diluting a
10-mg/L iron standard in iron-free deionized water.
Eight portions of the standard were tested according to the FerroZine method with the
following results:
Sample #
Result (mg/L)
0.009
0.010
0.009
0.010
0.008
0.011
0.010
0.009
Using a calculator program, the average concentration = 0.010 mg/L and the standard
deviation (s) = 0.0009 mg/L.
Based on the USEPA definition, calculate the MDL as follows:
MDL for iron test = 2.998 (t) x 0.0009 (s)
MDL = 0.003 mg/L (agrees with initial estimate)
Note: Occasionally, the calculated MDL may be very different from the estimate of the detection
limit. To test how reasonable the calculated MDL is, repeat the procedure using a standard near the
calculated MDL. The average result calculated for the second MDL derivation should agree with the
Page 41
Chemical Analysis
initial calculated MDL. Refer to 40 CFR, Part 136, Appendix B (7-1-94), pages 635637 for detailed
procedures to verify the MDL determination.
Run a laboratory blank containing deionized water without analyte through the test
procedure to confirm that the blank measurement is less than the calculated MDL. If the
blank measurement is near the calculated MDL, repeat the MDL procedure using a
separate blank for analysis for each portion of standard solution analyzed. Subtract the
average blank measurement from each standard and use the corrected standard values
to calculate the average and standard deviation used in the MDL.
3.3.3 Precision
Every chemical measurement has some degree of uncertainty. The quality of the entire
calibration curve determines the precision.
Uncertainty in chemical measurements may be due to systematic errors and/or random
errors. A systematic error is a mistake that is always the same for every measurement
made. For example, a blank can add to each measurement for a specific compound,
giving consistently high results (a positive bias). Random errors are different for every
test and can add either a positive or negative variation in response. Random errors are
most often caused by variation in analytical technique. Even with reliable reagents
developed to eliminate systematic errors, response variation occurs in all chemical
measurements.
3.3.4 Estimating precision

The method performance section in each procedure provides an estimate of test
precision. Most procedures use a replicate analysis estimate, based on real data.
For precision determined in this manner, the 95% confidence interval of the distribution is
reported.
In replicate analysis, the chemist prepares a specific concentration of the analyte in a
deionized water matrix. The standard is analyzed seven individual times on a single
instrument. The standard deviation is calculated and the 95% confidence interval of the
distribution is reported in the method. The reported value provides an estimate of the
scatter of results at a particular point in the calibration curve.
Precision estimates are based on a deionized water matrix. Precision on real samples
with varying matrices can be quite different from these estimates.
If the concentration obtained from running a standard solution is not as expected, refer to
Troubleshoot a test when results are in doubt.
3.3.5 Sensitivity
The definition of the sensitivity of a method is a change in concentration (Concentration)
for a 0.010 change in absorbance (Abs).
Use sensitivity when comparing different methods. For example, between three methods
for determining iron:
Iron Analysis Method
Page 42
Portion of curve
Abs
Concentration
FerroVer
Entire range
0.010
0.022 mg/L
FerroZine
Entire range
0.010
0.009 mg/L
TPTZ
Entire range
0.010
0.012 mg/L
Chemical Analysis
Notice that the FerroZine method has the greatest sensitivity of the three methods
because it will measure the smallest change in concentration. The technical definition of
sensitivity comes from a calibration curve with Abs on the x-axis and concentration on the
y-axis.
1. If the calibration is a line, the sensitivity is the slope of the line multiplied by 0.010.
2. If the calibration is a curve, the sensitivity is the slope of the tangent line to the curve
at the concentration of interest multiplied by 0.010.
The sensitivity value is also used as an estimate of the lower limit of the test. The value
may be used as a starting point in determining MDL.
3.4 Prepare a calibration curve

Note: Calibration curves are recommended when performing tests on different instruments or where
required by a regulator.
1. Prepare five or more standards of known concentration that cover the expected
range of the test. Run tests as described in the procedure on each prepared
standard. Then pour the customary volume of each known solution into a separate
clean sample cell of the type specified for the instrument.
2. Select the proper wavelength. Standardize (zero) the instrument using an untreated
water sample or a reagent blank, per procedure instructions.
3. Measure and record the absorbance of the known solutions within the time
constraints detailed in the procedure. To use absorbance vs. concentration, see the
section on Absorbance versus concentration calibration.
3.4.1 Absorbance versus concentration calibration

If absorbance values are measured, plot the results on linear graph paper. Plot the
absorbance value on the vertical axis and the concentration on the horizontal axis.
Plot increasing absorbance values from bottom to top. Plot increasing concentration
values from left to right. Values of 0.000 absorbance units and 0 concentration will begin
at the bottom left corner of the graph. A calibration table can be extrapolated from the
curve or the concentration values can be read directly from the graph. Or, determine an
equation for the line using the slope and y-intercept.
Alternately, use the User Program software in the spectrophotometer or a curve-fitting
program such as Excel to calculate the calibration curve.
3.5 Adapt procedures to other spectrophotometers

Test procedures may be used with more than one spectrophotometer, if calibration curves
are made that convert absorbance to concentration. Regardless of the
spectrophotometer used, prepare the sample and calibration standards following the
procedure and use the optimum wavelength used in the procedure.
To calibrate for a given analyte, a series of standards are prepared and measured to
establish the calibration curve. The absorbance vs. concentration is plotted as described
in the section on Absorbance versus concentration calibration. Points on the graph are
connected with a smooth line (curved or straight). If necessary, use the curve to make a
calibration table.
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Chemical Analysis
3.5.1 Select the best wavelength
When developing a new procedure or using procedures that are sensitive to wavelength,
select the wavelength where the instrument gives the greatest absorbance (see the
Select the best wavelength figure). Because procedures are usually developed to use the
best wavelength for the test, selecting the wavelength is not necessary for most stored
procedures.
3.5.1.1 Select the best wavelength on a spectrophotometer:

Note: When available, use of the Scan function is the easiest way to find the optimum wavelength.
1. Refer to the user manual for specific instructions for wavelength adjustments.
2. Select single wavelength adjustment.
3. Enter a wavelength in the range of interest.
Note: Sample color provides a good indication of what wavelength region to use. A yellow solution
absorbs light in the 400500 nm region. A red solution absorbs light between 500600 nm. A blue
solution absorbs light in the 600700 nm range.
4. Prepare the sample and blank for analysis. Fill the appropriate sample cells with the
blank and the reacted sample solution.
5. Place the blank in the cell holder. Zero the instrument.
6. Place the prepared sample into the cell holder. Read the absorbance level.
7. Increase the wavelength so it is at least 100 nm greater than the range of interest.
Re-zero as in step 5. Measure and record the absorbance of the sample.
8. Repeat, decreasing the wavelength by 50 nm. Re-zero, then measure and record the
absorbance at each increment. Continue this process throughout the wavelength
range of interest. Note the wavelength of greatest absorbance (see the Example
in Table 9).
Table 9 Example
Wavelength
Absorbance
550 nm
0.477
500 nm
0.762
450 nm
0.355
400 nm
0.134
9. Adjust the wavelength to 50 nm more than the highest absorbance point on the initial
search (step 8). Re-zero as in step 5.
Measure and record the absorbance. Repeat, decreasing the absorbance in 5-nm steps.
Re-zero, then measure and record the absorbance at each increment. Continue until the
entire range of interest is measured (see the Example in Table 10).
Table 10 Example
Page 44
Wavelength
Absorbance
520 nm
0.748
515 nm
0.759
510 nm
0.780
505 nm
0.771
500 nm
0.771
495 nm
0.651
490 nm
0.590
Chemical Analysis
Check to be sure there is enough difference in absorbance between samples with low
and high analyte concentrations by measuring two sample solutions that contain the
expected low and high concentrations of analyte at the optimum wavelength. The change
in absorbance caused by increases/decreases in concentration depends on the
sensitivity of the procedure and the chemistry. Chemistries with small absorbance
changes are less sensitive, but tend to have larger ranges. Chemistries with large
absorbance changes are more sensitive, but tend to have smaller ranges.
Select the best wavelength
1.0
Absorbance
500 505 510 515 520
400
0.0
450
500
550
600
650
700
Wavelength (nm)
Figure 11 Select the best wavelength
Page 45
Page 46
Section 4
Sample Pretreatment by Digestion

Several procedures require sample digestion before determining total metal content.
Digestion uses acid and heat to break organo-metallic bonds and free ions for analysis.
4.1 USEPA-approved digestions

For USEPA reporting, USEPA-approved digestions are required. There are two methods
for metals analysis: mild and vigorous.
4.1.1 USEPA mild digestion

1. Acidify the entire sample at the time of collection with concentrated nitric acid by
adding 5 mL of acid per liter (or quart) of sample.
2. Transfer 100 mL of well-mixed sample to a beaker or flask. Add 5 mL of distilled
1:1 hydrochloric acid (HCl).
3. Heat using a steam bath or hot plate until the volume has been reduced to 1520 mL.
Do not boil.
4. Filter the sample to remove any insoluble material.
5. Adjust the pH of the digested sample to a pH of 4 by adding 5.0 N Sodium Hydroxide
Standard Solution a drop at a time. Mix thoroughly and check the pH after each
addition.
6. Quantitatively transfer the sample to a 100-mL volumetric flask and dilute to volume
with deionized water. Continue with the procedure.
7. Process a reagent blank to match the sample.
4.1.2 USEPA vigorous digestion

For some samples mild digestion will not be sufficient. Use a vigorous digestion to make
sure that all the organo-metallic bonds are broken.
1. Use redistilled 1:1 Nitric Acid Solution to acidify the entire sample to a pH of less than
two. Do not filter the sample before digestion.
2. Transfer an appropriate sample volume (see the table on Vigorous digestion
volumes) into a beaker and add 3 mL of concentrated redistilled nitric acid.
3. Place the beaker on a hot plate and evaporate to near dryness, making certain the
sample does not boil.
4. Cool the beaker and add another 3 mL of the concentrated re-distilled nitric acid.
5. Cover the beaker with a watch glass and return it to the hot plate. Increase the
temperature of the hot plate so that a gentle reflux occurs. Add additional acid, if
necessary, until the digestion is complete (generally indicated when the digestate is
light in color or does not change color or appearance with continued refluxing).
Page 47

6. Again, evaporate to near dryness (do not bake) and cool the beaker. If any residue or
precipitate results from the evaporation, add redistilled 1:1 hydrochloric acid (5 mL
per 100 mL of final volume). See the table on Vigorous digestion volumes.
7. Warm the beaker. Adjust the sample to pH 4 by drop-wise addition of 5.0 N Sodium
Hydroxide Standard Solution; mix thoroughly and check the pH after each addition.
8. Quantitatively transfer the sample to a volumetric flask and dilute to volume with
deionized water. See the Vigorous digestion volumes table for the suggested final
volume.
9. Multiply the result by the correction factor in the Vigorous digestion volumes table.
10. Process a reagent blank to match the sample.
Table 11 Vigorous digestion volumes
Expected metal
concentration
Suggested sample
vol. for digestion
Suggested volume of
1:1 HCl
Suggested final
volume after
digestion
Correction factor
1 mg/L
50 mL
10 mL
200 mL
10 mg/L
5 mL
10 mL
200 mL
40
100 mg/L
1 mL
25 mL
500 mL
500
4.2 General Digesdahl digestion

Many samples may be digested using the Digesdahl Digestion Apparatus
(Catalog. No. 2313020). It is designed to digest samples such as oils, wastewater,
sludges, feeds, grains, plating baths, food and soils. In this procedure the sample is
oxidized by a mixture of sulfuric acid and hydrogen peroxide. Digestion of a dry sample
requires less than ten minutes, while liquid samples require about 1 minute/mL. The
digestion is done in a special flat-bottomed, 100-mL volumetric flask. Aliquots (sample
portions) are taken for analysis using colorimetric methods.
Procedures for digestion using the Digesdahl Digestion Apparatus are based on the type
and form of the sample and are found in the Digesdahl Digestion Apparatus Instruction
Manual, which is included with each Digesdahl Digestion Apparatus.
Digesdahl digestion is a process that yields a digest suitable for the determination of
metals, total phosphorus and total Kjeldahl nitrogen (TKN). It is faster than traditional
methods, but yields comparable accuracy and precision. The digest can be used with
colorimetric, turbidimetric or titrimetric tests.
Procedures for using the Digesdahl Digestion Apparatus vary with sample type. Sample
types include food products, feeds, grains, wastewater sludges, plating baths, plant
tissues, fertilizers, beverages and oils. Most procedures use a two-phase digestion
process involving concentrated sulfuric acid and 50% hydrogen peroxide. Sulfuric acid
dehydrates and chars the sample. Hydrogen peroxide is added through the capillary flow
funnel to complete decomposition. The analyst controls the digestion time (exposure to
the hydrogen peroxide) by varying the volume of hydrogen peroxide used.
Some samples are more difficult to digest completely. For example, resistant or refractory
materials such as nicotinic acid require several minutes of continued peroxide digestion
after clearing to obtain 100% nitrogen recovery. To make sure complete sample
digestion, consider variables such as sample size, solution temperature and sample
contamination. Refer to the Digesdahl Manual (Catalog No. 2313018) for complete
information.
Page 48

4.2.1 Frequently asked questions regarding digestion procedures
This section provides answers to common questions about digestion.
The reading on my instrument is over-range. What should I do?
The concentration range tables found in digestion procedures are only guidelines. Take a
smaller analysis volume and repeat the procedure. Record the new analysis volume and
use it in the calculation.
Do I have to prepare a reagent blank each time I use reagents with the same
lot number?
To decide, first determine the reading of the reagent blank by zeroing the instrument with
deionized or distilled water. If the reagent blank has an insignificant concentration reading
and the reagents have the same lot number, a reagent blank does not have to be
prepared every time. If the reagent blank shows a reading, either analyze it daily or
subtract the reading from the sample reading. If a reagent blank is not run daily, zero the
instrument with deionized water.
Do I have to follow the exact sample amount and the exact analysis volume given
for each procedure?
The sample amount and the analysis volume for each procedure are only suggested
guidelines. Digest any aqueous solution or suspension sample amount up to 40 mL. Solid
or organic liquid samples require less than 0.5 g of anhydrous materialas a routine
practice, 0.25 g of sample is used.
How can I refine the initial amount of sample required for digestion and the analysis
volume to be used?
The amount of sample to be digested is a critical aspect of the digestion. The aliquot size
of the digest to be used in the analysis is also very important. Tables are provided in each
method for determining amount of initial sample to be digested. In order to optimize the
specific test being performed, the following equations have been developed. Before using
these equations, please refer to the manual specifications for the sample type.
To use the equations, first determine the approximate concentration (in ppm or mg/L or
mg/kg). Next determine the range of the colorimetric test being used (e.g., 050 mg/L)
and the midpoint of the test range. (This midpoint range is optimum but can be lowered to
accommodate very low sample concentrations). The midpoint of the test range is
determined by subtracting the lower limit of the range from the higher limit and dividing
by 2.
After finishing these determinations, the following equation may be used:
BCD
A = ------------------------EF
Where:
A = approximate concentration of sample
B = midpoint of colorimetric test range
C = final volume of digest
D = final volume of analysis
E = sample amount to digest
F = analysis volume of digest
Using algebra, the following two equations are obtained:

equation (1)
BCD
E = ------------------------AF
equation (2)
BCD
F = ------------------------AE
Page 49

Both equations contain two unknown values, E and F and some trial and error may be
required to achieve the optimum values.
Using equation (1): If analyzing for copper using the CuVer method with an initial
sample containing approximately 150 ppm Cu, the amount of sample required for
digestion and the aliquot volume to be used can be determined as follows:
Determine the test range. In this example, the test range is assumed to be 05.0 ppm
and the midpoint is 2.5. When using the Digesdahl system, the final volume of digest is
100 mL and the procedure calls for a final analysis volume of 25 mL.
Therefore:
A = 150
B = 2.5
C = 100
D = 25
E and F are
unknown
Substituting values into equation (1) gives:

2.5 100 25
E = ------------------------------------150 F
or
41.7
E = ----------F
Since CuVer Copper Reagent is pH sensitive, a small analysis volume (0.5 mL) is desired
so that pH adjustment would not be necessary.
With this in mind, a 0.5-mL analysis volume would give:
41.7
E = ----------- = 83.4 mL digestion sample amount
0.5
Because the maximum digestion sample amount is 40 mL for Digesdahl digestions, a

0.5-mL analysis volume would not be acceptable for the range. (This is where trial and
error is necessary). Next, try a 5.0-mL analysis volume and the equation gives:
41.7
E = ----------- = 8.0 mL digestion sample amount
5.0
(Round to the nearest whole number for ease of measure).

From the calculation, an 8.0 mL sample amount would be digested and a 5.0-mL analysis
volume would be taken. A pH adjustment would be necessary before analysis.
Using equation (2): Equation (2) may be used when a minimum sample size is desired
or when a sample has already been digested for one parameter (such as copper) and
another parameter (such as zinc) also needs to be measured. Continuing the example for
copper, above, a zinc test may also be performed. The undigested sample contains
approximately 3 ppm zinc and the Zincon method is being used. The analysis volume can
be determined as follows.
In this example, the Zincon methods test range is assumed to be 02.5 ppm so the
midpoint of the range is 1.25. Therefore values are:
A=3
B = 1.25
C = 100
D = 50
E = 8 (as determined above)
substituting:
1.25 100 50
F = ---------------------------------------- = 260 mL
38
Page 50

This is an extreme example but it illustrates the need to compare the values of D and F to
assure that the analysis volume (F) does not exceed the final analysis volume (D). If F
exceeds D, the analysis cannot be done. A test with a more suitable range is necessary
or a larger sample may be digested for this test. Care must also be taken to assure that
the volume of digest taken for analysis (F) is greater than 0.1 mL because accurately
pipetting less than 0.1 mL is difficult.
As a comparison, assume that the concentration of zinc is 75 ppm (A = 75 instead of 3)
and substituting again will give:
1.25 100 50
F = ---------------------------------------- = 10.5 mL
75 8
In this case, the aliquot volume is less than the final analysis volume and analysis may be
done as stated in the procedure.
Why is the factor in the calculation step either 75, 2500 or 5000 depending on the
method used and where does the factor come from?
In all cases, the factor is a correction for sample dilution. For example, in some tests the
factor is 2500. The Digesdahl digestion total volume is 100 mL, the analysis total volume
is 25 mL and 100 x 25 = 2500. The mL units are not included with the factor because
they cancel out in the formula.
How do I report my total concentration on a dry basis when I am analyzing a slurry?
The sample must be analyzed for moisture content. See the figure for Determining dry
basis weight.
Determining dry basis weight
Figure 12 Determining dry basis weight

1
Weigh an aluminum dish and

record the weight as A.
Place the dish in the oven

(103105 C) for two hours.
Weigh the aluminum dish with

oven-dried sample. Record as C.
Note: The oven-dried material
generally is unsuitable for
additional testing and should be
discarded.
Weigh out approximately 2 g of

solid sample into the dish. Record
the exact weight added as B.
Cool to room temperature by

placing in a desiccator
Use the following formula to

calculate the sample on a dry
basis.
Test result (dry basis) =

C
A------------BA
Note: Multiply the test result on an
as is basis, by the factor above, to
report as dry basis.
Table 12 Required appartaus for dry basis weight

Description
Unit
Catalog. No.
Balance, general laboratory, 50-g
each
2610300
Page 51

Table 12 Required appartaus for dry basis weight (continued)
Desiccant, Drierite (without indicator)
454 g
2285901
Desiccator, vacuum (uses ceramic

plate)
each
2088800
100/pkg
2164000
each
1428900
240 VAC
each
1428902
Tongs, crucible
each
56900
Dish, moisture determination,

aluminum, 63 x 17.5 mm
Oven, laboratory
120 VAC
or
Table 13 Optional apparatus

Description
Unit
Catalog No.
Desiccator, without stopcock
each
1428500
4.2.2 Adjusting the pH

4.2.2.1 For a metals procedure
Note: If analyzing aliquots smaller than 0.5 mL, pH adjustment is unnecessary.
1. Determine the required volume of sample for analysis from the Sample and Analysis
Volume Tables following each digestion procedure. Determine the required volume of
sample for analysis from the Sample and Analysis Volume Tables following the
digestion procedure. Pipet this volume into a mixing graduated cylinder.
Note: Some methods require pipetting into a volumetric flask or a regular graduated cylinder.
2. Dilute to about 20 mL with deionized water.

3. Add one drop of 2,4 Dinitrophenol Indicator Solution (Cat. No. 186932).
4. Add one drop of 8 N Potassium Hydroxide (KOH) Standard Solution (Cat. No.
28232H), swirling between each addition, until the first flash of yellow appears (pH 3).
If analyzing for potassium, use 5 N sodium hydroxide (Cat. No. 245026) instead. Do
not use a pH meter if analyzing for potassium or silver.
5. Add one drop of 1 N KOH (Cat. No. 2314426). Stopper the cylinder and invert several
times to mix. If analyzing for potassium, use 1 N sodium hydroxide instead.
Note: Use pH paper to make sure that the pH is 3. If it is higher than 4, do not readjust with
acid; start over with a fresh aliquot.
6. Continue to add 1 N KOH in this manner until the first permanent yellow color
appears (pH 3.54.0).
7. View the cylinder from the top against a white background. Compare the cylinder to a
second cylinder filled to the same volume with deionized water.
Note: High iron content will cause precipitation (brown cloud) which will co-precipitate other
metals. Repeat this procedure with a smaller aliquot volume.
8. Add deionized water to the volume indicated in the colorimetric procedure for the
parameter under analysis.
9. Continue with the colorimetric procedure.
Page 52

4.2.2.2 For the total Kjeldahl Nitrogen colorimetric method
Consult the spectrophotometer or colorimeter procedure to complete the TKN analysis.
The following is only a guide to use if a procedure is not available.
1. Pipet an appropriate analysis volume into a mixing graduated cylinder.
2. Add one drop of TKN Indicator (Cat. No. 2251900).
3. Add one drop of 8 N KOH Standard Solution (Catalog No. 28232H), swirling between
each addition, until the first flash of pale blue appears (pH 3).
4. Add one drop of 1 N KOH (Cat. No. 2314426). Stopper the cylinder and invert several
times to mix.
Note: View the cylinder from the top against a white background. Compare the cylinder to a
second cylinder filled to the same volume with deionized water.
5. Continue to add 1 N KOH in this manner until the first permanent blue color appears.
6. Add deionized water to the volume indicated in the colorimetric procedure for the
parameter under analysis.
Continue with the colorimetric procedure.
Page 53
Page 54
Section 5
Water Management and Safety

This section provides guidelines for laboratory waste management. These guidelines are
only a summary of basic USEPA requirements and do not relieve the user from
complying with the complete regulations contained in the Code of Federal Regulations
(CFR). The regulations may change or additional state and local laws may apply; waste
generators are responsible for knowing and following all the laws and regulations that
apply to their operations.
5.1 Waste minimization

The most effective way to decrease waste management problems and expense is
through waste minimization. To do this:
Use the smallest sample size that will produce accurate results.
Where possible, choose methods that use reagents that pose fewer hazards.
Eliminate the need to dispose of out-dated materials by purchasing in smaller

quantities.
Use biodegradable detergents to clean glassware and apparatus unless solvents or

acids are specifically required.
5.2 Regulatory overview

The Resource Conservation and Recovery Act (RCRA) controls all solid waste disposal
with an emphasis on hazardous waste. Title 40 Code of Federal Regulations (CFR) part
260 contains the federal hazardous waste disposal regulations issued in accordance with
the RCRA. The regulations create a system to identify hazardous wastes and track waste
generation, transport and ultimate disposal from cradle to grave. Each facility involved in
hazardous waste management must be registered with the USEPA, with the exception of
conditionally exempt small quantity generators.
Federal regulations recognize three categories of generators with those generating larger
amounts of waste being under stricter control.
The categories are:
Conditionally Exempt Small Quantity Generator less than 100 kg (220 lb) per month
Small Quantity Generator between 100 kg (220 lb) and 1000 kg (2200 lb) per month
Large Quantity Generator greater than 1000 kg (2200 lb) per month.
5.3 Hazardous waste

5.3.1 Definition
For regulatory purposes, a hazardous waste is a material that is subject to special
consideration by the USEPA under 40 CFR 261. State or local authorities may also
designate additional materials as hazardous waste in their areas.
Many toxic compounds are not regulated, but improper management or disposal may
lead to legal problems under CERCLA (Superfund) or common law tort.
Page 55

The definition given by 40 CFR 261 defines a hazardous waste as a solid waste that is
not excluded from regulation and meets one or more of the following criteria:
It is a discarded commercial chemical product, off-specification species, container

residue or spill residue of materials specifically listed in 40 CFR 261.33 (P- and
U-codes);
It is a waste from a specific source listed in 40 CFR 261.32 (K-code);
It is a waste from a non-specific source listed in 40 CFR 261.31 (F-code);
and/or
It displays any of the following characteristics of hazardous waste:
ignitability
corrosivity
reactivity
toxicity
There are exceptions to these regulations; review the regulations to determine applicable
exclusions.
5.3.2 Sample codes

Hazardous wastes are managed by specific codes assigned in 40 CFR 261.20261.33.
These codes are provided to help identify hazardous waste. The generator is responsible
for making the actual waste code determination.
Selected characteristic waste codes for chemicals which may be generated using
methods for water analysis are given in the following table. A complete list of waste codes
is found in 40 CFR 261.20 through 40 CFR 261.33.
Table 14 Hazardous waste codes
Characteristic
Page 56
USEPA code
Chemical abstract services

(CAS) No.
Regulatory level
(mg/L)
Corrosivity
D002
na
na
Ignitability
D001
na
na
Reactivity
D003
na
na
Arsenic
D004
6440-38-2
5.0
100.0
Barium
D005
6440-39-3
Benzene
D018
71-43-2
0.5
Cadmium
D006
7440-43-9
1.0
Chloroform
D022
67-66-3
6.0
Chromium
D007
7440-47-3
5.0
Lead
D008
7439-92-1
5.0
Mercury
D009
7439-97-6
0.2
Selenium
D010
7782-49-2
1.0
Silver
D011
7440-22-4
5.0

5.3.3 How to determine if waste is hazardous
Federal laws do not require testing of material to decide if it is a hazardous waste. If the
product is not specifically listed in the regulations, apply product or generator knowledge
to decide if it is hazardous. Often, there is enough information on a Material Safety Data
Sheet (MSDS) to decide. Look for characteristics of a hazardous waste:
Flash point is below 60 C (140 F) or it is classified by DOT as an oxidizer (D001).
The pH of the material is 2 or12.5 (D002).
The material is unstable, reacts violently with water, may generate toxic gases when
mixed with water (D003).
It is toxic (D004D043).
Use the chemical composition data to decide if a material is toxic based on the
concentration of certain contaminants (Heavy metals and a number of organic
compounds). If the waste is a liquid, compare the concentration of contaminants to the
concentrations listed in 40 CFR 26. If the waste is a solid, analyze the sample by the
Toxicity Characteristic Leachability Procedure (TCLP) and then compare the results to the
concentrations in 40 CFR 261.24. Levels above the threshold amounts are considered
hazardous.
For more information on using the MSDS, see Material safety data sheets.
Some tests use or produce a number of chemicals that make the end product a
hazardous waste; for example, the COD tests and Nessler reagent. Hazardous waste
status may also result from substances present in the sample.
5.3.4 Disposal
Hazardous waste must be managed and disposed of according to federal, state and local
regulations. The waste generator is responsible for making hazardous waste
determinations. Analysts should check with their facilitys environmental compliance
department for specific instructions.
Most hazardous wastes should be handled by treatment, storage and disposal facilities
(TSDF) that have USEPA permits. In some cases, the generator may treat the hazardous
waste, but may need a permit from the USEPA and/or state agency. Laboratories are not
exempt from these regulations. If the facility is a Conditionally Exempt Small Quantity
Generator, special rules may apply. Check 40 CFR 261 to determine the laws and rules
that apply for a given generator.
The most common allowed treatment is elementary neutralization. This applies to wastes
that are hazardous only because they are corrosive or are listed only for that reason.
Neutralize acidic solutions by adding a base such as sodium hydroxide; neutralize basic
solutions by adding an acid such as hydrochloric acid. Slowly add the neutralizing agent
while stirring. Monitor the pH. When it is at or near 7, the material is neutralized and may
be flushed down the drain. Many generated wastes may be treated in this manner.
Other chemical or physical treatments such as cyanide destruction or evaporation may
require a permit. Check with the environmental department or local regulators to
determine which rules apply to each facility.
Laboratory chemicals may be mixed and disposed of with other hazardous wastes
generated at a facility. They may also be accumulated in accordance with 40 CFR 262.34
satellite accumulation rules. After collection they may be disposed of in a labpack. Many
environmental and hazardous waste companies offer labpacking services. They will
inventory, sort, pack and arrange for proper disposal of hazardous waste. Find
companies offering these services in the Yellow Pages under Waste Disposal
Hazardous or contact state and local regulators for assistance.
Page 57
5.4 Management of specific wastes

Recyling programs for some forms of laboratory waste are available through Hach
Company. Obtain information on recycling services by calling 1-800-227-4224 or by
visiting www.hach.com."
Several documents are also available to assist in managing generated waste. Obtain
available documents by calling 1-800-227-4224 or 970-669-3050 and requesting the
literature codes given:
Table 15 Waste management literature
Literature code
Title
9323
Mercury Waste Disposal Firms
9324
RCRA Waste Disposal Information
9325
COD Waste Disposal Information
9326
COD Heavy Metal Concentrations
5.4.1 Special considerations for Cyanide-containing materials

Several procedures in this manual use reagents that contain cyanide compounds. These
materials are regulated as reactive waste (D003) by the Federal RCRA. Instructions
provided with each procedure explain how to collect these materials for proper disposal.
It is imperative that these materials be handled safely to prevent the release of hydrogen
cyanide gas (an extremely toxic material with the smell of bitter almonds). Most cyanide
compounds are stable and can be safely stored for disposal, in highly alkaline solutions
(pH >11) such as 2 N sodium hydroxide. Never mix these wastes with other laboratory
wastes that may contain lower pH materials such as acids or even water.
If a cyanide-containing compound is spilled, avoid exposure to hydrogen cyanide gas.
Take the following steps to destroy the cyanide compounds in an emergency:
1. Use a fume hood, supplied air or self-contained breathing apparatus.
2. While stirring, add the waste to a beaker containing a strong solution of sodium
hydroxide and either calcium hypochlorite or sodium hypochlorite (household bleach).
3. Add an excess of hydroxide and hypochlorite. Let the solution stand for 24 hours.
4. Neutralize the solution and flush it down the drain with a large amount of water. If the
solution contains other regulated materials such as chloroform or heavy metals, it
may still need to be collected for hazardous waste disposal. Never flush untreated
hazardous wastes down the drain.
5.5 Resources
Many sources of information on proper waste management are available. The USEPA
has a hotline number for questions about the Resource Conservation and Recovery Act
(RCRA). The RCRA Hotline number is 1-800-424-9346. Copies of the appropriate
regulations are available. Federal hazardous waste regulations are found in 40 CFR
260-99. Obtain this book from the U.S. Government Printing Office or an alternate
vendor. Other documents that may be helpful to the hazardous waste manager in the
laboratory include:
Page 58
Task Force on Laboratory Waste Management. Laboratory Waste Management, A

Guidebook; American Chemical Society, Department of Government Relations and
Science Policy: Washington, DC 1994.
Task Force on Laboratory Waste Management. Waste Management Manual for

Laboratory Personnel; American Chemical Society, Department of Government
Relations and Science Policy: Washington, DC 1990.
Task Force on Laboratory Waste Management. Less is Better;

2nd ed.; American Chemical Society, Department of Government Relations and
Science Policy: Washington, DC 1993.
Committee on Chemical Safety. Safety in Academic Chemistry Laboratories, 5th ed.;

American Chemical Society: Washington, DC, 1990.
Armour, Margaret-Ann. Hazardous Laboratory Chemicals Disposal Guide; CRC

Press: Boca Raton, FL, 1991.
Environmental Health and Safety Managers Handbook; Government Institutes, Inc.:

Rockville, MD, 1988.
Lunn, G.; Sansone, E.B. Destruction of Hazardous Chemicals in the Laboratory; John
Wiley and Sons: New York, 1990.
National Research Council. Prudent Practices for Disposal of Chemicals from

Laboratories; National Academy Press: Washington, DC, 1983.
National Research Council. Prudent Practices for Handling Hazardous Chemicals in

Laboratories; National Academy Press: Washington, DC, 1981.
Environmental Protection Agency, Office of Solid Waste and Emergency Response.

The RCRA Orientation Manual; U.S. Government Printing Office: Washington, DC,
1991.
Environmental Protection Agency, Office of Solid Waste and Emergency Response.

Understanding the Small Quantity Generator Hazardous Waste Rules: A Handbook
for Small Business; U.S. Government Printing Office: Washington, DC, 1986.
5.6 Safety
Safety is the responsibility of every analyst. Many chemical procedures use potentially
hazardous chemicals and equipment; it is important to prevent accidents by practicing
good laboratory techniques. The following guidelines apply to water analysis and are not
intended to cover every aspect of safety.
5.6.1 Read labels carefully

Read each reagent label carefully. Pay particular attention to the precautions given.
Never remove or cover the label on a container while it contains reagent. Do not put a
different reagent into a labeled container without changing the label. When preparing a
reagent or standard solution, label the container clearly. If a label is hard to read, relabel
promptly according to the hazard communication program.
Warning labels also appear on some of the apparatus used with the test procedures. The
protective shields with the Digesdahl Digestion Apparatus point out potential hazards. Be
sure these shields are in place during use and observe the precautions on the label.
5.6.2 Protective equipment

Use the right protective equipment for the chemicals and procedures. The MSDS
contains this information. Protective equipment may include:
Eye protection, such as safety glasses or goggles, to protect from flying objects or
chemical splashes.
Gloves to protect skin from toxic or corrosive materials, sharp objects, very hot or very
cold materials or broken glass. Use tongs or finger cots when transferring hot
apparatus.
Laboratory coats or splash aprons to protect skin and clothing from splashes.
Footwear to protect feet from spills. Open toed shoes should not be worn in chemistry
settings.
Page 59
Respirators may be needed if adequate ventilation, such as fume hoods, are not
available. Use fume hoods when directed to do so by the procedure or as
recommended in the MSDS. For many procedures, adequate ventilation is enough if
there is plenty of fresh air and air exhaust to protect against unnecessary exposure to
chemicals.
5.6.3 First aid equipment and supplies

Most first aid instructions for chemical splashes in eyes or on skin call for thorough
flushing with water. Laboratories should have eyewash and shower stations. For field
work, carry a portable eyewash unit. Laboratories should also have appropriate fire
extinguishers and fume hoods.
5.6.4 General safety rules

Follow these rules when working with toxic and hazardous chemicals:
Never pipet by mouth. Always use a mechanical pipet or pipet bulb to avoid ingesting
chemicals.
Follow test procedures carefully and observe all precautionary measures. Read the
entire procedure before beginning.
Wipe up all spills promptly. Get proper training and have the right response equipment
to clean up spills. See the safety director for more information.
Do not smoke, eat or drink in an area where toxic or irritating chemicals are used.
Use reagents and equipment only as directed in the test procedure.
Do not use damaged labware and broken equipment.
Minimize all chemical exposures. Do not breathe vapors or let chemicals touch the
skin. Wash hands after using chemicals.
Keep work areas neat and clean.
Do not block exits or access to emergency equipment.
5.7 Material safety data sheets

Material safety data sheets (MSDS) describe the hazards of chemical products. This
section explains the information found on the MSDS and tells how to locate important
information for safety and waste disposal. The information provided on the MSDS applies
to the product as sold by a specific manufacturer. The properties of any mixtures obtained
by using this product will be different.
5.7.1 How to obtain an MSDS

The MSDS is shipped to a customer with the first order of any chemical product. A new
MSDS may be sent when the information on the data sheet is updated. Please review all
new MSDS for new information. To obtain another copy of an MSDS, call 1-800-227-4224
or download directly from www.hach.com/msdsinfo.htm.
5.7.2 Sections of an MSDS

Each MSDS has ten sections. The sections and the information included are
described below.
Header information
The manufacturer order number, MSDS date, change number, company address and
telephone number and emergency telephone numbers are listed at the top of the MSDS.
Page 60

5.7.2.1 Product identification
This section contains:
Product name
Chemical Abstract Services (CAS) number
Chemical name
Chemical formula, if appropriate
Chemical family to which the material belongs
5.7.2.2 Ingredients
This section lists each component in the product. It contains the following information for
each component:
PCT: Percent by weight of this component
CAS NO.: Chemical Abstract Services (CAS) registry number for this component
SARA: Superfund Amendments and Reauthorization Act, better known as the

Community Right to Know Law, informs if the component is listed in SARA 313. If the
component is listed and the facility uses more than the specified amount, report use to
the USEPA every year.
TLV: Threshold Limit Value. The maximum airborne concentration for an 8-hour
exposure that is recommended by the American Conference of Governmental
Industrial Hygienists (ACGIH).
PEL: Permissible Exposure Limit. The maximum airborne concentration for an 8-hour
exposure that is regulated by the Occupational Safety and Health Administration
(OSHA).
HAZARD: Physical and health hazards of the component are explained.
5.7.2.3 Physical data

The physical properties of the product are given in this section. They include the physical
state, color, odor, solubility, boiling point, melting point, specific gravity, pH, vapor density,
evaporation rate, corrosivity, stability and storage precautions.
5.7.2.4 Fire, explosion hazard and reactivity data

This section contains the flash point and flammable limits of the material. It also includes
how to fight fires if the material catches on fire. Key terms in this section include:
Flash point: The temperature at which a liquid will give off enough flammable vapor to
ignite.
Flammability and ignitability are usually defined by the flash point.
Lower Flammable Limit (LFL or LEL): The lowest concentration that will produce a fire
or flash when an ignition source is present.
Upper Flammable Limit (UFL or UEL): The vapor concentration in air above which the
concentration is too rich to burn.
NFPA Codes: The National Fire Protection Association (NFPA) has a system to rate
the degree of hazards presented by a chemical. These codes are usually placed in a
colored diamond. The codes range from 0 for minimal hazard to 4 for extreme hazard.
They are grouped into the following hazards: health (blue), flammability (red),
reactivity (yellow) and special hazards (white).
Page 61

5.7.2.5 Health hazard data
This section describes the pathways by which the chemical can enter the body (i.e.,
ingestion, inhalation, skin contact). It also gives acute (immediate) and chronic
(long-term) health effects. If the material causes cancer or genetic damage, it is stated in
this section.
5.7.2.6 Precautionary measures

This section contains special precautions for the material. These may include special
storage instructions, handling instructions, conditions to avoid and protective equipment
required to use this material safely.
5.7.2.7 First aid

First aid instructions for exposures to the chemical are given in this section. Be sure to
read this section before inducing vomiting in a victim. Some chemicals are better treated
by not inducing vomiting. Seek prompt medical attention for all chemical exposures.
5.7.2.8 Spill and disposal procedures

This section explains safe practices for cleaning up and disposing of spilled material. For
more information, see Hazardous waste. The waste generator is ultimately
responsible for meeting the federal, state and local laws that apply to each facility.
5.7.2.9 Transportation data

Domestic and International shipping information is provided in this section. It gives
shipping name, hazard class and ID number of the product.
5.7.2.10 References
This section lists the reference materials used to write the MSDS.
Following the Reference section, the product will be listed as having SARA 313
chemicals or California Proposition 65 List Chemicals, if applicable. Also found here is
any special information about the product.
5.7.3 OSHA chemical hygiene plan

The Occupational Safety and Health Administration (OSHA) enforces laws controlling
exposure to hazardous chemicals in laboratories. These regulations are in Title 29 CFR
1910.1450. The regulations apply to all employers who use hazardous chemicals and
require employers to develop and use a written Chemical Hygiene Plan and to appoint a
qualified person as the Chemical Hygiene Officer.
Page 62
Section 6
International Guideline Comparison

The following table shows a comparison between international drinking water and FDA
bottled water guidelines:
Table 16 Comparison of international drinking water and FDA bottled water guidelines1
Parameter
Aluminum
0.050.2 mg/L7
0.2 mg/L
0.2 mg/L
0.2 mg/L
0.5 mg/L
No standard
1.5 mg/L
Antimony
0.006 mg/L
0.01 mg/L
0.002 mg/L8
0.005 mg/L
Arsenic
0.05 mg/L
0.025 mg/L
0.05 mg/L
0.01 mg/L
0.01 mg/L
0.05 mg/L
Barium
2.0 mg/L
1.0 mg/L
No standard
No standard
0.7 mg/L
2.0 mg/L
Boron
5.0 mg/L
1.0 mg/L
0.2 mg/L8
0.3 mg/L
Cadmium
0.005 mg/L
0.005 mg/L
0.005 mg/L
0.01 mg/L
0.003 mg/L
0.005 mg/L
Chloride
250 mg/L7
250 mg/L
250 mg/L
200 mg/L
250 mg/L
0.1 mg/L
0.05 mg/L
0.05 mg/L
0.05 mg/L
0.05 mg/L
0.1 mg/L
% positive
0 or MPN 1
1 MF
15 cu7
15 cu
20 mg Pt-Co/L
5 cu
15 cu
<15 cu
1.3 mg/L7
1.0 mg/L
2.0 mg/L
1.0 mg/L
12 mg/L
1.0 mg/L
Ammonium
Chromium
Coliforms, total
Organisms/100
mL
Coliforms (E. coli)
Organisms/100
mL
Color
Copper
Canada3
maximum
acceptable
concentration
EEC4
maximum
admissible
concentration
Japan5
maximum
admissible
concentration
Bottled Water
U.S. Federal
Drug
Administration
level
USEPA2
maximum
contaminant
level (MCL)
WHO6
guideline
Cyanides
0.2 mg/L
0.2 mg/L
0.05 mg/L
0.01 mg/L
0.07 mg/L
Fluoride
2.0-4.0 mg/L7
1.5 mg/L
0.7-1.5 mg/L
0.8 mg/L
1.5 mg/L
50 mg/L
300 mg/L
Hardness
Iron
0.3 mg/L7
0.3 mg/L
0.2 mg/L
0.3 mg/L
0.3 mg/L
Lead
0.015 mg/L
0.01 mg/L
0.01 mg/L
0.05 mg/L
0.01 mg/L
0.005 mg/L
Manganese
0.05 mg/L
0.05 mg/L
0.05 mg/L
0.010.05 mg/L
0.10.5 mg/L
Mercury
0.002 mg/L
0.001 mg/L
0.001 mg/L
0.0005 mg/L
0.001 mg/L
0.002 mg/L
Molybdenum
0.07 mg/L
0.07 mg/L
0.1 mg/L
0.02 mg/L
0.01 mg/L8
0.02 mg/L
Nitrate/Nitrite, total
10.0 mg/L as N
10.0 mg/L as N
10 mg/L as N
Nitrates
10.0 mg/L as N
10.0 mg/L as N
50 mg/L
10 mg/L as N
50 mg/L as
NO3-
Nitrites
1 mg/L as N
3.2 mg/L
0.1 mg/L
10 mg/L
3 mg/L as
NO2-
1 mg/L as N
Odor
3 TON9
2 dilution no.
@ 12 C;
3 dilution no.
@ 25 C.
3 TON
pH
Nickel
6.58.5
6.58.5
6.59.5
5.88.6
6.58.5
Phosphorus
5 mg/L
No Standard
Phenols
0.002 mg/L
0.5 g/L
C6H5OH
0.005 mg/L
Page 63
International Guideline Comparison

Table 16 Comparison of international drinking water and FDA bottled water guidelines1 (continued)
Parameter
USEPA2
maximum
contaminant
level (MCL)
Canada3
maximum
acceptable
concentration
EEC4
maximum
admissible
concentration
Japan5
maximum
admissible
concentration
WHO6
guideline
Bottled Water
U.S. Federal
Drug
Administration
level
Potassium
12 mg/L
No Standard
Selenium
0.05 mg/L
0.01 mg/L
0.01 mg/L
0.01 mg/L
0.01 mg/L
0.05 mg/L
Silica Dioxide
10 mg/L
No Standard
Silver
0.1 mg/L7
0.05 mg/L
0.01 mg/L
No standard
No standard
Solids, total
dissolved
500 mg/L7
500 mg/L
No standard
500 mg/L
1000 mg/L
Sodium
75-150 mg/L
200 mg/L
200 mg/L
Sulfate
250 mg/L7
500 mg/L
250 mg/L
No Standard
250 mg/L
Turbidity
0.5-5 NTU
1 NTU
4 JTU
12 units
5 NTU
<5 NTU
5 mg/L7
5.0 mg/L
No Standard
1.0 mg/L
3.0 mg/L
Zinc
1 Contact
the local regulatory agency for the most current information.
2 United
States Environmental Protection Agency.
3 These
limits are established by Health Canada.
4 In
the EEC (European Economic Community), limits are set by the European Committee for Environmental Legislation.
5 In
Japan, limits are established by the Ministry of Health and Welfare.
6 World
7 U.S.
Health Organization.
Secondary MCL.
8 Identified
as a parameter to be regulated in the future.
9 Threshold
Page 64
Odor Number.
Section 7
Definitions of USEPA Approved and Accepted
7.1 USEPA approved

The United States Environmental Protection Agency (USEPA) establishes limits for
maximum contamination levels of certain constituents in water. It also requires that
specific methodology be used to analyze for these constituents. Sometimes the USEPA
develops these methods; more often, the USEPA evaluates methods developed by
manufacturers, professional groups, and public agencies such as:
American Public Health Association
American Water Works Association
Water Environmental Federation
American Society for Testing and Materials
United States Geological Survey
Association of Official Analytical Chemists
When a method meets the USEPA criteria, it is approved. All USEPA approved methods
are cited in the Federal Register and compiled in the Code of Federal Regulations.
USEPA-approved methods may be used for reporting results to the USEPA and other
regulatory agencies.
7.2 USEPA accepted

Some procedures are equivalent to USEPA approved methods. Even though minor
modifications exist, the USEPA has reviewed and accepted certain procedures for
reporting purposes. These methods are not published in the Federal Register, but are
referenced to the equivalent USEPA method in the procedure.
Page 65
Definitions of USEPA Approved and Accepted
Page 66

Sample cells
2495402
2401906
1353702
1993500
2095000
2095100
2410212
2410222
2427606
4822800
2629250
5940506
2612602
4864302
2122800

Page 67 of 68
Instruments and adapters
DR 5000
DR 2800 and DR 2700
DR2500
DR2400
LZV583
LZV584
LZV585
LZV646
LZV479
5940400
5912200

Page 68 of 68
Procedures for Analysis
Page 69
Page 70
Acid-Base, DT, 8200 and 8233
Acid-Base
DOC316.53.01165
Acid Determination and Base Determination
Method 8200 and 8233
1 to 4000 meq/L
Digital Titrator
Scope and Application: For water, wastewater and seawater.
Test preparation
Before starting the test:

Four drops of Phenolphthalein Indicator Solution1 can be substituted for the Phenolphthalein Indicator Powder Pillow.
A pH meter can be used in place of the indicators. The end point for the titration is pH 8.3.
To change the result from meq/L to normality (N), divide the result in meq/L by 1000.
For added convenience when stirring, use the TitraStir stirring apparatus1.
1
See Optional reagents and apparatus.
Collect the following items:

Description
Quantity
Phenolphthalein Indicator Powder Pillow
1 pillow
Ttitration cartridge (see Range-specific information)
1 cartridge
Digital titrator
Delivery tube for digital titrator
Graduated cylinder
Erlenmeyer flask, 250-mL
See Consumables and replacement items for reorder information.
Acid determination (Method 8200)
See
Table 1
1. Select a sample
volume and sodium
hydroxide titration
cartridge from the Rangespecific information table.
2. Insert a clean delivery

tube into the titration
cartridge. Attach the
cartridge to the titrator.
3. Turn the delivery knob

to eject air and a few drops
of titrant. Reset the
counter to zero and wipe
the tip.
4. Use a graduated
cylinder or pipet to
measure the sample
volume from the Rangespecific information table.
Acid-Base
Page 71
Acid-Base
Acid determination (Method 8200)
5. Transfer the sample

into a clean, 250-mL
Erlenmeyer flask. If the
sample volume is less
than 100 mL, dilute to
approximately 100 mL with
deionized water.
6. Add the contents of

one Phenolphthalein
Indicator Powder Pillow.
Swirl to mix. The solution
should be colorless.
7. Place the delivery tube

into the solution and swirl
the flask. Turn the knob on
the titrator to add titrant to
the solution. Continue to
swirl the flask and add
titrant until a light pink
color forms and persists
for 30 seconds.
Write down the number of
digits displayed on the
counter.
8. Use the multiplier in

the Range-specific
information table to
calculate the
concentration:
digits x multiplier =
meq/L acid
Example: 100 mL of
sample was titrated with
the 8.00 N cartridge and
250 digits were used to
reach the endpoint. The
concentration is 250 x 0.1
= 25 meq/L acid
Base determination (Method 8233)
See
Table 1
1. Select a sample
volume and acid titration
Acid-Base
Page 72
2. Use a graduated
measure the sample
volume from theRangespecific information table.


the tip.
Acid-Base
Base determination (Method 8233)

deionized water.

one Phenolphthalein
Swirl to mix. The solution
should be pink.

titrant until the solution is
colorless.
counter.

the Range-specific
calculate the
concentration:
meq/L base
Example: 100 mL of
= 25 meq/L base
Table 17 Range-specific information
Range (meq/L)
Sample volume (mL)
Titration cartridge1 (N)
14
100
1.600
0.02
410
50
1.600
0.04
1040
100
8.00
0.1
2080
50
8.00
0.2
0.5
Multiplier
50200
20
8.00
100400
10
8.00
1.0
200800
8.00
2.0
5002000
8.00
5.0
10004000
8.00
10.0
For acid determinations, use a sodium hydroxide titration cartridge. For base determinations, use a hydrochloric acid or sulfuric acid cartridge.
Interferences
Color or turbidity can mask the color change of the end point. Use a pH meter instead of the color
indicators and titrate to a pH of 8.3 to duplicate the phenolphthalein end point.
Acid-Base
Page 73
Acid-Base
Sample collection, preservation and storage
Collect samples in clean plastic or glass bottles. Fill completely and cap tightly.
Prevent excessive agitation or prolonged exposure to air.
Complete the test procedure as soon as possible after collection for best accuracy. The
sample can be stored for at least 24 hours if cooled to 4 C (39 F) or below.
Warm to room temperature before the test is started.
Accuracy check
The standard solution method can be used to confirm analytical technique and reagent
performance.
Standard solution method
Complete the following test to make sure the concentration of the titrant is accurate.
Required for accuracy check:
For acid determination0.500 N Sulfuric Acid Standard Solution
For base determinationAlkalinity Standard Solution, 0.500 N Na2CO3
1. Add the standard solution to an Erlenmeyer flask:
If using the 1.600 N Titration Cartridge, pipet 1.0 mL of the standard solution into the
Erlenmeyer flask and dilute to approximately 100 mL with deionized water.
If using the 8.00 N Titration Cartridge, pipet 5.0 mL of the standard solution into the
Erlenmeyer flask and dilute to approximately 100 mL with deionized water.
2. Add one Phenolphthalein Powder Pillow and swirl to mix.

3. Follow the test procedure to titrate the standard to the end point. The expected digits to reach
the end point are 250.
Summary of method
A measured amount of sample is treated with a phenolphthalein colorimetric indicator and then
titrated with a strong acid or base to a pH of 8.3. The amount of titrant used is directly proportional
to the milliequivalents of acid or base in the sample. These titrations also can be performed using
a pH meter instead of a colorimetric indicator.
Acid-Base
Page 74
Acid-Base
Consumables and replacement items

Required reagents
Description
Hydrochloric Acid Titration Cartridge, 8.00 N
Phenolphthalein Indicator Powder Pillows
Sodium Hydroxide Titration Cartridge, 1.600 N
Quantity/Test
Unit
Catalog number
varies
each
1439001
1 pillow
100/pkg
94299
varies
each
1437901
varies
each
1438101
Sulfuric Acid Titration Cartridge, 1.600 N
varies
each
1438901
Sulfuric Acid Titration Cartridge, 8.00 N
varies
each
1439101
Required apparatus
Description
Quantity/Test
Digital Titrator
Flask, Erlenmeyer, graduated, 250-mL
Unit
Catalog number
each
1690001
each
50546
Graduated cylinderselect one or more based on range:

each
50837
each
50838
each
50840
each
50841
each
50842
Recommended standards
Description
Unit
Catalog number
Alkalinity Standard Solution, 0.500 N Na2CO3, 10-mL ampules
16/pkg
1427810
Sulfuric Acid Standard Solution, 0.500 N
100 mL
212132
Description
Unit
Catalog number
Stir bar, octagonal 28.6 mm x 7.9 mm
each
2095352
TenSette Pipet, 0.1 to 1.0 mL
each
1970001
500 mL
27249
Pipet, volumetric, Class A, 1 mL
each
1451535
each
1451536
1451537
Optional reagents and apparatus
Water, deionized
each
each
1451538
each
1451520
Pipet Filler, safety bulb
each
1465100
Bottle, sampling, poly, 500 mL
each
2087079
pH meter
each
Delivery Tube, 180 hook
5/pkg
1720500
TitraStir stir plate, 115 Vac
each
1940000
each
1940010
Stir Bar, Octagonal 28.6 mm x 7.9 mm
each
2095352
Acid-Base
Page 75
FOR TECHNICAL ASSISTANCE, PRICE INFORMATION AND ORDERING:

In the U.S.A. Call toll-free 800-227-4224
Outside the U.S.A. Contact the HACH office or distributor serving you.
On the Worldwide Web www.hach.com; E-mail techhelp@hach.com
Hach Company, 2007, 2010, 2012. All rights reserved. Printed in the U.S.A.
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Acid-Base, BT, 8288 and 8289
Acid-Base
DOC316.53.01148
Sodium Hydroxide for meq/L of Acid

Sulfuric Acid for meq/L of Base
(0 to 25,000 meq/L)
Method 8288
Method 8289
Buret Titration
Scope and Application: For water, wastewater and seawater
Test preparation

Read the entire procedure before starting the test.
Four drops of Phenolphthalein Indicator Solution can be substituted for the Phenolphthalein Indicator
Powder Pillow.
To determine the normality of the sample, divide the milliequivalents per liter obtained by 1000.

Description
Phenolphthalein indicator powder pillow
Sodium hydroxide standard solution (acid test)
varies1
Sulfuric acid standard solution (base test)
varies1
Buret clamp
Buret, Class A
Graduated cylinder
Quantity
varies1
Erlenmeyer flask
Support Stand
See Consumables and replacement items.
Acid-Base
Page 77
Acid-Base
Sodium hydroxide acid determination (Method 8288)
See
Table 1
1. Select the sample

volume that corresponds
to the expected acid
concentration in
milliequivalents per liter
(meq/L) or normality (N)
from the Sample volume
selection for expected acid
concentration table.
2. Fill a 25-mL buret to

the zero mark with the
appropriate Sodium
Hydroxide Solution.
3. Measure the sample

volume from the Sample
volume selection for
expected acid
concentration table using
a graduated cylinder or
pipet.

one Phenolphthalein
Indicator Powder Pillow
and swirl to mix. The
solution should be
colorless.
6. Swirl the flask while

titrating until a light pink
color forms and persists
for 30 seconds.
7. Calculate:
4. Pour the sample into a

250-mL Erlenmeyer flask.
Dilute to 50100 mL with
deionized water, if
necessary.
mL NaOH Titrant
Multiplier used = meq/L of
Acid
Table 18 Sample volume selection for expected acid concentration

Range
(meq/L)
(N)
Sample Volume
(mL)
Sodium Hydroxide Standard

Solution Titrant (N)
Catalog
Number
Multiplier
0.4
010
00.010
50
0.020
19353
10100
0.0100.100
25
0.100
19153
100500
0.1000.500
50
1.000
104553
20
5002500
0.5002.50
10
1.000
104553
100
250025000
2.5025.0
1.000
104553
1000
Acid-Base
Page 78
Acid-Base
Sulfuric acid base determination (Method 8289)
See
Table 2

to the expected base
concentration in
milliequivalents per liter
(meq/L) or normality (N)
from the Sample volume
selection for expected
base concentration table.

appropriate Sulfuric Acid
Standard Solution.

volume from Table 2 using
a graduated cylinder or
pipet.

one Phenolphthalein
and swirl to mix. The
solution should turn pink.
6. Swirl the flask while

titrating just until the
solution turns colorless.
7. Calculate:

Dilute to 50100 mL with
deionized water, if
necessary.
mL Sulfuric Acid Titrant X

Multiplier used = meq/L of
Base
Table 19 Sample volume selection for expected base concentration

Range
(meq/L)
Sample Volume (mL)
Sulfuric Acid Standard Solution

Titrant (N)
(N)
Catalog Number Multiplier
010
00.010
50
0.020
20353
10100
0.0100.100
25
0.100
20253
0.4
4
100500
0.1000.500
50
1.000
127053
20
5002500
0.5002.50
10
1.000
127053
100
250025000
2.5025.0
1.000
127053
1000
Acid-Base
Page 79
Acid-Base
Interferences
Highly colored or turbid samples may mask the color change at the end point. Use a pH meter for
these samples. Titrate to pH 8.3 to duplicate the colorimetric phenolphthalein end point.
Sampling and storage

Collect samples in clean plastic or glass bottles. Fill completely and cap tightly. Minimize agitation
or prolonged exposure to air. Samples may be stored at least 24 hours by cooling to 4 C (39 F)
or below if they cannot be analyzed immediately. Warm to room temperature before analyzing.
Accuracy check
For acid determinations, use a 20-mL volumetric pipet to measure Sulfuric Acid Standard Solution
of the same normality as the Sodium Hydroxide Standard Solution being used. Pipet the sulfuric
acid into an Erlenmeyer flask. Dilute to the 100-mL mark with deionized water. Perform the test; 20
mL of sodium hydroxide should be required.
For base determinations, use a 20-mL volumetric pipet to measure Sodium Hydroxide Standard
Solution of the same normality as the Sulfuric Acid Standard Solution being used. Pipet sodium
hydroxide into an Erlenmeyer flask. Dilute to the 100-mL mark with deionized water. Perform the
test; 20 mL of sulfuric acid should be required.
If incorrect results are obtained, refer to Method Performance in the Water Analysis Guide.
Summary of method
A measured amount of sample is treated with a phenolphthalein colorimetric indicator and then
titrated with a strong acid or base to pH 8.3. The amount of titrant used is directly proportional to
the milliequivalents of acid or base in the sample. These titrations also can be performed using a
pH meter instead of a colorimetric indicator.
Acid-Base
Page 80
Acid-Base

Required reagents
Description
Water, deionized
Quantity/test
Unit
1 pillow
100/pkg
Catalog number
94299
varies
4L
27256
For acid, pick one or more based on range:

Sodium Hydroxide Standard Solution, 0.020 N
1L
19353
1L
19153
1L
104553
1L
20353
1L
20253
1L
127053
Catalog number
For base, pick one or more based on range:
Required apparatus
Description
Quantity/test
Unit
Buret clamp, double
each
32800
Buret, Class A, 25-mL
each
2636540
Pick one or more based on sample volume:

each
50837
each
50838
each
50840
each
50841
each
50546
Pipet, volumetric, Class A, 1-mL
each
1451535
each
1451538
Support stand
each
56300
Description
Unit
Catalog number
Pipet, 20-mL, volumetric
each
1451520
Pipet filler, Safety bulb
each
1465100
16232
Optional items
Phenolphthalein Indicator Solution, 5 g/L100 mL
Acid-Base
Page 81

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Acid-Base, BT, 8219
Acidity, Methyl Orange
DOC316.53.01150
Sodium Hydroxide with a Buret1

(0 to 10,000 mg/L as CaCO3)
Method 8219
Buret Titration

1
Adapted from Standard Methods for the Examination of Water and Wastewater.
Test preparation

Avoid excessive agitation of the sample to prevent the loss of dissolved gases such as carbon dioxide, hydrogen sulfide
or ammonia.
Six drops of Phenolphthalein Indicator Solution may be substituted for the Phenolphthalein Powder Pillow.
When determining Methyl Orange Acidity, six drops of Bromphenol Blue Indicator Solution can be substituted for the
Bromphenol Blue Indicator Powder Pillow. Methyl Orange Indicator Solution can also be substituted for the indicator. When
using the Methyl Orange Indicator Solution, the end point color change then becomes red to orange.
The Methyl Orange and Phenolphthalein Acidity procedures can be run sequentially if both values are desired. First, titrate to
pH 3.7 and record the amount of sodium hydroxide used. Then titrate to pH 8.3.
Phenolphthalein acidity in mg/L CaCO3 x 0.0584 = grains per gallon

Description
Bromphenol Blue indicator powder pillow
Sodium hydroxide standard solution, 0.020 N
1
varies1
Buret clamp
Buret, Teflon plug, Class A
Graduated cylinder
Quantity
varies1
Erlenmeyer flask
Funnel, Micro
Support Stand
See Consumables and replacement items on page 86.

Page 83
Buret titration (Method 8219)
See
Table 1
1. Select a sample
expected concentration
table that corresponds to
the expected acidity
concentration in mg/L as
calcium carbonate
(CaCO3).
2. Use a graduated
measure the sample
volume.

into a 250-mL Erlenmeyer
flask. Dilute to about
50-mL with deionized
water if necessary.

the zero mark with 0.020 N
Sodium Hydroxide
Standard Solution.
6. While swirling the

flask, titrate the sample
with 0.020 N Sodium
Hydroxide Standard
Solution until the solution
color changes from yellow
to pure green (pH 3.7).
7. Calculate:

one Bromphenol Blue
Swirl to mix. (Omit this
step when using a
pH meter.)
mL Titrant multiplier
used = mg/L methyl
orange acidity as CaCO3
Table 20 Sample volume selection for expected concentration

Range
(mg/L as CaCO3)
Sample Volume (mL)
Sodium Hydroxide
Multiplier
11000
50
19353
20
8002000
25
19353
40
20005000
10
19353
100
400010000
19353
200
Interferences
these samples. Titrate to pH 3.7.

Page 84

Add a drop of 0.1 N Sodium Thiosulfate Standard Solution to remove residual chlorine that may
interfere with the indicator.

Collect samples in clean plastic or glass bottles. Fill completely and cap tightly. Avoid excessive
agitation and prolonged exposure to air. Samples should be analyzed as soon as possible after
collection but can be stored at least 24 hours by cooling to 4 C (39 F) or below. Warm to room
temperature before analyzing.
Accuracy check
End point confirmation
To determine the correct end point, dissolve the contents of a Buffer Powder Pillow, pH 3.7, in 50
mL of deionized water in a 250-mL Erlenmeyer flask. Add the contents of one Bromphenol Blue
Indicator Powder Pillow and swirl to mix. Titrate the prepared water test samples to this same
color.
Standard additions method (Sample spike)
The standard additions method check can be performed as follows:
4. Use a TenSette Pipet to add 0.1 mL of Sulfuric acid standard solution 0.500 N to a prepared
sample titrated to the end point.
5. Swirl to mix and titrate again to the end point. Note the amount of additional titrant used.
6. Make 0.2-mL and 0.3-mL acid additions, titrating to the end point after each addition. The mL
of titrant required should increase by 2.5 mL for each 0.1-mL increment of acid added. If these
increases do not occur, refer to the Water Analysis Guide for the Standard Additions.
Sodium Hydroxide Standard Solution slowly absorbs carbon dioxide when exposed to air, causing
a partial loss of strength. Check the solution frequently (monthly) by titrating 50 mL of Potassium
Acid Phthalate Standard Solution as 100 mg/L CO2 and using a Bromphenol Blue Indicator
Powder Pillow. The titration should require 5.68 mL of Sodium Hydroxide Standard Solution. If the
volume required for the titration is greater than 5.88 mL, discard the solution and replace with a
fresh supply.
Summary of method
Acidity is classified by the pH value of the titration end point. Various pH indicators are used
depending on the pH end point selected. Acidity also can be determined by using a pH meter to
follow the solution pH to the correct end point value as the standard base is added.

Page 85

Required reagents
Description
Quantity/test
Unit
Catalog number
1 pillow
100/pkg
1455099
varies
1L
19353
Water, Deionized
varies
500 mL
27249
Quantity/test
Unit
Catalog number
Buret, Teflon Plug, Class A, 25-mL
each
2636540
Buret Clamp, double
each
32800
Bromphenol Blue Powder Pillows
Required apparatus
Description
Select one or more based on sample volume:

each
50837
each
50838
each
50840
each
50841
each
50546
each
1451537
each
1451538
Support Stand
each
56300
Funnel, Micro
each
2584335
Unit
Catalog number
Required standards
Descripiton
Buffer Powder Pillows, pH 3.7
Potassium Acid Phthalate Standard Solution, 100-mg/L as CO2
100 mL
212132
25
1455168
100 mL
226142
Optional items
Description
Catalog number
Meter, pH, SenION 1 with Platinum pH electrode
5170010
Pipet Filler
1465100
Sodium Thiosulfate Standard Solution, 0.1 N

32332
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Acid-Base, BT, 8010
Acidity, Phenolphthalein
DOC316.53.01149
Sodium Hydroxide with a Buret1

Method 8010
Buret Titration

1
Adapted from Standard Methods for the Examination of Water and Wastewater, 23/0 B (4a).
Test preparation

Avoid excessive agitation of the sample to prevent the loss of dissolved gases such as carbon dioxide, hydrogen sulfide or
ammonia.
Six drops of Phenolphthalein Indicator Solution may be substituted for the Phenolphthalein Powder Pillow.
The Methyl Orange and Phenolphthalein Acidity procedures can be run sequentially if both values are desired. First, titrate to
pH 3.7 and record the amount of sodium hydroxide used. Then titrate to pH 8.3.
Phenolphthalein acidity in mg/L x 0.0584 = grains per gallon

Description
Sodium hydroxide standard solution, 0.020 N
1
varies1
Buret clamp
Buret, Teflon plug, Class A
Graduated cylinder
Quantity
varies1
Erlenmeyer flask
Funnel, Micro
Support Stand
Page 87
See
Table 1
1. Select a sample
the expected acidity
calcium carbonate
(CaCO3).
2. Use a graduated
measure the sample
volume.

water if necessary.

Sodium Hydroxide
Standard Solution.

with 0.020 N Sodium
Hydroxide Standard
Solution until the solution
color changes from
colorless to a light pink
that persists for 30
seconds (pH 8.3).
7. Calculate:

one Phenolphthalein
step if you are using a
pH meter.)
used = mg/L
phenolphthalein acidity
as CaCO3

Range
(mg/L as CaCO3)
Sample Volume (mL)
Sodium Hydroxide
Multiplier
11000
50
19353
20
8002000
25
19353
40
20005000
10
19353
100
400010000
19353
200
Page 88
Interferences
these samples. Titrate to pH 8.3.
Add a drop of 0.1 N Sodium Thiosulfate Standard Solution to remove residual chlorine that may
interfere with the indicator.
Pretreat samples that contain significant amounts of hydrolyzable metals such as iron, aluminum
or manganese as follows:
1. Adjust the sample taken in step 1 of the procedure to pH 4.0 or less (if necessary) by adding
5.0-mL increments of 0.020 N Sulfuric Acid Standard Solution. Record the amount of acid
added. Use a pH meter or appropriate indicator to determine when pH 4.0 is reached. Remove
the pH electrodes after completing this step if a pH meter is used.
2. Using a 1-mL glass serological pipet and pipet filler, add five drops of 30% Hydrogen Peroxide
Solution.
3. Boil the solution for two to five minutes.
4. Cool to room temperature and titrate to pH 8.3 as described in the procedure beginning with
step 3 of the procedure.
5. Subtract the number of milliliters of 0.020 N Sulfuric Acid Standard Solution added from the
number of milliliters of 0.020 N Sodium Hydroxide Standard Solution used in the titration
before multiplying the volume of sodium hydroxide by the multiplier used in step 7 of the
procedure.

agitation and prolonged exposure to air. Samples should be analyzed as soon as possible after
collection but can be stored at least 24 hours by cooling to 4 C (39 F) or below. Warm to room
temperature before analyzing.
Accuracy check
To determine the correct end point, dissolve the contents of a Buffer Powder Pillow, pH 8.3, in 50
mL of deionized water in a 250-mL Erlenmeyer flask. Add the contents of one Phenolphthalein
Indicator Powder Pillow and swirl to mix. Titrate the prepared water test samples to this same
color.
The standard additions method check can be performed as follows:
1. Use a TenSette Pipet to add 0.1 mL of Sulfate standard solution 0.500 N to a prepared sample
titrated to the end point.
2. Swirl to mix and titrate again to the end point. Note the amount of additional titrant used.
3. Make 0.2-mL and 0.3-mL acid additions, titrating to the end point after each addition. The mL
of titrant required should increase by 2.5 mL for each 0.1-mL increment of acid added. If these
increases do not occur, refer to the Water Analysis Guide for Standard Additions.
Sodium Hydroxide Standard Solution slowly absorbs carbon dioxide when exposed to air, causing
a partial loss of strength. Check the solution frequently (monthly) by titrating 50 mL of Potassium
Acid Phthalate Standard Solution as 100 mg/L CO2and using a Phenolphthalein Indicator Powder
Pillow. The titration should require 5.68 mL of Sodium Hydroxide Standard Solution. If the volume
required for the titration is greater than 5.88 mL, discard the solution and replace with a fresh
supply.
Page 89
Summary of method
Acidity is classified by the pH value of the titration end point. Various pH indicators are used
depending on the pH end point selected. Acidity also can be determined by using a pH meter to
follow the solution pH to the correct end point value as the standard base is added.

Required reagents
Description
Phenolphthalein Powder Pillows
Quantity/test
Unit
Catalog number
94299
1 pillow
100/pkg
varies
1L
19353
Water, Deionized
varies
500 mL
27249
Quantity/test
Unit
Catalog number
Buret, Teflon Plug, Class A, 25-mL
each
2636540
Buret Clamp, double
each
32800
Required apparatus
Description

each
50837
each
50838
each
50840
each
50841
each
50546
each,
1451537
each
1451538
Pipet Filler, Safety bulb
each
1465100
Support Stand
each
56300
Funnel, Micro
each
2584335
Required standards
Description
Unit
Catalog number
100 mL MDB
212132
Buffer Powder Pillows, pH 8.3, 50 mL
25/pkg
89868
100 mL
226142
Optional items
Description
Unit
Hydrogen Peroxide Solution. 30 %

Meter, pH, SensION 1 with Platinum pH electrode
Catalog number
473 mL
14411
each
5170010
Phenolphthalein Indicator Solution
100 mL MDB
16232
Pipet, 1-mL glass serological pipet
each
53235
each
1465100
100 mL MDB
32332
Pipet Filler

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Acidity, DT, 8201 and 8202
Acidity
Methyl Orange and Phenolphthalein (Total) Acidity
10 to 4000 mg/L CaCO3
DOC316.53.01164
Digital Titrator
Test preparation
Avoid excess agitation when collecting and swirling the sample to prevent the loss of gases such as carbon dioxide,
hydrogen sulfide and ammonia.
Six drops of Bromphenol Blue Indicator Solution1 can be substituted for the Bromphenol Blue Indicator Powder Pillow.
A pH meter can be used in place of the indicators. The end point for methyl orange acidity is pH 3.7. The end point for
phenolphthalein acidity is pH 8.3.
1

Description
Quantity
Bromphenol Blue Indicator Powder Pillow
1 pillow
1 pillow
Sodium Hydroxide Titration Cartridge (see Range-specific information)
1 cartridge
Digital Titrator
Delivery Tube for Digital Titrator
Graduated Cylinder
Erlenmeyer Flask, 250-mL
Acidity
Page 91
Acidity
Methyl orange acidity (Method 8201)
See
Table 1
1. Select a sample
volume and titration
cartridge from the Rangespecific information.


the tip.
4. Use a graduated
measure the sample
volume from the Rangespecific information.

deionized water.

one Bromphenol Blue
Swirl to mix.

titrant until the color
changes from yellow to
blue-violet (pH 3.7).

the Range-specific
calculate the
concentration:

counter.
Acidity
Page 92
mg/L as CaCO3 methyl
orange acidity
Example: 100 mL of
= 25 mg/L as CaCO3
Acidity
Phenolphthalein (total) acidity (Method 8202)
1. Measure a second
portion of the sample from
the Range-specific
information table into a
clean, 250-mL Erlenmeyer
flask. If the sample volume
is less than 100 mL, dilute
to approximately 100 mL
with deionized water.

one Phenolphthalein
Swirl to mix.

changes from colorless to
a light pink color that
persists for 30 seconds.
counter.

Range-specific information
table to calculate the
concentration:
mg/L as CaCO3
phenolphthalein acidity
Example: 100 mL of
= 250 mg/L as CaCO3

Range (mg/L as CaCO3)
Sample volume (mL)
Titration cartridge (N NaOH)
1040
100
0.1600
0.1
40160
25
0.1600
0.4
100400
100
1.600
1.0
200800
50
1.600
2.0
Multiplier
5002000
20
1.600
5.0
10004000
10
1.600
10.0
Interferences
Interfering substances lists substances that can interfere with this test.
Table 23 Interfering substances

Interfering substance
Interference level
Chlorine
Chlorine may interfere with the indicators. Add one drop of 0.1 N Sodium Thiosulfate to the
sample to remove chlorine before starting the test.
Color or turbidity
Color or turbidity can mask the color change of the end point. Use a pH meter instead of the
color indicators and titrate to a pH of 3.7 for methyl orange acidity or pH 8.3 for
phenolphthalein acidity.
Acidity
Page 93
Acidity
Table 23 Interfering substances (continued)
Interference level
Hydrolyzable metals
Samples that contain hydrolyzable metals such as iron, manganese or aluminum should be
pretreated before the test for phenolphthalein acidity is started.
1. Measure the sample volume into the flask.
2. Use the Digital Titrator and a Sulfuric acid cartridge of the same normality as the one
being used for the Acidity test to adjust the sample pH to pH 4 or less. Write down the
number of digits of acid that was added to lower the pH.
3. Add five drops of 30% hydrogen peroxide solution.
4. Boil the solution for 25 minutes.
5. Cool the solution to room temperature.
6. Add the phenolphthalein indicator and titrate the sample.
7. Subtract the number of digits of acid that were added in step 2 from the number of digits
that were used to reach the end point in step 6. Multiply this number by the multiplier in
Range-specific information to find the phenolphthalein acidity in the sample.
Complete the test procedure as soon as possible after collection for best accuracy. The
sample can be stored for at least 24 hours if cooled to 4 C (39 F) or below.
Accuracy check
Use a buffer pillow with the same pH as the end point with the indicator to make sure the end point
color is accurate.
Methyl orange acidityAdd 50 mL of deionized water to a flask. Add one pH 3.7 buffer
powder pillow and one Bromphenol Blue Indicator Powder Pillow and swirl to mix. Use this
solution for comparison during the titration with the sample.
Phenolphthalein acidityAdd 50 mL of deionized water to a flask. Add one pH 8.3 buffer

powder pillow and one Phenolphthalein Indicator Powder Pillow and swirl to mix. Use this
solution for comparison during the titration with the sample.
Standard additions method (sample spike)

Ampule breaker
TenSette Pipet, 0.11.0 mL
1. Open the standard solution.

2. Use the TenSette Pipet to add 0.1 mL of the standard to the titrated sample. Swirl to mix.
3. Titrate the spiked sample to the end point. Write down the amount of titrant that was used to
reach the end point.
4. Use the TenSette Pipet to add 0.2 mL of standard to the titrated sample. Swirl to mix.
Acidity
Page 94
Acidity
8. Each 0.1 mL of standard that was added will use approximately 25 digits of the 1.600 N
titration cartridge or 250 digits of the 0.1600 N titration cartridge to reach the endpoint. If more
or less titrant was used, there can be an interference (see Interferences) or the concentration
of the titrant has changed.
Summary of method
Bromphenol blue or phenolphthalein indicator is used to titrate the sample with sodium hydroxide
to a colorimetric end point. Bromphenol blue gives a better end point than methyl orange indicator.
Titration to pH 3.7 determines strong mineral acidity (also referred to as methyl orange acidity),
whereas the pH 8.3 phenolphthalein end point includes weaker acid species as well and
represents the total acidity. The results are expressed in mg/L as calcium carbonate (CaCO3) at a
specified pH.

Required reagents
Description
Quantity/Test
Unit
Catalog number
(1) Bromphenol Blue Powder Pillows
1 pillow
100/pkg
1455099
(1) Phenolphthalein Indicator Powder Pillows
Acidity Reagent Set (approximately 100 tests)
2272800
1 pillow
100/pkg
94299
(1) Sodium Hydroxide Titration Cartridge, 0.1600 N
varies
each
1437701
varies
each
1437901
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50546

each
50838
each
50840
each
50841
each
50842
Unit
Catalog number
100 mL
212132
Description
SUlfuric Acid Standard Solution, 0.500 N H2SO4, 10-mL
Acidity
Page 95
Acidity

Description
Unit
Catalog number
25/pkg
1455168
25/pkg
89868
each
2095352

Pipet tips
each
1970001
50/pkg
2185696
TitraStir Stir Plate, 115 VAC
each
1940000
each
1940010
500 mL
27249
Sulfuric Acid DT cartridge, 0.1600
Water, deionized
each
1438801
1438901
Sulfuric Acid DT cartridge, 1.600
each
each
1451538
each
1451520
each
1465100
Bottles, sampling, poly, 500 mL
each
2087079
Bromphenol Blue indicator solution
100 mL MDB
1455232
Phenolphthalein Indicator solution, 5 g/L
100 mL MDB
16232
pH meter
each
Delivery tube, 180 hook
5/pkg
1720500

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Alachlor
Alachlor
DOC356.53.01001
Immunoassay Method1
Method 10202
Scope and Application: For water

1
This test is semi-quantitative. Results are expressed as greater or less than the threshold value used.
Test preparation
How to use instrument-specific information
The Instrument-specific information table displays requirements that may vary between
instruments. To use this table, select an instrument then read across to find the corresponding
information required to perform this test.
Table 24 Instrument-specific information

Instrument
DR 6000
Adapter
DR 5000
A23618
DR 3900
LZV846(A)
DR3800, DR 2800, DR 2700
LZV583

This method analyzes for Alachlor in water. Sample calibrators and reagents are added to cuvettes coated with Alachlorspecific antibodies. The color that develops is then measured and compared with the color measurements of the calibrators.
The test requires about 30 minutes for complete analysis. As many as 20 cuvettes (18 samples and 2 calibrators) can be run
simultaneously.
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes, and other
apparatus before beginning the analysis.
Timing is critical; follow instructions carefully.
A consistent technique when mixing the cuvettes is critical to this test. The best results come from using the cuvette
rack and mixing as described in Using the 1-cm MicroCuvette Rack. Cuvettes can be mixed individually, but test results may
not be as consistent.
Handle the cuvettes carefully. Scratches on the inside or outside may cause erroneous results. Carefully clean the outside of
the cuvettes with a clean absorbent cloth or tissue before placing them into the instrument.
Antibody cuvettes and enzyme conjugate are made in matched lots. Do not mix reagent lots.
To avoid damaging the Color Developing Solution, do not expose it to direct sunlight.
The cuvette rack is designed to be inverted with the cuvettes in place. This is especially helpful when running many samples
at once; the cuvettes can remain in the rack and be processed together until they are read in the spectrophotometer.
Twenty Antibody Cuvettes are provided with each reagent set. One Antibody Cuvette will be used for each calibrator and
each sample. Cuvettes are not reusable.
Protective nitrile gloves are recommended for this procedure.
Alachlor
Page 97
Alachlor

Description
Quantity
Alachlor Reagent Set
Caps, flip spout
Marker, laboratory
Rack, for 1-cm Micro Cuvettes
Wipes, disposable
Pipet,
TenSette,
1
1
0.11.0 mL
Pipet tip for 19700-01, TenSette Pipet
Immunoassay for Water
Single Wavelength
OK
1. Press
SINGLE WAVELENGTH
Press OPTIONS and the

button.
Enter 450 NM and press
OK.
Insert an adapter if
required (Instrumentspecific information). Refer
to the user manual for
orientation.
Alachlor
Page 98
2. Label an Antibody
Cuvette for each calibrator
and each sample to be
tested.
3. Insert the cuvettes into

the rack snugly.
4. Pipet 0.5 mL of each

calibrator into the
appropriately labeled
cuvette.
Use a new pipette tip for
each calibrator.
Alachlor
Immunoassay for Water (continued)

sample to be tested into
the appropriately labeled
cuvette.
6. Immediately pipet
0.5 mL of Alachlor
Enzyme Conjugate into
each cuvette.
7. Set the instrument

timer for 20:00 minutes.
Press OK to start a 20minute reaction time.

each sample.
Use the same pipette tip

for each sample.
Immediately mix the

contents of the cuvettes
for 30 seconds using the
technique described
in Using the 1-cm
MicroCuvette Rack.
9. At the end of the

20-minute period, discard
the contents of all the
cuvettes into an
appropriate waste
container.
10. Wash each cuvette

forcefully and thoroughly
four times with deionized
water. Empty the rinse
water into the waste
container.
8. After 10 minutes mix

the contents of the rack for
30 seconds using the
technique described
in Using the 1-cm
MicroCuvette Rack.
Ensure that most of the

water is drained from the
cuvettes by turning the
cuvettes upside down and
tapping them lightly on a
paper towel.
Alachlor
Page 99
Alachlor
Color Development
Important Note: Timing is critical. Follow instructions carefully.
11. With the cuvettes still

held snugly in the rack,
pipet 0.5 mL of Color
Developing Solution into
each Antibody Cuvette.
for each sample.

Press OK to start the
reaction period.
Mix, using the instructions
in Using the 1-cm
MicroCuvette Rack.
13. After 5 minutes, mix

the contents of the rack a
second time for a period of
same technique as
step 12.
Solutions will turn blue in
some or all of the cuvettes.

10-minute reaction period,
pipette 0.5 mL of Stop
Solution into each cuvette
in the same order as the
Color Developing Solution
was added in step 11.
Slide the rack for 20
seconds (Using the 1-cm
MicroCuvette Rack.)
Blue solutions will turn
yellow with the addition of
the Stop Solution.
repeatedly for this step.
Alachlor
Page 100
Alachlor
Measuring the Color
Zero
15. Label and fill a Zeroing

Cuvette with deionized
water. Wipe the outside of
all the cuvettes with a
tissue to remove water,
smudges, and fingerprints.
16. Insert the filled

Zeroing Cuvette into the
cell holder.
17. ZERO the instrument.

The display will show:
0.000 Abs
Orient the arrow in the

same direction for all
cuvettes.
18. Insert the first

calibrator into the cell
holder.
READ the results in (ABS)
for each calibrator
and sample. Record the
results.
Repeat this step for all

remaining calibrators and
samples.
See Interpreting and
reporting results for help
with interpretation of
results.
Using the 1-cm MicroCuvette Rack

The MicroCuvette rack (Figure 1) has been designed specifically to aid in achieving precise and
accurate results when using the immunoassay technique to analyze several samples at the same
time.
Figure 1 The 1-cm MicroCuvette Rack
Alachlor
Page 101
Alachlor
Loading the rackThe cuvette rack is designed so that it may be inverted with the cuvettes in
place. Identify each cuvette with a sample or calibrator number and insert all the cuvettes in the
rack before beginning the procedure. Fit the cuvettes snugly into the rack, but do not force them or
they may be difficult to remove and their contents may spill. The cuvettes should remain in place
when the rack is inverted and tapped lightly.
MixingSet the rack on a hard, flat surface that is at least twice the length of the rack. Hold the
rack by one end and vigorously slide it back and forth along its long axis for 30 seconds. The rack
should move through a distance equal to its own length in each direction.
Interpreting and reporting results

There is an inverse relationship between the concentration of Alachlor and the absorbance
reading. In other words, the higher the reading, the lower the concentration of Alachlor (see
Relative Alachlor concentration)
Table 25 Relative Alachlor concentration

If the sample reading is...
the sample Alachlor Concentration is...
less than calibrator reading
greater than the calibrator concentration
greater than calibrator reading
less than the calibrator concentration
Example
Readings:
0.1 ppb Alachlor Calibrator: 0.475 Abs
0.5 ppb Alachlor Calibrator: 0.245 Abs
Sample #1: 0.140 Abs
Interpretation
Sample #1Sample reading is less than the readings for both calibrators. Therefore the sample
concentration of Alachlor is greater than both 0.1 ppb and 0.5 ppb Alachlor.
Sample #2Sample reading is between the readings for the 0.1 ppb and 0.5 ppb Alachlor
calibrators. Therefore the sample concentration of Alachlor is between 0.1 ppb and 0.5 ppb.
Sample #3Sample reading is greater than the readings for both calibrators. Therefore the
sample concentration of Alachlor is less than both 0.5 ppb and 0.1 ppb.
Storing and handling reagents
Wear protective gloves and eyewear.
When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
reagents at a temperature of 4 C when not in use.
Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water
and seek immediate medical help.
Sensitivity
The Alachlor immunoassay test cannot differentiate between certain herbicides and metabolites,
but it detects their presence to differing degrees. The Required concentration for selected
chemicals table shows the required concentration for selected chemicals.
Alachlor
Page 102
Alachlor
Table 26 Required concentration for selected chemicals

Concentration to give a positive
response of 0.1 ppb Alachlor
Compound
Concentration to give a positive

response of 0.5 ppb Alachlor
Acetochlor
0.45 ppb
4 ppb
Butachlor
0.09 ppm
1 ppm
2 Chloro-2',6'-Diethylacetaniline
0.030 ppm
2 ppm
Metolachlor
0.085 ppm
2 ppm
2,6-Diethylaniline
0.3 ppm
9 ppm
Propachlor
0.72 ppb
12 ppb
Table 27 Compounds tested and not detected up to 10,000 ppb

Atrazine
2, 4-D
Aldicarb
Chlorpyrifos
Diazoton
Carbendazim
Carbofuran
Sample collection and storage

Collect samples in a clean glass bottle. Do not pre-rinse the bottle with the sample. If the
sample cannot be analyzed immediately, store the sample at 4 C. Samples may be kept for as
long as 14 days. Warm the samples to room temperature before analysis.
Water sample dilution

Other levels of Alachlor can be tested by diluting the sample and comparing the results to the 0.1
ppb Calibrator. Select the appropriate sample volume from the Sample volume and concentration
table, place it in a graduated mixing cylinder, and dilute it to 50 mL with deionized water.
Table 28 Sample volume and concentration

mL sample
Representative concentration using 0.1 ppb calibrator
0.5
10 ppb
1.0
5 ppb
2.5
2 ppb
5.0
1 ppb
Example:
Dilute 2.5 mL of sample to 50 mL with deionized water. Run the diluted sample in the procedure
along with the 0.1 ppb calibrator. If the absorbance of the diluted sample is less than the 0.1 ppb
calibrator, the concentration of the original sample is greater than 2 ppb.
Alachlor
Page 103
Alachlor
Summary of method
Immunoassay tests use antigen/antibody reactions to test for specific organic compounds in water
and soil. Alachlor-specific antibodies, attached to the walls of plastic cuvettes, selectively bind and
remove Alachlor from complex sample matrices. A prepared sample and a reagent containing
enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme) are added
to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and Alachlor compete
for binding sites on the antibodies. Samples with higher levels of analyte will have more antibody
sites occupied by Alachlor and fewer antibody sites occupied by the enzyme-conjugate molecules.
After incubation, the sample and unbound enzyme conjugate are washed from the cuvette and a
color-development reagent is added. The enzyme in the conjugate catalyzes the development of
color. Therefore, there is an inverse relationship between color intensity and the amount of
Alachlor in the sample. The resulting color is then compared with a calibrator to determine whether
the Alachlor concentration in the sample is greater or less than the threshold levels. Test results
are measured at 450 nm.

Required reagents
Description
Unit
Catalog Number
20 cuvettes
2813000
Description
Unit
Catalog Number
Caps, flip spout
2/pkg
2581802
Marker, laboratory
each
2092000
Pipet, TenSette, Pipet, 0.11.0 mL
each
1970001
1000/pkg
2185628
Alachlor Reagent
1
Set1
Immunoassay components are manufactured by Beacon Analytical Systems, Inc.
Required apparatus
Pipet Tips, for TenSette Pipet 1970001

each
4879900
Wipes, disposable
box
2097000
Unit
Catalog Number

Description
500 mL
27249
Glasses, Safety
each
2756800
2550502
Deionized
Water1
Gloves, Disposable, Nitrile, Medium1
each
Mixing Cylinder, graduated Class A, 50-mL1
each
2636341
Mixing Cylinder, graduated Class A, 25-mL1
each
2636340
50/pkg
2185696
Pipet Tips, for TenSette Pipet 19700-01

1
Other sizes available.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Alkalinity, DT, 8203
Alkalinity
DOC316.53.01166
Phenolphthalein and Total Alkalinity
Method 8203
10 to 4000 mg/L as CaCO3
Digital Titrator
Test preparation
Four drops of Bromcresol Green-Methyl Red Indicator Solution1 can be substituted for the Bromcresol Green-Methyl Red
meq/L Alkalinity = mg/L as CaCO3 50
1

Description
Quantity
Bromcresol Green-Methyl Red Indicator Powder Pillow
1 pillow
1 pillow
Sulfuric acid titration cartridge (see Range-specific information)
1 cartridge
Digital titrator
Graduated cylinder
Alkalinity
See
Table 1
1. Select a sample


the tip.
4. Use a graduated
measure the sample
Alkalinity
Page 105
Alkalinity
Alkalinity (continued)

deionized water.

one Bromcresol GreenMethyl Red Indicator
Powder Pillow. Swirl to
mix.

one Phenolphthalein
Swirl to mix.
If the solution turns pink,
proceed to step 7. If the
solution is colorless, the
Phenolphthalein (P)
alkalinity is zero. Proceed
to step 9.
10. Continue the titration

with sulfuric acid to a light
pink color.
counter.
Note: A pH meter may be
used to titrate to a specific
pH as required by sample
composition. See the
End point pH table.
Alkalinity
Page 106

changes from pink to
colorless.
counter.

the Rangespecific information table
to calculate the
concentration:
mg/L as CaCO3
total alkalinity
Example: 100 mL of
= 25 mg/L as CaCO3

the Rangespecific information table
to calculate the
concentration:
mg/L as CaCO3
P alkalinity
Example: 100 mL of
= 25 mg/L as CaCO3
Alkalinity

Sample volume (mL)
Titration cartridge (N H2SO4)
1040
100
0.1600
0.1
40160
25
0.1600
0.4
100400
100
1.600
1.0
200800
50
1.600
2.0
Multiplier
5002000
20
1.600
5.0
10004000
10
1.600
10.0
Table 30 End point pH

Sample composition
Total alkalinity
Phenolphthalein alkalinity
Alkalinity about 30 mg/L
pH 4.9
pH 8.3
pH 4.6
pH 8.3
pH 4.3
pH 8.3
Silicates or phosphates present
pH 4.5
pH 8.3
Industrial wastes or complex system
pH 4.5
pH 8.3
Routine or Automated Analyses
pH 4.5
pH 8.3
Interferences

Interference level
Chlorine
Chlorine at levels above 3.5 mg/L may cause a yellow-brown color when the Bromcresol
Green-Methyl Red Powder Pillow is added. Add one drop of 0.1 N Sodium Thiosulfate to the
sample to remove chlorine before starting the test.
Color or turbidity
color indicators and titrate to a pH of 8.3 for phenolphthalein acidity. For total alkalinity see
End point pH for the correct end point pH.
Prevent excessive agitation or prolonged exposure to air. Complete the test procedure as
soon as possible after collection for best accuracy.
The sample can be stored for at least 24 hours if cooled to 4 C (39 F) or below.
Alkalinity relationship table

Total alkalinity primarily includes hydroxide, carbonate and bicarbonate alkalinities. The
concentration of these alkalinities in a sample may be determined when the phenolphthalein and
total alkalinities are known (see Alkalinity relationships).
To use the table follow these steps:
g. Does the phenolphthalein alkalinity equal zero? If yes, use Row 1.
Alkalinity
Page 107
Alkalinity
h. Does the phenolphthalein alkalinity equal total alkalinity? If yes, use Row 2.
i.
Divide the total alkalinity by 2 to give one-half the total alkalinity.
j.
Select Row 3, 4 or 5 based on comparing the result of step c (one-half total alkalinity) with
the total alkalinity.
k. Perform the required calculations in the appropriate row, if any.

l.
Check your results. The sum of the three alkalinity types will equal the phenolphthalein
alkalinity.
For example:
A sample has 170 mg/L as CaCO3 phenolphthalein alkalinity and 250 mg/L as CaCO3 total
alkalinity. What is the concentration of hydroxide, carbonate and bicarbonate alkalinities?
The phenolphthalein alkalinity does not equal 0 (it is 170 mg/L), see step g.
The phenolphthalein alkalinity does not equal total alkalinity (170 mg/L vs. 250 mg/L), see step h.
One-half of the total alkalinity (250 g/L) equals 125 mg/L. Because the phenolphthalein alkalinity
(170 mg/L) is greater than one-half the total alkalinity (125 mg/L), select row 5.
The hydroxide alkalinity is equal to:
2 x 170 = 340
340 250 = 90 mg/L hydroxide alkalinity
The carbonate alkalinity is equal to:
250 170 = 80
80 x 2 = 160 mg/L carbonate alkalinity
The bicarbonate alkalinity equals 0 mg/L.
Check: (See step l)
90 mg/L hydroxide alkalinity + 160 mg/L carbonate alkalinity + 0 mg/L bicarbonate alkalinity =
250 mg/L
The above answer is correct; the sum of each type equals the total alkalinity.
Table 32 Alkalinity relationships

Row
Sample result
Phenolphthalein Alkalinity = 0
Phenolphthalein Alkalinity equal

to Total Alkalinity
Phenolphthalein Alkalinity less

than one-half of Total Alkalinity
Phenolphthalein Alkalinity equal

to one-half of Total Alkalinity
Phenolphthalein Alkalinity
greater than one-half of Total
Alkalinity
Alkalinity
Page 108
Hydroxide alkalinity
equals:
Carbonate alkalinity
equals:
Bicarbonate alkalinity
equals:
Total Alkalinity
Total Alkalinity
times 2
Total Alkalinity minus two

times Phenolphthalein
Alkalinity
Total Alkalinity
2 times Phenolphthalein
Alkalinity minus Total
Alkalinity
2 times the difference

between Total and
Alkalinity
Accuracy check
Use a buffer pillow with the same pH as the end point with the indicator to make sure the end point
color is accurate.
Phenolphthalein alkalinityAdd 50 mL of deionized water to a flask. Add one pH 8.3

buffer powder pillow and one Phenolphthalein Indicator Powder Pillow and swirl to mix.
Use this solution for comparison during the titration with the sample.
Total alkalinityAdd 50 mL of deionized water to a flask. Add one pH 4.5 buffer powder
pillow and one Bromcresol Green-Methyl Red Indicator Powder Pillow and swirl to mix.
Use this solution for comparison during the titration with the sample.

Alkalinity Voluette Ampule Standard Solution, 0.500 N
Ampule breaker
TenSette Pipet, 0.11.0 mL and Pipet Tips
1. Open the standard solution ampule.

4. Repeat steps 2 and 3, using two more additions of 0.1 mL. Titrate to the end point after each
addition.
titration cartridge or 250 digits of the 0.1600 N titration cartridge to reach the endpoint. If more
or less titrant was used, there may be an interference (see Interferences) or the concentration
of the titrant has changed.
Summary of method
The sample is titrated with sulfuric acid to a colorimetric end point corresponding to a specific pH.
Phenolphthalein alkalinity is determined by titration to a pH of 8.3, as evidenced by the color
change of phenolphthalein indicator and indicates the total hydroxide and one half the carbonate
present. M (methyl orange) or T (total) alkalinity is determined by titration to a pH between 4.3 and
4.9 and includes all carbonate, bicarbonate and hydroxide. Alternatively, total alkalinity end points
may be determined by using a pH meter and titrating to the specific pH required for the sample
composition.
Alkalinity
Page 109
Alkalinity

Required reagents
Description
Quantity/Test
Unit
Catalog number
(1) Bromcresol Green-Methyl Red Powder Pillows
1 pillow
100/pkg
1 pillow
100/pkg
94299
(1) Sulfuric Acid Titration Cartridge, 0.1600 N
varies
each
1438801
(1) Sulfuric Acid Titration Cartridge, 1.600 N
varies
each
1438901
Quantity/Test
Unit
Catalog number
each
1690001
each
50546
Alkalinity Reagent Set (approximately 100 tests)
2271900
94399
Required apparatus
Description
Digital Titrator

each
50838
each
50840
each
50841
each
50842
Unit
Catalog number
16/pkg
1427810
Unit
Catalog number
Description
Alkalinity Standard Solution, Voluette Ampule 0.500 N Na2CO3, 10-mL

Description
25/pkg
89568
25/pkg
89868
each
2095352
each
1970001
Water, deionized
500 mL
27249
each
1451538
each
1451520
each
1465100
each
2087079
Bromphenol Green-Methyl Red indicator solution
100 mL MDB
2329232
100 mL MDB
16232
pH meter
each
each
1940000
each
1940010

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Alkalinity, BT, 8221
Alkalinity
DOC316.53.01151
USEPA1 Buret Titration Method2

Method 8221
Buret Titration

1
USEPA Accepted
Adapted from Standard Methods for the Examination of Water and Wastewater, 2320 B
Test preparation

A pH meter is required for NPDES reporting and is recommended for best results.
Substitute six drops of Phenolphthalein Indicator Solution for the Phenolphthalein Indicator Powder Pillow if necessary
Substitute six drops of Bromcresol Green-Methyl Red Indicator Solution for the Bromcresol Green-Methyl Red Powder Pillow
if necessary.
Results in mg/L as CaCO3
17.12 = grains per gallon

Description
Bromcresol Green-Methyl Red indicator powder pillow
Sulfuric acid standard solution, 0.020 N
varies1
Buret clamp, double
Graduated cylinder
Quantity
varies1
Funnel, Micro
Support Stand
See Consumables and replacement items on page 116.
Alkalinity
Page 111
Alkalinity
See
Table 1
1. Select a sample
the expected alkalinity
calcium carbonate
(CaCO3).
2. Use a graduated
measure the sample
volume.

water if necessary.

one Phenolphthalein
step when using a
pH meter.)

Sulfuric Acid standard
solution.

until the solution color
changes from pink to
colorless (pH 8.3).
7. Calculate:

one Bromcresol GreenMethyl Red Indicator
Powder Pillow to the
titrated sample. Swirl to
mix.
If the solution is colorless

before titrating with sulfuric
acid, the phenolphthalein
alkalinity is zero.
Alkalinity
Page 112
used = mg/L
phenolphthalein alkalinity
as CaCO3.
Do not add indicator if a

pH meter is used.
Specific sample
composition may require
titration to a specific pH
(see the
Alkalinity relationship
table).
Alkalinity

until a light pink end point
is reached.
10. Calculate:
used = mg/L total alkalinity
as CaCO3.

Range
(mg/L as CaCO3)
Sample Volume (mL)
Sulfuric Acid
Multiplier
0500
50
20353
20
4001000
25
20353
40
10002500
10
20353
100
20005000
20353
200
The end points in the Alkalinity endpoints table are recommended for determining total alkalinity in
water samples of various compositions and alkalinity concentrations.
Table 34 Alkalinity endpoints

End point pH
Sample composition
Total Alkalinity
pH 4.9
pH 8.3
pH 4.6
pH 8.3
pH 4.3
pH 8.3
Silicates or phosphates present
pH 4.5
pH 8.3
Industrial wastes or complex system
pH 4.5
pH 8.3
Routine or Automated Analyses
pH 4.5
pH 8.3
Alkalinity
Page 113
Alkalinity
Total alkalinity primarily includes hydroxide, carbonate, and bicarbonate alkalinities. The
concentration of these types in a sample may be determined when the phenolphthalein and total
alkalinities are known ( Alkalinity relationship table).
Table 35 Alkalinity relationship

Row
Result of Titration
Phenolphthalein Alkalinity equal to 0
Phenolphthalein Alkalinity equal to Total

Alkalinity
Hydroxide Alkalinity
Equals:
Carbonate Alkalinity
Equals:
Bicarbonate
Alkalinity Equals:
Total Alkalinity
Total Alkalinity
times 2
Total Alkalinity minus

two times
Phenolphthalein
Alkalinity
Total Alkalinity
2 times
Phenolphthalein
Alkalinity minus Total
Alkalinity
2 times the difference

between Total and
3
Phenolphthalein Alkalinity less than
one-half of Total Alkalinity
4
Phenolphthalein Alkalinity equal to onehalf of Total Alkalinity
5
Phenolphthalein Alkalinity greater than
one-half of Total Alkalinity
Use the Alkalinity relationship table with the following procedure:

1. Does the phenolphthalein alkalinity equal zero? If yes, use Row 1.
2. Does the phenolphthalein alkalinity equal total alkalinity? If yes, use Row 2.
3. Divide the total alkalinity by 2 to calculate one-half the total alkalinity.
4. Select Row 3, 4 or 5 based on comparing the result of step c (one-half total alkalinity) with the
phenolphthalein alkalinity.
5. Perform the required calculations if any.
6. Check your results. The sum of the three alkalinity types will equal the total alkalinity.
Example:
A sample has 170 mg/L as CaCO3 phenolphthalein alkalinity and 250 mg/L as CaCO3 total
alkalinity. What is the concentration of hydroxide, carbonate, and bicarbonate alkalinities?
a. The phenolphthalein alkalinity does not equal zero but 170 mg/L.
b. The phenolphthalein alkalinity does not equal total alkalinity (170 mg/L vs. 250 mg/L).
c. One-half of the total alkalinity equals 125 mg/L.
d. Because the phenolphthalein alkalinity of 170 mg/L is greater than one-half the total
alkalinity of 125 mg/L, select Row 5.
The hydroxide alkalinity is equal to:
2 170 = 340
340 250 = 90 mg/L hydroxide alkalinity
The carbonate alkalinity is equal to:

250 170 = 80
80 2 = 160 mg/L carbonate alkalinity
Alkalinity
Page 114
Alkalinity
The bicarbonate alkalinity is equal to zero mg/L.
Check:
90 mg/L hydroxide alkalinity + 160 mg/L carbonate alkalinity + 0 mg/L bicarbonate alkalinity = 250 mg/L
The answer is correct.

The sum of each type equals the total alkalinity (250 mg/L).
Interferences
Chlorine at levels above 3.5 mg/L cause a yellow-brown color upon the addition of the Bromcresol
Green-Methyl Red Indicator Powder Pillow. Residual chlorine interference with the indicator may
be removed by adding a drop of 0.1 N Sodium Thiosulfate Standard Solution* before adding the
indicator.
these samples, titrating to pH 8.3 for phenolphthalein alkalinity and the appropriate pH (see the
Alkalinity endpoints table) for total alkalinity.

Collect samples in plastic or glass bottles. Fill completely and cap tightly. Avoid excessive agitation
and prolonged exposure to air. Samples should be analyzed as soon as possible after collection
but can be stored at least 24 hours by cooling to 4 C (39 F) or below. Warm to room temperature
before analyzing.
Accuracy check
To accurately determine the phenolphthalein alkalinity end point, mix the contents of one
Phenolphthalein Indicator Powder Pillow and the contents of one pH 8.3 Buffer Powder Pillow
with 50 mL of deionized water in a 250-mL Erlenmeyer flask. The resulting color is the end
point.
To accurately determine the total alkalinity end point, mix the contents of one pH 4.5 Buffer
Powder Pillow and the contents of one Bromcresol Green-Methyl Red Indicator Powder Pillow
with 50 mL of deionized water in a 250-mL Erlenmeyer flask. Titrate to a light pink color
change.
Standard additions method (Sample Spike)

Perform the standard additions method check as follows:
1. Break the top off an Alkalinity Voluette Ampule Standard Solution, 0.500 N.
2. Use the TenSette Pipet* to add 0.1 mL of standard to the sample titrated in step 6 or step 9.
Resume titration back to the same end point. Record the volume of titrant needed.
3. Repeat, using two more additions of 0.1 mL. Titrate to the end point after each addition.
4. The mL of titrant required should increase by 2.5 mL for each 0.1 mL increment of standard
added.
Summary of method
Alkalinity is expressed as P (phenolphthalein) alkalinity or as T (total) alkalinity. Both types are
determined by titration with a Sulfuric Acid Standard Solution to an end point evidenced by the
color change of an indicator solution or determined with a pH meter. The P alkalinity is determined
by titration to a pH of 8.3 and registers the total hydroxide and one half the carbonate present. The
T alkalinity is determined by titration to a pH of 4.5. The total alkalinity includes all carbonate,
bicarbonate and hydroxide alkalinity. Alternatively, total alkalinity end points may be determined by
using a pH meter and titrating to the specific pH required for the sample composition.
* See Consumables and replacement items on page 116.
Alkalinity
Page 115
Alkalinity

Required reagents
Description
Quantity/test
Unit
Bromcresol Green-Methyl Red Indicator Powder Pillows
1 pillow
100/pkg
Catalog number
94399
1 pillow
100/pkg
94299
varies
1L
20353
Catalog number
Required apparatus
Description
Quantity/test
Unit
Buret Clamp, double
each
32800
each
2636540

each
50837
each
50838
each
50840
each
50841
each
50546
Pipet, volumetric, Class A, 5-mL,
each
1451537
each
1451537
Pipet Filler, Safety Bulb
each
1465100
Ampule Breaker
each
2196800
Funnel, Micro
each
2584335
Support Stand
each
56300
Required standards
Description
Unit
Catalog number
Alkalinity Standard Solution, Voluette Ampules, 0.500 N, 10-mL
16/pkg
1427810
25/pkg
89568
25/pkg
89868
4L
27256
Unit
Catalog number
Water, deionized
Optional items
Description
32332
1970001
50/pkg
2185696
Tips for Tensette Pipet

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Aluminum, 8012
Aluminum
DOC316.53.01002
Aluminon Method1
Method 8012
(0.008 to 0.800 mg/L)
Powder Pillows
Scope and Application: For water and wastewater

1
Test preparation

Instrument
Sample cell
Cell orientation
DR 6000
2495402
Fill line faces right
DR 5000
2495402
Fill line faces user
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Digestion is required for determining total aluminum. Refer to the Digestion section of the Water Analysis Guide.
Clean all glassware with 6.0 N HCl and deionized water before use to remove contaminants from the glass.
Check the sample temperature. It must be between 2025 C (68 77 F) for accurate results.
The Pour-Thru Cell can be used if rinsed well with deionized water between the blank and prepared samples.
For more accurate results, determine a reagent blank value for each new lot of reagent. Follow the procedure using
deionized water in place of the sample. Subtract the reagent blank value from the final results or perform a reagent blank
adjust.

Description
Quantity
AluVer 3 Aluminum Reagent Powder Pillow
Ascorbic Acid Powder Pillow
Bleaching 3 Reagent Powder Pillow
50-mL graduated mixing cylinder with glass stopper
Sample cells (see Instrument-specific information)
Aluminum
Page 117
Aluminum
Aluminon method with powder pillows
Programs
10 Aluminum Alumin.
Start
1.
Select the test.
2. Fill the cylinder to the

50-mL mark with sample.
Add one Ascorbic Acid
Powder Pillow.
3. Add one AluVer 3

Aluminum Reagent
Powder Pillow. Insert the
stopper.
Refer to the user manual

for orientation.
Stopper. Invert several

times to dissolve the
powder.
An orange to orange-red
color will develop if
aluminum is present.
5. Invert repeatedly for

one minute to dissolve the
powder. Undissolved
powder will cause
inconsistent results.
6. Blank Preparation:
Pour 10 mL of the mixture
into a square sample cell.
7. Add one Bleaching 3

Reagent Powder Pillow to
the blank.
8. Start the instrument

timer.
9. Vigorously swirl the

cell for 30 seconds. The
solution should turn a light
to medium orange.

timer.
11. Prepared Sample:

Pour 10 mL of solution
from the cylinder into a
second square sample
cell.
12. Within five minutes

after the timer expires,
wipe and dry the blank and
place it into the cell holder.
required (Instrumentspecific information).
Aluminum
Page 118
A 15-minute reaction
period will begin.

timer.
A one-minute reaction
period will begin.
A 30-second reaction time

will begin.
Aluminum
Aluminon method with powder pillows (continued)
Zero

0.000 mg/L Al3+
Read
14. Immediately wipe and

dry the prepared sample
and place it into the cell
holder.
15. READ the results in

mg/L Al3+.
See the user manual for
instructions on changing
the chemical form to
Al2O3.
16. Clean the graduated

cylinder with soap and
brush immediately after
the test. Rinse with
deionized water.
Interferences
Interference level
Greater than 300 mg/L as CaCO3. Samples with greater than
300 mg/L acidity as CaCO3 must be treated as follows:
1.
2.
Acidity
3.
Alkalinity
Add one drop of 5.25 N Sulfuric Acid Standard Solution1

to change the solution from yellow back to colorless.
Continue with the test.
1000 mg/L as CaCO3. Interferences from higher alkalinity

concentrations can be eliminated by the following
pretreatment:
1. Add one drop of m-Nitrophenol Indicator Solution1 to
the sample taken in step 2, before adding ascorbic acid.
A yellow color indicates excessive alkalinity.
2.
Add one drop of m-Nitrophenol Indicator Solution1 to the

sample taken in step 2., before adding ascorbic acid.
Add one drop of 5.0 N Sodium Hydroxide Standard
Solution1. Stopper the cylinder. Invert to mix. Repeat as
often as necessary until the color changes from colorless
to yellow.
Add one drop of 5.25 N Sulfuric Acid Standard Solution1.

Stopper the cylinder. Invert to mix. If the yellow color
persists, repeat until the sample becomes colorless.
Continue with the test.
Fluoride
Interferes at all levels. See the Fluoride interference graph.
Iron
Greater than 20 mg/L
Phosphate
Polyphosphate
Polyphosphate interferes at all levels by causing negative

errors and must not be present. Before running the test,
polyphosphate must be converted to orthophosphate by acid
hydrolysis as described under the phosphorus procedures.
See Optional reagents and apparatus for reorder information.
Aluminum
Page 119
Aluminum
Fluoride interferes at all levels by complexing with aluminum. The actual aluminum
concentration can be determined using the Fluoride interference graph when the fluoride
concentration is known.
To use the fluoride interference graph:
1. Select the vertical grid line along the top of the graph that represents the aluminum reading
obtained in step 15.
2. Locate the point on the line where it intersects with the horizontal grid line that indicates how
much fluoride is present in the sample.
3. Extrapolate the true aluminum concentration by following the curved lines on either side of the
intersect point down to the true aluminum concentration.
For example, if the aluminum test result was 0.7 mg/L Al and the fluoride present in the sample
was 1 mg/L F, the point where the 0.7 grid line intersects with the 1 mg/L F grid line falls
between the 1.2 and 1.3 mg/L Al curves. In this case, the true aluminum content would be 1.27
mg/L.
mg/L F
mg/L Al3+ (Reading from instrument)
True Aluminum Concentration

Figure 1 Fluoride interference graph

Collect samples in clean glass or plastic containers. Preserve the sample by adjusting the pH to 2
or less with nitric acid (about 1.5 mL per liter). Preserved samples can be stored up to six months
at room temperature. Before analysis, adjust the pH to 3.54.5 with 5.0 N Sodium Hydroxide.
Correct the test result for volume additions.
Aluminum
Page 120
Aluminum
Accuracy check
Aluminum Voluette Ampule Standard, 50-mg/L Al
TenSette Pipet
Mixing cylinders, (3)
1. After reading test results, leave the sample cell (unspiked sample) in the instrument. Verify the
chemical form.
2. Select Options>More>Standard additions from the instrument menu.
3. Press OK to accept the default values for standard concentration, sample volume, and spike
volumes. Press EDIT to change these values. After values are accepted, the unspiked sample
reading will appear in the top row.
4. Open an Aluminum Voluette Ampule Standard, 50-mg/L Al.
5. Prepare three sample spikes. Fill three mixing cylinders* with 50 mL of sample. Use the
TenSette Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Analyze each sample spike as described in the procedure above, starting with the 0.1 mL
sample spike. Accept each standard additions reading by pressing READ. Each addition
should reflect approximately 100% recovery.
7. After completing the sequence, press GRAPH to view the best-fit line through the standard
additions data points, accounting for matrix interferences. Press IDEAL LINE to view
relationships between the sample spikes and the Ideal Line of 100% recovery.
1. Prepare a 0.4-mg/L aluminum standard solution as follows: Pipet 1.00 mL of Aluminum
Standard Solution, 100-mg/L as Al3+, into a 250-mL volumetric flask.
2. Dilute to the mark with deionized water. Prepare this solution daily. Follow the Aluminon
method with powder pillows test.
Or, use the following alternative procedure:
1. Using the TenSette Pipet, add 0.8 mL of solution from an Aluminum Voluette Ampule Standard
Solution (50-mg/L as Al) into a 100-mL volumetric flask.
2. Dilute to volume with deionized water. Follow the Aluminon method with powder pillows test.
3. To adjust the calibration curve using the reading obtained with the 0.4-mg/L aluminum
standard solution, select Options>More>Standard Adjust from the instrument menu.
4. Turn on the Standard Adjust feature and accept the shown concentration. If an alternate
concentration is used, enter the concentration and adjust the curve to that value.
* See Consumables and replacement items.
Aluminum
Page 121
Aluminum
Method performance
Program
Standard
Precision
95% Confidence Limits of
Distribution
Sensitivity
Concentration change
per 0.010 Abs change
10
0.40 mg/L Al3+
0.3850.415 mg/L Al3+
0.008 mg/L Al3+
Summary of method
Aluminon indicator combines with aluminum in the sample to form a red-orange color. The
intensity of color is proportional to the aluminum concentration. Ascorbic acid is added before the
AluVer 3 reagent to remove iron interference. To establish a reagent blank, the sample is split after
the addition of the AluVer 3. Bleaching 3 Reagent is then added to one-half of the split sample to
bleach out the color of the aluminum aluminon complex. The AluVer 3 Aluminum reagent,
packaged in powder form, shows exceptional stability and is applicable for fresh water
applications. Test results are measured at 522 nm.
Aluminum
Page 122
Aluminum
Required reagents
Description
Quantity/Test
Aluminum Reagent Set (100 Tests), includes:
Unit
Catalog number
2242000
(1) AluVer 3 Aluminum Reagent Powder Pillow
100/pkg
1429099
(1) Ascorbic Acid Powder Pillow
100/pkg
1457799
1429449
100/pkg
Hydrochloric Acid, 6.0 N
(1) Bleaching 3 Reagent Powder Pillow
varied
500 mL
88449
Water, deionized
varied
4L
27256
Quantity
Unit
Catalog number
each
189641
Unit
Catalog number
100 mL
1417442
Required apparatus
Description
Cylinder, graduated mixing, 50-mL, with glass stopper
Description
Aluminum Standard Solution, 100-mg/L as
Al3+
Aluminum Standard Solution, 10-mg/L as Al3+
100 mL
2305842
Aluminum Standard Solution, 10-mL Voluette Ampule, 50-mg/L as Al
16/pkg
1479210
each
2196800
Voluette Ampule Breaker

Description
Unit
Catalog number
Liqui-Nox Phosphate-free detergent
946 mL
2088153
m-Nitrophenol Indicator Solution
100 mL
247632
Nitric Acid Solution, 1:1
500 mL
254049
pH Paper, 014 pH range
100/pkg
2601300
each
1970001
Pipet Tips for 1970001
50/pkg
2185696
50 mL
245026
100 mL
244932
Pipet, TenSette, 0.11.0 mL
Test Tube Brush
each
69000
Volumetric Flask, Class A, 100-mL
each
1457442
each
1457446
Aluminum
Page 123

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Aluminum, 8326
Aluminum
DOC316.53.01003
Eriochrome Cyanine R Method1

(0.006 to 0.250 mg/L
Method 8326
Al3+)
Powder Pillows

1
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Clean all glassware with 6.0 N HCl and deionized water before use to remove contaminants from the glass.
deionized water in place of the sample. Subtract the reagent blank value from the final results or perform a reagent
blank adjust.

Description
ECR Reagent Powder Pillow
ECR Masking Reagent Solution
Hexamethylene-tetramine Buffer Reagent Powder Pillow
Quantity
1
1 drop
1
25-mL graduated mixing cylinder with glass stopper
Aluminum
Page 125
Aluminum
Eriochrome Cyanine R method with powder pillows
Programs
9 Aluminum ECR
Start
1. Select the test.

required (see Instrumentspecific information).
for orientation.
5. After the timer expires,

add one Hexamethylenetetramine Buffer Reagent
powder pillow.
2. Fill the 25-mL mixing

cylinder to the 20-mL mark
with sample. Add one ECR
Reagent Powder Pillow for
20-mL sample size.
3. Insert the stopper.

Invert several times to
completely dissolve
powder.
6. Insert the stopper.

Invert several times to
dissolve powder.
Put one drop of ECR
Masking Reagent Solution
into a clean square sample
cell.
A red-orange color will

develop if aluminum is
present.

timer.
A 30-second reaction
period will begin.
Undissolved reagent will

cause inconsistent results.
8. Pour 10 mL from the

mixing cylinder into the
blank cell. Swirl to mix.
The solution will begin to
turn yellow.
Zero
9. Prepared Sample: Fill

a second square sample
cell to the 10-mL mark with
the remaining solution in
the cylinder.
Aluminum
Page 126

timer.
A 5-minute reaction period
will begin.

wipe the blank then insert
it into the cell holder.

0.000 mg/L Al3+
This test uses a non-zero
intercept for the calibration
curve on some instrument
platforms.
Aluminum
Eriochrome Cyanine R method with powder pillows (continued)
Read
13. Immediately wipe and

dry the prepared sample
then insert it into the cell
holder.
14. Read the results in

mg/L Al3+.
instructions to change to
alternate form Al2O3.
If fluoride is present,
measure the fluoride
concentration and refer to
the Fluoride Concentration
(mg/L) table.
Interferences
Interference level and treatments
Acidity
Greater than 62 mg/L as CaCO3
Alkalinity
Ca2+
Cl
Greater than 1000 mg/L as Cl
Cr6+
0.2 mg/L (error is 5% of reading)
Cu2+
2 mg/L (error is 5% of reading)
Fe2+
Greater than 4 mg/L (error is positive and equals mg/L Fe2+ x 0.0075)
Fe3+
Greater than 4 mg/L (error is positive and equals mg/L Fe2+ x 0.0075)
See the Fluoride Concentration (mg/L) table.
Hexameta phosphate
0.1 mg/L as PO43 (error is 5% of reading)
Mg2+
Mn2+
NO2
Greater than 5 mg/L
NO3
pH
2.94.9 or 7.511.5. A sample pH between about 4.9 and 7.5 causes dissolved aluminum to
partially convert to colloidal and insoluble forms. This method measures much of that hardto-detect aluminum without any pH adjusting pretreatment as is necessary in some other
methods.
PO43 (ortho)
4 mg/L (error is 5% of reading)
Aluminum
Page 127
Aluminum
Polyphosphate
See procedure below.
SO42
Zn2+
Polyphosphate interference can be reduced by converting polyphosphate to orthophosphate by

the following steps:
1. Rinse a 50-mL graduated mixing cylinder and a 125-mL Erlenmeyer flask containing a
magnetic stir bar with 6 N hydrochloric acid. Rinse again with deionized water. This will
remove any aluminum present.
Note: Rinse two Erlenmeyer flasks if a reagent blank is used; see step 2.
2. Measure 50 mL deionized water into the 125-mL Erlenmeyer flask using the graduated
cylinder. This is the reagent blank. Because of the test sensitivity, this step must be done only
when any of the reagents used in the following pretreatment are replacedeven if the new
reagent has a matching lot number. When the pretreated sample has been analyzed, correct
for the aluminum concentration of the reagent blank. Refer to the instrument user manual.
3. Measure 50 mL sample into the 125-mL Erlenmeyer flask using the graduated cylinder. Use a
small amount of deionized water to rinse the cylinder contents into the flask.
4. Add 4.0 mL of 5.25 N Sulfuric Acid Standard Solution*.
5. Use a combination hot plate/stirrer to boil and stir the sample for at least 30 minutes. Add
deionized water as needed to maintain a sample volume of 20-40 mL. Do not boil dry.
6. Cool the solution to near room temperature.
7. Add 2 drops of Bromphenol Blue Indicator Solution*.
8. Add 1.5 mL of 12.0 N Potassium Hydroxide Standard Solution* using the calibrated, plastic
dropper provided. Swirl to mix. The solution color should be yellow or green but not purple. If
the color is purple, begin with step 1 again using an additional 1 mL Sulfuric Acid Standard
Solution in step 4.
9. While swirling the flask, add 1.0 N Potassium Hydroxide Solution*, a drop at a time, until the
solution turns a dirty green color.
10. Pour the solution into the graduated cylinder. Rinse the flask contents into the graduated
cylinder with deionized water to bring the total volume to 50 mL.
11. Use this solution in step 2. of the ECR method.
Note: Fluoride interference can be corrected by using the Fluoride Concentration (mg/L) table.
Example:
If the fluoride concentration is known to be 1.00 mg/L F and the ECR method gives a reading of
0.060 mg/L aluminum, what is the true mg/L aluminum concentration?
Intermediate values can be found by interpolation. Do not use correction graphs or charts found in
other publications.
Answer: 0.183 mg/L
* See Optional reagents and apparatus for reorder information
Aluminum
Page 128
Aluminum
Table 40 Fluoride Concentration (mg/L)

(mg/L)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.010
0.010
0.019
0.030
0.040
0.052
0.068
0.081
0.094
0.105
0.117
0.131
0.020
0.020
0.032
0.046
0.061
0.077
0.099
0.117
0.137
0.152
0.173
0.193
0.030
0.030
0.045
0.061
0.077
0.098
0.124
0.146
0.166
0.188
0.214
0.243
0.040
0.040
0.058
0.076
0.093
0.120
0.147
0.174
0.192
0.222
0.050
0.050
0.068
0.087
0.109
0.135
0.165
0.188
0.217
0.060
0.060
0.079
0.100
0.123
0.153
0.183
0.210
0.241
0.070
0.070
0.090
0.113
0.137
0.168
0.201
0.230
0.080
0.080
0.102
0.125
0.152
0.184
0.219
0.090
0.090
0.113
0.138
0.166
0.200
0.237
0.100
0.100
0.124
0.150
0.180
0.215
0.120
0.120
0.146
0.176
0.209
0.246
0.140
0.140
0.169
0.201
0.238
0.160
0.160
0.191
0.226
0.180
0.180
0.213
0.200
0.200
0.235
0.220
0.220
0.240
0.240
True Aluminum Concentration (mg/L) Al

Collect samples in clean glass or plastic containers. Preserve samples by adjusting the pH to 2 or
less with concentrated nitric acid (about 1.5 mL per liter). Preserved samples can be stored up to
six months at room temperature. Before analysis, adjust the pH to 2.94.9 with 12.0 N Potassium
Hydroxide Standard Solution* and/or 1 N Potassium Hydroxide Solution*. Correct the test result for
volume additions.
Accuracy check
Class A glassware (pipettes and volumetric flasks)
Aluminum Standard Solution, 100 mg/L

OR
Aluminum Voluette Ampule Standard Solution, 50-mg/L
Deionized water

Prepare a 0.100 mg/L aluminum standard solution as follows:
1. Using Class A glassware, pipet 1.00 mL of Aluminum Standard Solution 100 mg/L as Al3+, into
a 1000-mL volumetric flask.
2. Dilute to the mark with deionized water.
* See Optional reagents and apparatus for reorder information.
Aluminum
Page 129
Aluminum
3. Prepare this solution daily. Do the Eriochrome Cyanine R method with powder pillows. Go to
step 4.
OR
1. Add 2.0 mL of solution from an Aluminum Voluette Ampule Standard Solution (50-mg/L as Al)
into a 1000-mL volumetric flask.
2. Dilute to volume with deionized water. Prepare this solution daily.
3. Do the Eriochrome Cyanine R method with powder pillows test procedure.
5. Accept the shown concentration. If an alternate concentration is used, enter the actual
concentration.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
0.100 mg/L Al3+
0.0911.009 mg/L Al3+
0.002 mg/L Al3+
Summary of method
Eriochrome Cyanine R combines with aluminum in a sample to produce an orange-red color. The
intensity of color is proportional to the aluminum concentration. Test results are measured at
535 nm.

Required reagents
Description
Aluminum Reagent Set (100 Tests), includes:
ECR Reagent Powder Pillows
Hexamethylenetetramine Buffer Reagent Powder Pillows
ECR Masking Reagent Solution
Quantity/Test
Unit
Catalog number
2603700
100/pkg
2603849
100/pkg
2603999
1 drop
25 mL SCDB
2380123
Quantity
Unit
Catalog number
Required apparatus
Description
Cylinder, graduated mixing, 25-mL, with glass stopper
each
189640
Sample cell, 10 mL square, matched pair
2/pkg
2495402
Thermometer, 10 to 110 C
each
187701
Unit
Catalog number
100 mL
1417442
Description
Aluminum
Page 130
Aluminum
Description
Unit
Catalog number
100 mL
2305842
Aluminum Standard Solution, 10-mL Voluette Ampule, 50-mg/L as Al
16/pkg
1479210
4L
27256
Description
Unit
Catalog number
Ampule breaker for Voluette Standards
each
2196800
Water, deionized
Bromphenol Blue Indicator Solution

Cylinder, graduated mixing, 50-mL, Class A
Deionized Water
Hot Plate with Stirrer, 7 x 7 inches, 115 VAC
100 mL
1455232
each
2636341
500 mL
27249
each
50543
each
2881600
500 mL
88449
Nitric Acid 1:1
500 mL
254049
100/pkg
2601300
each
1970001
Pipet Tips for 1970001
50/pkg
2185696
Potassium Hydroxide Standard Solution, 12.0 N
100 mL
23032
Potassium Hydroxide Solution, 1.0 N
50 mL
2314426
50 mL
245026
Stir Bar, magnetic, octagonal, 22.2 x 7.9 mm
each
2095350

100 mL
244932
each
1457453
Aluminum
Page 131

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Arsenic, 8013
Arsenic
DOC316.53.01005
Silver Diethyldithiocarbamate Method1
Method 8013
(0 to 0.200 mg/L As)

Scope and Application: For water, wastewater, and seawater; distillation is required; USEPA accepted2 for
reporting for drinking and wastewater analysis (distillation required)
1
Procedure is equivalent to Standard Method 3500-As for drinking water analysis.
Test preparation

instruments. To use this table, select an instrument then read across to find the corresponding DR
3800, DR 2800, DR 2700information required to perform this test.

Instrument
Sample cell
Cell orientation
DR 6000
2612602
DR 5000
2612602
DR 3900
2612602
DR 3800, DR 2800, DR 2700
2612602

Create a user-entered program for arsenic. See step 1 and User programming.
Prepare the arsenic absorber solution as directed in Reagent preparation.
Perform a user-entered calibration for each new lot of arsenic absorber solution. See the Calibration section. Some
variations of the calibration procedure are possible.
In bright light conditions (e.g. direct sunlight) it may be necessary to close the cell compartment with the protective cover
during measurements.
Do not use the Pour-Thru Cell with this test.
The arsenic absorber in this test is a silver solution in pyridine. Both silver (D011) and pyridine (D038) are regulated
by the Federal RCRA as hazardous waste. In addition, the cotton ball soaked in lead acetate (D008) solution is a
hazardous waste. These materials should not be poured down the drain. Refer to a current MSDS sheet for proper
disposal.
Arsenic
Page 133
Arsenic
Collect the following items :

Description
Quantity
Apparatus (see Required apparatus)
Arsenic Standard Solution, 1000-mg/L As
varies
Hydrochloric Acid, ACS
25 mL
Lead Acetate Solution, 10%
1 mL
Potassium Iodide Solution, 20%
3 mL
Pyridine, ACS
50 mL
Sample Cells (see Instrument-specific information)
Silver Diethyldithiocarbamate
1g
Stannous Chloride Solution
1 mL
Water, deionized
varies
Zinc, 20-mesh, ACS
6g
User Programs
User Programs
Arsenic
Start
1. Perform the User

programming procedure.
Make note of the program
number.
Arsenic
Page 134
2. To run the test, press

USER PROGRAMS.
Select the test.

required (Table 1). Refer to
the user manual for
orientation.
3. Prepare the distillation

apparatus for arsenic
recovery. See the
Distillation Manual for
assembly instructions. Do
not connect to the
aspirator.
Place the distillation
apparatus under a fume
hood to vent toxic fumes.
4. Dampen a cotton ball

with 10% Lead Acetate
Solution. Insert it in the
gas scrubber. Be certain
that the cotton seals
against the glass.
Arsenic
6. Using a graduated
cylinder, pour 250 mL of
sample into the distillation
flask.
7. Turn on the power

switch. Set the stir control
to 5. Set the heat control
to 0.
cylinder, add 25 mL of
Hydrochloric Acid, ACS, to
the distillation flask.
9. Use a serological pipet

to add 1 mL of Stannous
Chloride Solution to the
flask.
10. Use a serological pipet

to add 3 mL of Potassium
Iodide Solution to the
flask. Cap.

timer.
12. When the timer

expires, weigh and add
6.0 g of 20-mesh zinc to
the flask. Cap
immediately.
13. Set the heat control

to 3.

timer.
15. When the timer

expires, set the heat
control to 1.
cylinder, pour 25-mL of
prepared arsenic absorber
solution (Reagent
preparation) into the
cylinder/gas bubbler
assembly.
Attach it to the distillation
apparatus.
A second 15-minute
reaction period will begin.
period will begin.

timer.
A third 15-minute reaction
period will begin.
Arsenic
Page 135
Arsenic
17. When the timer

expires, turn off the heater.
Remove the cylinder/gas
bubbler assembly as a
unit.
18. Rinse the gas bubbler

by moving it up and down
in the arsenic absorber
solution.

Fill a dry, 10-mL sample
cell with untreated arsenic
absorber solution.
Stopper.

Pour the reacted arsenic
absorber sample into a
sample cell.
23. Wipe the prepared

sample and insert it into
the cell holder.
20. Wipe the blank and

insert it into the cell holder.
Zero

The display will show the
intercept as calculated
from the user-entered
calibration curve. This will
probably be a non-zero
intercept.
READ the results.
Close the sample cell.
Interferences
Interference level
Antimony Salts
May interfere with color development.

Collect samples in acid washed glass or plastic bottles. Adjust the pH to 2 or less with sulfuric acid
(about 2 mL per liter)*. Preserved samples may be stored up to six months at room temperature.
* See Optional reagents and apparatus.
Arsenic
Page 136
Arsenic
Reagent preparation
Prepare the arsenic absorber solution as follows:
1. Weigh 1.00 g of silver diethyldithiocarbamate on an analytical balance.
2. Transfer the powder to a 200-mL volumetric flask. Dilute to volume with pyridine. Use pyridine
only in a fume hood and wear chemical resistant gloves. Read the MSDS before
using pyridine.
3. Mix well to dissolve. Store the reagent, tightly sealed, in an amber bottle. The reagent is stable
for one month if stored in this manner. Larger volumes of reagent can be prepared if the
reagent is used within one month.
Calibration
Standard preparation
Perform a new calibration for each lot of arsenic absorber solution.
1. Prepare a 10.0-mg/L arsenic working standard by pipetting 10.0 mL of Arsenic Standard
Solution, 1000 mg/L As into a 1000-mL volumetric flask.
2. Dilute to volume with deionized water.
3. Into three different 500-mL volumetric flasks, pipet 1.0, 2.0, and 10.0 mL of the 10.0 mg/L As
stock solution using Class A glassware.
4. Dilute to the mark with deionized water and mix thoroughly. These standards have
concentrations of 0.02, 0.04 and 0.20 mg/L As.
Note: Distill standards before making the calibration curve.
User programming
1. Press USER PROGRAMS on the main menu.
2. Press PROGRAM OPTIONS and NEW. Key any available program number (950999) to use for
arsenic testing. Press OK.
3. Fill in the appropriate fields using the touch screen when the field is highlighted. Use the
alphanumeric keys to name the User Program ARSENIC. Press NEXT to move to the next
screen. Set up the rest of the parameters as follows:
Program Type: Single Wavelength
Chemical Form: As
Units: mg/L
Wavelength: 520 nm
Concentration Resolution: 0.001
Calibration: Read Standards
4. After entering Read Standards, press NEXT>EXIT. Fill in the appropriate fields for each of the
following. Use the touch screen to activate the parameter and press EDIT to enter the data
entry screen. Set up the rest of the parameters as follows:
Timer1: 15 minutes
Upper Limit: 0.220 mg/L
Timer2: 15 minutes
Lower Limit: 0.020 mg/L
Timer3: 15 minutes
5. Press CALIBRATION: C = A + BA. Press EDIT.
Arsenic
Page 137
Arsenic
6. The Read Standards will be indicated. Enter the standard concentration values to be used to
perform the calibration: 0.00, 0.02, 0.04, and 0.20. To enter the concentration values press +
and enter the value followed by OK for each concentration value.
7. After the values are entered, press the UP arrow four times to move the cursor to the
0.00 concentration line.
8. Insert the 25-mL sample cell containing only unreacted arsenic absorber solution into the cell
holder. Press ZERO.
9. Press the DOWN arrow once to move to the next concentration. Insert the prepared sample in
the cell holder. Press READ to accept the absorbance value. Repeat steps for each standard.
Note: Standards must be distilled before absorbance values are measured.
10. Press GRAPH. Make sure FORCE ZERO is off.

11. If the graph is acceptable press DONE>EXIT.
12. Store Program? will appear on the display. Press YES.
The program is ready for use.
Some variations of the calibration procedure are possible. See the user manual for details.
Summary of method
Arsenic is reduced to arsine gas by a mixture of zinc, stannous chloride, potassium iodide, and
hydrochloric acid in a specially equipped distillation apparatus. The arsine is passed through a
scrubber containing cotton saturated with lead acetate for sulfide removal, and then into an
absorber tube containing silver diethyldithiocarbamate in pyridine. The arsenic reacts to form a red
complex which is read colorimetrically. This procedure requires a manual calibration. Test results

Required reagents
Description
Quantity/Test
Unit
Catalog number
Arsenic Standard Solution, 1000-mg/L As
varies
100 mL
1457142
Hydrochloric Acid, ACS
25 mL
500 mL
13449
Lead Acetate Solution, 10%
1 mL
100 mL
1458042
1456842
Potassium Iodide Solution, 20%
3 mL
100 mL
Pyridine, ACS
50 mL
500 mL
1446949
1g
25 g
1447624
Stannous Chloride Solution
1 mL
100 mL
1456942
Water, deionized
varies
4 liters
27256
6g
454 g
79501
Zinc, 20-mesh, ACS
Required apparatus
Description
Quantity
Unit
Catalog number
Balance, analytical, Zeta series, 80-g capacity
each
2936701
Balls, cotton
100/pkg
257201
Boat, weighing, 8.9-cm square
500/pkg
2179000
Arsenic
Page 138
Arsenic
Required apparatus (continued)
Description
Quantity
Unit
Catalog number
Bottle, amber, 237-mL, glass
6/pkg
714441
Cap, polypropylene, for amber bottle
6/pkg
2166706
50840
each
each
50846
Distillation Apparatus, arsenic accessories
set
2265400
Distillation Apparatus, general purpose accessories
set
2265300
Flask, volumetric, Class A, 1000-mL, with glass stopper
each
1457453
Flask, volumetric, Class A, 200-mL
each
1457445
each
1457449
1465100
each
Pipet, serological, 5-mL
each
53237
Pipet, volumetric, Class A, 1.00-mL
each
1451535
1451536
each
each
1451504
each
1451506
each
1451508
each
1451538
Select one based on available voltage:

Distillation Apparatus Heater, 115 VAC, 60 Hz
each
2274400
Distillation Apparatus Heater, 230 VAC, 50 Hz
each
2274402
Unit
Catalog number
each
189640

Description
Cylinder, mixing, 25-mL
Sulfuric Acid, 1.00 N
Gloves, chemical resistant, size 91
1
100 mL
127032
pair
2410104
Arsenic
Page 139

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Atrazine, 10050
Atrazine
DOC316.53.01006
Immunoassay1
Method 10050

1
Test preparation


Instrument
Adapter
DR 6000
DR 5000
A23618
DR 3900
LZV846(A)
DR 3800, DR 2800, DR 2700
LZV583

This method analyzes for Atrazine in water. Sample calibrators and reagents are added to cuvettes coated with Atrazinespecific antibodies. The color that develops is then measured and compared with the color measurements of the calibrators.
The test requires about 30 minutes for complete analysis. As many as 20 cuvettes (18 samples and 2 calibrators) can be run
simultaneously.
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes, and other
Atrazine
Page 141
Atrazine

Description
Quantity
Atrazine Reagent Set
Caps, flip spout
Marker, laboratory
Wipes, disposable
Pipet,
TenSette,
1
1
0.11.0 mL
Atrazine immunoassay method
Single Wavelength
OK
1. Press
Single Wavelength
button.
Enter 450 nm and press
OK.
required (see Instrumentspecific information). Refer
orientation.
Atrazine
Page 142
tested.

the rack snugly.

calibrator into the
appropriately labeled
cuvette.
each calibrator.
Atrazine
Atrazine immunoassay method (continued)

sample to be tested into
cuvette.
0.5 mL of Atrazine
Enzyme Conjugate into
each cuvette.

each sample.

for each sample.

cuvettes into an
appropriate waste
container.

container.
7. Press OPTIONS. Press

TIMER. Enter 20:00
minutes and press OK.
A 20-minute reaction time
will begin.

30 seconds (see Using the
1-cm MicroCuvette Rack.)
Immediately mix the

contents of the cuvettes
for 30 seconds (see Using
the 1-cm MicroCuvette
Rack.)
Ensure that most of the

paper towel.
Atrazine
Page 143
Atrazine
Color Development

each Antibody Cuvette.
for each sample.

timer for 10:00 minutes
Press OK to start a 10minute reaction time.
is Using the 1-cm
MicroCuvette Rack.

same technique.

was added in step 11. Use
the same pipette tip
seconds (see Using the 1cm MicroCuvette Rack.)
the Stop Solution.
Atrazine
Page 144
Atrazine
Zero

smudges, and fingerprints.

cell holder.
cuvettes.

0.000 Abs

holder.
READ the results. The
display will give an
absorbance reading.
Record the results for
each calibrator and
sample.
Repeat this step for all

remaining calibrators and
samples.
results.

The 1-cm MicroCuvette Rack has been designed specifically to aid in achieving precise and
accurate results when using the immunoassay technique to analyze several samples at the
same time.
Atrazine
Page 145
Atrazine
Loading the RackThe cuvette rack is designed so that it may be inverted with the cuvettes in

There is an inverse relationship between the concentration of Atrazine and the absorbance
reading. In other words, the higher the reading, the lower the concentration of Atrazine. See the
Relative Atrazine concentration table.
Table 44 Relative Atrazine concentration

the sample Atrazine Concentration is...
Example
Readings:
0.5 ppb Atrazine Calibrator: 0.475 Abs
3.0 ppb Atrazine Calibrator: 0.245 Abs
Interpretation
concentration of Atrazine is greater than both 0.5 ppb and 3.0 ppb Atrazine.
Sample #2Sample reading is between the readings for the 0.5 ppb and 3.0 ppb Atrazine
calibrators. Therefore the sample concentration of Atrazine is between 0.5 ppb and 3.0 ppb.
sample concentration of Atrazine is less than both 3.0 ppb and 0.5 ppb.
Storing and Handling Reagents

1. Wear protective gloves and eyewear.
2. When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
3. Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
4. If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water
Sensitivity
The Atrazine immunoassay test cannot differentiate between certain triazines and metabolites, but
it detects their presence to differing degrees. The Required concentrations for selected chemicals
Atrazine
Page 146
Atrazine
table shows the required concentration for selected chemicals. The Compounds tested but not
detectable up to 10,000 ppb table shows compounds not detectable at 10,000 ppb.
Table 45 Required concentrations for selected chemicals

Compound
Concentration to give a positive result at 3 ppb (in ppb)
Ametryne
Atrazine
Atrazine, de-ethylated
115
Atrazine, de-isopropyl
714
Cyanazine
460
Cyromazine
1200
Prometon
Prometryne
0.7
Propazine
2.3
Simetryne
5.4
Simazine
37
Terbuthylazine
91
Terbutryne
8.3
Table 46 Compounds tested but not detectable up to 10,000 ppb

Alachlor
2, 4-D
Aldicarb
Diaminoatrazine
Carbendazim
Melamine
Carbofuran
Metolachlor
Collect samples in a clean glass bottle.
Do not pre-rinse the bottle with the sample.
If the sample cannot be analyzed immediately, store the sample at 4 C.
Samples may be kept for as long as 14 days.
Warm stored samples to room temperature before analysis.
Atrazine
Page 147
Atrazine
Water sample dilution

Other levels of Atrazine can be tested by diluting the sample and comparing the results to the 0.1
ppb Calibrator. Select the appropriate sample volume from the Sample volume and concentration
table, place it in a graduated mixing cylinder, and dilute it to 50 mL with deionized water.
Table 47 Sample volume and concentration

mL sample
Representative concentration using 0.1 ppb calibrator
0.5
10 ppb
1.0
5 ppb
2.5
2 ppb
5.0
1 ppb
Example:
Dilute 2.5 mL of sample to 50 mL with deionized water. Run the diluted sample in the procedure
along with the 0.1 ppb calibrator. If the absorbance of the diluted sample is less than the 0.1 ppb
calibrator, the concentration of the original sample is greater than 2 ppb.
Summary of method
and soil. Atrazine-specific antibodies, attached to the walls of plastic cuvettes, selectively bind and
remove Atrazine from complex sample matrices. A prepared sample and a reagent containing
enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme) are added
to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and Atrazine compete
for binding sites on the antibodies. Samples with higher levels of analyte will have more antibody
sites occupied by Atrazine and fewer antibody sites occupied by the enzyme-conjugate molecules.
color. Therefore, there is an inverse relationship between color intensity and the amount of
Atrazine in the sample. The resulting color is then compared with a calibrator to determine whether
the Atrazine concentration in the sample is greater or less than the threshold levels. Test results
Atrazine
Page 148
Atrazine

Required reagents
Description
Unit
Cat. No.
20 cuvettes
2762700
Description
Unit
Cat. No.
Caps, flip spout
2/pkg
2581802
Marker, laboratory
each
2092000
each
1970001
1000/pkg
2185628
Atrazine Reagent Set1

1
Required apparatus

each
4879900
Wipes, disposable
box
2097000

Description
Atrazine Reagent Set
Glasses, safety
Gloves, disposable, nitrile, medium1
Unit
Cat. No.
100 cuvettes
2762710
each
2756800
100/pkg
2550502
Graduated Mixing Cylinder, 50-mL
each
2636341
Graduated Mixing Cylinder, 25-mL
each
2636340
50/pkg
2185696
Water, deionized
500 mL
27249
Other sizes are available.
Atrazine
Page 149

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Barium, 8014
Barium
DOC316.53.01007
Turbidimetric Method1
Method 8014
(2 to 100 mg/L)
Powder Pillows
Scope and Application: For water, wastewater, oil-field water, and seawater
1
Adapted from Snell and Snell, Colorimetric Methods of Analysis, Vol. II, 769 (1959).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Perform a standard curve adjustment or a new calibration for each new lot of reagent. See Standard solutions and
Calibration standard preparation.
blank adjust.
Filter highly colored or turbid water samples using a funnel1 and filter paper1. Large amounts of color or turbidity will interfere
and cause high readings.
If samples cannot be analyzed immediately, refer to Sample collection, preservation and storage. Adjust the pH of preserved
samples before analysis.
The Pour-Thru and Sipper Cell modules cannot be used with this procedure.
1

Description
Quantity
BariVer 4 Barium Reagent Powder Pillows
Barium
Page 151
Barium
Turbidimetric method with powder pillows
Stored Programs
20 Barium
Start
1. Select the test.

2. Fill a square sample

cell with 10 mL of sample.

for orientation.
3. Prepared Sample:
Add the contents of one
BariVer 4 Barium
the cell. Swirl to mix.
If barium is present, a
white turbidity will develop.

timer.
A five-minute reaction
period will begin.
Do not disturb the
sample during the
reaction period.
If the reagent does not

dissolve readily in the
sample, mix the sample
and reagent in a 25-mL
graduated mixing cylinder
before pouring into the
sample cell.
Fill another square sample
6. When the timer

expires, wipe the blank
and insert the blank into
the cell holder.
7. Wipe and the

prepared sample and
insert the prepared sample
into the cell holder.
ZERO the instrument. The
READ the results in mg/L
display will show:
Ba2+.
0 mg/L Ba2+
Interferences
Interference levels and treatments
Calcium
10,000 mg/L as CaCO3
Magnesium
Barium
Page 152
8. Clean the sample cell

immediately after each
test. Use soap, water and
a brush to prevent a film of
barium sulfate from
forming inside the cell.
Barium
Silica
500 mg/L
Sodium Chloride
130,000 mg/L as NaCl
Strontium
Interferes at any level. If present, the total concentration between barium and strontium may
be expressed as a PS (Precipitated by Sulfate). While this does not distinguish between
barium and strontium, it gives an accurate indication of scaling tendency.
Highly buffered samples or

extreme sample pH
May exceed the buffering capacity of the reagents and require sample pretreatment.

Collect samples in an acid cleaned glass or plastic container. Adjust the pH to 2 or less with nitric
acid* (about 2 mL per liter). Preserved samples can be stored up to six months at room
temperature. Before analysis, adjust the pH to 5 with 5.0 N sodium hydroxide*. Correct the test
result for volume additions.
Standard solutions
Prepare a 90.0-mg/L barium standard solution as follows:
1. Pipet 9.00 mL of Barium Standard Solution, 1000-mg/L, into a 100-mL volumetric flask.
3. Prepare this solution daily. Follow the Turbidimetric method with powder pillows procedure.
4. To adjust the calibration curve using the reading obtained with the standard solution, select
Options>More>Standard Adjust from the instrument menu.
5. Turn on the Standard Adjust feature and accept the displayed concentration. If an alternate
Accuracy check
Barium Standard Solution, 1000-mg/L Ba
Pipet, TenSette, 0.11.0 mL and tips

chemical form.
3. Default values for standard concentration, sample volume, and spike volumes can be
accepted or edited. After values are accepted, the unspiked sample reading will appear in the
top row. See the user manual for more information.
4. Open a Barium Standard Solution, 1000-mg/L Ba.
5. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to the unspiked sample. Touch
the timer icon. After the timer expires, read the result. Press READ to accept the reading.
Barium
Page 153
Barium
6. Prepare a 0.2 mL sample spike by adding 0.1 mL of standard to the 0.1 mL sample spike.
Touch the timer icon. After the timer expires, read the result. Press READ to accept
the reading.
Touch the timer icon. After the timer expires, read the result. Press READ to accept
the reading.
8. Each addition should reflect approximately 100% recovery.
additions data points, accounting for the matrix interferences. Press IDEAL LINE to view the
relationship between the sample spikes and the "Ideal Line" of 100% recovery.
Calibration standard preparation

Prepare calibration standards containing 10, 20, 30, 50, 80, 90, and 100 mg/L Ba as follows:
1. Into seven different 100-mL Class A volumetric flasks, pipet 1, 2, 3, 5, 8, 9, and 10 mL of the
1000-mg/L Barium Standard Solution using Class A glassware.
2. Dilute to the mark with deionized water. Mix thoroughly.
3. Using the turbidimetric method and the calibration procedure described in the user manual,
generate a calibration curve from the standards prepared above.
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
20
DR 5000
30 mg/L Ba
2535 mg/L Ba
1 mg/L Ba
Summary of method
The BariVer 4 Barium Reagent Powder combines with barium to form a barium sulfate
precipitate, which is held in suspension by a protective colloid. The amount of turbidity present
caused by the fine white dispersion of particles is directly proportional to the amount of barium
present. Test results are measured at 450 nm.

Required reagents
Description
BariVer
4 Barium Reagent Powder Pillows
Quantity/Test
Unit
Catalog number
100/pkg
1206499
Quantity
Unit
Catalog number
Required apparatus
Description
Beaker, 50-mL
each
50041H
2/pkg
2495402
Barium
Page 154
Barium
Description
Barium Standard Solution, 1000-mg/L Ba
Water, deionized
Unit
Catalog number
100 mL
1461142
4L
27256
Unit
Catalog number
100/pkg
189457

Description
Filter Paper for Funnel, 12.5 cm
Funnel, 65 mm
Liqui-Nox Phosphate-free Detergent
each
108367
946 mL
2088153
Nitric Acid 1:1
500 mL
254049
100/pkg
2601300
each
1970001
Pipet Tips for TenSette Pipet 1970001
50/pkg
2185696
1000/pkg
2185628
100 mL
245032
Sodium Hydroxide, 5.0 N

Test Tube Brush
each
69000
each
1457442
Barium
Page 155

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Benzotriazole/Tolyltriazole
UV Photolysis Method1
DOC316.53.01008
Method 8079
(Benzotriazole 1.0 to 16.0 mg/L)

(Tolyltriazole 1.0 to 20.0 mg/L)
Powder Pillows
Scope and Application: For cooling or boiler water.

1
Adapted from Harp, D., Proceedings 45th International Water Conference, 299 (October 2224, 1984)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Avoid fingerprints on the quartz surface of the lamp. Rinse the lamp and wipe with a soft, clean tissue between tests.
If sample contains nitrite or borax (sodium borate), adjust the pH to 46 with 1 N sulfuric acid.
If the sample contains more than 500 mg/L hardness (as CaCO3), add 10 drops of Rochelle Salt Solution before
adding reagent.

Description
Quantity
Triazole Reagent powder pillows
Square bottle, 25-mL
Ultra-violet lamp with power supply
UV safety goggles
Page 157
UV Photolysis method for powder pillows
Stored Programs
30 Benzotriazole
730 Tolyltriazole
Start
1. Select the test.

2. Prepared Sample:
Fill a marked mixing bottle
to the 25 mL line with
sample.

one Triazole Reagent
Powder Pillow.

timer. A 5-minute reaction
period will begin.
7. When the timer

expires, turn the lamp off.
Remove the lamp from the
bottle. Swirl the bottle to
mix thoroughly.

for orientation.
5. Insert the ultraviolet

lamp into the mixing bottle.
Turn on the UV lamp.
Low results will occur if

photolysis (lamp on) takes
place for more or less than
five minutes.
A yellow color will form if
triazole is present.
Page 158
4. Put on UV safety
goggles.
Swirl to dissolve
completely.
8. Fill a square sample

cell with 10 mL of reacted
(prepared) sample.
UV Photolysis method for powder pillows (continued)
Fill another square sample
10. Insert the blank into

the cell holder.
11. Insert the prepared

sample into the cell holder.
ZERO the instrument.

Benzotriazole or mg/L
Tolyltriazole.

0.0 mg/L Benzotriazole
or
0.0 mg/L Tolyltriazole
Interferences
Interference level
Acrylates (as methyl acrylate)
Alum
Borate (as sodium tetraborate)
Greater than 4000 mg/L. Adjust the pH to 46 with 1 N sulfuric acid1.
Chlorine (as Cl2)
Chromium (as chromate)
Copper
Hardness
Greater than 500 mg/L as CaCO3. Add 10 drops of Rochelle Salt Solution1
before adding reagent.
Iron
Lignosulfonates
Magnesium
Molybdenum (as molybdate)
Nitrite
Greater than 4000 mg/L. Adjust the pH to 46 with 1 N sulfuric acid1.
Phosphonates (AMP or HEDP)
Sulfate
Zinc
Strong oxidizing or reducing agents
Interfere at all levels
Page 159

The most reliable results are obtained when samples are analyzed as soon as possible after
collection.
Accuracy check
Benzotriazole Standard Solution, 500 mg/L

1. After reading test results, leave the sample cell (unspiked sample) in the instrument.
4. Open a 500-mg/L Benzotriazole Standard Solution*.
5. Prepare three sample spikes. Fill three mixing cylinders with 25 mL of sample. Use the
and mix thoroughly.
6. Follow the UV Photolysis method for powder pillows test procedure for each of the spiked
samples, starting with the 0.1 mL sample spike. Accept each standard addition result by
pressing READ. Each addition should reflect approximately 100% recovery.
7. After completing the sequence, select GRAPH to view the best-fit line through the standard
additions data points, accounting for matrix interferences. Select IDEAL LINE to view
UV Lamp Check
To verify the ultraviolet lamp (normal life equals 5000 hours) is working properly, perform the
following test:
1. Prepare a 5.0 mg/L benzotriazole standard solution by pipetting 10.0 mL of Benzotriazole
Standard Solution, 500-mg/L benzotriazole, into a 1-liter volumetric flask. Dilute to volume.
2. Analyze according to the above procedure. If the result is significantly below 5 mg/L, replace
the lamp.
Method performance
Program
Precision
Distribution
Sensitivity
10 mg/L benzotriazole
9.710.3 benzotriazole
0.2 mg/L benzotriazole
12 mg/L tolyltriazole
11.612.4 mg/L tolyltriazole
0.2 mg/L tolyltriazole
Instrument
Standard
30
DR 5000
730
DR 5000
Page 160
Summary of method
Benzotriazole or tolyltriazole, used in many applications as corrosion inhibitors for copper and
copper alloys, are determined by a proprietary catalytic ultraviolet (UV) photolysis procedure
requiring less than 10 minutes to perform. Test results are measured at 425 nm.

Required reagents
Description
Triazole Reagent Powder Pillows
Quantity/Test
Unit
Catalog number
100/pkg
2141299
Catalog number
Required apparatus
Description
Quantity
Unit
UV Safety Goggles
each
2113400
Bottle, Square, with 25 mL mark
each
1704200
2/pkg
2495402
Lamp Kit, UV, with power supply, 115 VAC, 60 Hz
each
2704500
Lamp Kit, UV, with power supply, 230 VAC, 50 Hz
each
2704502
Description
Benzotriazole Standard Solution, 500-mg/L
Unit
Catalog number
100 mL
2141342

Description
Flask, Volumetric, Class A, 1000 mL
Pipet Bulb
Unit
Catalog number
each
1457453
100/pkg
2601300
each
1465100
each
1970001
50/pkg
2185696
1000/pkg
2185628
Sulfuric Acid, 1 N
100 mL
127032
Rochelle Salt Solution
29 mL
172533
each
1451538
Volumetric pipet, Class A, 10-mL
Page 161

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Boron, 8015
Boron
DOC316.53.01009
Carmine Method1
Method 8015
(0.2 to 14.0 mg/L)
Powder Pillows

1
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

All labware must be completely dry. Excess water will cause low results.
Use the BoroVer 3 Reagent with adequate ventilation. See Reagent preparation.
Do not cap the sample cells or the Erlenmeyer flasks at any time during sample preparation or reaction time. Samples may
be capped immediately prior to placing in the instrument.
Sulfuric acid may contain residual moisture; this will cause low results. Make sure the sulfuric acid is suitable by running a
known boron standard before running any unknown samples.
Wear safety goggles and gloves during this test.
Boron
Page 163
Boron
Description
Quantity
BoroVer 3 Reagent Powder Pillow
Cylinders, graduated, 50-mL and 100 mL
1 of each
Pipet Bulb
Pipet, volumetric, 2.0 mL
Sulfuric Acid, Concentrated
75 mL
Water, deionized
2.0 mL
Carmine method with powder pillows
Stored Programs
40 Boron
Start
1. Select the test.

for orientation.
Boron
Page 164
2. Using a 100-mL
graduated cylinder,
measure 75 mL of
concentrated sulfuric acid.
Pour the acid into a
3. With good ventilation,

add the contents of one
BoroVer 3 Reagent
Powder Pillow to the flask.
Swirl to mix. Allow up to
five minutes for the
powder to dissolve
completely.
Accurately pipet 2.0 mL of
deionized water into a
Boron
Carmine method with powder pillows (continued)
5. Prepared Sample:
Accurately pipet 2.0 mL of
sample into another 125mL Erlenmeyer flask.
6. Using a 50-mL
graduated cylinder, add
35 mL of the solution
prepared in step 3 to each
Erlenmeyer flask.

timer.
period will begin.
8. When the timer

expires, pour at least
10 mL from each flask into
separate square sample
cells.
Swirl to mix completely.
Zero

insert the blank into the
cell holder.

0.0 mg/L B
Read
11. Wipe and insert the

prepared sample into the
cell holder.

mg/L B.

Collect samples in clean polyethylene or polypropylene bottles, or alkali-resistant,
boron-free glass.
Reagent preparation
To prepare additional BoroVer 3/Sulfuric Acid Solution, mix one BoroVer 3 Reagent Powder Pillow
per 75 mL of concentrated sulfuric acid, adding the powder pillows individually while stirring.
Preparation of this solution generates gaseous HCl when the indicator pillow is added to the
sulfuric acid. Use of a fume hood or other well-ventilated lab area is strongly advised. This solution
is stable for up to 48 hours if stored in plastic containers. Do not store in borosilicate glassware
(Pyrex or Kimax) for longer than one hour; the solution may leach boron from these
containers.
The BoroVer 3/Sulfuric Acid Solution is highly acidic. Refer to the current MSDS for safe
handling and disposal instructions.
Accuracy check
Boron Standard Solution, 1000-mg/L as B
Pipet Bulb
Volumetric pipet, 5-mL
Mixing cylinder, 25-mL

1. Prepare a 250 mg/L boron standard as follows:
Boron
Page 165
Boron
a. Pipet 5.00 mL of Boron Standard Solution, 1000 mg/L as B into a mixing cylinder.
b. Pipet 15.00 mL of demineralized water into the cylinder, stopper and mix thoroughly.
2. After reading test results, leave the unspiked sample in the instrument. Verify the chemical
form.
3. Select Options>More>Standard Additions from the instrument menu.
and mix thoroughly.
6. Follow the Carmine method with powder pillows test. Analyze 2.0 mL of each sample spike
starting with the 0.1 mL sample spike. Accept each standard additions reading by pressing
READ. Each addition should reflect approximately 100% recovery.
relationship between the sample spikes and the Ideal Line of 100% recovery.
Standard Solution Method
Boron Standard Solution, 1000-mg/L B
Volumetric flask, 200-mL
Volumetric pipet, Class A and bulb
Deionized water
1. Prepare a Boron Standard Solution, 10-mg/L as B. solution as follows:

a. Use Class A glassware to pipet 2.00 mL of the Boron Standard Solution, 1000-mg/L B,
into a 200-mL volumetric flask.
b. Dilute to volume with deionized water.
c. Swirl to mix. Follow the Boron test procedure.
Boron
Page 166
Boron
Method performance
Program
Standard
Precision
Distribution
Sensitivity
Concentration change per 0.010 Abs
change
40
7.6 mg/L B
7.57.7 mg/L B
0.14 mg/L B (at 0.2 mg/L)

0.16 mg/L B (at 7.0 mg/L)
0.18 mg/L B (at 14.0 mg/L)
Summary of Method
Boron is determined by its reaction with carminic acid in the presence of sulfuric acid to produce a
reddish to bluish color. The amount of color is directly proportional to the boron concentration. Test
results are measured at 605 nm.
Boron
Page 167
Boron

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
1417099
Sulfuric Acid, ACS, concentrated
75 mL
2.5 L
97909
Water, deionized
2.0 mL
4L
27256
Quantity/Test
Unit
Catalog number
each
50841
each
50842
each
50543
each
50546
Pipet, volumetric, 2.0-mL, Class A
each
1451536
BoroVer 3 Boron Reagent Powder Pillows
Required apparatus
Description
Safety Bulb
each
1465100
2/pkg
2495402
Unit
Catalog number
100 mL
191442
Description
Unit
Catalog number.
Cylinder, mixing, 25 mL
each
2088640
Gloves, chemical resistant, size 9 to 9.5
pair
24101041
100/pkg
2601300
Description
Boron Standard Solution, 1000 mg/L as B

each
1970001
Pipet Tips, for 1970001
50/pkg
2185696
1000/pkg
2185628
each
1451537
Pipet, volumetric, 5.00 mL, Class A
each
1451539
Safety Goggles, vented
each
2550700
each
1457445
Other sizes available

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Boron, LR, 10061
Boron
DOC316.53.01010
Azomethine-H Method1
Method Number
LR (0.02 to 1.50 mg/L as B)
Powder Pillows
Scope and Application: For testing low levels of boron (boric acid or borates) in drinking water, cooling water,
industrial process waters or wastewaters.
1
Adapted from ISO Method 9390 Harp, D.L., Analytica Chimica Acta, 346(1997), pp. 373379.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2410212
DR 5000
2410212
DR 3900
2410212
DR 3800, DR 2800, DR 2700
2410212

For best results, match two cells using the Cell matching procedure.
Sample temperature should be 2224 C (7275 F) for most accurate results.
If outside this range, measure and record the sample temperature. See Sample temperature compensation.

Description
BoroTrace Reagent Set Includes:
EDTA Solution, 1M
Quantity
20 drops
BoroTrace #2 Reagent Powder Pillow
BoroTrace #3 Reagent Powder Pillow
Water, Ultra-pure, aldehyde-free

Sample cell (see Instrument-specific information)
25 mL
2
Boron
Page 169
Boron
Azomethine-H method
Stored Programs
45 Boron, LR
Start
1. Select the test.

Fill a clean plastic sample
ultra-pure water.
Boron
Page 170
4. Add ten drops of

EDTA Solution, 1 M, to
each cell. Cap and invert
each cell twice to mix.
If the sample is colored or

turbid, sample
pretreatment may
be required See the
Interfering substances and
suggested treatments
table.

for orientation.
5. Open one
BoroTrace 2 Reagent
powder pillow and add the
contents to the
prepared sample.

a second clean plastic
sample cell to the 25-mL
mark with sample.
6. Cap the prepared

sample and shake to
dissolve the powder.
Proceed immediately with
steps 7 through 10.

timer.
A ten-minute reaction
period will begin.
8. Continue shaking
vigorously for 30 seconds,
or until all powder is
dissolved
Let the cell sit capped for
the remaining reaction
period.
Boron
Azomethine-H method (continued)
9. During the reaction

period, add the contents of
a BoroTrace 2 Reagent
blank.
10. Cap the blank and

shake vigorously until the
powder is dissolved.

BoroTrace 3 Reagent
Powder Pillow to each cell.
Cap and shake to
dissolve.

BoroTrace 3 Reagent
stops the reaction.
Read
Zero

0.00 mg/L B

the cell holder.

mg/L B.
To correct for sample
temperature, refer to the
Sample temperature
compensation table. To
change to alternate form
H3BO3, refer to the
instrument user manual.
Cell matching procedure

1. Rinse and fill two cells with deionized water.
2. Wipe each cell with a soft cloth or tissue to remove liquid or fingerprints.
3. Using one of the cells, set the instrument absorbance at 410 nm to zero.
4. Read the absorbance of the other cell.
5. Cells that read within 0.002 absorbance are matched.
Boron
Page 171
Boron
Interferences
The substances listed in the Interfering substances table have been tested for interference and
found not to interfere up to the indicated levels (in mg/L). The Interfering substances and
suggested treatments table lists suggested treatments for interferences:

Interference level
Aluminum (3+)
10
Benzotriazole
20
Biocides:
Carbamate-type
120
Isothiazolin-type
120
Quat-type
90
Thiocyanate-type
60
Calcium
1000 (as CaCO3)
Chloride
2500
Copper (2+)
20
Magnesium
1000 (as CaCO3)
Manganese (7+)
Molybdate
(Mo6+)
60
Phosphonates, AMP
20
Phosphonates, HEDP
20
Polyacrylates
20 (as Acumer 1000, 1100)
Polymaleic Acid
40 (as Belclene 200)
Silica
120
Sulfate
1800
Sulfite
40
Tolyltriazole
20
Zinc (2+)
10
Table 55 Interfering substances and suggested treatments

Alkalinity >500 mg/L (+ or )
1.
2.
Adjust sample pH to between 57 using 1.0 N Sulfuric Acid Solution1.

Continue with step 4 of the Azomethine-H method procedure.
1.
Zero the instrument (0.00 mg/L B) using Ultra-Pure Water2. Measure and
record the apparent concentration, in mg/L B, due to the sample color.
Subtract the apparent concentration from the result in step 15 of the
Azomethine-H method procedure.
Color (+)
2.
Halogen disinfectants in the sample can produce a red-color after the addition
of BoroTrace 2 Reagent. To eliminate this interference:
1. Add 1 pillow Dechlorinating Reagent1 to 25 mL each of Ultra-Pure Water2
Halogens (Bromine or Chlorine) all levels (+)
and sample.
2. Cap and shake to dissolve.
3. Continue with step 4 of the test procedure.
Boron
Page 172
Boron
Table 55 Interfering substances and suggested treatments (continued)
Iron (Fe3+ or Fe2+), above 8 mg/L (+)
High levels of iron in the sample can produce a red-color after the addition of
BoroTrace 2 Reagent. To compensate, increase the amount of EDTA2 from
10 drops to 15 drops to be added to each cell (step 4). Alternatively, dilute the
sample with Ultra-Pure Water and continue with step 5 of the test procedure.
Correct the results (step 15) using the appropriate dilution factor.
1.
2.
3.
Nitrites, all levels (+)
4.
5.
Add 0.1 gram scoop Sulfamic Acid1 to 25 mL each Ultra-Pure Water and
sample in plastic cells.
Cap and shake to dissolve.
Uncap and wait 5 minutes.
Add 5 N Sodium Hydroxide Reagent1 solution to each to adjust pH to 58
using pH paper.
Continue with step 4 of the test procedure.
Filter the sample through a 3 m membrane1 prior to testing. Do not use a

glass fiber filter.
Turbidity (+)
1
See Required reagents.

Collect samples in clean polyethylene bottles. Do not use borate-based detergents or soaps to
clean sample containers or labware used for this method. After use, thoroughly rinse all plastic
containers with deionized water, allow to air dry, and keep covered.
Sample temperature compensation

The reaction chemistry is very dependent on the sample temperature. Calibrations are performed
at 23 C (73 F). If the sample temperature is outside the range of 2224 C (7275 F), multiply
the results, in mg/L, by the appropriate multiplier (see the Sample temperature multipliers table).
Table 56 Sample temperature multipliers

Sample Temp.
Sample Temp.
Multiplier
Multiplier
41
0.70
20
68
0.94
45
0.73
25
77
1.04
10
10
0.78
26
79
1.06
12
54
0.81
27
81
1.08
14
57
0.84
28
82
1.10
16
61
0.87
29
84
1.12
18
64
0.91
30
86
1.15
Accuracy check
1000-mg/L Boron Standard Solution
100-mL plastic volumetric flask with stopper
Deionized water
Boron
Page 173
Boron
Pipet bulb
3. Accept the default values for standard concentration, sample volume, and spike volumes.
After the values are accepted, the unspiked sample reading will appear in the top row. See the
user manual for more information.
4. Prepare a 50.0-mg/L boron standard:
a. Pipet 5.0 mL of a 1000-mg/L Boron Standard Solution into a 100-mL plastic volumetric
flask.
b. Dilute with deionized water.
c. Insert a stopper and invert to mix.
TenSette Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of the diluted standard, respectively, to
each sample and mix thoroughly.
6. Follow the Azomethine-H method test procedure for each of the spiked samples, starting with
the 0.1 mL sample spike. Measure each of the spiked samples in the instrument.
7. Select GRAPH to view the results. Select IDEAL LINE (or best-fit) to compare the standard
addition results to the theoretical 100% recovery.
Boron Standard Solution, 1000 mg/L as B
Deionized water
Plastic volumetric flask, 1000-mL
Plastic pipet
1. Prepare a 1.0-mg/L B standard:

a. Use a plastic pipet to transfer 1.0 mL of Boron Standard Solution, 1000 mg/L as B, into a
1000-mL plastic volumetric flask.
b. Dilute to volume with deionized water.
c. Insert a stopper and invert to mix.
* See Optional reagents and apparatus
Boron
Page 174
Boron
Method performance
Program
Standard
Precision
Distribution
Sensitivity
45
0.92 mg/L B3+
0.870.97 mg/L B
0.01 mg/L B
Summary of method
Azomethine-H, a Schiff base, is formed by the condensation of an aminonaphthol with an
aldehyde by the catalytic action of boron. Test results are measured at 410 nm.

Required reagents
Description
BoroTrace Reagent Set, includes:
EDTA Solution, 1 M
BoroTrace #2 Reagent Powder Pillows
BoroTrace #3 Reagent Powder Pillows
Water, Ultra-pure, Aldehyde-free
Quantity/Test
Unit
Catalog number
2666900
20 drops
50 mL SCDB
2241926
100/pkg
2666669
100/pkg
2666799
25-mL
500 mL
2594649
Quantity/Test
Unit
Catalog number
12/pkg
2410212
Required apparatus (powder pillows)

Description
Sample cell, 1-inch square plastic, with cap
Description
Boron Standard Solution, 1000-mg/L as B
Unit
Catalog number
100 mL
191442
Unit
Catalog number

Description
Dechlorinating Reagent, Powder Pillows
Filter Holder, 25 mm
Membrane, filter, 3 m, 25 mm
pH Paper, 1.011.0
Pipet, tips for 1970001 TenSette Pipet
each
2088640
100/pkg
1436369
each
246800
25/pkg
2594025
5 rolls/pkg
39133
each
1970001
50/pkg
2185696
each
1451537
Pipet, volumetric, Class A, 1- mL
each
1451537
Safety Bulb
each
1465100
Sodium Hydroxide Reagent, 5.0 N, 50 mL
each
245026
Boron
Page 175
Boron
Description
Unit
Catalog number
Spoon, measuring, 0.1 g
each
51100
Sulfamic Acid, 454 g
each
234401
Sulfuric Acid Solution 1.0N, 100 mL
each
127032
Syringe, 30 cc Luer-lock
each
2225800
Thermometer, non-mercury, 10 to +225 C
each
1451535
Volumetric Flask, Class A 1000-mL
each
1451535

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Bromine, 8016
Bromine
DOC316.53.01011
DPD Method1
Method 8016
Powder Pillows or AccuVac Ampuls
(0.05 to 4.50 mg/L)
Scope and Application: For testing bromine residuals (including hypobromite, hypobromous acid and
bromamines) used as disinfectants in process waters, treated water, estuary water, and seawater
1
Adapted from Standard Methods for the Examination of Water and Wastewater
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Analyze samples immediately. Do not preserve for later analysis.
blank adjust.
If the sample temporarily turns yellow after reagent addition, dilute a fresh sample and repeat the test. A slight loss of
bromine may occur because of the dilution. Multiply the result by the dilution factor.
Sample cell orientation may vary. Refer to the instrument user manual for correct cell orientation.
Wipe the outside of sample cells before each insertion into the instrument cell holder. Use a damp towel followed by a dry
one to remove fingerprints or other marks.
Bromine
Page 177
Bromine
Description
Quantity
Powder Pillow Test:

DPD Total Chlorine Reagent Powder Pillows
AccuVac Test:
DPD Total Chlorine Reagent AccuVac Ampuls
Beaker, 50-mL
Stopper, 18 mm tube
See Consumables and replacement items on page 183 for reorder information.
DPD Powder Pillows
Stored Programs
50 Bromine
Start
1. Select the test.

for orientation.
2. Fill a sample cell with

10 mL of sample.
3. Prepared Sample:
DPD Total Chlorine
sample cell.
Swirl for 20 seconds to
mix.
A pink color will develop if
bromine is present.
Bromine
Page 178

timer.
A three-minute reaction
period will begin.
Perform steps 56 during
the reaction period.
Bromine
DPD Powder Pillows (continued)
Fill a second sample cell
with 10 mL of sample.


0.00 mg/L Br2
7. Within three minutes

wipe the prepared sample
and insert it into the cell
holder.
Br2.
DPD AccuVac Ampuls
Stored Programs
50 Bromine
Start
1. Select the test.

for orientation.
Fill a round 1-inch sample
3. Prepared Sample:
Collect at least 40 mL of
sample in a 50-mL beaker.
4. Quickly invert the

Ampul several times
to mix.
Fill a DPD Total Chlorine

Reagent AccuVac Ampul
with sample. Keep the tip
immersed while the Ampul
fills completely.

bromine is present.
Bromine
Page 179
Bromine
DPD AccuVac Ampuls (continued)

timer.

period will begin.
Perform step 6 during the

reaction period.
Bromine
Page 180

0.00 mg/L Br2

wipe the Ampul and insert
READ the results in
mg/L Br2.
Bromine
Interferences
Interference level
Acidity
Greater than 150 mg/L CaCO3. May not develop full color or color may fade instantly.
Neutralize to pH 6 7 with 1 N Sodium Hydroxide1. Determine amount to be added on
separate sample aliquot, then add the same amount to the sample being tested. Correct for
volume addition.
Alkalinity
Neutralize to pH 6 7 with 1 N Sulfuric Acid1. Determine amount to be added on separate
sample aliquot, then add the same amount to the sample being tested. Correct for volume
addition.
Chlorine
Interferes at all levels
Chlorine Dioxide
Chloramines, organic
May interfere
Hardness
No effect at less than 1000 mg/L as CaCO3
Iodine
Manganese, Oxidized
(Mn4+, Mn7+) or
Chromium, Oxidized (Cr6+)
1.
Adjust sample pH to 6 7.
2.
3.
Add 3 drops Potassium Iodide1 (30-g/L) to a 25-mL sample.

Mix and wait one minute.
4.
5.
6.
Add 3 drops Sodium Arsenite1, 2 (5-g/L) and mix.

Analyze 10 mL of the treated sample as described in the procedure.
Subtract the result of this test from the original analysis to obtain the correct bromine
concentration.
Monochloramine
Ozone
Peroxides
May interfere
Extreme sample pH or highly

buffered samples
Adjust to pH 67.
Samples treated with sodium arsenite for manganese or chromium interferences will be hazardous wastes as regulated by the Federal RCRA
for arsenic (D004).

Collect samples in clean, dry glass containers. If sampling from a tap, allow the water to flow at
least 5 minutes to ensure a representative sample. Avoid excessive agitation and exposure to
sunlight when sampling. Allow several volumes of water to overflow the container and cap the
container so there is no headspace above the sample. If sampling with a DR cell, rinse the cell
several times with the sample, then carefully fill to the 10-mL mark. Proceed with the analysis
immediately.
Method performance
Precision
Distribution
Sensitivity
2.80 mg/L Br2
2.752.85 mg/L Br2
0.05 mg/L Br2
2.80 mg/L Br2
2.712.89 mg/L Br2
0.05 mg/L Br2
Program
Standard
50
55
Bromine
Page 181
Bromine
Summary of method
Bromine residuals react with iodide and DPD (N,N-diethyl-p-phenylenediamine) to form a pink
color which is proportional to the total bromine concentration. Test results are measured at 530
nm.
Bromine
Page 182
Bromine

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
2105669
25/pkg
2503025
Quantity
Unit
Catalog number
2405200
OR
Required apparatus
Description
AccuVac snapper
each
Beaker, 50-mL
each
50041H
Sample cell, 10 mL round, 25 x 54 mm
each
2122800
6/pkg
2427606
2/pkg
2495402
Unit
Catalog number
Description
Chlorine Standard Solution, 2-mL PourRite Ampule, 2530 mg/L
20/pkg
2630020
20/pkg
1426820
Chlorine Standard Solution, 10-mL Voluette Ampule, 5075 mg/L
16/pkg
1426810
PourRite Ampule Breaker, 2-mL
each
2484600
Voluette Ampule Breaker, 10-mL
each
2196800
Unit
Catalog number
each
2405200

Description
AccuVac Snapper
Chlorine Demand-Free Water
500 mL
2641549
each
2088640
each
189641
1000/pkg
2105628
300/pkg
2105603
pH Paper,0 14 pH range
100/pkg
2601300
Potassium Iodide, 30 g/L
100 mL
34332
Sodium Arsenite, 5 g/L
100 mL
104732
Sodium Hydroxide, 1 N
100 mL
104532
Sulfuric Acid, 1 N
100 mL
127032
6/pkg
173106
Stopper for 18 mm tube
Bromine
Page 183

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Cadmium, 8017
Cadmium
DOC316.53.01012
Dithizone Method1
Method 8017
(0.7 to 80.0 g/L)
Powder Pillows
Scope and Application: For water and wastewater; digestion is required to determine total cadmium.
1
Adapted from Standard Methods for the Examination of Water and Wastewater (method 3500 Cd D).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

deionized water instead of the sample.
Clean all glassware with 6 N Hydrochloric Acid Solution and rinse with deionized water.
Sample cell orientation may vary. Refer to the instrument user manual for correct cell orientation.
Cloudy and turbid samples may require filtering before running the test. Report results as g/L soluble cadmium. Use glass
membrane type filter to avoid loss of cadmium by adsorption onto the filter paper.
If samples cannot be analyzed immediately, see Sample collection, preservation and storage. Adjust the pH of preserved
The Flow Cell and Sipper Modules cannot be used with this procedure.
The DithiVer powder will not completely dissolve in the chloroform. For further notes see Dithiver solution preparation and
storage.
Read the MSDS before testing. Spilled reagent will affect test accuracy and is hazardous to skin and other materials.
Goggles and gloves are recommended.
Cadmium
Page 185
Cadmium

Description
Quantity
Citrate buffer powder pillows
Chloroform
30 mL
DithiVer Metals Reagent powder pillows
Potassium Cyanide
0.1 g
Sodium Hydroxide solution, 50%
20 mL
Cotton balls
Clippers
Cylinder, 25-mL graduated
Cylinder, 50-mL graduated mixing
Funnel, 500-mL separatory
Sample Cells
Spoon, measuring, 0.1-g
Support ring (4-inch) and stand (5 x 8-inch base)
Dithizone method with powder pillows

DANGER
Cyanide is a deadly poison. Use a fume hood. Maintain cyanide solutions at pH 11 or greater to prevent
formation of cyanide gas.
Stored Programs
60 Cadmium, Dithizone
Start
1. Select the test.

for orientation.
Cadmium
Page 186
2. Fill a 250-mL
graduated cylinder to the
Pour the sample into a
500-mL separatory funnel.

one Buffer Powder Pillow
for heavy metals, citrate
type. Stopper the funnel
and shake to dissolve.
4. DithiVer Solution
Preparation:
Add 30 mL of chloroform
to a 50-mL mixing
graduated cylinder. Add
the contents of one
DithiVer Metals Reagent
Powder Pillow. Stopper the
cylinder. Invert several
times to mix.
Cadmium
Dithizone method with powder pillows (continued)
5. Add 20 mL of 50%
Sodium Hydroxide
Solution to the funnel.
6. Add a 0.1-g scoop of

potassium cyanide to the
funnel. Stopper. Shake
vigorously for 15 seconds.
7. Remove the stopper.

timer.

timer and allow the funnel
to stand undisturbed until
the timer expires.

Insert a cotton plug
the size of a pea into the
delivery tube of the funnel
and slowly drain the
bottom (chloroform) layer
into a dry 25-mL sample
cell (the prepared sample).
Stopper.
Close the stopcock and

shake the funnel
vigorously during the
1-minute time period.
The bottom (chloroform)

layer will be orange to pink
if cadmium is present.
Start the instrument timer.

will begin.
8. Add 30 mL of the
DithiVer solution to
the 500-mL separatory
funnel. Stopper, invert, and
open stopcock to vent.
Close the stopcock and
shake funnel once or
twice; vent again.

Fill a dry sample cell with
at least 10 mL of
chloroform. Stopper.
The cadmium-dithizone
complex is stable for more
than one hour if the
sample cell is kept tightly
capped and out of direct
sunlight.
Cadmium
Page 187
Cadmium
Dithizone method with powder pillows (continued)
Zero

the cell holder.

Read


g/L cadmium.
0.0 g/L Cd
Interferences
Interference level
Highly buffered samples or extreme sample pH
May exceed the buffering capacity of the reagents and

require sample pretreatment.
Bismuth
Greater than 80 mg/L. See treatment below.
Copper
Mercury
All levels. See treatment below.
Silver
To eliminate interference from the metals listed in the Interfering substances table, insert the
following steps into the procedure after step 4.
1. Measure approximately 5 mL of the DithiVer solution into the separatory funnel. Stopper the
funnel, invert and open the stopcock to vent. Close the stopcock and shake the solution
vigorously for 15 seconds. Allow the funnel to stand undisturbed until the layers separate
(about 30 seconds). A yellow, red, or bronze color in the bottom (chloroform) layer confirms
the presence of interfering metals. Draw off and collect the bottom (chloroform) layer for
proper disposal.
2. Repeat the extraction with fresh 5-mL portions of the DithiVer solution (discarding the bottom
layer each time) until the bottom layer shows a pure dark green color for three successive
extracts. Extractions can be repeated several times without appreciably affecting the amount
of cadmium in the sample.
3. Extract the solution with several 2- or 3-mL portions of pure chloroform to remove any
remaining DithiVer, collecting the bottom layer each time for proper disposal.
4. Continue with step 5 of the procedure.
5. In step 8, substitute 28.5 mL of DithiVer solution for the 30 mL.
6. Continue with step 9 of the procedure.
Cadmium
Page 188
Cadmium
Table 61 Substances that do not interfere

Aluminum
Lead
Antimony
Magnesium
Arsenic
Manganese
Calcium
Nickel
Chromium
Tin
Cobalt
Zinc
Iron

Collect samples in an acid-washed glass or plastic containers. Adjust the pH to 2 or less with nitric
acid (about 2 mL per liter). Store preserved samples up to six months at room temperature. Adjust
the pH to 2.5 with 5.0 N sodium hydroxide before analysis. Correct the test result for
volume additions.
Dithiver solution preparation and storage

Store DithiVer Powder Pillows away from light and heat. A convenient way to prepare this solution
is to add the contents of 16 DithiVer Metals Reagent Powder Pillows to a 500-mL bottle of
chloroform and invert several times until well mixed (carrier powder may not dissolve completely).
Store dithizone solution in an amber glass bottle. This solution is stable for 24 hours.
Accuracy check
Cadmium Voluette Ampule Standard, 25-mg/L Cd
TenSette Pipet, 0.11.0 mL and tips
5. Use the TenSette Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of standard, respectively to three
250-mL samples and mix each thoroughly.
6. Follow the Dithizone method with powder pillows test procedure for each of the spiked
samples, starting with the 0.1 mL sample spike. Measure each of the spiked samples in the
instrument.
Cadmium Standard Solution, 100-mg/L Cd.
Deionized water
100-mL Volumetric flask
Cadmium
Page 189
Cadmium
Class A volumetric pipet and bulb
1. Prepare a 5.0-mg/L cadmium standard solution:

a. Pipet 5.00 mL of Cadmium Standard Solution, 100-mg/L Cd, into a 100-mL volumetric
flask.
b. Dilute to the mark with deionized water. Prepare this solution daily.
2. Pipet 2.00 mL of the 5.0-mg/L Cadmium Standard Solution into 248 mL of deionized water in a
500-mL separatory funnel. This is a 40-g/L cadmium solution. Perform Dithizone method with
powder pillows on this solution beginning with step 3 of the procedure.
3. To adjust the calibration curve using the reading obtained with the standard solution, elect
Method performance
Program
Standard
Precision
Distribution
Sensitivity
60
40.0 g/L Cd
39.340.7 g/L Cd
0.73 g/L
Summary of method
The dithizone method is designed for the determination of cadmium in water and wastewater. The
DithiVer Metals Reagent is a stable powder form of dithizone. Cadmium ions in basic solution react
with dithizone to form a pink to red cadmium-dithizonate complex, which is extracted with
chloroform. Test results are measured at 515 nm.
Pollution prevention and waste management

Both chloroform (D022) and cyanide (D003) solutions are regulated as hazardous wastes by the
Federal RCRA. Do not pour these solutions down the drain. Chloroform solutions and the cotton
plug used in the delivery tube of the separatory funnel should be collected for disposal with
laboratory solvent waste. Collect the cyanide solution as a reactive waste. Be sure that cyanide
solutions are stored in a caustic solution with a pH >11 to prevent potential release of hydrogen
cyanide gas. See the current MSDS for disposal information.

Required reagents
Description
Cadmium Reagent Set
Quantity/Test
Unit
Catalog number
100/pkg
2242200
Includes:(1) 14202-99, (1) 14458-17, (1) 12616-99, (1) 767-14, (4) 2180-49, (1) 2572-01
Buffer Powder Pillows, citrate
Chloroform, ACS
DithiVer Metals Reagent Powder Pillows
100/pkg
40 mL
4L
1420299
1445817
100/pkg
1261699
Potassium Cyanide
0.1 g
125 g
76714
Sodium Hydroxide Solution, 50%
20 mL
500 mL
218049
Cadmium
Page 190
Cadmium
Required reagents (continued)
Description
Cotton Balls, absorbent
Quantity/Test
Unit
Catalog number
100/pkg
257201
Quantity
Unit
Catalog number
96800
Required apparatus
Description
Clippers, for opening powder pillows
each
each
50840
each
50846
Cylinder, graduated, mixing, 50-mL
each
189641
Funnel, separatory, 500-mL
each
52049
Sample cell, 25 mL square, matched pair with cap
2/pkg
2612602
each
51100
Support Ring, 4"
each
58001
Support Ring Stand, 5" x 8" base
each
56300
Description
Unit
Catalog number
Cadmium Standard Solution, 100-mg/L Cd
100 mL
1402442
Chloroform, ACS
500 mL
1445849
Hydrochloric Acid Solution, 6.0 N
500 mL
88449
100 mL MDB
245032
59 mL SCDB
245026
4L
27256
Water, deionized
Cadmium
Page 191
Cadmium

Description
Unit
each
50837
Filter Discs, glass, 47 mm
100/pkg
253000
Filter Holder, glass, for 47-mm filter
each
234000
each
50549
each
54649
each
1457442
each
1457446
each
1457453
Gloves, chemical resistant, size 9 to 9.51
pair
2410104
Goggles, safety, vented
each
2550700
Hot Plate, 3-in. diameter, 120 VAC, 50/60 Hz
each
1206701
pH Paper, pH 1.0 to 11.0
Catalog number
5 rolls/pkg
39133
each
1465100
each
53236
Pipet, TenSette, 0.1 to 1.0 mL
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451536
each
1451503
each
1451506
each
1451508
each
1451509
each
1451538
each
1451520
Tongs, crucible, 9-inch
each
56900

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Carbon Dioxide, DT, 8205
Carbon Dioxide
DOC316.53.01167
Digital Titrator Method Using Sodium Hydroxide

10 to 1000 mg/L CO2
Method 8205
Digital Titrator
Scope and Application: For water and seawater.
Test preparation
Avoid excess agitation when collecting and swirling the sample to prevent the loss of carbon dioxide from the sample. The
sample is measured directly in the Erlenmeyer flask to avoid agitation..
For more accurate results, check the calibration of the Erlenmeyer flask. Fill a graduated cylinder with the sample volume of
deionized water. Pour the water into the Erlenmeyer flask and make a mark at the correct level.
A pH meter can be used in place of the indicator. The end point pH is 8.3.
1

Description
Quantity
1 pillow
Sodium hydroxide titration cartridge (see Range-specific information)
1 cartridge
Digital titrator
Carbon dioxide
See
Table 1
1. Select a sample


the tip.
4. Collect a water sample

directly into the titration
flask by filling to the
appropriate mark.
Carbon Dioxide
Page 193
Carbon Dioxide
Carbon dioxide (continued)

one Phenolphthalein
Swirl to mix.
If a pink color forms, no
carbon dioxide is present.

changes from colorless to
a light pink color that
persists for 30 seconds
(pH 8.3).
counter.

the Range-specific
calculate the
concentration:
mg/L as CO2
Example: 100 mL of
= 50 mg/L CO2

Range (mg/L as CO2)
Sample volume (mL)
Titration cartridge (N NaOH)
Multiplier
1050
200
0.3636
0.1
20100
100
0.3636
0.2
100400
200
3.636
1.0
2001000
100
3.636
2.0
Interferences

Interference level
Other acids
Other acid components in the sample will be titrated and interfere directly in this test.
Color or turbidity
color indicators and titrate to a pH of 8.3.
Carbon Dioxide
Page 194
Carbon Dioxide
Complete the test procedure as soon as possible after collection for best accuracy.
The sample can be stored for at least 24 hours if cooled to 4 C (39 F) or below.
Accuracy check
Carbon Dioxide Voluette Ampule Standard Solution, 10,000 mg/L CO2
Ampule breaker

titration cartridge or 5 digits of the 3.636 N titration cartridge to reach the endpoint. If more or
less titrant was used, there may be an interference (see Interferences) or the concentration of
the titrant has changed.
Summary of method
Acidity due to carbon dioxide in a sample is titrated with sodium hydroxide to a phenolphthalein
end point. Strong acids are assumed to be absent or of insignificant concentration.
Carbon Dioxide
Page 195
Carbon Dioxide

Required reagents
Description
Quantity/Test
Unit
Catalog number
1 pillow
100/pkg
94299
varies
each
1437801
varies
each
1438001
Carbon Dioxide Reagent Set (approximately 100 tests)
2272700
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50543
each
50546
Description
Carbon Dioxide Standard Solution,
Voluette
Ampule, 10,000-mg/L as CO2, 10-mL
Unit
Catalog number
16/pkg
1427510
Unit
Catalog number

Description
Phenolphthalein Indicator Solution, 5-g/L
100 mL MDB
16232
100 mL
226142
each
2095352
each
1970001
Tips for Tensette
50/pkg
2185696
Water, deionized
500 mL
27249
each
108146
Bottle, sampling, poly, 500 mL
each
2087079
pH meter
each
Thermometer, -10 to 220 C
each
2635700
Ampule Breaker
each
2196800
TitraStir Apparatus, 115 Vac
each
1940000
each
1940010
Digital Titrator delivery tips
5/pkg
1720500
Cylinder, graduated, 250 mL

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Carbon Dioxide, BT, 8223
Carbon Dioxide
Buret Titration Method1
0 to 250 mg/L
DOC316.53.01152
Method 8223
Buret Titration

1
Test preparation
Avoid excess agitation when collecting and swirling the sample to prevent the loss of carbon dioxide. Measure the sample
directly in the Erlynmeyer flask to avoid agitation.
For more accurate results, check the calibration of the Erlenmeyer flask. Fill a graduated cylinder with the sample volume of
deionized water. Pour the water into the Erlenmeyer flask and mark the correct level with a wax pencil or permanent marker.
The concentration of the sodium hydroxide standard solution will slowly decrease. To prevent deterioration, keep the bottle
tightly sealed after use. Fill the buret immediately before the test is started and discard the remaining solution after the test.
Follow the Accuracy check each month to make sure the solution will give accurate results.
A pH meter can be used in place of the indicators. The end point pH is 8.3.
For added convenience when stirring, use the TitraStir stirring apparatus.
1

Description
Quantity
1
1 bottle
Buret, Class A, 25-mL, with support stand
Erlenmeyer flask
Carbon Dioxide
Page 197
Carbon Dioxide
Buret titration
See
Table 1
1. Select a sample
volume and flask from the
table.
5. Titrate the sample

while gently swirling the
flask until a light pink color
forms and persists for 30
seconds.

the zero mark with
0.0227 N Sodium
Hydroxide Standard
Solution.
3. Fill a clean
Erlenmeyer flask to the
selected volume. If
collected from a faucet,
allow the sample to
overflow several times and
then pour off the excess
sample.

one Phenolphthalein
and mix gently.
6. Calculate:
mL titrant used x multiplier = mg/L as CO2
Example: 100 mL of sample was titrated and 15 mL of
titrant was used to reach the endpoint. The
concentration is 15 x 10 = 150 mg/L as CO2

Range (mg/L as CO2)
Sample volume (mL)
Flask size
0125
200
250
100250
100
125
10
Multiplier
Interferences

Interference level
Color or turbidity
phenolphthalein indicator and titrate to a pH of 8.3.
Acids
Acids other than carbonic acid will be titrated and interfere directly.
Carbon Dioxide
Page 198
Carbon Dioxide

agitation or prolonged exposure to air. Complete the test procedure as soon as possible after
collection for best accuracy. The sample can be stored for at least 24 hours if cooled.
Accuracy check
The standard additions method can be used to find if the sample has an interference. The
standard solution method can be used to confirm analytical technique and reagent performance.
Carbon Dioxide Voluette Ampule Standard Solution, 10,000-mg/L as CO2
Ampule breaker
TenSette Pipet, 0.11.0 mL and Pipet Tips.

8. Each 0.1 mL of standard that was added will use 1.0 mL of titrant to reach the endpoint. If
more or less titrant was used, there may be an interference (see Interferences) or the
concentration of the titrant has changed (see Standard solution method).
A sodium hydroxide standard solution will slowly absorb carbon dioxide from the atmosphere,
which forms carbonic acid and neutralizes some of the hydroxide. When sealed in plastic bottles,
the sodium hydroxide concentration will decrease by approximately 1% per year. Complete the
following test to make sure the concentration is accurate.
1. Add 50.0-mL of Potassium Acid Phthalate Standard Solution, 100-mg/L as CO2 to an

Erlenmeyer flask.
2. Add the phenolphthalein indicator and swirl to mix.
3. Titrate the standard to the end point with the sodium hydroxide standard solution. The titration
should require 5.00 mL of sodium hydroxide solution. If the volume is greater than 5.25 mL,
discard the sodium hydroxide and replace it with a fresh supply.
Note: To correct for a reduction in strength, divide 5.00 by the number of mL required for this titration.
Multiply all sample results by this factor.
Carbon Dioxide
Page 199
Carbon Dioxide
Summary of method
The analysis for dissolved carbon dioxide in water is similar to that for acidity. A water sample is
titrated to a phenolphthalein end point at pH 8.3 with a sodium hydroxide standard solution. Strong
mineral acids are assumed to be absent or their effect to be negligible.

Required reagents
Description
Quantity/Test
Unit
Catalog number
1 pillow
100/pkg
94299
varies
1L
19253

Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
2636540
Buret Clamp, double
each
32800
each
50543
each
50546
Support Stand
each
56300
Description
Carbon Dioxide Standard Solution,
Voluette
Ampule, 10,000-mg/L as CO2, 10-mL
Unit
Catalog number
16/pkg
1427510
100 mL
226142
Unit
Catalog number

Description
Phenolphthalein Indicator Solution, 5-g/L
100 mL
16232
each
1970001
50/pkg
2185696
Water, deionized
500 mL
27249
each
108146

Sampling Bottle with cap, low density polyethylene, 500 mL
12/pkg
2087079
Thermometer, Non-Mercury, -10 to 225 C
each
2635700
Voluette Ampule breaker 10 mL
each
2196800
each
1940000
each
1940010
each
2095352
Other sizes are available

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chelant, Free, DT, 8352
Chelant, Free
Digital Titrator Using Magnesium Chloride
0 to 20.0 mg/L as CaCO3
DOC316.53.01168
Method 8352
Digital Titrator
Scope and Application: For industrial waters.
Test preparation
All apparatus must be extremely clean and rinsed frequently with acid and deionized water to remove any hardness present
on the plastic or glass.
Filter turbid samples with filter paper1 and a funnel.
Four drops of ManVer Hardness Indicator Solution1 or a 0.1 g scoop of ManVer 2 Hardness Indicator Powder1 can be used
in place of the ManVer 2 Hardness Indicator Powder Pillow.
For best results, measure a reagent blank. Use 50 mL of deionized water in place of the sample. Subtract the number of
digits that were used for the reagent blank from the number of digits that were used for titrating the sample.
Results may be expressed as mg/L tetrasodium EDTA. (Digits required x 0.38 = mg/L as Na4 EDTA)
1

Description
Quantity
Hardness 1 Buffer Solution
1 bottle
ManVer 2 Hardness Indicator Powder Pillow
1 pillow
Magnesium Chloride Titration Cartridge, 0.0800 M
1 cartridge
Digital titrator
Graduated cylinder
Chelant, Free
Page 201
Chelant, Free
Free chelant

8. Hold the Digital

Titrator with the cartridge
tip pointing up. Turn the
delivery knob to eject a
few drops of titrant. Reset
the counter to zero and
wipe the tip.
9. Use a graduated
cylinder to measure
100 mL of sample into a

one ManVer 2 Hardness
Swirl to mix.

changes to red-violet.
13. Calculate:
If the solution turns blue,

free chelant is present. If
the solution turns red, a
chelant deficiency exists.

digits on the counter.
Chelant, Free
Page 202
10. Add two full droppers

(2 mL) of Hardness 1
Buffer Solution to the flask.
Swirl to mix.
digits x 0.1 = mg/L Free Chelant as CaCO3

Example: 100 mL of sample was titrated with the
0.0800 M cartridge and 120 digits were used to reach
the endpoint. The concentration is 120 x 0.1 = 12 mg/L
as CaCO3
Chelant, Free
Interferences

Interference level
Orthophosphate
Causes a slow end point.
Polyphosphate
Must be absent for accurate results.
pH
If chelant residual in boiler water is being analyzed, adjust the pH before adding the Buffer
Solution, Hardness 1 as follows:
1. Measure 100 mL of sample into a flask.
2. Add 2 drops of Phenolphthalein Indicator Solution.
3. Add 5.25 N Sulfuric Acid Standard Solution by drops until the solution changes from pink
to colorless. Write down the number of drops that were added. Discard this sample.
4. To a fresh 100-mL portion of sample, add the same number of drops of 5.25 N Sulfuric
Acid Standard Solution before adding the buffer in step 10 of the test.
Collect samples in clean plastic or glass bottles.
Filter the sample if turbid with filter paper and a funnel.
Accuracy check
EDTA Standard Solution, 0.035 N
6. Each 0.4 mL of standard that was added will use approximately 70 digits of the titration
cartridge to reach the endpoint. If more or less titrant was used, there can be an interference
(see Interferences) or the concentration of the titrant has changed.
Summary of method
Chelant residual is determined by titration with a standard solution of magnesium chloride at
pH 10. The end point is determined by a color change from blue to red-violet.
Chelant, Free
Page 203
Chelant, Free

Required reagents
Description
Quantity/Test
Unit
Catalog number
2 mL
100 mL
42432
1 pillow
100/pkg
85199
varies
each
2062501
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50543
each
50842
Unit
Catalog number
100 mL
2349932
Unit
Catalog number
Delivery tubes, with 180 hook
5/pkg
1720500
5/pkg
4157800
Filter paper, folded, 12.5 cm
100/pkg
189457
each
108367

ManVer 2 Hardness Indicator Powder Pillows
Magnesium Chloride Titration Cartridge, 0.0800 M
Required apparatus
Description
Description
EDTA Standard Solution, 0.035 N

Description
Funnel, analytical, poly, 65 mm
113 g
28014
ManVer Hardness Indicator Solution
ManVer 2 Hardness Indicator Powder
100 mL
42532
each
2095352
each
1970001
each
1940000
each
1940010

Water, deionized
500 mL
27249
50/pkg
2185696
1000/pkg
2185628

Phenolphthalein indicator solution, 5 g/L
15 mL SCDB
16236
Sulfuric acid standard solutions, 5.25 N
100 mL MDB
244932

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chelant, Total, DT, 8350
Chelant, Total
Bismuth Nitrate Method
0 to 40.0 mg/L as Na4 EDTA
DOC316.53.01169
Method 8350
Digital Titrator
Scope and Application: For industrial waters.
Test preparation
Filter turbid samples with filter paper and a funnel.
For best results, measure a reagent blank. Use 50 mL of deionized water in place of the sample. Subtract the number of
digits that were used for the reagent blank from the number of digits that were used for titrating the sample.

Description
Methylthymol Blue Indicator Powder Pillow
Ascorbic Acid Powder Pillow
Bismuth Nitrate Titration Cartridge, 0.0200 M
Quantity
1 pillow
1 pillow
1 cartridge
1 bottle
Digital titrator
Graduated cylinder, 100-mL
Chelant, Total
Page 205
Chelant, Total
Total chelant

15. Hold the Digital

wipe the tip.
16. Use a graduated

cylinder to measure 50 mL
of sample into a 125-mL
Erlenmeyer flask.

one Ascorbic Acid
Swirl to mix.

one Methylthymol Blue
Swirl to mix.
19. If the solution in the

flask is yellow, add one
drop of 5.25 N Sulfuric
Acid Standard Solution.

blue-green. Titrate slowly
as the end point is
approached.
21. Calculate:
If the solution is blue, add

5.25 N Sulfuric Acid
Standard solution by drops
until the color changes to
yellow. Add one additional
drop.
digits x 0.188 = mg/L total

chelant as Na4 EDTA
Example: 50 mL of sample
was titrated and 120 digits
were used to reach the
endpoint. The
concentration is
120 x 0.188 = 22.3 mg/L
total chelant as Na4 EDTA

counter.
Interferences
Interference from ferric iron (Fe3+) is minimized by the addition of ascorbic acid. Approach the end
point slowly in samples that contain ferric iron because the reduced iron decreases the sharpness
of the color change.
Chelant, Total
Page 206
Chelant, Total
Filter the sample if turbid with filter paper and a funnel.
Summary of method
Total chelant is determined by titrating an acidic sample with bismuth nitrate in the presence of
methylthymol blue indicator. The end point is indicated by a color change from yellow to
blue-green.

Required reagents
Description
Quantity/Test
Unit
Catalog number
1 pillow
100/pkg
1457799
Bismuth Nitrate Titration Cartridge, 0.0200 M
varies
each
2434501
Methylthymol Blue Indicator Powder Pillows
1 pillow
100/pkg
2284799
varies
100 mL MDB
244932
Ascorbic Acid Powder Pillows
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50543
Cylinder, graduated, poly, 100-mL
each
108142
5/pkg
1720500
5/pkg
4157800
Unit
Catalog number

Description
Filter paper, folded, 12.5 cm
100/pkg
189457
each
108367
each
2095352
each
1940000
each
1940010
Water, deionized
500 mL
27249
Sampling bottle
250 mL
2087076
Chelant, Total
Page 207

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloramine (Mono) and Free Ammonia, 10200
Chloramine (Mono);
Nitrogen, Free Ammonia
DOC316.53.01016
Indophenol Method1
Method 10200
(0.010.50 mg/L NH3N; 0.044.50 mg/L Cl2)
Powder Pillows
Scope and Application: For determining Free Ammonia and Monochloramine simultaneously in finished
chloraminated water.
1
U.S. Patent 6,315,950
Test preparation


Instrument
Sample cell
Cell orientation
Adapter
DR 6000
4864302
Orientation key toward user
A23618
DR 5000
4864302
A23618
DR 3900
4864302
Orientation key inserted in adapter slot
LZV846 (A)
DR 3800, DR 2800, DR 2700
5940506
1-cm (flat) path aligned with the arrow on the adapter
LZV585 (B)

For more accurate chloramine results, determine a reagent blank value for each new lot of reagent, using deionized water in
place of the sample. Subtract the reagent blank value from the final results or perform a reagent blank adjust.
Dispose of reacted solutions according to local, state and federal regulation. Use the guidance given on the Material Safety
Data Sheets to dispose of unreacted reagents. Consult local regulatory agencies for further disposal information.

Description
Free Ammonia Reagent Solution
Quantity
1 drop
Monochlor F Reagent Pillows
Chloramine (Mono); Nitrogen, Free Ammonia

Page 209
Indophenol method, powder pillows
Stored Programs
66 Monochloramine LR
Zero
M
Start
M
FA
1. Select the
Monochloramine test.
5. Remove the cell and

Monochlor-F pillow to the
sample for
monochloramine
measurement.
2. Fill two 1-cm cells to

the 10-mL line with
sample.
Label one cell Free
Ammonia and one cell
Monochloramine.
6. Cap and shake the

cell about 20 seconds to
dissolve.
A green color will develop
if monochloramine is
present.

Page 210
3. Wipe the
monochloramine cell and
See Instrument-specific
information for cell
orientation

0.00 mg/L Cl2
FA
7. Add one drop of Free

Ammonia Reagent
Solution to the cell for Free
Ammonia measurement.
8. Cap the reagent bottle

to maintain reagent
performance and stability.

Indophenol method, powder pillows (continued)
Read
M
FA
9. Cap and invert the

Free Ammonia cell to mix.

timer.
If the sample becomes

cloudy by the end of the
reaction period, pretreat
the sample and retest. See
the Interferences section.

will begin.
11. When the timer

expires, wipe the
Monochloramine cell and
Color development time

depends on sample
temperature. For accurate
results allow the full
reaction period to occur.
See the Color
development table

mg/L Monochloramine
(as Cl2).
Leave the cell in the
instrument.
Exit
Stored Programs
Zero
388 N, Ammonia Free
FA
Start
13. Select the Free

Ammonia test.
If Display Lock is on, the
display will show Store
Data?. Press YES or NO
and continue.
14. With the

monochloramine sample
still in the cell holder,

one Monochlor F pillow to
the cell for the Free
Ammonia measurement.
The reaction period in step

10 must be complete
before adding the
Monochlor F to the cell for
Free Ammonia
measurement.
0.00 mg/L NH3N f

Remove the
monochloramine cell.
FA

dissolve the reagent.
A green color will form if
monochloramine or
ammonia is present.

Page 211

Indophenol method, powder pillows (continued)
Read
FA

timer.
will begin.
18. When the timer

expires, insert the vial into
the cell.

mg/L NH3N f.
Samples colder than 18 C

will require additional time.
See the Color
development table.
Interferences
This method is intended for finished, chloraminated drinking water samples that have a
measurable combined (total) chlorine disinfectant residual. Samples where the disinfectant
residual has disappeared and samples which exhibit a chlorine demand may produce low
ammonia test results. Blanks and ammonia standards analyzed without a disinfectant residual
must be prepared using high quality, reagent grade water.
The substances listed in the Non-interfering substances table do not interfere in free ammonia
determination at or below the stated concentration:
Table 68 Non-interfering substances

Substance
Maximum level tested
Aluminum
0.2 mg/L Al
Chloride
1200 mg/L Cl
Copper
1 mg/L Cu
Iron
0.3 mg/L Fe
Manganese
0.05 mg/L Mn
Nitrate
10 mg/L NO3N
Nitrite
1 mg/L NO2N
Phosphate
2 mg/L oPO4
Silica
100 mg/L SiO2
Sulfate
1600 ppm as SO42
Zinc
5 ppm Zn

Page 212

Samples containing high levels of both Total Hardness and Alkalinity may become cloudy after the
addition of the Free Ammonia Reagent Solution. If this occurs by the end of the first reaction
period, the sample for Free Ammonia measurement must be pretreated as follows:
Note: The sample for Monochloramine measurement does not need pretreatment.
1. Measure 10 mL of sample into the cell for Free Ammonia measurement.

2. Add the contents of one Hardness Treatment Reagent Powder Pillow to the sample.
3. Cap the cell and invert until the reagent is dissolved.
4. Remove the cap.
5. Continue with the analysis at step 2 using the pretreated sample as the Free Ammonia cell.

Test results are strongly influenced by sample temperature. Both reaction periods in the
procedure are the same and depend on the temperature of the sample. The reaction periods
indicated in the procedure are for a sample temperature of 1820 C (6468 F). Adjust both
reaction periods according to the Color development table. Samples can be read up to 15 minutes
after the listed development times.
Table 69 Color development

Sample temperature (C)
Sample temperature (F)
Development time (minutes)
41
10
45
47
10
50
12
54
14
57
16
61
18
64
20
68
23
73
2.5
25
77
greater than 25
greater than 77

Collect samples in clean glass bottles. Most reliable results are obtained when samples are
analyzed as soon as possible after collection.
Accuracy check (Monochloramine, Program 66)

Buffer Powder Pillow, pH 8.3
Nitrogen, Ammonia Standard Solution, 100-mg/L as NH3N
Chlorine Solution Ampules, 5070 mg/L
100-mL Class A volumetric flask

Page 213
Pipet, Volumetric 2 mL and pipet bulb
Organic-free water

Important Note: Because of the strong buffer used in the preparation of this standard, it cannot be
used for accuracy verification of the Free Ammonia test.
To check test accuracy, prepare the following 4.5-mg/L (as Cl2) monochloramine standard
immediately before use.
1. Add the contents of one Buffer Powder Pillow, pH 8.3 to about 50-mL of organic-free water in a
clean 100-mL Class A volumetric flask. Swirl to dissolve the powder.
2. Using a Class A volumetric pipet, transfer 2.00 mL of Nitrogen, Ammonia Standard Solution,
100-mg/L as NH3N into the flask.
3. Dilute to volume with organic-free water, cap and mix thoroughly. This is a 2.00-mg/L buffered
ammonia standard.
4. Pipet 50.00 mL of the buffered ammonia standard into a clean 100-mL beaker.
Add a stir bar.
5. Obtain a recent lot of Chlorine Solution Ampules, 5070 mg/L, and note the actual free
chlorine concentration for this lot.
6. Calculate the amount of Chlorine Solution to be added to the ammonia standard using the
following equation:
455
mL chlorine solution required = ---------------------------------------------------------------------free chlorine concentration
7. Open an ampule and use a glass Mohr pipet to add the calculated amount of Chlorine Solution
slowly to the ammonia standard, while mixing at medium speed on a stir-plate.
8. Allow the monochloramine solution to mix for 1 minute after all Chlorine Solution is added.
9. Quantitatively transfer the monochloramine solution to a clean 100-mL Class A volumetric
flask. Dilute to the mark with organic-free water, cap, and mix thoroughly. This is a nominal
4.5-mg/L (as Cl2) monochloramine standard.
10. Use this standard within 1 hour of preparation. Analyze according to the Low Range
Monochloramine procedure described above.
11. To adjust the calibration curve using the reading obtained with the 4.5-mg/L standard solution,
select Options>More>Standard Adjust from the instrument menu.
Accuracy check (Free Ammonia, Program 388)

Ammonium Nitrogen Standard, 10 mg/L as NH3-N
100-mL plastic volumetric flask with stopper
50-mL mixing cylinders, three
Deionized water

Page 214

Dilution water is required when testing a diluted sample and preparing standard solutions. Dilution
water must be free of ammonia, chlorine and chlorine demand. A convenient source is a
recirculating, deionizer system with carbon filtration which produces 18 megaohm-cm water.
Standard Additions Method
chemical form.
4. Prepare three spiked samples. Measure 50 mL of sample into three 50-mL mixing cylinders.
5. Use the TenSette Pipet to add 0.3, 0.6, and 1.0 mL of Ammonium Nitrogen Standard, 10 mg/L
as NH3-N to the three samples. Mix well.
6. Analyze each spiked sample starting with the 0.3 mL sample spike. Follow all steps in Method
10200. Accept each standard additions reading by pressing READ. Each addition should
reflect approximately 100% recovery.
additions data points, accounting for matrix interferences. Press IDEAL LINE to view the
1. Prepare a 0.20 mg/L ammonia nitrogen standard by diluting 2.00 mL of the Ammonia Nitrogen
Standard Solution, 10 mg/L, to 100 mL with dilution water. Or, using the TenSette Pipet,
prepare a 0.20 mg/L ammonia nitrogen standard by diluting 0.4 mL of a Ammonia Nitrogen
Voluette Standard Solution, 50 mg/L as NH3N, to 100 mL with dilution water. Analyze the
Standard Solution, following all steps in Method 10200.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
66
2.60 mg/L Cl2
2.582.62 mg/L Cl2
0.04 mg/L Cl2
388
0.20 mg/L NH3N
0.190.21 mg/L NH3N
0.01 mg/L NH3N

Page 215
Summary of method
Monochloramine (NH2Cl) and free ammonia (NH3 and NH4+) can exist in the same water sample.
Added hypochlorite combines with free ammonia to form more monochloramine. In the presence
of a cyanoferrate catalyst, monochloramine in the sample reacts with a substituted phenol to form
an intermediate monoimine compound. The intermediate couples with excess substituted phenol
to form a green-colored indophenol, which is proportional to the amount of monochloramine
present in the sample. Free ammonia is determined by comparing the color intensities, with and
without added hypochlorite. Test results are measured at 655 nm.

Page 216

Required reagents
Description
Free Ammonia Reagent Set, includes:
Quantity/Test
Unit
Catalog number
50/pkg
2879700
1 drop
4 mL SCDB
2877336
100/pkg
2802299
Unit
Catalog number
(1) 2802299, (1) 2877336

Recommended standards and reagents

Description
Buffer, pH 8.3 Powder Pillows
25/pkg
89868
16/pkg
1426810
20/pkg
1426820
20/pkg
2630020
Hardness Treatment Reagent Pillows (1 per test)
50/pkg
2882346
Nitrogen Ammonia Standard Solution, 10 mg/L as NH3N
500 mL
15349
Nitrogen Ammonia Standard Ampule, 50 mg/L as NH3N, 10 mL
16/pkg
1479110
500 mL
2406549
Chlorine Standard Solution, 2-mL
PourRite
Ampule, 2530 mg/L
PourRite Ampule Breaker, for 2-mL ampules
each
248460
Voluette Ampule Breaker, for 10-mL ampules
each
2196800
500-mL
2641549
Description
Unit
Catalog number
Beaker, 100 mL, Polypropylene
each
108042
Beaker, 100 mL, Glass
each
50042H
Water, Organic-free
Cylinder, 50 mL, mixing
each
2088641
Flask, volumetric, Class A, 100 mL
each
1457442
Free Ammonia Reagent Set
250/pkg
2879701
Monochloramine/Free Ammonia Spec Check Kit
each
2507500
each
1465100
each
1970001
50/pkg
2185696
1000/pkg
2185628
Pipet, Mohr, Glass, 10 mL
each
2093438
each
1451536
each
1451541
Scissors
each
2883100
Stir Bar, octagonal
each
2095352
2881200
Stirrer, magnetic
each
each
187701
Wipers, Disposable, 30 x 30 cm, 280/box
box
2097000

Page 217

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloramine (Mono), HR, TNT, 10172
Chloramine (Mono)
DOC316.53.01014
Indophenol Method1
Method 10172
HR (0.1 to 10.0 mg/L Cl2)
Test N Tube
Scope and Application: Chlorinated wastewater.

1
Patent Pending
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield into compartment #2 before performing this test. See the
blank adjust.
Dispose of reacted solutions according to local, state and federal regulations. Use the guidance given on the Material Safety

Description
Quantity
High-range Monochloramine Diluent Vials
Monochlor F Reagent Pillow
Funnel, micro
Light Shield (see Instrument-specific information)
Pipet, Mohr glass, 2.00 mL and pipet bulb
Test Tube Rack
Chloramine (Mono)
Page 219
Chloramine (Mono)
Indophenol method for monochloramine
Stored Programs
67 Monochlora. HR TNT
Zero
Start
20. Select the test.

Insert an adapter or light
shield if required
(Instrument-specific
information).
for orientation.
21. Remove the cap from

one HR Monochloramine
Diluent vial. Use a glass
pipet to add 2.0 mL
sample to the vial. Re-cap
and invert several times to
mix.
22. Wipe the vial and

insert it into the 16 mm cell
holder.

0.0 mg/L Cl2
Read
24. Remove the vial from

the holder. Using a microfunnel, add the contents of
one Monochlor F pillow to
the sample. Cap and
shake the cell about
20 seconds to dissolve.

timer.
will begin.

re-insert the vial into the
cell holder.

mg/L Cl2.
Interferences
The substances in the Non-interfering substances table have been tested for interference and do
not interfere at or below the indicated levels. The Interfering substances table describes suggested
treatments for interfering substances.

Interfering substance and effects
Interference level
Ozone
Above 1 mg/L
Usually does not coexist with monochloramine.
Sulfide
Turns a rust color if present.
Thiocyanate
Above 0.5 mg/L
This method will tolerate up to 2 mg/L.
Chloramine (Mono)
Page 220
Recommended treatment
Chloramine (Mono)

Substance
Maximum level tested
Alanine
1 mg/L N
Aluminum
10 mg/L
Bromide
100 mg/L Br
Bromine
15 mg/L Br2
Calcium
1000 mg/L as CaCO3
Chloride
18,000 mg/L Cl
Chlorine Dioxide
5 mg/L ClO2
Chromium (III)
5 mg/L Cr
Copper
10 mg/L Cu
Cyanide
10 mg/L CN
Dichloramine
10 mg/L as Cl2
Fluoride
5 mg/L F
Free Chlorine
10 mg/L Cl2
Glycine
1 mg/L N
Iron (II)
10 mg/L Fe2+
Iron (III)
10 mg/L Fe3+
Magnesium
1000 mg/L as CaCO3
Manganese (VII)
10 mg/L
Lead
10 mg/L Pb
Nitrate
100 mg/L N
Nitrite
50 mg/L N
Phosphate
100 mg/L PO4
Silica
100 mg/L SiO2
Sulfate
2600 mg/L SO42
Sulfite
50 mg/L SO32
Tyrosine
1 mg/L N
Urea
10 mg/L N
Zinc
5 mg/L Zn

Analyze samples for monochloramine immediately after collection. Rinse the sample container
several times with sample, letting the container overflow each time. If sampling from a tap, let the
water flow for at least 5 minutes, then cap the container so that there is no head space (air) above
the sample.
Chloramine (Mono)
Page 221
Chloramine (Mono)
Accuracy check
Chlorine Solution Ampules, 5070 mg/L Cl2
Pipet, Volumetric, 2 mL and pipet bulb
Organic-free water
ammonia standard.
4. Pipet 50.00 mL of the buffered ammonia standard into a clean 100-mL beaker.
Add a stir bar.
following equation:
455
slowly to the ammonia standard, while mixing at medium speed on a stir plate.
10. Use this standard within 1 hour of preparation. Follow the Indophenol method for
monochloramine test.
Chloramine (Mono)
Page 222
Chloramine (Mono)
Method performance
Program
Standard
Precision
Distribution
Sensitivity
67
5.9 mg/L Cl2
5.66.2 mg/L Cl2
0.1 mg/L Cl21
Use the LR Chloramine (Mono) test for concentrations below 4.5 mg/L Cl2.
Summary of Method
The sample is first diluted in a Test N Tube. In the presence of a cyanoferrate catalyst,
monochloramine (NH2Cl) in the sample reacts with a substituted phenol to form an intermediate
monoimine compound. The intermediate couples with excess substituted phenol to form a greencolored indophenol, which is proportional to the amount of monochloramine present in the sample.
Test results are measured at 655 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2805145
(50) HR Monochloramine Diluent Vials1
50/pkg
Funnel, micro
each
2584335
50/pkg
2802246
Quantity
Unit
Catalog number
Pipet, Mohr, glass 2.00 mL
each
2093636
Piper Filler, safety bulb
each
1465100
12
each
1864100
Unit
Catalog number
HR Monochloramine Test N Tubes, 50 tests, includes:
Not sold separately
Required apparatus
Description
Test Tube Rack
Recommended standards and apparatus

Description
25/pkg
89868
Chlorine Solution Voluette Ampule, 5075 mg/L
16/pkg
1426810
Chlorine Solution 2 mL PourRite Ampule, 5075 mg/L
20/pkg
1426820
Chlorine Solution 2 mL PourRite Ampule, 2530 mg/L
20/pkg
2630020
500 mL
2406549
each
2484600
each
2196800
500 mL
2641549
Water, organic-free
Chloramine (Mono)
Page 223
Chloramine (Mono)

Description
Unit
Catalog number
Beaker, 100 mL
each
50042H
Clippers
each
96800
each
1457442
Pipet, Mohr, glass, 5-mL
each
2093437
each
1970001
Pipet Tips for TenSette Pipet
50/pkg
2185696
1000/pkg
2185628
each
1451536
each
1451541
Stir bar, octagonal
each
2095352
Stirrer, magnetic
each
2881200
Shears
each
2369400

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloramine (Mono), LR, 10171
Chloramine (Mono)
DOC316.53.01015
Indophenol Method1
Method 10171
LR (0.04 to 4.50 mg/L Cl2)
Powder Pillows
Scope and Application: Chloraminated drinking water and chlorinated wastewater

1
U.S. Patent 6,315,950
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
4864302
Adapter
A23618
DR 5000
4864302
DR 3900
4864302
Orientation key away from user
LZV846 (A)
A23618
DR 3800, DR 2800, DR 2700
5940506
LZV585 (B)

adjust.
To determine chloramine (mono) and free ammonia on the same sample, use Method 10200 Nitrogen, Free Ammonia and
Chloramine (Mono)
Dispose of reacted solutions according to local, state and federal regulation. Use the guidance given on the Material Safety

Description
Quantity
Monochlor F Reagent Pillow
Sample Cell (see Instrument-specific information)
Chloramine (Mono)
Page 225
Chloramine (Mono)
Indophenol method, powder pillows
Stored Programs
Zero
Start
1. Select the test.

2. Fill the cell to the

10-mL line with sample.
3. Wipe the cell and


0.00 mg/L Cl2

for orientation.

Monochlor-F pillow to the
sample.
Chloramine (Mono)
Page 226

dissolve.

timer.
will begin. Samples colder
than 18 C will require
additional time. See the
table.

insert the vial into the cell.
Cl2.
Chloramine (Mono)
Interferences
The substances listed in the Non-interfering substances table have been tested for interference
and do not interfere at or below the indicated levels. The Interfering substances table suggests
treatments for interferences.

Interference level
Alanine
1 mg/L N
Aluminum
10 mg/L Al
Bromide
100 mg/L Br
Bromine
15 mg/L Br2
Calcium
1000 mg/L as CaCO3
Chloride
18,000 mg/L Cl
Chlorine Dioxide
5 mg/L ClO2
Chromium (III)
5 mg/L Cr3+
Copper
10 mg/L Cu
Cyanide
10 mg/L CN
Dichloramine
10 mg/L as Cl2
Fluoride
5 mg/L F
Free Chlorine
10 mg/L Cl2
Glycine
1 mg/L N
Iron (II)
10 mg/L Fe2+
Iron (III)
10 mg/L Fe3+
Lead
10 mg/L Pb
Nitrate
100 mg/L N
Nitrite
50 mg/L N
Phosphate
100 mg/L PO4
Silica
100 mg/L SiO2
Sulfate
2600 mg/L SO42+
Sulfite
50 mg/L SO32
Tyrosine
1 mg/L N
Urea
10 mg/L N
Zinc
5 mg/L Zn

Interfering substance and effects
Magnesium
Interference level
Recommended treatment
Above 400 mg/L CaCO3
Add 5 drops Rochelle Salt Solution prior to testing.

OR: use the high range (HR) test.
Use the HR test; it will tolerate up to 10 mg/L.
Manganese (+7)
Above 3 mg/L
Ozone
Above 1 mg/L
Sulfide
Turns a rust color if present.
Thiocyanate
Above 0.5 mg/L
This method will tolerate up to 2 mg/L.
Chloramine (Mono)
Page 227
Chloramine (Mono)

Test results are strongly influenced by sample temperature. The reaction period indicated in the
procedure is for a sample temperature of 1820 C (6468 F). Adjust the reaction period
according to Table 76. Samples can be read up to 15 minutes after the listed development time.
Table 76 Color development time

Sample temperature
Development time (minutes)
C
40
10
42
48
10
50
12
54
14
58
16
61
18
68
20
73
23
75
2.5
25
77
greater than 25
greater than 77

Analyze samples for monochloramine immediately after collection. Rinse the sample container
several times with sample, letting the container overflow each time. If sampling from a tap, let the
water flow for at least 5 minutes, then cap the container so that there is no head space (air) above
the sample.
Accuracy check
Chlorine Solution Ampules, 5070 mg/L
Pipet, Volumetric, 2 mL and pipet bulb
Organic-free water
Chloramine (Mono)
Page 228
Chloramine (Mono)
ammonia standard.
4. Pipet 50.00 mL of the buffered ammonia standard into a clean 100-mL beaker. Add a stir bar.
following equation:
455
slowly to the ammonia standard, while mixing at medium speed on a stir-plate.
10. Use this standard within 1 hour of preparation. Analyze according to the Low Range
Monochloramine procedure described above.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
66
2.60 mg/L Cl2
2.582.62 mg/L Cl2
0.04 mg/L Cl2
Summary of method
In the presence of a cyanoferrate catalyst, monochloramine (NH2Cl) in the sample reacts with a
substituted phenol to form an intermediate monoimine compound. The intermediate couples with
excess substituted phenol to form a green-colored indophenol, which is proportional to the amount
of monochloramine present in the sample. Test results are measured at 655 nm.
Chloramine (Mono)
Page 229
Chloramine (Mono)

Required reagents
Description
Quantity/Test
Unit
Cat. No.
50/pkg
2802246
Unit
Cat. No.
Description
Buffer Powder Pillows pH 8.3
25/pkg
89868
Chlorine Standard Solution,10-mL Voluette Ampule 5075 mg/L
16/pkg
1426810
Chlorine Standard Solution, 2-mL PourRite Ampule 5075 mg/L
20/pkg
1426820
20/pkg
2630020
PourRite
Ampule 2530 mg/L
500 mL
2406549
1L
2354153
500-mL
2641549
Unit
Cat. No.
Organic-free Water

Description
Beaker, glass, 100 mL
each
50042H
each
1457442
100/pkg
2802299
Monochlor F Reagent Powder Pillows

Monochloramine/Free Ammonia Spec Check Kit
each
2507500
Pipet, Mohr, glass, 5-mL
each
2093437
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451536

Stir Bar, octagonal
each
1451541
100/pkg
2601300
each
2484600
29 mL DB
172533
each
2095352
2881200
Stirrer, magnetic
each
Shears
each
2369400
Thermometer, non-mercury, 10 to +225 C
each
2635700
each
2196800

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloride, 8113
Chloride
DOC316.53.01017
Mercuric Thiocyanate Method

(0.1 to 25.0 mg/L
Method 8113
Cl)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Filter turbid samples with moderately rapid filter paper and a funnel before analysis.
Both the sample and the blank will contain mercury (D009) at a concentration regulated as a hazardous waste by the Federal
RCRA. Do not pour these solutions down the drain.
Refer to the MSDS sheet for safe handling and disposal of hazardous waste. Gloves are recommended.

Description
Quantity
Ferric Ion Solution
1 mL
Mercuric Thiocyanate Solution
2 mL
Deionized Water
10 mL
Pipet tips for 0.1 to 1.0 mL TenSette pipet
Chloride
Page 231
Chloride
Mercuric Thiocyanate method for Chloride
Stored Programs
70 Chloride
Start
1. Select the test.

2. Prepared Sample:
Fill a sample cell with
10 mL of sample.
Fill another sample cell
with 10 mL of deionized
water.
4. Pipet 0.8 mL of
Mercuric Thiocyanate
Solution into each sample
cell. Note: Use 1.0 mL
with DR 5000.
6. Pipet 0.4 mL of Ferric

Ion Solution into each
sample cell. Note: Use 0.5
mL with DR 5000.
7. Swirl to mix. An
orange color will develop if
chloride is present.

timer.

for orientation.
5. Swirl to mix.
Zero

wipe the blank and insert it
Chloride
Page 232

0.0 mg/L Cl
A two-minute reaction time

will begin.
Read

the cell holder.

mg/L Cl.
Chloride
Interferences
Table 78 Interfering substances and levels
Extreme pH
The pH should be about 2 after adding reagents.

If the sample is strongly acidic or alkaline, adjust a portion of sample before testing to a pH of
about 7. Use either 5.0 N Sodium Hydroxide Standard Solution1 or a 1:5 dilution of perchloric acid.
Use pH paper; most pH electrodes will contaminate the sample with chloride.

Collect samples in glass or plastic containers. Samples can be stored for at least 28 days at room
temperature.
Accuracy check
Chloride Standard Solution, 1000-mg/L
50 mL mixing cylinders (3)
TenSette Pipet and tips

4. Prepare three sample spikes. Fill three 50 mL mixing cylinders with 50 mL of sample. Use the
TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of 1000-mg/L Chloride Standard Solution,
respectively, to the cylinders and mix each thoroughly.
5. Analyze a 10 mL portion of each sample spike as described in the Mercuric Thiocyanate
method for Chloride test, starting with the 0.1 mL sample spike. Each addition should reflect
approximately 100% recovery.
Chloride Standard Solution, 1000-mg/L
Volumetric Flask, 500-mL
Pipet, Class A, 10 mL
1. Prepare a 20.0-mg/L chloride standard solution.

a. Pipet 10.00 mL of Chloride Standard Solution, 1000-mg/L, into a 500-mL volumetric flask.
b. Dilute to the mark with deionized water. Follow the Mercuric Thiocyanate method for
Chloride test.
Chloride
Page 233
Chloride
Method performance
Program
Standard
Precision
Distribution
Sensitivity
Portion of curve
70
20.0 mg/L Cl
17.922.1 mg/L Cl
0.1 mg/L Cl
1.0 mg/L
Cl
10.0 mg/L
0.6 mg/L Cl
20.0 mg/L
0.3 mg/L
Summary of method
Chloride in the sample reacts with mercuric thiocyanate to form mercuric chloride and liberate
thiocyanate ion. Thiocyanate ions react with the ferric ions to form an orange ferric thiocyanate
complex. The amount of this complex is proportional to the chloride concentration. Test results are
measured at 455 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Chloride Reagent Set, includes:
250 tests/pkg1
2319800
(1) Ferric Ion Solution
1 mL
100 mL
2212242
(1) Mercuric Thiocyanate Solution
2 mL
200 mL
2212129
10 mL
4L
27256
Water, deionized
1
This test requires a reagent blank. The number of tests shown refers to any combination of samples and blanks.
Required apparatus
Description
Pipet,
TenSette,
Quantity
0.1 to 1.0 mL

Unit
Catalog number
1970001
each
varies
50/pkg
2185696
2/pkg
2495402
Unit
Catalog number
500 mL
18349
Description
Chloride Standard Solution, 1000-mg/L Cl
Chloride
Page 234
Chloride

Description
Chloride Standard Solution, 10-mL Voluette Ampule, 12,500-mg/L Cl
Chloride Standard Solution, 100-mg/L Cl
Filter Paper, funnel, 125 mm
Funnel, poly, 75 mm
Catalog number
16/pkg
1425010
1L
2370853
each
189641
100/pkg
69257
each
108368
pair
2410104
Perchloric Acid, ACS
680 g
75765
pH Paper, 1.011.0 pH range, 15 foot roll
5/pkg
39133
1000/pkg
2185628
each
1451538
each
1465100
Gloves, chemical resistant, size 99.51

1
Unit
50 mL
245026
each
2196800
Other sizes available
Chloride
Page 235

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloride, DT, 8206
Chloride
DOC316.53.01170
Mercuric Nitrate Method
Method 8206
10 to 8000 mg/L as Cl
Digital Titrator
Test preparation
mg/L sodium chloride = mg/L chloride x 1.65
meq/L chloride = mg/L chloride / 35.45
1

Description
Quantity
Diphenylcarbazone Indicator Powder Pillow
1 pillow
Mercuric Nitrate titration cartridge (see Range-specific information)
1 cartridge
Digital titrator
Graduated cylinder
Chloride
See
Table 1
1. Select a sample

3. Hold the Digital

wipe the tip.
4. Use a graduated
measure the sample
volume from the Rangespecific information table
in a 250 mL Erlenmeyer
flask.
Chloride
Page 237
Chloride
Chloride (continued)
5. If the sample volume


one Diphenylcarbazone
Swirl to mix.
Results will still be
accurate if a small amount
of powder does not
dissolve.

light pink.
counter.

the Range-specific
calculate the
concentration:
mg/L Cl
Example: 100 mL of
= 25 mg/L Cl

Range (mg/L as Cl)
Sample volume (mL)
Titration cartridge (N Hg(NO3)2)
Multiplier
1040
100
0.2256
0.1
40160
25
0.2256
0.4
100400
100
2.256
1.0
200800
50
2.256
2.0
5002000
20
2.256
5.0
10004000
10
2.256
10.0
20008000
2.256
20.0

Collect samples in clean plastic or glass bottles. The sample can be stored for up to 7 days
before the analysis.
Chloride
Page 238
Chloride
Interferences

Interference level
Bromide
Interferes directly and is included in the test result.
Chromate
Concentrations above 10 mg/L interfere with this method.
Ferric iron
Concentrations above 10 mg/L interfere with this method.
Iodide
pH
Neutralize strongly alkaline or acidic samples to a pH of 2 to 7 with 5.25 N sulfuric acid or

5.0 N sodium hydroxide. If a pH meter is used in the pH adjustment, use a separate sample
to find the correct amount of acid or base to use. Then add the same amount of acid or base
to the sample to be tested. pH electrodes will contaminate the sample.
Sulfide
Complete the following steps to remove sulfide interference:

1. Add the contents of one Sulfide Inhibitor Reagent Powder Pillow to approximately
125 mL of sample.
2. Mix for one minute.
3. Filter through folded filter paper.
4. Use the filtered sample in the chloride test procedure.
Sulfite
Concentrations above 10 mg/L interfere with this method. Eliminate sulfite interference by
adding three drops of Hydrogen Peroxide, 30%, to the sample before the test is started.
Accuracy check
Use the standard additions method to find if the sample has an interference and to confirm
analytical technique.
Chloride Voluette Ampule Standard Solution, 12,500-mg/L Cl
Ampule breaker

8. Each 0.1 mL of standard that was added will use approximately 12.5 digits of the 2.256 N
titration cartridge or 125 digits of the 0.2256 N titration cartridge to reach the endpoint.
If more or less titrant was used, the problem can be due to user technique, an interference
(see Interferences) or a problem with reagents or apparatus.
Chloride
Page 239
Chloride
Summary of method
When using Mercuric Nitrate Standard Solution, the sample is titrated under acidic conditions in
the presence of diphenylcarbazone indicator. Upon addition of a slight excess of mercuric ion, a
pink-purple complex is formed with the indicator, signaling the end point.

Required reagents
Description
Quantity/Test
Unit
1 pillow
100/pkg
83699
(1) Mercuric Nitrate Titration Cartridge, 0.2256 N
varies
each
1439301
(1) Mercuric Nitrate Titration Cartridge, 2.256 N
varies
each
92101
Chloride Reagent Set (approximately 100 tests):

(2) Diphenylcarbazone Powder Pillows
Catalog number
2272600
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50546

each
50838
each
50840
each
50841
each
50842
Delivery tubes w/ 180 hook
each
1720500
each
4157800
Unit
Catalog number
Description
Chloride Standard Solution, Voluette Ampule, 12,500-mg/L Cl, 10-mL
Voluette breaker
Chloride
Page 240
16/pkg
1425010
2196800
Chloride
Description
Filter paper, 12.5 cm
Hydrogen Peroxide, 30%, ACS
Unit
Catalog number
100/pkg
69257
each
108367
473 mL
14411
100 mL MDB
245032
each
2095352
Sulfide Inhibitor Reagent Powder Pillow
100/pkg
241899
100 mL MDB
244932
each
1970001
each
1940000
each
1940010

Water, deionized
500 mL
27249
2601300
pH Test Strip , 014 pH
100/pkg
2601300
Pipet tips
100/pkg
2185628
Pipet tips
50/pkg
2185696
Chloride standard solution, 1000 mg/L
500 mL
18349
Sampling bottle
250 mL
2087076
each
96800
pH paper
Clipper for Medium PWD PLWS
Chloride
Page 241

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloride, DT, 8207
Chloride
DOC316.53.01171
Silver Nitrate Method
Method 8207
10 to 10,000 mg/L as Cl
Digital Titrator
Test preparation
mg/L sodium chloride = mg/L chloride x 1.65
meq/L chloride = mg/L chloride / 35.45

Description
Quantity
Chloride 2 Indicator Powder Pillow
1 pillow
Silver Nitrate titration cartridge (see Range-specific information)
1 cartridge
Digital titrator
Graduated cylinder
Chloride
See
Table 1
1. Select a sample

3. Hold the Digital

wipe the tip.
4. Use a graduated
measure the sample
into a 250 mL Erlenmeyer
flask.
Chloride
Page 243
Chloride
Chloride (continued)

deionized water.

one Chloride 2 Indicator
mix.
of powder does not
dissolve.

red-brown.
counter.

the Range-specific
calculate the
concentration:
mg/L Cl
Example: 100 mL of
= 25 mg/L Cl

Titration cartridge (N AgNO3)
Range (mg/L as Cl)
Sample volume (mL)
1040
100
0.2256
0.1
25100
40
0.2256
0.25
1.0
Multiplier
100400
50
1.128
2501000
20
1.128
2.5
10004000
1.128
10.0
250010,000
1.128
25.0

Collect samples in clean plastic or glass bottles. The sample can be stored for up to 7 days
before the analysis.
Interferences

Interference level
Bromide
Cyanide
Iron
Concentrations above 10 mg/L mask the end point.
Chloride
Page 244
Chloride
Interference level
Iodide
Orthophosphate
Concentrations above 25 mg/L will precipitate the silver.
pH
Neutralize strongly alkaline or acidic samples to a pH of 2 to 7 with 5.25 N sulfuric acid or

5.0 N sodium hydroxide. If a pH meter is used in the pH adjustment, use a separate sample
to find the correct amount of acid or base to use. Then add the same amount of acid or base
to the sample to be tested. pH electrodes will contaminate the sample.
Sulfide
Complete the following steps to remove sulfide interference:

1. Add the contents of one Sulfide Inhibitor Reagent Powder Pillow to approximately
125 mL of sample.
2. Mix for one minute.
3. Filter through folded filter paper.
4. Use the filtered sample in the chloride test procedure.
Sulfite
Concentrations above 10 mg/L interfere with this method. Eliminate sulfite interference by
adding three drops of Hydrogen Peroxide, 30%, to the sample before the test is started.
Accuracy check
Use the standard additions method to determine whether the sample has an interference and
confirm analytical technique.
Chloride Voluette Ampule Standard Solution, 12,500-mg/L Cl
Ampule breaker

8. Each 0.1 mL of standard that was added will use approximately 12.5 digits of the 2.256 N
titration cartridge or 25 digits of the 1.128 N titration cartridge to reach the endpoint.
Chloride
Page 245
Chloride
Summary of method
The sample is titrated with Silver Nitrate Standard Solution in the presence of potassium chromate
(from the Chloride 2 Indicator Powder). The silver nitrate reacts with the chloride present to
produce insoluble white silver chloride. After all the chloride has been precipitated, the silver ions
react with the excess chromate present to form a red-brown silver chromate precipitate, marking
the end point of the titration.

Required reagents
Description
Quantity/Test
Unit
1 pillow
50/pkg
105766
(1) Silver Nitrate Titration Cartridge, 0.2256 N
varies
each
1439601
(1) Silver Nitrate Titration Cartridge, 1.128 N
varies
each
1439701
Chloride Reagent Set (approximately 100 tests):

(2) Chloride 2 Indicator Powder Pillows
Catalog number
2288000
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50546

each
50838
each
50840
each
50841
each
50842
each
1720500
each
4157800
Description
Chloride Standard Solution, Voluette Ampule, 12,500-mg/L Cl, 10-mL
Voluette breaker
Chloride
Page 246
Unit
Catalog number
16/pkg
1425010
2196800
Chloride

Description
Filter paper, 12.5 cm
Unit
Catalog number
100/pkg
69257
each
108367
473 mL
14411
100 mL MDB
245032
each
2095352
100/pkg
241899
100 mL MDB
244932
each
1970001
each
1940000
each
1940010

Water, deionized
500 mL
27249
pH Test Strip, 014 pH
100/pkg
2601300
Pipet tips
100/pkg
2185628
Pipet tips
50/pkg
2185696
Chloride standard solution, 1000 mg/L
500 mL
18349
Sampling bottle
250 mL
2087076
Dropper, glass
5/pkg
1419705
Chloride
Page 247

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chloride, BT, 8225
Chloride
DOC316.53.01153
USEPA1 Silver Nitrate Buret Titration Method2
Method 8225
0 to 25,000 mg/L as Cl
Buret Titration

1
USEPA accepted for NPDES reporting when 0.0141 N silver nitrate standard solution is used.
Adapted from Standard Methods for the Examination of Water and Wastewater, (Standard Method 4500 CI- B).
Test preparation
Adjust highly acidic or alkaline samples to a pH between pH 7 and 9. Use pH paper to measure the pH. A pH meter will
contaminate the sample.
To calculate the result as mg/L sodium chloride (NaCl): mg/L chloride x 1.65 = mg/L sodium chloride
A small amount of silver nitrate is used to make the red-brown color in step 6. For most accurate results, follow the procedure
using 100 mL of deionized water in place of the sample. Titrate this solution and note the volume of titrant required. For all
samples, subtract this volume of titrant before calculating the mg/L chloride.

Description
Quantity
Chloride 2 Indicator Powder Pillow
Silver Nitrate Standard Solution (see Range-specific information)
1 bottle
Erlenmeyer flask, 250 mL
Graduated cylinder
Buret titration
See
Table 1

volume and silver nitrate
standard solution from
Range-specific
information.

Silver Nitrate Standard
Solution.
3. Use a graduated
measure the sample
volume from Rangespecific information.

Chloride
Page 249
Chloride
Buret titration (continued)

one Chloride 2 Indicator
Powder Pillow. Swirl
to mix.

while swirling the flask
until the color changes
from yellow to red-brown.
7. Calculate:
mL titrant used x multiplier = mg/L chloride as Cl
Example: 100 mL of sample was titrated with the
0.0141 N silver nitrate solution and 15 mL of titrant was
used to reach the endpoint.
The chloride concentration is: 15 x 5 = 75 mg/L as Cl

Cl)
Sample volume (mL)
Silver nitrate concentration
0125
100
0.0141 N
100250
50
0.0141 N
10
200500
25
0.0141 N
20
5001250
100
0.141 N
50
Range (mg/L as
Multiplier
10002500
50
0.141 N
100
250010,000
25
0.141 N
200
500025,000
10
0.141 N
500
Interferences
An interfering substance can mask the end point. A dilution can reduce the interference to a level
at which the substance does not interfere. If an interference is suspected, use a smaller amount of
fresh sample and repeat the test.

Interference level
Bromide
Cyanide, bromide and iodide interfere directly and are titrated as chloride.
Cyanide
Iodide
Iron
Iron concentrations over 20 mg/L will mask the end point.
Orthophosphate
Orthophosphate concentrations over 25 mg/L will cause a precipitate to form.
Sulfide
To remove interference from sulfide, add one Sulfide Inhibitor Reagent Powder Pillow to
approximately 125 mL of the sample, mix for one minute and filter through filter paper.
Sulfite
To remove interference from at least 10 mg/L sulfite, add 3 drops of 30% hydrogen peroxide
to 100 mL of sample before starting the test.
Chloride
Page 250
Chloride

Collect samples in clean plastic or glass bottles. Samples can be stored in sealed containers.
Accuracy check
Use the standard additions method to find if the sample has an interference. Use the standard
solution method to make sure that the user has followed the test correctly and that the reagents
are good.
Chloride Voluette Ampule Standard, 12,500-mg/L as Cl
Ampule breaker
Procedure for use with the 0.0141 N titrant:

8. Each 0.1 mL of standard that was added should use 2.5 mL of titrant to reach the endpoint. If
more or less titrant was used, there can be an interference (see Interferences) or the
Chloride
Page 251
Chloride
A silver nitrate standard solution will slowly decompose with exposure to light. Complete the
following test to make sure the concentration is accurate.
Sodium Chloride Standard Solution, 1000-mg/L as Cl
100-mL Class A volumetric flask (for use with 0.0141 N titrant only)

1. Add 10.0 mL of the sodium chloride standard solution, 1000-mg/L as Cl, to a 100-mL Class A
volumetric flask. Dilute to 100 mL with deionized water and mix fully. This solution has a
concentration of 100 mg/L chloride.
1. Add 100.0 mL of the diluted sodium chloride standard solution, 100-mg/L as Cl, to an
Erlenmeyer flask.
2. Add the Chloride 2 indicator and swirl to mix.
3. Titrate the standard to the end point with the 0.0141 N silver nitrate titrant and calculate the
result. If the result is more than 105 mg/L chloride, discard the silver nitrate titrant and replace
it with a fresh supply.
1. Add 100.0 mL of the sodium chloride standard solution, 1000-mg/L as Cl, to an Erlenmeyer
flask.
2. Add the Chloride 2 indicator and swirl to mix.
3. Titrate the standard to the end point with the 0.141 N silver nitrate titrant and calculate the
result. If the result is more than 1050 mg/L chloride, discard the silver nitrate titrant and
replace it with a fresh supply.
Summary of method
Silver nitrate is used as the titrant and potassium chromate as the indicator. Silver nitrate first
reacts selectively with the chloride in the sample to produce insoluble white silver chloride. After all
the chloride has been precipitated, the silver nitrate reacts with the chromate to form an orange or
red-brown silver chromate precipitate.
Chloride
Page 252
Chloride

Required reagents
Description
Quantity/Test
Unit
Catalog number
1 pillow
50/pkg
105766
Chloride 2 Indicator Powder Pillows

Titrantselect one or more based on range:
Silver Nitrate Standard Solution, 0.0141 N
varies
1L
31653
Silver Nitrate Standard Solution, 0.141 N
varies
500 mL
1255149
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
2636540
Buret Clamp, double
each
32800
each
50546
50838

each
each
50840
each
50841

Support Stand
each
50842
each
56300
Unit
Catalog number
16/pkg
1425010
Description
Chloride Standard Solution,
Voluette
Ampule, 12,500-mg/L as
Sodium Chloride Standard Solution, 1000-mg/L as Cl

Voluette Ampule breaker, 10 mL
Cl,
10-mL
500 mL
18349
each
2196800
Chloride
Page 253
Chloride

Description
Catalog number
69257
Filter Paper, 12.5 cm diameter
100/pkg
Hydrogen Peroxide, 30%
473 mL
14411
100/pkg
241899
each
1970001

Water, deionized
500 mL
27249
each
1970010
50/pkg
2199796
250/pkg
2199725
each
1970001
50/pkg
2185696
1000/pkg
2185628

Pipet Tips, for TenSette Pipet
Unit
19700011
100/pkg
2601300
12/pkg
2087076
Flask, Class A volumetric, 100 mL
each
1457442
Dropper, glass
5/pkg
1419705
Clippers for powder pillows
each
66800

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Dioxide, 10126
Chlorine Dioxide
DOC316.53.01021
DPD Method1
Method 10126
Powder Pillows and AccuVac Ampuls
(0.04 to 5.00 mg/L)
Scope and Application: For water and wastewater. USEPA accepted for reporting for drinking water analysis.2
1
Procedure is equivalent to Standard Methods, 18 ed., 4500 ClO2 D.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Analyze samples immediately because chlorine dioxide is unstable and volatile. See Sample collection, preservation and
storage.
deionized water instead of the sample. Subtract the reagent blank value from the final results or perform a reagent blank
adjust.
After adding the DPD Free Chlorine Powder Pillow to the sample, a pink color will develop if chlorine dioxide is present.
If the chlorine dioxide concentration in the sample exceeds the upper limit of the test, the color may fade or the sample may
turn yellow. Dilute the sample with high quality water that is chlorine demand-free, and repeat the test. Some loss of chlorine
dioxide may occur. Multiply the result by the appropriate dilution factor.
Chlorine Dioxide
Page 255
Chlorine Dioxide

Description
Quantity
Powder Pillow Test:

DPD Free Chlorine powder pillow, 10-mL
Glycine Reagent
4 drops
AccuVac Test:
DPD Free Chlorine Reagent AccuVac Ampuls
Glycine Reagent
16 drops
Beaker, 50-mL
DPD method, powder pillows
Stored Programs
76 Chlro Diox DPD
Start
1. Select the test.

for orientation.
Chlorine Dioxide
Page 256
Fill a sample cell with 10
mL of sample. Close the
sample cell.
3. Prepared Sample:
Close the sample cell.

Chlorine Dioxide
DPD method, powder pillows (continued)
Zero

0.00 mg/L ClO2
6. Add four drops of

Glycine Reagent to the
sample. Swirl to mix.

one DPD Free Chlorine
prepared sample cell.
Swirl the sample for
20 seconds to mix.
8. Wait 30 seconds for

any undissolved powder to
settle.
Immediately proceed to
step 9.
Read
9. Within one minute of

adding the DPD reagent,
wipe the sample cell and

mg/L ClO2.
DPD method, AccuVac Ampuls
Stored Programs
77 Chlor Diox DPD AV
Start
1. Select the test.

for orientation.
Fill a round sample cell
with 10-mL of sample.


0.00 mg/L ClO2
4. Prepared Sample:
Fill a 50-mL beaker with
40 mL of sample.
Add 16 drops of Glycine
Reagent to the sample in
the beaker. Swirl gently to
mix.
Chlorine Dioxide
Page 257
Chlorine Dioxide
DPD method, AccuVac Ampuls (continued)
Read
5. Fill a DPD Free

Chlorine Reagent
AccuVac Ampul with
sample. Keep the tip
fills completely.

Ampul several times to
mix. Wait 30 seconds for
any undissolved powder to
settle.

adding the sample, wipe
the Ampul and insert it into
the cell holder.

mg/L ClO2.
Interferences
Interference level
Greater than 150 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize
Acidity
to pH 6 7 with 1 N Sodium Hydroxide1. Determine amount to be added on a separate sample

aliquot, then add the same amount to the sample being tested. Correct for volume addition.
Greater than 250 mg/L CaCO3. Color may not develop fully or may fade instantly. Neutralize to
Alkalinity
pH 6 7 with 1 N Sulfuric Acid1. Determine amount to be added on a separate sample aliquot, then
add the same amount to the sample being tested. Correct for the volume addition.
Bromine, Br2
Interferes at all levels.
Chlorine, Cl2
May interfere at levels greater than 6 mg/L. Additional glycine may be able to compensate for
this interference.
May interfere.
Flocculating agents
High levels of most flocculating agents can be tolerated. This tolerance is decreased if chlorine
is present. See the information about metals in this table. In the presence of 0.6 mg/L Cl2,
Al(SO4)3 (< 500 mg/L) and FeCl2 (<200 mg/L) may be tolerated.
Hardness
No effect at less than 1000 mg/L as CaCO3.
Iodine, I2

Oxidized manganese interferes at all levels. Oxidized chromium interferes at levels greater than
2 mg/L. To remove the interferences:
5. Adjust sample pH to 6 7.
Manganese, oxidized
(Mn4+, Mn7+) or
Chromium, oxidized (Cr6+)
Chlorine Dioxide
Page 258
6.
7.
Add 3 drops Potassium Iodide1 (30 g/L) to a 25-mL sample.

8. Add 3 drops Sodium Arsenite1, 2 (5 g/L) and mix.

9. Analyze 10 mL of the treated sample as described in the procedure.
10. Subtract the result of this test from the original analysis to obtain the correct chlorine
dioxide concentration.
Chlorine Dioxide
Interference level
Metals
Various metals may interfere by combining with the glycine needed to remove the chlorine
interference. Metal interference is limited except when chlorine is present. In the presence of
0.6 mg/L Cl2, both copper (>10 mg/L) and nickel (>50 mg/L) interfere. Other metals may also
interfere, depending on their ability to prevent glycine from reacting with any Cl2 in the sample.
It may be necessary to add more glycine to overcome this interference.
Monochloramine
Causes a gradual drift to higher readings. When read within 1 minute after reagent addition,
3 mg/L monochloramine causes less than a 0.1 mg/L ClO2 increase in the reading.
Ozone
Interferes at levels greater than 1.5 mg/L.
Peroxides
May interfere.
Extreme sample pH
Adjust to pH 67.
Highly buffered samples
Adjust to pH 67.
Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). Refer to a
current MSDS for proper disposal instructions.

Analyze samples for chlorine dioxide immediately after collection. Chlorine dioxide is a strong
oxidizing agent and is unstable in natural waters. It reacts rapidly with various inorganic
compounds, but oxidizes organic compounds more slowly. Many factors, including reactant
concentrations, sunlight, pH, temperature, and salinity influence decomposition of chlorine dioxide
in water.
Avoid plastic containers since these may have a large chlorine dioxide demand. Pretreat glass
sample containers to remove any chlorine or chlorine dioxide demand by soaking in a dilute bleach
solution (1 mL commercial bleach to 1 liter of deionized water) for at least one hour. Rinse
thoroughly with deionized or distilled water. If sample containers are rinsed thoroughly with
deionized or distilled water after use, only occasional pretreatment is necessary.
A common error in testing for chlorine dioxide is not obtaining a representative sample. If sampling
from a tap, let the water flow for at least 5 minutes to ensure a representative sample. Let the
container overflow with the sample several times, then cap the sample containers so there is no
headspace (air) above the sample. If sampling with a sample cell, rinse the cell several times with
the sample, then carefully fill to the 10-mL mark. Perform the chlorine dioxide analysis
immediately.
Accuracy check
Preparing chlorine dioxide standards is difficult and dangerous. In addition, these standards are
both explosive and volatile! Only a trained chemist should prepare the standards using appropriate
safety equipment and precautions. The manufacturer does not recommend preparation of chlorine
dioxide standards. If independent standard preparation is required, please see the instructions in
Standard Methods for the Examination of Water and Wastewater, Part 4500-ClO2 Chlorine
Dioxide, under the headings "Stock chlorine dioxide solution" and "Standard chlorine dioxide
solution". Prepare a chlorine dioxide standard.
Chlorine Dioxide
Page 259
Chlorine Dioxide
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
76
DR 5000
3.00 mg/L ClO2
2.893.11 mg/L ClO2
0.04 mg/L ClO2
77
DR 5000
3.00 mg/L ClO2
2.913.09 mg/L ClO2
0.04 mg/L ClO2
Summary of method
Chlorine dioxide reacts with DPD (N, N-diethyl-p-phenylenediamine) to the extent of one-fifth of its
total available chlorine content, corresponding to reduction of chlorine dioxide to chlorite. The
resulting pink color intensity is proportional to the ClO2 in the sample. Chlorine interference is
eliminated by adding glycine, which converts free chlorine to chloroaminoacetic acid, but has no
effect on chlorine dioxide at the test pH. Test results are measured at 530 nm.

Required reagents
Description
Quantity/Test
Unit
100/pkg
2105569
4 drops
29 mL
2762133
Chlorine Dioxide DPD/Glycine Reagent Set (100 tests), includes:

(1) DPD Free Chlorine Reagent Powder Pillows, 10-mL
(1) Glycine Reagent
Catalog number
2770900
OR
2771000
Chlorine Dioxide DPD/Glycine AccuVac Ampul Reagent Set (25 tests), includes:
(1) DPD Free Chlorine Reagent AccuVac Ampuls
(1) Glycine Reagent
25/pkg
2502025
16 drops
29 mL
2762133
Required apparatus
Description
Quantity
Unit
Catalog number
AccuVac Snapper
each
2405200
Beaker, 50-mL
each
50041H
6/pkg
173106
each
2122800
6/pkg
2427606
2/pkg
2495402
Description
Voluette

Water, organic-free
Chlorine Dioxide
Page 260
Ampule, 5075 mg/L
Unit
Catalog number
16/pkg
1426810
each
2196800
500 mL
2641549
Chlorine Dioxide

Description
AccuVac Vials, for sample blanks
Unit
Catalog number
25/pkg
2677925
DPD Free Chlorine Reagent Powder Pillows, 10-mL
1000/pkg
2105528
2105503
300/pkg
100 mL
34332
100 mL
104732
100 mL
104532
each
2270800
Standard Methods Book, most current edition

Sulfuric Acid, 1 N, 100 mL
25/pkg
173125
each
127032
Chlorine Dioxide
Page 261

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Dioxide, HR, 8138
Chlorine Dioxide
DOC316.53.01019
Direct Reading Method
Method 8138
HR (5 to 1000 mg/L)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Chlorine dioxide is unstable and volatile. Analyze samples immediately.
Gloves and goggles are recommended.

Description
Water, deionized
Quantity
10 mL
2
Chlorine Dioxide
Page 263
Chlorine Dioxide
Direct Reading method
Stored Programs
75 Chlor Diox HR
Zero
Start
1. Select the test.

Fill a sample cell to the 10- insert it into the cell holder.
mL mark with deionized
water.

0 mg/L ClO2

for orientation.
Read
5. Prepared Sample:
to the 10-mL mark with
sample.

the cell holder.

mg/L ClO2.
Interferences
Because this is a Direct Reading test, no known interferences exist.
Sample collection, storage and preservation

in water.
Chlorine Dioxide
Page 264
Chlorine Dioxide
immediately.
Accuracy check
solution". Prepare a 500-mg/L chlorine dioxide standard.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
75
469 mg/L ClO2
459479 mg/L ClO2
5 mg/L ClO2
Summary of method
Chlorine dioxide, a yellow gas, can be measured directly in a water solution. Test results are
measured at 445 nm.

Required reagents and apparatus
Description
Water, deionized
Quantity/Test
Unit
10 mL
4L
Catalog number
27256
2/pkg
2495402
Unit
Catalog number
pair
2410104
Optional apparatus
Description
Gloves, chemical resistant, size
99.51
each
2550700
each
2270800
Chlorine Dioxide
Page 265

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Dioxide, LR, 8065
Chlorine Dioxide
DOC316.53.01022
Chlorophenol Red Method1
Method 8065
LR (0.01 to 1.00 mg/L)

1
Adapted from Harp, Klein and Schoonover, Jour. Amer. Water Works Assn., 73 387388 (1981).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

For most accurate results, analyze each portion of sample at the same temperature.
A TenSette Pipet may be used to dispense Chlorine Dioxide Reagent 1 and Chlorine Dioxide Reagent 3

Description
Quantity
Chlorine Dioxide Reagent 1
2 mL
2 mL
2 mL
Dechlorinating Reagent Pillows
Cylinder, graduated mixing, 50 mL
Pipet Filler, with safety bulb
Chlorine Dioxide
Page 267
Chlorine Dioxide
Powder Pillows
Stored Programs
72 Chlor Diox CPR LR
Start
1. Select the test.

2. Fill two 50-mL mixing

cylinders to the 50-mL
mark with sample.
3. Use a volumetric pipet

and pipet filler to add 1.0
mL of Chlorine Dioxide
Reagent 1 to each
cylinder. Stopper.
4. Invert several times to

mix.

to add exactly 1.00 mL of
Chlorine Dioxide Reagent
2 to each cylinder.
Stopper.

mix.

and pipet filler to add
1.0 mL of Chlorine Dioxide
Reagent 3 to each
cylinder. Stopper.

for orientation.
Dechlorinating Reagent
Powder Pillow to one
cylinder. (This is
the blank).
Stopper and invert several
times until dissolved.
The second cylinder,
which does not receive
dechlorinating reagent, is
the prepared sample.
Chlorine Dioxide
Page 268
Chlorine Dioxide
Powder Pillows

mix.
10. Pour 10 mL from each

cylinder into two
sample cells.


the cell holder.

0.00 mg/L ClO2
READ the results in

mg/L ClO2.
Interferences
Highly acidic or alkaline water
May require 2.0 mL each of Chlorine Dioxide Reagent 1 and

Chlorine Dioxide Reagent 3 instead of 1.0 mL
ClO
Greater than 5.5 mg/L
Greater than 6 mg/L
ClO3
Greater than 6 mg/L
CrO42
ClO2
Fe3+
Greater than 5 mg/L
Hardness
Ozone
Turbidity
Greater than 1000 NTU
Analyze samples for chlorine dioxide immediately after collection.
Chlorine dioxide is a strong oxidizing agent and is unstable in natural waters. It reacts rapidly
with various inorganic compounds, but oxidizes organic compounds more slowly. Many
factors, including reactant concentrations, sunlight, pH, temperature and salinity influence
decomposition of chlorine dioxide in water.
Avoid plastic containers since these may have a large chlorine dioxide demand.
Pretreat glass sample containers to remove any chlorine or chlorine dioxide demand by
soaking in a dilute bleach solution (1 mL commercial bleach to 1 liter of deionized water) for at
least one hour. Rinse thoroughly with deionized or distilled water. If sample containers are
rinsed thoroughly with deionized or distilled water after use, only occasional pretreatment is
necessary.
Chlorine Dioxide
Page 269
Chlorine Dioxide
A common error in testing for chlorine dioxide is not obtaining a representative sample. If
sampling from a tap, let the water flow for at least 5 minutes to ensure a representative
sample. Let the container overflow with the sample several times, then cap the sample
containers so there is no headspace (air) above the sample.
Accuracy check
solution". Prepare a 0.50-mg/L chlorine dioxide standard.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
72
0.53 mg/L ClO2
0.500.55 mg/L ClO2
0.01 mg/L ClO2
Summary of method
Chlorine Dioxide (ClO2) is determined by its combination with chlorophenol red at pH 5.2 to form a
colorless complex. The net effect is bleaching of the color in an amount proportional to the chlorine
dioxide concentration. The method is specific for ClO2 and is unreactive to other active chlorine or
moderate oxidizing compounds. Test results are measured at 575 nm.
Chlorine Dioxide
Page 270
Chlorine Dioxide

Required reagents
Description
Chlorine Dioxide Reagent Set (100 Tests), includes:
(2) Chlorine Dioxide Reagent 1
Quantity/Test
Unit
each
Catalog number
2242300
2 mL
100 mL
2070042
2 mL
100 mL
2070142
2 mL
100 mL
2070242
100/pkg
1436369
Catalog number
(1) Dechlorinating Reagent Powder Pillows
Required apparatus
Description
Quantity
Unit
Cylinder, graduated mixing, 50-mL
each
189641
each
1451535
each
1465100
2/pkg
2495402
Unit
Catalog number

Description
each
1970001
50/pkg
2185696
1000/pkg
2185628
Standard Methods book, current edition
each
2270800
100/pkg
2601300
Pipet,
TenSette,
0.1 to 1.0 mL
Chlorine Dioxide
Page 271

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Dioxide, Amaranth, Europe only
Chlorine Dioxide
DOC356.53.01018
Amaranth Method1
(20 to 500 g/L)
Scope and Application: For water, drinking water
1
This method is under license of Elf Atofina. Reagent sets for this method are only available in Europe.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Chlorine dioxide is unstable and volatile. Analyze samples immediately. See Sample collection, preservation and storage.
For most accurate results, analyze each portion of sample at the same temperature.
For best precision, measurement of the reagent with a volumetric pipet or high precision pipettor is recommended.
A TenSette Pipet may be used to dispense Chlorine Dioxide Reagent A.

Description
Quantity
Chlorine Dioxide Reagent Set
Volumetric Flask, 25-mL plastic
Syringe, 1-mL with needle
Chlorine Dioxide
Page 273
Chlorine Dioxide
Amaranth method
Stored Programs
78 Chlor Diox, Amaranth
Start
1. Select the test.

for orientation.
5. Prepared Sample:
Add 1.0 mL of Chlorine
Dioxide Reagent A into a
second 25-mL volumetric
flask.
Using the syringe and
needle provided, add
1.0 mL of Chlorine Dioxide
Reagent A into a 25-mL
volumetric flask.
3. Fill the volumetric flask

to the mark with deionized
water. Stopper. Invert at
least 7 times to mix.
4. Pour 10 mL from the

volumetric flask into a
10 mL sample cell.
6. Fill the second

volumetric flask to the
mark with the sample.
Stopper. Invert at least 7
times to mix.

timer. A 1-minute reaction
period will begin.
8. Prepared Sample:
Pour 10 mL from the
second volumetric flask
into a second sample cell.
Use a volumetric pipet and

pipet filler or a TenSette
Pipet to add this reagent.
Zero


0 g/L ClO2
Chlorine Dioxide
Page 274
Read
11. When the timer

expires, wipe the prepared
the cell holder.

g/L ClO2.
Chlorine Dioxide
Interferences
Interference level
ClO
ClO2
ClO3
CrO42
Fe3+
Hardness
Ozone
Turbidity
Greater than 1000 NTU

in water.
headspace (air) above the sample. Perform the analysis immediately.
Accuracy check
solution". Prepare a 0.25-mg/L (250-g/L) chlorine dioxide standard.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
78
250 g/L ClO2
192308 g/L ClO2
24 g/L ClO2
Chlorine Dioxide
Page 275
Chlorine Dioxide
Summary of method
Chlorine dioxide (ClO2) is determined by its combination with Amaranth. Color intensity decreases
as the level of chlorine dioxide increases. Test results are measured at 521 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
LYW240
Quantity
Unit
Catalog number
LZC140
Chlorine Dioxide Reagent Set (100 Tests)1

1
Available only in Europe.
Required apparatus
Description
each
(2) Flask, volumetric, 25-mL
each
(1) Syringe, 1-mL, with needle
each
Description
Unit
Catalog number
each
1970001
50/pkg
2815696
1000/pkg
2185628
Chlorine Dioxide Tool
Set1,
includes:
Available only in Europe.
each
1465100
each
1451535
Standard Methods Book (most current edition)

Water, organic-free

each
2270800
500 mL
2641549
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Dioxide, MR, 8345
Chlorine Dioxide
DOC316.53.01020
Direct Reading Method
Method 8345
MR (1 to 50 mg/L)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800
2495402

Gloves and goggles are recommended.

Description
Water, deionized
Quantity
10 mL
2
Chlorine Dioxide
Page 277
Chlorine Dioxide
Direct Reading method
Stored Programs
73 Chlor Diox MR
Zero
Start
1. Select the test.

Fill a sample cell to the 10- insert it into the cell holder.
mL mark with deionized
water.

0 mg/L ClO2

for orientation.
Read
5. Prepared Sample:
sample.

the cell holder.

mg/L ClO2.

in water.
immediately.
Chlorine Dioxide
Page 278
Chlorine Dioxide
Accuracy check
dioxide standards. If independent standard preparation is required, see the instructions in
solution". Prepare a 25.0-mg/L chlorine dioxide standard.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
75
43 mg/L ClO2
4145 mg/L ClO2
0.3 mg/L ClO2
Summary of method
Chlorine dioxide, a yellow gas, can be measured directly in a water solution. Test results are
measured at 360 nm.

Description
Water, deionized
Quantity/Test
Unit
Catalog number
10 mL
4L
27256
2/pkg
2495402
Optional apparatus
Description
Unit
Catalog number
Gloves, chemical resistant, size 99.51
pair
2410104
each
2550700
each
2270800
Chlorine Dioxide
Page 279

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Demand/Requirement
DOC316.53.01146
Method 10223
DPD Reagent 1
Scope and Application: For determining the chlorine demand and the chlorine requirement in drinking water
production. For establishing chlorine demand constants and establishing historical background data on raw water
quality. For determining chlorine demand on distributed waters.
1
Adapted from Standard Methods for the Examination of Water and Wastewater, Section 2350
Test preparation

Important Note: Read Getting started and all procedure steps before performing this test.
Develop a chlorine demand plan to detail the number of sample doses, the concentration of chlorine dose additions and
length of chlorine contact time. See Chlorine demand test plan and Getting started.
Precondition sample containers, test bottles and labware to be chlorine demand free. See Treatment of analysis labware.
Allow time for samples to equilibrate to the temperature indicated in the test plan before beginning the test.

Description
Quantity
DPD Free Chlorine Reagent PP, 10-mL or 25-mL
Varies
Chlorine Dosing Solution Ampules
Varies
Sample Bottles and Caps
Bottle Labels
pH Meter
Thermometer
Stir Plate
Stir Bar Magnets
Sample Cells, 10 mL 1-inch square or 1-cm/10 mL
Spectrophotometer or Colorimeter
Page 281
DPD Reagent
1. Complete a Chlorine
demand test plan.
Measure and record the
temperature and pH of the
sample water to be tested.
5. Turn off the stirrer and

fill the bottle until it
overflows with sample.
Cap and invert to mix.
Record the start time and
put the sample bottle in
the dark or wrap with foil.
Each 0.1 mL of chlorine
dosing solution added will
add approximately 1.0 mg/
L Cl2 to the sample.
2. Prepare 6 chlorine
demand-free bottles.
Rinse each bottle with
sample and fill each
118-mL bottle with
approximately 100 mL of
the sample to be tested.
3. Use tweezers or tongs

to insert a stir bar magnet
into each bottle. Set
Bottle #1 on a stir plate
and stir gently. A small
vortex should be visible on
the surface of the liquid.
Label the bottles 1 to 6.
Do not handle the stir

bar with fingers. Bare
fingers will add chlorine
demand to the sample.
6. Calculate the actual

amount of chlorine added.
See Chlorine addition
calculation for the formula
and an example.
The amounts of Dosing
Solution added may be
increased or decreased
based on the expected
organic level of the sample
water and chlorine
contact time.
Page 282
4. Open a Chlorine
Dosing Solution Ampule.
Using a TenSette Pipet,
add 0.1 mL of the chlorine
solution to Bottle #1 while
stirring. Immerse the end
of the pipet tip under the
water to dispense the
chlorine. Mixing while
adding the chlorine is
imperative to avoid highly
localized areas of chlorine
concentration.
Repeat steps 46
Method 8021
or
Method 10069
7. Repeat Steps 46 for

bottles 2 through 6.
Increase the amount of
chlorine added in
increments of 0.1 mL. See
Incremental addition of
Cl2 dosing solution and
Getting started. Stagger
the chlorine additions if the
contact time is expected to
be less than 30 minutes.
This allows time to perform
the chlorine analysis on
each sample bottle at the
specified contact time.
8. After the prescribed

contact is completed,
analyze the samples for
Free Chlorine using DPD
Free Chlorine Reagent.
Use Method 8021 if the
desired chlorine residual is
below 2.0 mg/L Cl2 or
Method 10069 if residuals
of greater than 2.0 mg/L
Cl2 are required. Follow
the procedure supplied
with the
spectrophotometer or
colorimeter being used.
DPD Reagent (continued)
9. Subtract the residual

chlorine determined in
Step 8 from the original
amount of chlorine added
to each bottle to determine
the chlorine demand:
Cl2 Demand = Cl2 added
concentration (mg/L) Cl2
residual measured
concentration (mg/L).1
10. Determine the chlorine

requirement (chlorine
dosage) needed to meet
the operating goal:
Cl2 Requirement = Cl2
Demand + Cl2 Residual
Required
Report the chlorine

demand according to the
goals of the study.
Example: The sample

required a dose of
3.0 mg/L chlorine to
achieve a free chlorine
residual of 1.1 mg/L
chlorine after 2 hours at
20 C and pH 8.1.
Example: The sample

dosed at 6.0 mg/L
consumed 4.1 mg/L
chlorine after 2 hours at
20 C and pH 8.1.
1
Report the chlorine

requirement according to
the goals of the study.
Some bottles will have no residual chlorine if the chlorine demand exceeded the amount of chlorine added. Choose a bottle that has a chlorine
residual to determine the demand. See Chlorine demand results.
Chlorine addition calculation

Use the following formula to calculate the concentration of the chlorine added in step 6.
0.1 mL volume of standard added ampule certificate value mg/L Cl 2
mg/L Cl 2 = ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------125 mL
Example:
0.1 mL certificate value of 1250 mg/L Cl
mg/L Cl 2 = ----------------------------------------------------------------------------------------------------------------2125 mL
mg/L Chlorine = 1.0
Page 283
Incremental reagent addition

Incremental addition of Cl2 dosing solution defines the incremental addition of a 1250 mg/L
chlorine dosing solution.
Table 93 Incremental addition of Cl2 dosing solution

Bottle #
Cl2 dosing solution added (mL)
Increases sample concentration (in mg/L Cl2) by
0.1
1.0
0.2
2.0
0.3
3.0
0.4
4.0
0.5
5.0
0.6
6.0
Chlorine demand results

Select the sample bottle that most closely fits the following criteria to calculate the
chlorine demand.
1. Residual chlorine measured is less than the chlorine dose added (0.03* mg/L).
2. Residual chlorine measured is greater than 0.03* mg/L.
3. Chlorine dose added is most similar to the dosage range expected in the field.
Criteria 1 and 2 ensure that the chlorine residual and demand are greater than the detection limit
of the DPD method used for determining the chlorine residual. If no sample portion satisfies all
criteria, repeat the test and adjust the chlorine doses accordingly.
Chlorine demand test plan

Chlorine demand test plans are developed for many purposes. The purpose should be well
defined and documented. This will develop the specific details of the plan, with the goal of
establishing reproducible test conditions to obtain reproducible data. This data is useful in
characterizing and optimizing a water treatment operation. Purposes for developing a chlorine
demand test plan might be:
Characterize the water system to establish a historical data baseline.
This baseline with chlorine demand date can be used to troubleshoot water quality problems,
provide background information for new employees and can provide additional support for
monitoring changes in water quality. The plan would include:
The standard elements of temperature, water pH, chlorine dose rate and chlorine
contact time.
Additional specific details to allow other analysts to reproduce the plan.
Characterize the chlorine demand of an influent raw water source.

Data obtained to establish chlorine demand data for use in understanding the effects of water
source changes, blending operations and seasonal weather variations. The plan would include:
Source water description, sample location, time of year, specific or unusual

weather events
* The minimum detection limit for DPD Chlorine Method 8021 when calculating the concentrations by difference
(1.412 x 0.02 mg/L).
Page 284
Additional complementary tests to be run, such as TOC, turbidity or UV-254, in addition to

the standard temperature, pH, chlorine dose rate and contact time.
Track the reduction in chlorine demand as water moves through the treatment process.
Data obtained would be used to establish a baseline for monitoring the effects of treatment
changes, seasonal water temperatures and overall changes in chlorine demand. The plan
would include:
Specific sampling locations
Treatment practices in use and flow rates
Getting started
Before starting this procedure, determine:
The magnitude of the chlorine demand present in the water to be tested.
Which chlorine method to use to determine chlorine residual.
First time users of this method or users evaluating a new water source should perform a screening
test to determine an approximate chlorine demand level before performing a full chlorine demand
test series.
1. Add 0.5 mL and 1.0 mL of Chlorine Dosing Solution to a 125-mL water sample.
2.
Hold for the contact time indicated in the test plan and then analyze the chlorine residual.
3. Use the chlorine residual values to determine the specific dose requirements in the chlorine
demand test plan. As a rule, use the HR DPD Chlorine Method (Method 10069) for raw water
samples or where the desired chlorine residual will be greater than 2.0 mg/L chlorine and use
the LR DPD Chlorine Method (Method 8021) for low chlorine demand waters, such as treated
waters or samples where the desired chlorine residual is less than 2.0 mg/L chlorine.
Chlorine demand procedure modification

The chlorine demand procedure is an operationally defined procedure. The procedure is defined
by the user and may be modified to meet the specific requirements of the sample or the process
operation. Run chlorine demand studies under the range of conditions expected in the field. Use
the basic test protocol while changing contact time, temperature, sample pH and chlorine
concentrations. Total Chlorine or Monochloramine can be determined as required from the
residual measured at the end of the prescribed contact time.
Use the following guidelines if modifications are made:
Make smaller chlorine concentration additions by using a larger sample size. A 237-mL bottle
(contains 250 mL when filled to overflowing) is available for low chlorine demand applications.
Each 0.1 mL of chlorine dosing solution added will increase the chlorine concentration by
approximately 0.5 mg/L. Substitute 250 mL for 125 mL in the above equation. A lower
concentration Chlorine Standard Solution, 5075 mg/L as Cl2 is also available for testing low
chlorine demand waters.
High chlorine demand waters require larger additions of chlorine. Use 0.2 mL, 0.4 mL, 0.6 mL,
etc., to spike the bottles in steps 4 and 7 in the procedure.
Wrap sample bottles made of clear colorless glass in foil to protect from light or kept in the
dark during the contact time.
Sample pH can be modified or standardized by adding a fixed amount of a pH buffer solution

to each bottle. Prepare a reagent blank bottle using organic free water. Add the same amount
of buffer to this blank, add a known amount of chlorine and carry the blank through the
procedure. Add only enough buffer to give the desired pH. This will check the chlorine demand
(if any) that was added by the buffer. Subtract the chlorine demand of the blank from the
sample chlorine demand values.
Page 285
Chlorine demand tests that have an extended contact time will require temperature control if
the sample temperature is significantly different from the analysis environment. Use a
refrigerator, water bath or incubator as required. It is important to control and document these
variables in order to be able to duplicate the chlorine demand procedure on future samples.
Treatment of analysis labware

Glassware used in this test must be chlorine demand-free. Treat all glassware with a dilute
solution of chlorine bleach prepared by adding 0.5 mL of commercial bleach to 1 liter of water.
Alternatively, the sample bottles may be treated by adding 2.0 mL of the Chlorine Dosing Solution
to each 125-mL bottle and filling to overflowing with deionized water. Soak glassware in this
solution for at least one hour. After soaking, rinse the glassware with copious amounts of chlorine
demand-free water before filling with sample.

Most reliable results are obtained on fresh low solid samples that are analyzed immediately.
Samples may be stored up to 24 hours at 4 C. Warm the samples to the required temperature
before running the chlorine demand test.
Summary of method
The chlorine demand of a water sample is defined as the difference between the concentration of
chlorine added to the sample and the concentration of the chlorine residual remaining at the end of
a predetermined contact time. The chlorine demand is a function of chlorine concentration, sample
temperature, contact time and sample pH.
The chlorine requirement is the amount of chlorine required to achieve a predetermined chlorine
residual at a prescribed contact time, pH and temperature.
Chlorine demand is caused by a complex set of reactions. Chlorine reacts with dissolved or
suspended organic materials in the water to form stable chlorinated organic compounds such as
trihalomethanes, haloacetic acids or other chlorinated organic compounds. Some of these
compounds (trihalomethanes) are referred to as disinfection by-product (DBPs) and are regulated
under the Disinfection/Disinfection By-Products Rule; other chlorinated organics contribute to taste
and odor problems. As a general rule, the lower the chlorine demand the lower the amounts of
DBPs formed and less taste and odor problems occur. Chlorine also is reduced by inorganic
reductants present such as ferrous, manganous, nitrite, sulfide and sulfite ions. Ammonia present
in the water also consumes chlorine to form chloramines.
Chlorine demand is significantly impacted by the physical and chemical characteristics of the
water sample. Chlorine demand studies ran at 10 C will be considerably different than studies ran
at 20 C. It is imperative that the sample temperature, pH and chlorine dose be accurately
measured and recorded. It is difficult to extrapolate chlorine demand data from one water source
to another. Demand studies need to be performed directly on the water source of interest. This
provides the information required to establish chlorine demand constants, to provide usable
historical data and to provide the test requirements for making repeatable and meaningful chlorine
demand measurements.
Page 286

Required reagents
Description
DPD Free Chlorine Reagent Powder Pillows, 10 mL
OR
Catalog number
2105569
1407099
Chlorine Dosing Solution Ampules, 11901310 mg/L as Cl2, 10-mL ampules, 16/pkg
2504810
Required Apparatus
Description
Catalog number
Bottles, Amber Glass, 118-mL 6/pkg
714424
Caps, Black, PP Teflon liner, 12/pkg
2401812

Description
Catalog number
Ampule Breaker, for Voluette Ampules
2196800
Bottles, Amber Glass, 237 mL, 6/pkg
714441
714463
Buffer Powder Pillows, pH 6.86, 15/pkg
1409895
1407995
89868
Buffer Solution, pH 7.0, demand-free, 500 mL
2155353
Caps, for 714441 Bottles, 6/pkg
2166706
2371026
Chlorine Standard Solution Ampules, 10 mL, 5075 mg/L as Cl2, 16/pkg
1426810
DPD Free Chlorine AccuVacs, 25/pkg
2502025
DPD Free Chlorine SwifTest Dispenser with reagents
2802300
DPD Total Chlorine Reagent Powder Pillows, 25 mL
1406499
DPD Total Chlorine Reagent PP, 10 mL
2105969
DPD Total Chlorine SwifTest Dispenser with reagents
2802400
Graduated Cylinder, plastic, 100 mL
108142
Incubator, Model 205, 110 V, 0 to 40 C
2616200
Labels, PolyPaper, 1.5 x 3 inches, 120/pkg
2091502
Monochlor F Reagent Powder Pillows, 10 mL
2802299
Page 287
Optional reagents and apparatus (continued)
Description
Catalog number
Sension 2 Portable pH/ISE Meter, with electrode

Sodium Hydroxide Standard Solution, 0.100N, 500 mL
5172510
19153
Standard Methods Handbook
2270800
Stir Bar, Teflon-coated, 2.22 cm x 0.48 cm
4531500
Stir Plate, 120 V, 4.25 x 4.25 inches
2881200
Sulfuric Acid Std. Solution, 0.100 N, 500 mL
20253
TenSette Pipet, 0.1 - 1.0 mL
1970001
Tips for TenSette Pipet, 0.1-1.0 mL, 50/pkg
2185696
Thermometer, Double Scale, 20 to 110 C (0230 F)
2095911
Tweezers
1428200
Water, Organic-Free 500 mL
2641549

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Free, 10059
Chlorine, Free
DOC316.53.01026
DPD Rapid Liquid Method1
Method 10059
(0.02 to 2.00 mg/L)
Pour-Thru Cell
Scope and Application: For treated water.

1
Test preparation


Instrument
DR 6000
DR 5000
DR 3900
DR 3800, DR 2800, DR 2700
Pour-thru Kit
Cell orientation
Adapter
LQV175.99.20002
Arrow faces right
LZV479
LQV157.99.10002
5940400
Align cell flow arrows with arrows on cell

compartment
1-inch (round) path in line with the arrow on

the adapter
LZV585 (B)

Refer to the instrument User Manual for Pour-Thru cell and module assembly and installation.
Protect the Pour-Thru Cell from contamination when not in use by inverting a small beaker over the top of the glass funnel.
The indicator reagent must be prepared in advance. See Reagent preparation.
Make sure the pour-thru cell is completely seated in the sample cell compartment.

Description
Quantity
DPD Indicator Powder
varies
Free Chlorine Indicator Solution
1 mL
Free Chlorine Buffer Solution
1 mL
Cylinder, glass, mixing, 100-mL
Dispenser, Adjustable Volume
Pour-Thru Cell Module and Cell (see Instrument-specific information)
Chlorine, Free
Page 289
Chlorine, Free
Free Chlorine, rapid liquid method
Stored Programs
82 Chlorine, F&T RL
Zero
Start
1. Select the test.

(See Instrument-specific
information).
5. Add 1.0 mL of Free

Chlorine Buffer Solution to
a clean, dry 100-mL glass
mixing cylinder using the
Dispenser.
Chlorine, Free
Page 290
2. Install the Pour-Thru

Cell module and cell. See
Instrument-specific
information for adapter
and cell orientation.
3. Pour approximately
50 mL of sample into the
Pour-Thru Cell.
4. When the flow stops,

press ZERO. The display
will show: 0.00 mg/L Cl2.
6. Add 1.0 mL of
prepared Free Chlorine
Indicator Solution to the
same mixing cylinder
using the dispenser. Swirl
to mix the reagents.
Proceed to step 7
immediately.
7. Carefully fill the mixing

with sample.
8. Stopper the cylinder

and gently invert it twice to
mix. Proceed to step 8
immediately.
Chlorine, Free
Free Chlorine, rapid liquid method (continued)
Read
9. Fill the funnel of the

Pour-Thru Cell with the
reacted sample from the
mixing cylinder.
10. After the flow stops,

press READ. Results will
appear in mg/L Cl2.
11. Flush the Pour-Thru

Cell with at least 50-mL of
deionized water
immediately after use.
(It is not necessary to pour

the entire sample into the
Pour-Thru Cell;
approximately half of the
sample may be
discarded.)
Reagent preparation
The Free Chlorine Indicator Solution must be prepared before use. Using a powder funnel, add the
contents of one 24 g bottle of DPD Powder* to one 473-mL bottle of Free Chlorine Indicator
Solution*. Invert several times and swirl until the powder is completely dissolved. A pale pink color
may develop, but should not affect results.
This solution will give accurate results for at least one month after mixing when stored at 2025 C
(6877 C). Write the date of preparation on the Indicator Solution Bottle. Discard any remaining
solution after one month. Use of this reagent after one month may result in high reagent blanks
and low values at high concentration. Do not combine fresh reagent with previously
mixed reagent.
Interferences
Interference level
Alkalinity

addition.
Bromine, Br2
Hardness
Levels below 1000 mg/L as CaCO3 will not interfere.
Iodine, I2
* See Required reagents.
Chlorine, Free
Page 291
Chlorine, Free
Manganese, oxidized (Mn4+,

Mn7+) or Chromium, oxidized
(Cr6+)
Interference level
1.
Adjust sample pH to 6 7 with 1.000 N Sulfuric Acid1.
2.
3.
Add 9 drops Potassium Iodide (30 g/L)1 to an 80-mL sample.

Mix and wait 1 minute.
4.
5.
6.
Add 9 drops Sodium Arsenite1, 2 (5 g/L) and mix.

Analyze the treated sample as described in the procedure above.
Subtract the result of this test from the original analysis to obtain the correct
concentration.
Monochloramine (NH2Cl)
Samples containing monochloramine will cause a gradual drift to higher chlorine readings.
When read within one minute of reagent addition, 3.0 mg/L monochloramine will cause an
increase of less than 0.1 mg/L in the free chlorine reading.
Ozone
See Optional standards and apparatus.
Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by the Federal RCRA for arsenic (D004). Refer
to the current MSDS for safe handling and disposal instructions.

Samples must be analyzed immediately and cannot be preserved for later analysis.
A common testing error is introduced if the analyst does not obtain a representative sample. If
sampling from a tap, let the water flow for at least five minutes to ensure a representative sample.
Let the container overflow with the sample several times, then cap the sample container so there is
no headspace (air) above the sample. Perform the chlorine analysis immediately.
Avoid plastic containers since these may have a chlorine demand.
Pre-treat glass sample containers to remove any chlorine demand by soaking in a dilute bleach
solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse thoroughly
If sample containers are rinsed thoroughly with deionized water after use, only occasional
pretreatment is necessary. A pre-treated glass BOD bottle with a ground-glass stopper makes an
ideal sample container for chlorine collection.
Treating analysis labware

Glassware used in this test must be chlorine demand-free. Fill the 100-mL mixing cylinder and
sample container with a dilute solution of chlorine bleach prepared by adding 1 mL of commercial
bleach to 1 liter of water. Soak in this solution at least one hour. After soaking, rinse thoroughly
with deionized water and allow to dry before use. If the mixing cylinder is thoroughly rinsed with
deionized water and allowed to dry after each use, only occasional pretreatment is necessary. Do
not use the same mixing cylinder for Free and Total Chlorine analysis.
Treat the Pour-Thru Cell similarly with dilute bleach and let stand for several minutes. Rinse
several times with deionized water.
Cleaning the Pour-Thru Cell

The Pour-Thru Cell may accumulate a buildup of colored reaction products, especially if the
reacted solutions are allowed to remain in the cell for long periods after measurement. Remove
the buildup by rinsing the cell with 5.25 N Sulfuric Acid followed by several rinsings with deionized
water.
Chlorine, Free
Page 292
Chlorine, Free
Accuracy check
Chlorine Voluette Ampule Standard Solution, 50 to 75-mg/L Cl2

3. Default values for standard concentration, sample volume and spike volumes can be accepted
or edited. Enter the chlorine concentration from the ampule package. After values are
accepted, the unspiked sample reading will appear in the top row. See the user manual for
more information.
4. Open a Chlorine Voluette Ampule Standard Solution, 50 to 75-mg/L Cl2.
5. Prepare three sample spikes. Use the TenSette Pipet to add 0.3, 0.6 and 0.9 mL of standard
to three 80-mL samples, respectively. Swirl gently to mix.
sample spike. Accept each standard additions value. Each addition should reflect
Method performance
Program
Standard
Precision
Distribution
Sensitivity
82
1.18 mg/L Cl2
1.171.19 mg/L Cl2
0.02 mg/L Cl2
Summary of method
Chlorine in the sample as hypochlorous acid or hypochlorite ion (free chlorine or free available
chlorine) immediately reacts with DPD (N,N-diethyl-p-phenylenediamine) indicator to form a pink
color which is proportional to the chlorine concentration. Test results are measured at 530 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2556900
varies
24 g
2297255
Free Chlorine Indicator Solution
1 mL
473 mL
2314011
Free Chlorine Buffer Solution
1 mL
473 mL
2314111
Free Chlorine Reagent Set, includes:
Chlorine, Free
Page 293
Chlorine, Free
Required apparatus
Description
Quantity
Unit
Catalog number
Cylinder, mixing graduated, 100-mL, glass
each
2636342
Dispenser, adjustable-volume, 1.0 5.0 mL
each
2563137
Powder Funnel
each
2264467
Description
Unit
Catalog number
Ampule, 50-75 mg/L, 10-mL
16/pkg
1426810
Chlorine Standard Solution, Pour-Rite Ampule, 50-75 mg/L, 2-mL
20/pkg
1426820
Chlorine Standard Solution,
Voluette
OR
each
2196800
PourRite Ampule breaker 2-mL
each
2484600
4L
27256
Unit
Catalog number
Water, deionized
Optional standards and apparatus

Description
each
1970001
50/pkg
2185696
1000/pkg
2185628
pH Paper, 0 - 14 pH range
100/pkg
2601300
100 mL
34332
100 mL
104732
Sulfuric Acid, 1 N
100 mL
127032
100 mL
244953
Pipet,
TenSette 0.11.0
mL

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Free, TNT, 10102
Chlorine, Free
DOC316.53.01024
DPD Method1
Method 10102
(0.09 to 5.00 mg/L)
Test N Tube Vials
Scope and Application: For testing higher levels of free chlorine (hypochlorous acid and hypochlorite ion) in
drinking water, cooling water and industrial process water.
1
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

DR 3900, DR 3800, DR 2700 and DR 2800: Install the light shield in Cell Compartment #2 before performing this test.
Analyze samples immediately. Do not preserve samples for later analysis.
blank adjust.
After adding sample to the Test N Tube, a pink color will develop if free chlorine is present.

Description
Quantity
Test N Tube DPD Free Chlorine Reagent
Wipes, disposable
varies
Chlorine, Free
Page 295
Chlorine, Free
DPD method for Test N Tube vials
Stored Programs
89 CHlorine F&T TNT
Zero
Start
1. Select the test.

Fill an empty Test N Tube
vial to the top of the label
with sample.

insert it into the 16 mm cell
holder.

0.00 mg/L Cl2
Read
5. Remove the cap from

a Free Chlorine DPD Test
N Tube. Add 10 mL of
sample to the tube. Fill the
vial to the top of the label.
6. Prepared Sample:
Cap and invert slowly at
least 10 times to dissolve
the powder. Invert by
turning the vial upside
down, then returning it to
an upright position. Ten
inversions should take at
least 30 seconds for
complete recovery.
7. Immediately wipe the

sample and insert it in the
16 mm cell holder.

mg/L Cl2.
Interferences

Acidity
Neutralize to pH 6 7 with1 N sodium hydroxide1. Determine amount to be added on separate

addition.
Alkalinity
Neutralize to pH 67 with 1 N sulfuric acid1. Determine amount to be added on separate sample

Bromine, Br2
Chlorine Dioxide, ClO2
May interfere
Chlorine, Free
Page 296
Chlorine, Free
Table 97 Interfering substances and levels (continued)
Hardness
Iodine, I2
Manganese, oxidized
(Mn4+, Mn7+) or Chromium,
oxidized (Cr6+)
Add 3 drops potassium iodide1 (30-g/L) to a 25-mL sample.
Add 3 drops sodium arsenite1,2 (5-g/L) and mix.
Subtract the result from this test from the original analysis to obtain the correct chlorine
concentration in the sample.
For conventional free chlorine disinfection (beyond the breakpoint), typical monochloramine
concentrations are very low. If monochloramine is present in the sample, its interference in the
free chlorine test depends on the sample temperature, relative amount of monochloramine to
free chlorine and the time required to do the analysis. Typical interference levels of
monochloramine as mg/L Cl2 in the free chlorine test are listed below (1 minute test time).
Sample Temp. C (F)
NH2Cl
(as Cl2)
Monochloramine
5 (41)
10 (50)
20 (68)
30 (83)
1.2 mg/L
+0.15
0.19
0.30
0.29
2.5 mg/L
+0.35
0.38
0.55
0.61
3.5 mg/L
+0.38
0.56
0.69
0.73
Note: Determine Monochloramine levels using Hach method 10200.

Ozone, O3
Peroxides
May interfere

buffered samples
Adjust to pH 67 using acid (Sulfuric Acid1, 1.000 N) or base (Sodium Hydroxide1, 1.00 N).
Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). Refer to
reagent MSDS for disposal instructions.
Analyze samples for chlorine immediately after collection. Free chlorine is a strong oxidizing
agent and it is unstable in natural waters. It reacts rapidly with various inorganic compounds
and more slowly oxidizes organic compounds. Many factors, including reactant
concentrations, sunlight, pH, temperature and salinity influence decomposition of free chlorine
in water.
Avoid plastic containers since these may have a large chlorine demand.
Pretreat glass sample containers to remove any chlorine demand by soaking in a dilute bleach
solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse
deionized or distilled water after use, only occasional pre-treatment is necessary.
A common error in testing for chlorine is not obtaining a representative sample. If sampling
container overflow with the sample several times, then cap the sample containers so there is
Chlorine, Free
Page 297
Chlorine, Free
Accuracy check
Chlorine PourRite Ampule Standard*, 5075 mg/L Cl2
TenSette Pipet 0.1 1.0 mL and tips
Ampule Breaker

1. After reading test results, leave the sample cell (unspiked sample) in the instrument. Verify
that the units displayed are in mg/L.
3. A keypad will appear. Enter the average chlorine concentration shown on the label of the
standard solution. Press OK. A summary of the Standard Additions procedure will appear.
Press OK.
4. Open a Chlorine PourRite Ampule Standard*, 5075 mg/L Cl2.
5. Use the TenSette Pipet to add 0.1 mL to a 10-mL sample. Mix thoroughly.
6. Analyze the standard addition sample as described in the procedure above. Accept the
standard additions reading by pressing READ. The addition should reflect approximately 100%
recovery.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
89
2.68 mg/L Cl2
2.632.73 mg/L Cl2
0.03 mg/L Cl2
Summary of method
Chlorine, Free
Page 298
Chlorine, Free

Required reagents
Description
Test N Tube DPD Free Chlorine Reagent
Quantity/Test
Unit
Catalog number
50/pkg
2105545

Description
Unit
Catalog number
20/pkg
1426820
Ampule, 2530 mg/L
20/pkg
2630020
16/pkg
1426810
PourRite
Ampule, 5075 mg/L
PourRite
each
2196800
each
2484600
Unit
Catalog number

Description
280/pkg
2097000
each
1970001
50/pkg
2185696
100 mL
127032
100 mL
104532
100/pkg
2601300
each
2635700
Wipes,
Pipet,
disposable
TenSette,
0.11.0 mL
Thermometer, Non-Mercury, -10 to 225C

Test Tube Rack
1000/pkg
2185628
each
1864100
Potassium Iodide, 30-g/L
100mL
34332
Sodium Arsenite, 5-g/L
100mL
104732
Chlorine, Free
Page 299

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Free, 8021
Chlorine, Free
DOC316.53.01023
USEPA DPD Method1
Method 8021
(0.02 to 2.00 mg/L)
Scope and Application: For testing free chlorine (hypochlorous acid and hypochlorite ion) in water, treated
waters, estuary and seawater. USEPA accepted for reporting for drinking water analyses.2
1
Procedure is equivalent to USEPA and Standard Method 4500-Cl G for drinking water.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

If the test over-ranges, dilute the sample with a known volume of high quality, chlorine demand-free water and repeat the
test. Some loss of chlorine may occur due to the dilution. Multiply the result by the dilution factor. Alternatively, samples with
high chlorine concentrations may be analyzed directly without dilution by using Method 10069, Chlorine, Free HR, or Method
10245, Chlorine Free MR .
The SwifTest Dispenser for Free Chlorine can be used in place of the powder pillow in step 4.
The sample cell shown is a generic representation. Refer to Instrument-specific information for the correct sample cell and
adapter configuration.
An empty AccuVac ampule can be used as a blank in place of the sample cell in Step 2.
Do not use the same sample cells for free and total chlorine. If trace iodide from the total chlorine reagent is carried over into
the free chlorine determination, monochloramine will interfere. It is best to use separate, dedicated sample cells for free and
total chlorine determinations.
Chlorine, Free
Page 301
Chlorine, Free

Description
Quantity
Powder Pillow Test:

AccuVac Test:
Beaker, 50-mL
Powder pillow procedure
Stored Programs
80 Chlorine, F&T PP
Start
1. Select the test.

10 mL of sample.

chlorine is present.
Proceed to step 6
immediately.
Chlorine, Free
Page 302

for orientation.
5. Swirl the sample cell

for 20 seconds to mix.

0.00 mg/L Cl2

adding the reagent, insert
the prepared sample into
the cell holder.
Results are in mg/L Cl2.
4. Prepared Sample:
Fill a second cell with
10 mL of sample.
DPD Free Chlorine
sample cell.
Chlorine, Free
AccuVac Ampuls procedure
Stored Programs
85 Chlorine, F&T AV
Start
1. Select the test.


mix. Wipe off any liquid or
fingerprints.
10-mL of sample.

display will show:
0.00 mg/L Cl2
4. Prepared Sample:
Collect at least 40 mL
of sample in a 50-mL
beaker.
Fill a DPD Free Chlorine
fills completely.
6. Within one minute

after sample addition, wipe
the AccuVac Ampul and
READ the results in
mg/L Cl2
Chlorine, Free
Page 303
Chlorine, Free
Interferences
Acidity
Greater than 150 mg/L CaCO3. May not develop full color or
color may fade instantly. Neutralize to pH 67 with
1 N Sodium Hydroxide. Determine amount to be added on
separate sample aliquot, then add the same amount to the
sample being tested. Correct for volume addition.
Alkalinity
Greater than 250 mg/L CaCO3. May not develop full color or
color may fade instantly. Neutralize to pH 6 7 with 1 N
Sulfuric Acid. Determine amount to be added on separate
sample aliquot, then add the same amount to the sample being
tested. Correct for volume addition.
Bromine, Br2
May interfere
Hardness
Iodine, I2

1.
2.
Manganese, Oxidized
(Mn4+, Mn7+) or Chromium, Oxidized (Cr6+)
3.
4.
5.
6.
Add 3 drops Sodium Arsenite 1 (5-g/L) and mix.

Analyze 10 mL of the treated sample as described in
the procedure.
Subtract the result from this test from the original
analysis to obtain the correct chlorine concentration.
Causes a gradual drift to higher readings. When read within

1 minute after reagent addition, 3 mg/L monochloramine
causes less than a 0.1 mg/L increase in the reading.
Monochloramine
Add 3 drops Potassium Iodide (30-g/L) to a 10-mL
sample.
Ozone
Peroxides
May interfere
Extreme sample pH or highly buffered samples
Adjust to pH 67 using acid (Sulfuric Acid, 1.000 N) or base

(Sodium Hydroxide, 1.00 N).
Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). See the
current MSDS for proper disposal of hazardous material.
Chlorine, Free
Page 304
in water.
Chlorine, Free
no headspace (air) above the sample. If sampling with a sample cell, rinse the cell several
times with the sample, then carefully fill to the 10-mL mark. Perform the chlorine analysis
immediately.
Accuracy check
Breaker, PourRite Ampules
3. Enter the average chlorine concentration shown on the label of the ampule container.
4. A summary of the standard additions procedure will be displayed. Press OK to accept the
default values for standard concentration, sample volume and spike volumes. After the values
are accepted, the unspiked sample reading will appear in the top row.
5. Open one Voluette ampule standard.
6. Prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of standard to three 10-mL portions
of fresh sample.
Note: For AccuVac Ampuls, add 0.4 mL, 0.8 mL and 1.2 mL of standard to three 50-mL portions of
fresh sample.
7. Follow the test procedure for each of the spiked samples using the powder pillows or AccuVac
ampules, starting with the smallest sample spike. Measure each of the spiked samples in the
instrument.
Note: If results are not within acceptable limits ( 10%), be sure that the sample volumes and sample spikes
are measured accurately. The sample volumes and sample spikes that are used should agree with the
selections in the standard additions menu. If all procedures are followed correctly but the standard
additions results are not within acceptable limits, the sample may contain an interference.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
80
1.25 mg/L Cl2
1.231.27 mg/L Cl2
0.02 mg/L Cl2
85
1.25 mg/L Cl2
1.211.29 mg/L Cl2
0.02 mg/L Cl2
Chlorine, Free
Page 305
Chlorine, Free
Summary of method
color, the intensity of which is proportional to the chlorine concentration. Test results are measured
at 530 nm.
Chlorine, Free
Page 306
Chlorine, Free

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
2105569
2502025
Catalog number
OR
Required apparatus
Description
Quantity
Unit
Beaker, 50-mL
each
50041H
AccuVac Snapper
each
2405200
2122800
each
6/pkg
2427606
2/pkg
2495402
Description
PourRite Ampule breaker, 2-mL
Unit
Catalog number
20/pkg
2630020
each
2484600
Unit
Catalog number

Description
Chlorine-demand Free Water
2641549
each
2088640
189641
each
100 mL
104532
Sulfuric Acid, 1 N
100 mL
127032
Potassium Iodide, 30-g/L
100 mL
34332
Sodium Arsenite, 5-g/L
100 mL
104732
each
2802300
each
1970001
SwifTest Dispenser for Free Chlorine1

Pipet,
TenSette,
Pipet, 0.1 - 1.0 mL
50/pkg
2185696
1000/pkg
2185628
100/pkg
2601300
each
2196800
AccuVac, vials for sample blanks
25/pkg
2677925
20/pkg
1426820
16/pkg
1426810
1000/pkg
2105528
300/pkg
2105503
DPD Free Chlorine Reagent, 10 mL, SwifTest Dispenser refill vial
250 tests
2105560
each
2635300
SpecCheck Secondary Standard Kit, Chlorine DPD, 02.0 mg/L Set

1
500 mL
Includes one vial of 2105560 for 250 tests
Chlorine, Free
Page 307

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, free, BT, 8334
Chlorine, Free
USEPA1 Amperometric Buret Titration Method2
0.5 mg/L and above
DOC316.53.01155
Method 8334
Buret Titration
Scope and Application: For water and wastewater.

1
USEPA accepted; 40 CFR Part 141, Section 141.74.
Adapted from Standard Methods for the Examination of Water and Wastewater (4500 Cl-D).
Test preparation
Chlorine can be lost from the sample during sample collection. Review the precautions in Sample collection, preservation
and storage before the test is started.
Use only a 50-mm stir bar. The wrong size can cause the loss of chlorine, unstable readings and loss of method sensitivity,
especially when measuring low level chlorine concentrations.
For added convenience when stirring, use the TitraStir apparatus.
When a new probe is placed in service or when the probe has not been used recently, prepare it according to the Probe
Stabilization instructions in the Amperometric Titrator Instruction Manual.

Description
Phenylarsine Oxide Solution, 0.00564 N
Phosphate Buffer Solution, pH 7
Quantity
1 bottle
1 mL
Amperometric Buret Titrator System
Beaker, 250-mL
Chlorine, Free
Page 309
Chlorine, Free
Buret titration
1. Fill the 5-mL automatic

buret to the zero mark with
0.00564 N Phenylarsine
Oxide (PAO) Solution.
2. Put a 50-mm stir bar

into a 250-mL beaker.
Use a graduated cylinder
to measure 200 mL of
sample. Add the sample to
the beaker.
3. If the pH is less than 6

or greater than 7.5, add
1.0 mL of pH 7 Phosphate
Buffer Solution to make
4. Place the beaker of

prepared sample on the
TitraStir titration stand and
turn on the stirring motor.
Put the tip of the probe
fully into the prepared
sample. The platinum
wires must be submerged.
If a stir plate other than the
TitraStir is used, set the
speed for moderate
mixing. Do not adjust the
speed after this point.
5. Turn the BIAS control

knob to adjust the value on
the display to
approximately 1.00.
The BIAS adjustment
controls the slope of the
titration curve. The actual
value is not important.
Only the relative value as
the titration continues is
important. A precise
adjustment is not
necessary.
Chlorine, Free
Page 310
6. Dispense the titrant

into the beaker in small
increments while
monitoring the values on
the Amperometric Titrator.
The values will decrease.
7. Continue dispensing
slowly. Near the end point
of the titration, write down
the value on the display
and the corresponding
total volume of titrant that
was added. Read the
volume to the nearest 0.01
mL. Add a small amount of
titrant and wait several
seconds for a stable value.
Write down the value.

by recording at least three
points on the downward
sloping curve and at least
three points after the end
point has been reached.
The value on the display
will not change
significantly after the
end point.
Chlorine, Free
9. Make a graph of the

titration. Plot the values
from the amperometric
titrator on the vertical axis
volume of titrant on the
horizontal axis.
10. Draw the two best

intersecting lines through
the points as shown
above. Find the volume of
titrant to the nearest
0.01 mL at the intersection
of the two lines. This is the
mL titrant end point. This
volume is equivalent to the
free chlorine concentration
in mg/L.
mL titrant =
mg/L free chlorine as Cl2
Interferences
Refer to the Amperometric Titrator Instruction Manual for a discussion of sources of errors and
interferences using the amperometric methods.

Start the chlorine analysis immediately after the samples are collected. Chlorine is a strong
oxidizing agent and is not stable in natural waters. Chlorine reacts quickly with various inorganic
compounds and slowly oxidizes organic compounds. Many factors such as sample composition,
sunlight, pH, temperature and salinity can cause the decomposition of chlorine in water.
Do not use plastic containers because plastic can react with and consume chlorine. Pretreat glass
sample containers to remove any chlorine demand by soaking in a dilute bleach solution (1 mL
commercial bleach to 1 liter of demineralized water) for at least 1 hour. Rinse thoroughly with
demineralized or distilled water. If sample containers are rinsed thoroughly with demineralized or
distilled water after use, only occasional pre-treatment is necessary.
Do not use the same sample containers for free and total chlorine. If trace iodide from the total
chlorine reagent is carried over into the free chlorine determination, monochloramine will interfere.
It is best to use separate, dedicated sample containers for free and total chlorine determinations.
A common error in testing for chlorine is introduced when a representative sample is not obtained.
If sampling from a tap, let the water flow for at least 5 minutes before sample collection. Let the
sample container overflow with the sample several times, then cap the container so that there is no
headspace (air) above the sample. Start the chlorine analysis immediately.
Chlorine, Free
Page 311
Chlorine, Free
Summary of method
Free chlorine is measured by a titration at pH 7 with PAO solution to the amperometric end point.
The amperometric titration method has greater sensitivity and accuracy when compared to
colorimetric methods. Refer to the Amperometric Titrator Instruction Manual for more information.

Required reagents
Description
Quantity/Test
Unit
varies
1L
Catalog number
199953
1 mL
100 mL MDB
2155332
Description
Unit
Catalog number
Amperometric Buret Titrator System, 115 VAC
each
1930010
Beaker, 250-mL
each
50046H
Graduated Cylinder, 250-mL
each
50846
Stir bar, 50 mm
each
2095355
TitraStir apparatus, 115 VAC
each
1940000
Required apparatus

each
1940010
100/pkg)
2601300
Unit
Catalog number

Description
16/pkg
1426810
20/pkg
1426820
20/pkg
2630020
Voluette Ampules,
5075 mg/L
Voluette Ampule breaker 10-mL
each
2196800
each
2484600
500 mL
27249
Water, deionized

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Free, HR, 10069
Chlorine, Free
DOC316.53.01025
DPD Method1
Method 10069
HR (0.1 to 10.0 mg/L as Cl2)
Powder Pillows
Scope and Application: For testing higher levels of free chlorine (hypochlorous acid and hypochlorite ion) in
drinking water, cooling water and industrial process waters
1
Test preparation


Instrument
Sample cell
Cell orientation
Adapter
DR 6000
4864302
A23618
DR 5000
4864302
A23618
DR 3900
4864302
Orientation key away from user
A23618
DR 3800, DR 2800, DR 2700
5940506
1-cm (flat) path in line with the indicator arrow on the

adapter
LZV585 (B)

If the chlorine concentration is less than 2 mg/L, use Method 8021, program number 80.

Description
Quantity
DPD Free Chlorine Reagent Powder Pillows
Chlorine, Free
Page 313
Chlorine, Free
Multi-path Cell
Stored Programs
88 Chlorine F&T HR
Zero
Start
1. Select the test.

2. Fill the sample cell to

the 5-mL line with sample.


0.0 mg/L Cl2

for orientation.

DPD Free Chlorine
powder pillow for 25-mL
samples to the sample.

dissolve reagent.
chlorine is present.

READ the results in
mg/L Cl2.
Interferences
Interference level
Acidity
Neutralize to pH 67 with1 N Sodium Hydroxide1. Determine amount to be added on separate

addition.
Alkalinity
Bromine, Br2

addition.
May interfere
Iodine, I2
Chlorine, Free
Page 314
Chlorine, Free

(Cr6+)
Interference level
1.
2.
3.

4.
5.
6.

concentration.
For conventional free chlorine disinfection (beyond the breakpoint), typical monochloramine
concentrations are very low. If monochloramine is present in the sample, its interference in
the free chlorine test is dependent on sample temperature, relative concentration of
monochloramine to free chlorine and the time required to perform the analysis.
Typical interference levels of NH2Cl (1 minute test time, interference as mg/L Cl2):
Monochloramine (NH2Cl)
Sample Temperature C ( F)
NH2Cl Level
(as Cl2)
5 (41)
10 (50)
20 (68)
30 (86)
1.2 mg/L
+0.15
0.19
0.30
0.29
2.5 mg/L
+0.35
0.38
0.55
0.61
3.5 mg/L
+0.38
0.56
0.69
0.73
5.0 mg/L
+0.68
0.75
0.93
1.05
Note: Determine Monochloramine levels using Hach method 10200.

Ozone
Peroxides
May interfere

buffered samples
Adjust to pH 67 using acid (Sulfuric Acid1) or base (Sodium Hydroxide1).

Analyze samples for chlorine immediately after collection. Free chlorine is a strong oxidizing agent
and reacts rapidly with various compounds. Many factors such as sunlight, pH, temperature and
sample composition will influence decomposition of free chlorine in water.
Avoid plastic containers since these may have a large chlorine demand. Pretreat glass sample
containers to remove any chlorine demand by soaking in a dilute bleach solution (1 mL
commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse thoroughly with deionized
or distilled water. If sample containers are rinsed thoroughly with deionized or distilled water after
use, only occasional pre-treatment is necessary.
Do not use the same sample cells for free and total chlorine. If trace iodide from the total chlorine
reagent is carried over to the free chlorine test, monochloramine could interfere. It is best to use
separate, dedicated sample cells for free and total chlorine determinations.
A common error in testing for chlorine is not obtaining a representative sample. If sampling from a
tap, let the water flow for at least 5 minutes to ensure a representative sample. Let the container
overflow with the sample several times, then cap the sample container so there is no headspace
(air) above the sample. If sampling with a sample cell, rinse the cell several times with the sample,
then carefully fill to the 5-mL mark. Proceed with the chlorine test immediately.
Chlorine, Free
Page 315
Chlorine, Free
Accuracy check
more information.
4. Open a Chlorine PourRite Ampule Standard, 5075 mg/L.
5. Prepare three sample spikes. Fill three mixing cylinders* with 5-mL of sample. Using the
TenSette Pipet*, add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Analyze each standard addition sample as described in the procedure above. Accept each
standard additions reading by pressing READ. Each addition should reflect approximately
100% recovery.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
88
5.4 mg/L Cl2
5.35.5 mg/L Cl2
0.04 mg/L Cl2
Summary of method
The range of analysis using the DPD method for free chlorine can be extended by adding more
indicator in proportion to sample volume. Thus, a larger fill powder pillow of DPD Free Chlorine
Reagent is added to a 5-mL sample portion.
chlorine) immediately reacts with DPD (N, N-diethyl-p-phenylenediamine) indicator to form a pink
Chlorine, Free
Page 316
Chlorine, Free

Required reagents
Description
DPD Free Chlorine Reagent Powder Pillows for 25-mL samples
Quantity/Test
Unit
Catalog number
100/pkg
1407099
Description
PourRite
PourRite
Unit
Catalog number
Ampules, 5075 mg/L
20/pkg
1426820
Ampules, 2530 mg/L
20/pkg
2630020
Chlorine Standard Solution, 10-mL Voluette Ampules, 5075 mg/L
16/pkg
1426810
Spec Gel Secondary Standard Kit, Chlorine DPD, 010 mg/L
4/pkg
2893300
each
2196800
each
2484600
Unit
Catalog number
Cylinder, mixing, 25 mL tall form
each
2088640
each
1970001
50/pkg
2185696
100/pkg
2601300
each
2635700

Description
1000/pkg
2185628
1000/pkg
2105528
300/pkg
2105503
100 mL
34332
100 mL
104732
Sodium Hydroxide, 1N
100 mL
104532
Sulfuric Acid, 1N
100 mL
127032
Chlorine, Free
Page 317

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine Free, Indophenol, 10241
Chlorine, Free
DOC316.53.01256
Indophenol1
Method 10241
(0.04 to 4.50 mg/L Cl2)
Powder Pillows
Scope and Application: For determining residual free chlorine levels in the presence of manganese, chloramines
and other oxidants which interfere with DPD colorimetric, DPD titrimetric, and amperometric methods for free
chlorine. For use in potable water, chlorinated drinking water, swimming pool water and treated wastewater
effluent.
1
Patent pending.
Test preparation

The Instrument-specific information table shows requirements that can vary between instruments.
To use this table, select an instrument, then read across to find the corresponding information
required to perform this test.

Instrument
Sample cell
Cell orientation
Adapter
DR 6000
4864302
A23618
DR 5000
4864302
DR 3900
4864302
LZV846 (A)
DR 3800, DR 2800, DR 2700
5940506
1-cm (flat) path aligned with arrow on adapter
LZV585 (B)

Put the protective cover or lid over the cell compartment during measurements.
This method uses Program 66, Monochloramine LR.
The sample and reagent from one analysis can contaminate other analyses and interfere with the test results. Be sure to
rinse the cells and caps several times with deionized water or with the sample water to be tested before each test.
Do not switch the sample cell caps between the Blank and Sample during the analysis.
Tap sample cells lightly on a hard surface or slowly invert the cells to remove air bubbles from the cell walls.
Be sure to keep the cap on the sample cells when not used to avoid ammonia contamination.

Description
Quantity
Freechlor F Reagent Solution
5 drops
Chlorine, Free
Page 319
Chlorine, Free
Collect the following items: (continued)
Description
Quantity
Sample cell (refer to Instrument-specific information)
Indophenol Method
Stored Programs
Start
1. Select the test. Insert

the correct adapter (refer
to Instrument-specific
information).
2. Fill 2 sample cells to

the 10-mL line with
sample. Mark one cell as
the Blank. Mark the other
cell as the Sample.
3. Add 5 drops of the

Freechlor F Reagent to the
Sample cell.
4. Cap and invert the cell

to mix.
Zero

one Monochlor F Reagent
Blank cell and to the
Sample cell. Cap and
shake both cells 20
seconds to dissolve.
Chlorine, Free
Page 320
6. Press TIMER>OK. A
5-minute reaction will start.
If the sample temperature
is less than 18 C (64 F),
refer to Table 103 for the
correct reaction time.
7. When the timer stops,

invert the Blank cell to
mix. Insert the cell into the
cell holder.

0.00 mg/L Cl2
Chlorine, Free
Indophenol Method (continued)
9. Invert the Sample cell

to mix. Insert the cell into
the cell holder. READ the
results in mg/L Cl2.
Color reaction time

Use Table 103 to find the reaction time when the sample temperature is less than 18 C (64 F).
The color is stable for 30 minutes after the reaction time is reached. If a temperature measurement
is not available, read the sample after the color is stable.
Table 103 Color development based on sample temperature

Sample Temperature
Reaction Time (minutes)
C
41
10
45
47
10
50
12
54
14
57
16
61
18
64
20
68
23
73
2.5
25
77
greater than 25
greater than 77
Interferences
The substances listed in Table 104 have been tested for interference and do not interfere at or
below the indicated levels. Refer to Table 105 for substances that do interfere with the test.

Interference level
Alanine
1 mg/L N
Aluminum
10 mg/L Al3+
Bromide
100 mg/L Br-
Chlorine, Free
Page 321
Chlorine, Free
Table 104 Non-interfering substances (continued)
Interference level
Bromine
15 mg/L Br2
Calcium
1000 mg/L as CaCO3
Chloride
18,000 mg/L Cl-
Chlorine Dioxide
5 mg/L ClO2
Chromium (III)
5 mg/L Cr3+
Copper
10 mg/L Cu
Cyanide
10 mg/L CN-
Dichloramine
10 mg/L as Cl2
Fluoride
5 mg/L F-
Glycine
1 mg/L N
Iodine
4 mg/L I2
Iron (II)
10 mg/L Fe2+
Iron (III)
10 mg/L Fe3+
Lead
10 mg/L Pb
Manganese (7+)
3 mg/L MnO4
Nitrate
100 mg/L NO3N
Nitrite
50 mg/L NO2N
Oxone1
(potassium
peroxomonopersulfate)
30 mg/L
Phosphate
100 mg/L PO43-
Silica
100 mg/L SiO2
Sulfate
2600 ppm SO42-
Tyrosine
1 mg/L N
Urea
10 mg/L N
Zinc
5 mg/L Zn2+
Oxone is a registered trademark of E.I. du Pont de Nemours & Co., Inc.
Interference level
Ozone1
> 1 mg/L O3
Sulfide1
> 0.5 mg/L S2-
This compound does not usually exist with free chlorine.
Chlorine, Free
Page 322
concentrations, sunlight, pH, temperature and salinity influence the decomposition of free
chlorine in water.
Avoid plastic containers because they can have a large chlorine demand.
Chlorine, Free
no headspace (air) above the sample. If sampling with a sample cell, rinse the cell several
times with the sample, then carefully fill to the 10-mL mark. Perform the chlorine analysis
immediately.
Chlorine, Free
Page 323
Chlorine, Free
Testing applications
Finished chlorinated drinking waters and distributions systems
Finished waters contain free chlorine and various levels of organic chloramines and inorganic
contaminants. It is generally assumed that the free chlorine has reacted with all easily oxidizable
species present and the remaining free chlorine is likely present in a steady-state equilibrium.
Replicate analyses for free chlorine on this type of water should give equivalent results. It is
especially important when testing water where free chlorine residual levels are low to observe all
precautions that refer to sample cell cleanliness, water temperature and sampling techniques.
At breakpoint
These waters may contain a mixture of free chlorine, chloramines and nuisance residuals
depending on water temperature, mixing efficiencies, sampling location and distance beyond the
theoretical breakpoint. It is important to note that the water can be in a state of "dynamic
equilibrium" and the chemical speciation can change rapidly, especially if one is at or near
breakpoint. The chemical speciation can change dynamically in both the Blank cell and the
Sample cell. The test must be conducted immediately on these types of samples. Test results may
be difficult to replicate on duplicate samples depending on the dynamics of the water. Test results
are best used to identify free chlorine trends and to monitor changes due to different mixing
efficiencies, sampling locations, temperature changes, increased chlorine feed rates, etc.
In chloramination kinetic studies
These waters will contain a mixture of free chlorine and chloramines depending on water
temperature, mixing efficiencies, sampling locations, feed rates for chlorine and ammonia and
contact time. It is important to note that the water is in a state of "dynamic equilibrium" and the
chemical speciation can change rapidly depending on water conditions. The chemical speciation
can change dynamically in both the Blank cell and the Sample cell. The test must be conducted
immediately on these types of samples. Test results may be difficult to replicate on duplicate
samples depending on the dynamics of the water. Test results are best used to identify free
chlorine trends and to monitor changes based on changes in mixing efficiencies, sampling
locations, water temperature changes, increased chlorine feed rates, etc.
With other oxidants such as Oxone, permanganate, chlorine dioxide, bromine and iodine
It is assumed that the free chlorine residual has stabilized in the presence of the other oxidants.
Replicate analyses for free chlorine on this type of water should give equivalent results. The levels
of alternate oxidants that can be present without interference have been tested only in laboratory
bench studies (refer to Table 104). Field data for free chlorine in the presence of these oxidants is
not available.
Accuracy check
Important Note: This procedure is only valid for stabilized or equilibrated free chlorine samples.
PourRite Ampule Breaker
Pipet tips
2. Select standard additions from the instrument menu (if available). Refer to the instrument user
manual for specific instructions. Refer to step 9 if a standard additions menu is not available.
3. Enter the average chlorine concentration from the label or certificate that is enclosed with the
standard solution ampules.
Chlorine, Free
Page 324
Chlorine, Free
5. Open one standard solution ampule.
6. Use the TenSette Pipet to prepare 3 spiked samples: add 0.1 mL, 0.2 mL, and 0.3 mL of
standard to three 10-mL portions of fresh sample.
7. Follow the Indophenol Method test procedure for each of the spiked samples, starting with the
0.1 mL sample spike. Measure each of the spiked samples in the instrument.
9. If a standard additions menu is not available, calculate the percent recovery:
c. Put the spiked sample in the cell holder and press READ.
d. Calculate the concentration of chlorine that was added to the sample:
0.1 mL concentration of chlorine standard (mg/L Cl2 )
mg/L chlorine added = ----------------------------------------------------------------------------------------------------------------------------------------------10.1 mL
e. Find the expected result of the spiked sample from the sum of the unspiked sample result
plus the concentration of chlorine that was added (step d).
f.
Calculate the percent recovery from the actual (step c) and the expected (step e) results.
Note: If results are not within acceptable limits ( 10%), be sure that the sample volumes and sample spikes
are measured accurately. The sample volumes and sample spikes that are used should agree with the
selections in the standard additions menu. If all procedures are followed correctly but the standard
additions results are not within acceptable limits, the sample may contain an interference.
Method performance
In a single laboratory with a standard solution of 3.51 mg/L chlorine (as Cl2) and a single lot of
reagent with a single instrument (DR 5000), a single operator obtained a standard deviation of
0.04 mg/L Cl2.
Summary of method
An ammonia solution at a pH of 8.3 is added to a sample containing free chlorine. The free
chlorine is immediately converted into monochloramine (NH2Cl). The monochloramine is then
determined by the indophenol method using Monochlor F Reagent. In this method, the
monochloramine reacts with a substituted phenol in the presence of a cyanoferrate catalyst to form
an intermediate monoimine compound. The intermediate compound couples with excess
substituted phenol to form a green-colored indophenol compound, which is proportional to the
amount of free chlorine present in the sample. A sample blank containing Monochlor F Reagent
compensates for background color from the reagent and sample. Test results are measured with a
spectrophotometer at 655 nm or with a colorimeter at 610 nm.
Chlorine, Free
Page 325
Chlorine, Free

Required reagents
Description
Quantity/Test
Unit
Freechlor F Reagent Solution
5 drops
50-mL SCDB
Catalog number
2964926
100/pkg
2802299
Description
Unit
Catalog number
20/pkg
2630020
Ampule, 5075 mg/L
20/pkg
1426820
16/pkg
1426810
Description
Unit
Catalog number
Ampule Breaker, 2-mL PourRite Ampules
each
2484600
Ampule Breaker Kit, 10-mL Volulette Ampules
each
2196800
each
1970001
50/pkg
2185696
1000/pkg
2185628
PourRite
Ampule, 2530 mg/L
PourRite
Clippers (shears)
each
2369400
Thermometer, non-mercury, 10 to 225 C
each
2635700
188/pkg
2932800
Wipers, disposable, 28 x 37 cm

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total, 8167
Chlorine, Total
DOC316.53.01027
USEPA1 DPD Method2
Method 8167
(0.02 to 2.00 mg/L)
Scope and Application: For testing residual chlorine and chloramines in water, wastewater, estuary water and
seawater; USEPA-accepted1 for reporting for drinking and wastewater analyses.
1
Procedure is equivalent to USEPA method and Standard Method 4500-Cl G for drinking water and wastewater analyses.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Samples must be analyzed immediately and cannot be preserved for later analysis
If the test overranges, dilute the sample with a known volume of high quality, chlorine demand-free water and repeat the test.
Some loss of chlorine may occur due to the dilution. Multiply the result by the dilution factor. Or, analyze samples with high
chlorine concentrations directly without dilution by using Method 10070, Chlorine, Total HR, or Method 10250, Chlorine Total,
MR.
For chloramination disinfection control, use Method 10172, Chloramine (Mono), Low Range (program number 66) or High
Range (program number 67).
After adding reagent a pink color will develop.
The SwifTest Dispenser1 for Total Chlorine can be used in place of the powder pillow in step 3.
An AccuVac ampule for Blanks can be used as a blank in place of the sample cell in step 2.
1
Optional reagents and apparatus.
Chlorine, Total
Page 327
Chlorine, Total

Description
Quantity
Powder Pillow Test:

DPD Total Chlorine Reagent powder pillow, 10-mL
AccuVac Test:
Collect at least 40 mL of sample in a 50-mL beaker
DPD Total Chlorine Reagent
AccuVac
40 mL
Ampul
Beaker, 50-mL
DPD method for powder pillows
Stored Programs
80 Chlorine, F&T PP
Start
1. Select the test.

with 10-mL of sample.

10 mL of sample.

timer.
DPD Total Chlorine Powder A three-minute reaction
period will begin. Perform
Pillow to the sample cell.
Swirl the sample cell for 20 steps 5 and 6 during this
time period.
seconds to mix.
3. Prepared Sample:
6. Wipe the blank sample 7. Within three minutes

cell and insert it into the cell after the timer expires,
holder.
holder.
display will show:
0.00 mg/L Cl2
Cl2.
Chlorine, Total
Page 328
Chlorine, Total
DPD method for AccuVac Ampuls
Stored Programs
85 Chlorine, F&T AV
Start
1. Select the test.

10-mL of sample.
3. Prepared Sample:
6. Wipe the blank sample

cell and insert it into the
cell holder.

wipe the AccuVac Ampul
holder.

for orientation.

timer.
period will begin. Perform
steps 6 and 7 during this
time period.
display will show:

0.00 mg/L Cl2.

fills completely.

mix. Wipe off any liquid or
fingerprints.
Cl2.
Interferences
Interfering Substance
Interference Levels and Treatments

Greater than 150 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize to
Acidity
Alkalinity
pH 6 7 with 1 N sodium hydroxide1. Determine amount to be added on separate sample aliquot, then
add the same amount to the sample being tested. Correct for volume addition.
Greater than 300 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize to
pH 67 with 1 N sulfuric acid1. Determine amount to be added on separate sample aliquot, then add
the same amount to the sample being tested. Correct for volume addition.
Bromine, Br2
Chlorine Dioxide
May interfere
Hardness
Chlorine, Total
Page 329
Chlorine, Total
Iodine, I2
Manganese, Oxidized
(Mn4+, Mn7+) or
Chromium, Oxidized
(Cr6+)
1.
2.
3.
Add 3 drops potassium iodide1 (30 g/L) to a 25-mL sample.

4.
5.
6.
Add 3 drops sodium arsenite1, 2 (5 g/L) and mix.

Subtract the result from this test from the original analysis to obtain the correct
chlorine concentration.
Ozone
Peroxides
May interfere
Extreme sample pH or
Highly buffered samples
Adjust to pH 6 7 using acid (Sulfuric Acid1, 1.000 N) or base (Sodium Hydroxide1, 1.00 N).
for arsenic (D004). Reference the current MSDS for more information on proper disposal of these materials.
Analyze samples for chlorine immediately after collection. Chlorine is a strong oxidizing agent
and it is unstable in natural waters. It reacts rapidly with various inorganic compounds and
more slowly oxidizes organic compounds. Many factors, including reactant concentrations,
sunlight, pH, temperature and salinity influence decomposition of chlorine in water.
Do not use the same sample cells for free and total chlorine. If trace iodide from the total
chlorine reagent is carried over into the free chlorine determination, monochloramine will
interfere. It is best to use separate, dedicated sample cells for free and total chlorine
determinations.
container overflow with the sample several times, cap the sample containers so there is no
headspace (air) above the sample. If sampling with a sample cell, rinse the cell several times
with the sample, then carefully fill to the 10-mL mark.
Perform the chlorine analysis immediately.
Accuracy check
Chlorine Voluette Ampule Standard, 2530 mg/L Cl2.
Ampule Breaker

Chlorine, Total
Page 330
Chlorine, Total
more information.
4. Open a LR Chlorine Voluette Ampule Standard, 2530 mg/L Cl2.
TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively to three 10-mL
samples and mix each thoroughly.
Note: For AccuVac Ampuls, fill three mixing cylinders with 50-mL of sample and spike with 0.4 mL, 0.8
mL and 1.2 mL of standard. Transfer 40 mL from each of the three mixing cylinders to three 50-mL
beakers*. Analyze each standard addition sample as described in the procedure above. Accept
each standard additions reading by pressing Read. Each addition should reflect approximately
100% recovery.
6. Analyze each sample spike as described in the procedure above, starting with the smallest
sample spike. Accept each standard additions reading by pressing Read. Each addition
Method performance
Program
Standard
Precision
Distribution
Sensitivity
80
1.25 mg/L Cl2
1.231.27 mg/L Cl2
0.02 mg/L Cl2
85
1.25 mg/L Cl2
1.211.29 mg/L Cl2
0.02 mg/L Cl2
Summary of method
Chlorine can be present in water as free chlorine and as combined chlorine. Both forms can exist
in the same water and be determined together as the total chlorine. Free chlorine is present as
hypochlorous acid and/or hypochlorite ion. Combined chlorine exists as monochloramine,
dichloramine, nitrogen trichloride and other chloro derivatives. The combined chlorine oxidizes
iodide in the reagent to iodine. The iodine and free chlorine reacts with DPD (N,N-diethyl-pphenylenediamine) to form a pink color which is proportional to the total chlorine concentration. To
determine the concentration of combined chlorine, run a free chlorine test and a total chlorine test.
Subtract the results of the free chlorine test from the total chlorine test to obtain the combined
chlorine concentration. Test results are measured at 530 nm.
Chlorine, Total
Page 331
Chlorine, Total

Required reagents
Description
DPD Total Chlorine Reagent Powder Pillows, 10-mL
Quantity/Test
Unit
Catalog number
100/pkg
2105669
25/pkg
2503025
OR
Required apparatus
Description
Quantity
Unit
Catalog number
AccuVac snapper
each
2405200
Beaker, 50-mL
each
50041H
Description
Unit
Catalog number
Chlorine Standard Solution, 2-mL Pour-Rite Ampule, 2530 mg/L
20/pkg
2630020
Chlorine Standard Solution, 2-mL PourRite Ampules, 5075 mg/L
20/pkg
1426820
16/pkg
1426810
each
2196800
each
2484600
Description
Unit
Catalog number
Beakers, 50 mL
each
50041H
500 mL
2641549
each
2088640
each
189641
Chlorine Demand-Free Water
Deionized Water
4L
27256
100 mL
34332
100 mL
104732
100 mL
104532
Sulfuric Acid, 1 N
100 mL
127032
SwifTest Dispenser for Total Chlorine
each
2802400
each
1970001
50/pkg
2185696
1000/pkg
2185628
100/pkg
2601300
25/pkg
2677925
1000/pkg
2105628
300/pkg
2105603
DPD Total Chlorine Reagent, 10 mL, SwifTest Dispenser refill vial
250 tests
2105660
each
2635300
SpecCheck Secondary Standard Kit, Chlorine DPD, 0 - 2.0 mg/L Set
Chlorine, Total
Page 332
Chlorine, Total
Chlorine, Total
Page 333

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total
DOC316.53.01030
DPD Rapid Liquid Method1
Method 10060
(0.02 to 2.00 mg/L)
Pour-Thru Cell
Scope and Application: For treated water.

1
Test preparation


Instrument
DR 6000
Pour-thru Kit
Cell orientation
Adapter
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
LQV157.99.10002
Align cell flow arrows with arrows on cell compartment
5940400
1-inch (round) path aligned with arrow on the adapter
LZV585 (B)
DR 3800, DR 2800, DR 2700

Refer to Reagent preparation
Make sure the Pour-Thru cell is completely seated in the sample cell compartment.

Description
Quantity
varies
Total Chlorine Indicator Solution
1 mL
Total Chlorine Buffer Solution
1 mL
Cylinder, glass, mixing, 100-mL
Pour-Thru Module and Cell (See instrument-Specific Information)
Chlorine, Total
Page 335
Chlorine, Total
DPD rapid liquid method for Pour-Thru Cell
Stored Programs
82 Chlorine F&T RL
Zero
Start
1. Select the test.

Pour-Thru Cell.

display will show:

0.00 mg/L Cl2.
Refer to Instrumentspecific information for

Pour-thru cell orientation.
5. Add 1.0 mL of
prepared Total Chlorine
Indicator Solution to the
same mixing cylinder
using the Repipet Jr.
Dispenser. Swirl to mix the
reagents. Proceed to
step 6 immediately.
Chlorine, Total
Page 336
6. Carefully fill the mixing

with sample. Stopper the
cylinder and gently invert it
twice to mix. Proceed to
step 7 immediately.

timer.
A two-minute reaction
period will begin.
Complete steps 8 and 9
within two minutes after
the timer expires.
4. Add 1.0 mL of Total

Chlorine Buffer Solution to
a clean, dry 100-mL glass
mixing cylinder using the
Repipet Jr. Dispenser.
8. When the timer

expires, fill the funnel of
the Pour-Thru Cell with the
mixing cylinder.
It is not necessary to pour
the entire sample into the
Pour-Thru Cell;
approximately half of the
sample may be discarded.
Chlorine, Total
DPD rapid liquid method for Pour-Thru Cell (continued)
Read

READ the results in
mg/L Cl2.

deionized water
Reagent preparation
The Total Chlorine Indicator Solution must be prepared before use. Using a powder funnel, add the
contents of one 24 g bottle of DPD Powder to one 473-mL bottle of Total Chlorine Indicator
Solution*. Invert several times and swirl until the powder is completely dissolved. A pale pink color
may develop, but should not affect results.
This solution will give accurate results for at least one month after mixing when stored at 2025 C
(6877 F). Write the date of preparation on the Indicator Solution Bottle. Discard any remaining
solution after one month. Use of this reagent after one month may result in high reagent blanks
and low values at high concentration. Do not combine fresh reagent with previously
mixed reagent.
Interferences

Greater than 700 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize
Alkalinity
to pH 6 7 with 1 N Sulfuric Acid1. Determine amount to be added on separate sample aliquot, then
add the same amount to the sample being tested. Correct for volume addition.
Bromine, Br2
Hardness
Levels below 1000 mg/L as CaCO3 will not interfere.
Hexavalent Chromium
Levels greater than 1 mg/L will cause a positive interference.
Iodine, I2
Manganese, oxidized
(Mn4+, Mn7+) or
Chromium, oxidized
(Cr6+)
Ozone
1.
2.
3.

4.
5.
6.

Subtract the result of this test from the original analysis to obtain the correct concentration.
Chlorine, Total
Page 337
Chlorine, Total
Samples must be analyzed immediately and cannot be preserved for later analysis.
A common testing error is introduced if the analyst does not obtain a representative sample. If
sampling from a tap, let the water flow for at least five minutes to make sure that it is a
representative sample. Let the container overflow with the sample several times, then cap the
sample container so there is no headspace (air) above the sample. Perform the chlorine
analysis immediately.
Avoid plastic containers since these may have a chlorine demand.
Pre-treat glass sample containers to remove any chlorine demand by soaking in a dilute
bleach solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse
thoroughly with deionized water. If sample containers are rinsed thoroughly with deionized
water after use, only occasional pretreatment is necessary. A pre-treated BOD bottle with a
ground-glass stopper is an ideal sample container for chlorine collection.

with deionized water and allow to dry before use. If the mixing cylinder is thoroughly rinsed with
deionized water and allowed to dry after each use, only occasional pretreatment is necessary. Do
not use the same mixing cylinder for Free and Total Chlorine analysis.

water.
Accuracy check
Required for accuracy check
Chlorine Voluette Ampule Standard Solution, 50 to 75-mg/L Cl2
Ampule Breaker

more information.Open a Chlorine Voluette Ampule Standard Solution, 50 to 75-mg/L Cl2.
4. Prepare three sample spikes. Use the TenSette Pipet to add 0.3, 0.6 and 0.9 mL of standard to
three 80-mL samples, respectively. Swirl gently to mix.
Chlorine, Total
Page 338
Chlorine, Total
Method performance
Program
Standard
Precision
Distribution
82
1.18 mg/L Cl2
1.171.19 mg/L Cl2
Sensitivity
Point of curve
Concentration
Entire range
0.02 mg/L Cl2
Summary of method
Chlorine can be present in water as free available chlorine and as combined available chlorine.
Both forms can exist in the same water and can be determined together as the total available
chlorine. Free chlorine is available as hypochlorous acid and/or hypochlorite ion. Combined
chlorine exists as monochloramine, dichloramine, nitrogen trichloride and other chloro derivatives.
The combined chlorine oxidizes iodide in the reagent to iodine. The iodine reacts with DPD (N,Ndiethyl-p-phenylenediamine) indicator along with free chlorine present in the sample to form a pink
color which is proportional to the total chlorine concentration. To determine the concentration of
combined chlorine, run a free chlorine test and a total chlorine test. Subtract the free chlorine
results from the results of the total chlorine test to obtain combined chlorine. Test results are
measured at 530 nm.

Required reagents
Description
Rapid Liquid Total Chlorine Reagent Set, includes:
DPD Indicator Powder, 24 g
Quantity/Test
Unit
Catalog number
2557000
varies
each
2297255
Total Chlorine Indicator Solution
1 mL
473 mL
2263411
Total Chlorine Buffer Solution
1 mL
473 mL
2263511
Catalog number
Required apparatus
Description
Quantity
Unit
Cylinder, mixing graduated, 100-mL, glass
each
2636342
Dispenser, Adjustable volume, 1.0-mL 5.0 mL
each
2563137
Funnel, powder
each
2264467
Description
Unit
Catalog number
Ampule, 5075 mg/L, 10-mL
16/pkg
1426810
Chlorine Standard Solution, Pour-Rite Ampule, 5075 mg/L, 2-mL
20/pkg
1426820
Voluette
OR
Chlorine, Total
Page 339
Chlorine, Total
Description
Unit
Catalog number
Water, deionized
4L
27256
Unit
Catalog number
20/pkg
2630020
100/pkg
2601300
each
1970001

Description

50 pkg
2185696
1000/pkg
2185628

34332
each
2484600
100 mL
104732
Sulfuric Acid, 1 N
100 mL
127032
1000 mL
244953
each
2196800
Voluette Ampule Breaker

1
100 mL

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total, TNT, 10101
Chlorine, Total
DOC316.53.01028
DPD Method1
Method 10101
(0.09 to 5.00 mg/L)
Test N Tube Vials
Scope and Application: For testing higher levels of total (free plus combined) chlorine in drinking water, treated
wastewater, cooling water or industrial process water
1
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

For DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield before performing this test.
Analyze samples immediately. Do not preserve samples for later analysis.
adjust.
For chloramination disinfection control, use Method 10172, Chloramine (Mono), High Range.
After adding sample to the Test N Tube, a pink color will develop if free chlorine is present.

Description
Quantity
Test N Tube DPD Total Chlorine Reagent
Chlorine, Total
Page 341
Chlorine, Total
DPD method for Test NTubes
Stored Programs
89 Chlorine F&T TNT
Start
1. Select the test.
Fill an empty Test N Tube

vial to the top of the label
with sample.
3. Wipe the outside of

the vial to remove
fingerprints and other
marks.

the cell holder.
7. Cap and slowly invert

at least 10 times to
dissolve the powder. (Ten
inversions should take at
least 30 seconds. One
inversion equals turning
the vial upside down, then
returning it to an upright
position.)

timer.
Zero

0.00 mg/L Cl2
6. Prepared Sample:
Remove the cap from a
Total Chlorine DPD Test N
Tube. Add 10 mL of
sample to the tube. (Fill
the vial to the top of the
label.)
Read
9. When the timer

expires, wipe the outside
of the vial that contains the
prepared sample.
Chlorine, Total
Page 342
10. Insert the sample in

the cell holder.

mg/L Cl2.
period will begin.
Chlorine, Total
Interferences
Table 111 Interfering Substances and Levels
Interference Levels and Treatment
Acidity
sample aliquot, then add the same amount to the sample being tested. Correct for volume addition.
Alkalinity
Neutralize to pH 67 with 1 N Sulfuric Acid1. Determine amount to be added on separate sample
Bromine, Br2
May interfere
Hardness
Iodine, I2
Manganese, oxidized
(Mn4+, Mn7+)
or
Chromium, oxidized (Cr6+)
1.
2.
3.

4.
5.
6.

Ozone, O3
Peroxides
May interfere
Extreme sample pH or
highly buffered samples
Adjust to pH 6 7 using acid (Sulfuric Acid1, 1.000 N) or base (Sodium Hydroxide1, 1.00 N).
for arsenic (D004). See the current reagent MSDS for safe disposal instructions.
Analyze samples for chlorine immediately after collection. Free chlorine and combined
chlorine are strong oxidizing agents and are unstable in natural waters. Many factors,
including reactant concentrations, sunlight, pH, temperature and salinity influence
decomposition of free chlorine in water.
A common error in testing for chlorine is obtaining an unrepresentative sample. If sampling

Chlorine, Total
Page 343
Chlorine, Total
Accuracy check
Chlorine PourRite Ampule Standard, 50-75 mg/L Cl2
Ampule Breaker

more information.
4. Open a Chlorine PourRite Ampule Standard, 50-75 mg/L Cl2.
5. Use the TenSette Pipet to add 0.1 mL of standard to a 10-mL sample and mix thoroughly.
6. Analyze the standard addition sample as described in the procedure above. Accept the
standard additions reading. The addition should reflect approximately 100% recovery.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
89
2.68 mg/L Cl2
2.632.73 mg/L Cl2
0.03 mg/L Cl2
Summary of method
Chlorine can be present in water as free chlorine and as combined chlorine. Both forms can exist
in the same water and be determined together as the total chlorine. Free chlorine is present as
hypochlorous acid and/or hypochlorite ion. Combined chlorine exists as monochloramine,
dichloramine, nitrogen trichloride and other chloro derivatives.
Free or combined chlorine oxidizes iodide in the reagent to iodine. The iodine and chlorine react
with DPD (N,N-diethyl-p-phenylenediamine) to form a pink color, which is proportional to the total
chlorine concentration. To determine the concentration of combined chlorine, run a free chlorine
test and a total chlorine test. Subtract the results of the free chlorine test from the total chlorine test
to obtain the combined chlorine concentration. Test results are measured at 530 nm.
Chlorine, Total
Page 344
Chlorine, Total

Required reagents
Description
Test N Tube DPD Total Chlorine Reagent
Quantity/Test
Unit
Catalog number
50/pkg
2105645
Description
Unit
Catalog number
20/pkg
1426820
each
2484600

Description
Unit
each
1970001
50/pkg
2185696
1000/pkg
2185628
100/pkg
2601300
each
2196800
each
1864100

Test Tube Rack
Catalog number
100 mL
34332
Sodium Arsenite 5 g/L
100 mL
104732
100 mL
104532
100 mL
127032
16/pkg
1426810
20/pkg
2630020
Chlorine, Total
Page 345

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total, HR, 10070
Chlorine, Total
DOC316.53.01029
USEPA DPD Method1
Method 10070
HR (0.1 to 10.0 mg/L as Cl2)
Powder Pillows
Scope and Application: For testing higher levels of total chlorine (free and combined) in drinking water, cooling
water and industrial process waters
1
USEPA accepted for reporting drinking water analyses. Procedure is equivalent to USEPA, Standard Method 4500-Cl-G for Drinking Water
and Wastewater.
Test preparation


Instrument
Sample cell
Cell orientation
Adapter
DR 6000
4864302
DR 5000
4864302
DR 3900
4864302
LZV846 (A)
DR 3800, DR 2800, DR 2700
5940506
LZV585 (B)

If the chlorine concentration is less than 2 mg/L, use Method 8167, program number 80.
After adding the reagent, a pink color will develop if chlorine is present.

Description
Quantity
Sample Cell (See Instrument Specific Information)
Chlorine, Total
Page 347
Chlorine, Total
DPD method for powder pillows
Stored Programs
88 Chlorine F&T HR
Zero
Start
1. Select the test.

2. Fill the sample cell to

the 5-mL line with sample.

insert it into the cell holder
information.

0.0 mg/L Cl2

for orientation.

DPD Total Chlorine
powder pillow for 25-mL
samples to the sample.

dissolve reagent.

timer.
will begin.

sample into the
Instrument-specific
information
READ the results in
mg/L Cl2.
Interferences

Acidity

addition.
Alkalinity
Neutralize to pH 67 with 1 N Sulfuric Acid1. Determine amount to be added on separate sample

Bromine, Br2
May interfere
Chlorine, Total
Page 348
Chlorine, Total
Iodine, I2

(Cr6+)
1.
Adjust sample pH to 67 with 1.000 N Sulfuric Acid1.
2.
3.

4.
5.
6.

Subtract the result of this test from the original analysis to obtain the correct concentration.
Ozone
Peroxides
May interfere

buffered samples
Adjust to pH 6 7 using acid (Sulfuric Acid1) or base (Sodium Hydroxide1).
Analyze samples for chlorine immediately after collection. Free and combined chlorine are
strong oxidizing agents and react rapidly with various compounds. Many factors such as
sunlight, pH, temperature and sample composition will influence decomposition of chlorine in
water.
Do not use the same sample cells for free and total chlorine. If trace iodide from the total
chlorine reagent is carried over to the free chlorine test, monochloramine could interfere. It is
best to use separate, dedicated sample cells for free and total chlorine determinations.
A common error in testing for chlorine is obtaining a representative sample. If sampling from a
tap, let the water flow for at least 5 minutes to ensure a representative sample. Let the
container overflow with the sample several times, then cap the sample container so there is no
headspace (air) above the sample. If sampling with a sample cell, rinse the cell several times
with the sample, then carefully fill to the 5-mL mark. Proceed with the chlorine test
immediately.
Accuracy check
Chlorine PourRite Ampule Standard, 5075 mg/L

Chlorine, Total
Page 349
Chlorine, Total
more information.
4. Open a Chlorine PourRite Ampule Standard, 5075 mg/L*.
5. Prepare three sample spikes. Fill three mixing cylinders with 5-mL of sample. Using the
TenSette Pipet*, add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Analyze each standard addition sample as described in the procedure above. Accept each
standard additions reading by pressing Read. Each addition should reflect approximately
100% recovery.
Method performance
Program
Standard
Precision
Distribution
88
5.4 mg/L Cl2
5.35.5 mg/L
Sensitivity
Point of Curve
Concentration
Entire range
0.1 mg/L Cl2
Use Method 8167 to test chlorine concentrations at levels typically less than 2 mg/L or Method
8370 to test chlorine concentrations at levels typically less than 500 g/L.
Summary of method
The range of analysis using the DPD method for total chlorine can be extended by adding more
indicator in proportion to sample volume. Thus, a larger fill powder pillow of DPD Total Chlorine
Reagent is added to a 5-mL sample portion.
The combined chlorine oxidizes iodide in the reagent to iodine. The iodine reacts with DPD (N, Ndiethyl-p-phenylenediamine) along with free chlorine present in the sample to form a pink color
which is proportional to the total chlorine concentration. Test results are measured at 530 nm.

Required Reagents
Description
DPD Total Chlorine Reagent Powder Pillows for 25-mL samples
Quantity/Test
Unit
Catalog number
100/pkg
1406499
Unit
Catalog Number
Recommended Standards
Description
20/pkg
1426820
Chlorine Standard Solution, 10-mL Voluette ampules, 5075 mg/L
16/pkg
1426810
Chlorine, Total
Page 350
Chlorine, Total

Description
Unit
Catalog number
Ampule breaker, Voluette, 10 mL
each
2196800
Ampule breaker, PourRite, 2-mL
each
2484600
each
189640
each
1970001
50/pkg
2185696
1000/pkg
2185628
100/pkg
2601300
100 mL
34332
100 mL
104732
100 mL
104532
Sulfuric Acid, 1 N
100 mL
127032
20/pkg
2630020
1000/pkg
1406428

1
Chlorine, Total
Page 351

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total
DOC316.53.01032
USEPA DPD Method1
Method 10014
ULR (2 to 500 g/L as Cl2)
Pour-Thru Cell and OriFlo Filtration
Scope and Application: For testing trace levels of chlorine and chloramines in treated domestic and industrial
wastewater; USEPA accepted for reporting for wastewater analysis2
1
U.S. Patent 5,362,650 covers the procedure. U.S. Patent 5,549,816 covers the OriFlo Filtration System.
Test preparation


Pour-thru Kit
Cell orientation1
Adapter
DR 6000
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
LQV157.99.10002
5940400
LZV585 (B)
Instrument
DR 3800, DR 2800, DR 2700

1
Align for long path

Analyze samples immediately. Samples containing chlorine cannot be preserved for later analysis.
A reagent blank value for a combined lot of indicator/buffer reagent solutions should be determined at least once a day. If
sample color or turbidity fluctuates frequently during the day, determine a reagent blank for each sample. Refer to Treating
Analysis Labware on page 7.
The reagent blank value is normally less than 5 g/L. If the value is greater than 5 g/L, an interfering substance may be
present in the blanking water or the DPD Indicator may be degrading. If there is doubt about the reagents, repeat the reagent
blank determination using chlorine-demand-free water for the sample. Blanks up to 5 g/L may be used.
Use a new filter for each test. Using an unspecified filter may give low analysis results or inability to filter the required volume.
Ampules contain more than 1.0 mL of solution for ease of transfer. Discard excess reagent in the ampule.
Use forceps to handle filters.
Chlorine, Total
Page 353
Chlorine, Total

Description
Quantity
ULR Chlorine Buffer Solution, 1.5-mL ampules
1 mL
DPD Indicator Solution for ULR Chlorine, 1.5-mL ampules
1 mL
Blanking Reagent for ULR Chlorine
1 mL
Membrane Filters, 3-micron, 25-mm
OriFlo Assembly
Beaker, 250 mL
Cylinder, graduated mixing, 50-mL.
Pipet Tips
Deionized water
Varies
Ampule Breaker
Pour-Thru Module and cell
DPD method for Pour-Thru Cell
Stored Programs
86 Chlorine Total, ULR
Start
1. Select the test.

Chlorine, Total
Page 354

cell with 50 mL of
deionized water.
3. Unscrew the cap from

the OriFlo plunger
assembly. Be sure that the
O-ring is properly seated
in the cap.
4. Install a new, 3-micron

filter (white) into the cap
well. Wet the filter with a
few drops of deionized
water. Reassemble and
hand-tighten the cap onto
the plunger.
Chlorine, Total
DPD method for Pour-Thru Cell (continued)
5. Open one ULR

Chlorine Buffer Solution
Ampule.
6. Using a TenSette
Pipet and a clean tip,
transfer 1.0 mL of buffer
from the ampule to a
clean, treated 50-mL
graduated mixing cylinder.
7. Open one ampule of

DPD Indicator Solution for
Ultra Low Range Chlorine.
8. Use a TenSette Pipet

and a clean tip to transfer
1.0 mL of indicator from
the ampule to the
Swirl to mix.
Proceed to step 9 within
one minute.
9. Prepared Sample:
Avoiding extra agitation,
carefully fill the cylinder to
the 50-mL mark with
sample. Stopper the
cylinder. Gently invert it
twice to mix.
10. Press TIMER>OK.

time will begin. Perform
steps 1116 during this
period.
Measure the reacted
sample 36 minutes after
mixing the sample and
reagents. If less than three
minutes elapses, the
reaction with chloramines
may be incomplete. A
reading after six minutes
may result in higher
reagent blank values.
11. Push the valve button

on the OriFlo barrel
assembly in (closed
position). Place the barrel
assembly into its stand.
Pour approximately 50 mL
of the original sample into
the barrel.
12. Insert the plunger into

the barrel and slowly push
the plunger down with
even pressure, until the
plunger is fully seated.
The lower ring on the

barrel assembly
represents about a 50-mL
volume.
Chlorine, Total
Page 355
Chlorine, Total
Zero
13. Pour the filtered

sample from the plunger
reservoir into the
Pour-Thru Cell.


0 g/L Cl2.
15. Pull the barrel valve

button out to the open
position. Pull the plunger
up to separate it from the
barrel assembly. Discard
the remaining unfiltered
sample.
16. Push the barrel valve

button to the closed
position. Place the barrel
assembly into its stand.
A new membrane may be

required for very turbid
samples. Alternatively, use
a second Quick Filter unit
with a new membrane filter
installed.
17. When the timer

expires, pour the contents
of the mixing cylinder into
the barrel.
18. Insert the plunger into

the barrel and slowly push
the plunger down with
even pressure, until the
plunger is fully seated.
19. Pour the filtered,

plunger reservoir into the
Pour-Thru Cell.

deionized water
READ the results in g/L

chlorine.
Subtract the reagent blank

value (See Determining
the reagent blank value)
from the sample value
obtained in step 19.
If a dechlorinating agent
(e.g., sulfite or sulfur
dioxide) is present, the
sample result, corrected
for the reagent blank, will
read 0 or a slightly
negative value.
Chlorine, Total
Page 356
Chlorine, Total
Determining the reagent blank value
Stored Programs
86 Chlorine Total ULR
Start
1. Select the test.

Make sure that the reagent
blank setting is off.
information.

module.
Flush the Pour-Thru cell
water.
3. Collect about 100 mL

of deionized or tap water
in a clean, 250-mL beaker.

to add 1.0 mL of Blanking
Reagent to the beaker.
Swirl several times to mix.
The Blanking Reagent
removes chlorine and
chloramines from the
water.
Note: The solution from step
4 will be used in Step 10.
5. Access the general

timer and set it for five
minutes. Start the timer.

open one ampule of ULR
Chlorine Buffer Solution.

1.0 mL of buffer from the
ampule a clean 50 mL
mixing graduated cylinder.

Chlorine, Total
Page 357
Chlorine, Total
Determining the reagent blank value (continued)

the ampule to the cylinder.
Swirl to mix the reagents.
one minute.

50-mL mark with
dechlorinated water from
step 4. Cap and invert
twice to mix. Save the
remaining water for
step 12.

timer.
14. When the timer

of the cylinder into the
Pour-Thru Cell. READ the
results in g/L chlorine.
15. Use this value to

correct the sample result
obtained in this procedure.
time will begin.

period, flush the Pour-Thru
Cell with the remainder of
original dechlorinated
water from step 10.
Zero


0 g/L Cl2.
Chlorine, Total
Page 358

details on saving the
reagent blank value.

deionized water
Chlorine, Total
Interferences
Bromine, Br2
May interfere
Copper, Cu2+
Greater than 1000 g/L
Iodine, I2

(Cr6+)
1.
2.
3.

4.
5.
6.

concentration.
Nitrite, NO2 (uncommon in

clean waters)
Ozone
Peroxides
May interfere

buffered samples
Adjust to pH 67
mg/L nitrite
Apparent g/L chlorine
2.0 mg/L
3 g/L
5.0 mg/L
5 g/L
10.0 mg/L
7 g/L
15.0 mg/L
16 g/L
20.0 mg/L
18 g/L
Analyze samples for chlorine immediately after collection. Chlorine is a strong oxidizing agent
and it is unstable in natural waters. It reacts rapidly with various inorganic compounds and
more slowly oxidizes organic compounds. Many factors, including reactant concentrations,
sunlight, pH, temperature and salinity influence decomposition of chlorine in water.
Avoid plastic containers. These may have a large chlorine demand.
solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour.
Rinse thoroughly with deionized or distilled water. If sample containers are rinsed thoroughly
with deionized or distilled water after use, only occasional pre-treatment is necessary.
no head space (air) above the sample.
Chlorine, Total
Page 359
Chlorine, Total

Glassware used in this test must be chlorine demand-free. Fill the mixing cylinder and sample
container with a dilute solution of chlorine bleach prepared by adding 1 mL of commercial bleach
to 1 liter of water. Soak in this solution at least one hour. After soaking, rinse thoroughly with
deionized water and allow to dry before use.
Cleaning the Pour-Thru cell

the buildup by rinsing the cell with 5.25 N Sulfuric Acid* followed by several rinsings with
deionized water.
Accuracy check
Low Range Chlorine Voluette Ampule Standard Solution, 25 to 30-mg/L

(25,000 to 30,000 g/L) Cl2
TenSette Pipet and Pipet Tips
Ampule Breaker
Standard additions method (samples spike)

accepted or edited. Enter the chlorine concentration from the ampule package. After values
are accepted, the unspiked sample reading will appear in the top row. See the user manual for
more information.
4. Snap the top off a Low Range Chlorine Voluette Ampule Standard Solution, 25 to 30-mg/L
(25,000 to 30,000 g/L).
5. Prepare three sample spikes. Use the TenSette Pipet to add 0.1, 0.2, and 0.3 mL of standard
to three 50-mL samples, respectively. Swirl gently to mix.
Chlorine, Total
Page 360
Chlorine, Total
Method performance
Program
86
Standard
295 g/L Cl2
Precision
Distribution
290300 g/L Cl2
Sensitivity
Portion of Curve
Concentration
Entire range
17 g/L Cl2
Summary of method
Several modifications to the normal DPD chlorine method are necessary to measure trace levels
of chlorine. The Pour-Thru Cell must be used in the spectrophotometer. Liquid reagents are also
required. The reproducible optics of the Pour-Thru Cell give more stable readings than is possible
with movable sample cells, resulting in more stable measurements.
It is essential that interfering sample turbidity is removed using a 3-micron membrane filter. To
avoid chlorine loss, the filtration is done after reacting the DPD with the chlorine in the sample. The
filter used has been specifically selected to avoid retention of the colored product. Sample color is
compensated by zeroing the spectrophotometer on a filtered sample.
The reagents are packaged in ampules and sealed under argon gas to ensure stability. Use of
liquid reagents eliminates any slight turbidity that might be caused by using powdered reagents.
Due to the possible oxidation of the reagents (which could give a positive chlorine reading in the
blank), a reagent blank must be determined at least once a day for each lot of reagent used. This
reagent blank value is subtracted from the sample result and the corrected value is the actual
Chlorine, Total
Page 361
Chlorine, Total

Required reagents
Description
Quantity/Test
ULR Chlorine Reagent Set (approximately 20 tests), includes:
Unit
Catalog number
2563000
1 mL
20/pkg
2493120
1 mL
20/pkg
2493220
1 mL
29 mL
2493023
varies
4L
27256

Water deionized
Required apparatus
Description
Quantity
ULR Chlorine Apparatus Set, includes:
Unit
Catalog number
2595600
Membrane Filters, 3-micron, 25-mm
25/pkg
2594025
OriFlo Assembly
4966000
each
Beaker, 250-mL
each
50046H
Breaker, ampule
each
2484600
each
189641
each
1970001
50/pkg
2185696
Unit
Catalog number
20/pkg
2630020
Description
Voluette

Description
Forceps, flat square tips, 115 mm
Sulfuric Acid, 1 N 100 mL

Unit
Catalog number
each
1453700
100 mL MDB
34332
100 mL
104732
100 mL MDB
127032
1000 mL
244953
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total
DOC316.53.01031
USEPA1 DPD Method2
Method 8370
ULR (2 to 500 g/L as Cl2)
Pour-Thru Cell
Scope and Application: For detecting trace levels of chlorine and chloramines in clean waters relatively free of
color and turbidity; USEPA accepted for reporting for drinking water analysis.
1
USEPA accepted
U.S. Patent 5,362,650
Test preparation


Instrument
DR 6000
Pour-thru Kit
Cell orientation
Adapter
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
LQV157.99.10002
5940400
LZV585 (B)
DR 3800, DR 2800, DR 2700

Analyze samples immediately. Samples containing chlorine cannot be preserved for later analysis.
A reagent blank value for a combined lot of indicator/buffer reagent solutions should be determined at least once a day. If
sample color or turbidity fluctuates frequently during the day, determine a reagent blank for each sample.
Ampules contain more than 1.0 mL of solution for ease of transfer. Discard excess reagent in the ampule.
Refer to Treating analysis labware.

Description
Quantity
1 mL
1 mL
1 mL
Beaker, 250 mL
Cylinder, graduated mixing, 50-mL.
Chlorine, Total
Page 363
Chlorine, Total
Description
Quantity
Pour-Thru Module and cell (See Instrument Specific Information)
Ampule Breaker
See Consumables and Replacement Items for reorder information.
DPD method for Pour-Thru Cell
Stored Programs
Zero
Start
1. Select the test.

5. Open one ULR

Chlorine Buffer Solution
Ampule.
2. Pour at least 50 mL of
sample into the Pour-Thru
Cell.
6. Using a TenSette
Pipet and a clean tip,
transfer 1.0 mL of buffer
from the ampule to a
clean, treated 50-mL

start the instrument timer.
period will begin. This time
allows turbidity or solids to
settle and ensures a stable
reading.

4. When the timer

expires, ZERO the
instrument.
0 g/L

the ampule to the
Swirl to mix.
one minute.
Chlorine, Total
Page 364
Chlorine, Total
Read
9. Prepared Sample:
Avoiding extra agitation,
carefully fill the cylinder to
the 50-mL mark with
sample. Stopper the
cylinder. Gently invert it
twice to mix.

timer.
time will begin.
Measure the reacted
sample 34 minutes after
mixing the sample and
reagents. If less than three
minutes elapses, the
reaction with chloramines
may be incomplete. A
reading after four minutes
may result in higher
reagent blank values.
11. Pour the contents of

the graduated mixing
cylinder into the Pour-Thru
cell.
12. When the timer

expires, READ the results
in g/L chlorine.
If a dechlorinating agent
(e.g. sulfite or sulfur
dioxide) is present, the
sample result (corrected
for the reagent blank) will
read 0 or a slightly
negative value.

deionized water
Chlorine, Total
Page 365
Chlorine, Total
Determining the reagent blank value
Stored Programs
Start
1. Select the test.

Make sure that the reagent
blank setting is off.

module.

information.
Flush the Pour-Thru cell

water.
5. Access the general

timer and set it for five
minutes. Start the timer.

open one ampule of ULR
Chlorine Buffer Solution.
Chlorine, Total
Page 366
3. Collect about 100 mL

of deionized or tap water
in a clean, 250-mL beaker.

to add 1.0 mL of Blanking
Reagent to the beaker.
Swirl several times to mix.
The Blanking Reagent
removes chlorine and
chloramines from the
water. Note: This
solution will be used in
Step 10.

1.0 mL buffer from the
ampule to a clean 50 mL
mixing graduated cylinder.

Chlorine, Total
Determining the reagent blank value (continued)

the ampule to the cylinder.
Swirl to mix the reagents.
one minute.

50-mL mark with
dechlorinated water from
step 4. Cap and invert
twice to mix. Save the
remaining water for
step 12.
Zero
Read


0 g/L Cl2.
14. When the timer

of the cylinder into the
Pour-Thru Cell. READ the
results in g/L chlorine.

timer.
time will begin.
15. Use this value to

correct the sample result
obtained in this procedure.
details on saving the

period, flush the Pour-Thru
Cell with the remainder of
original dechlorinated
water from step 10.

deionized water
Interferences
Bromine, Br2
May interfere
Copper, Cu2+
Iodine, I2
Iron (Fe3+)
Chlorine, Total
Page 367
Chlorine, Total

(Cr6+)

1.
2.
3.

4.
5.
6.

concentration.
Nitrite, NO2 (uncommon in

clean waters)
Ozone
Peroxides
May interfere

buffered samples
Adjust to pH 67
mg/L nitrite
Apparent g/L chlorine
2.0 mg/L
3 g/L
5.0 mg/L
5 g/L
10.0 mg/L
7 g/L
15.0 mg/L
16 g/L
20.0 mg/L
18 g/L
Analyze samples for chlorine immediately after collection. Many factors, including reactant
concentrations, sunlight, pH, temperature and salinity influence decomposition of chlorine
in water.
A common error in testing for chlorine is failure to obtain a representative sample. If sampling

with deionized water and allow to dry before use.
Chlorine, Total
Page 368
Chlorine, Total
Cleaning the Pour-Thru cell

water.
Accuracy check
Low Range Chlorine PourRite Ampule Standard Solution, 25 to 30-mg/L (25,000 to 30,000
g/L) Cl2
Ampule Breaker

more information.
4. Open a Low Range Chlorine Voluette Ampule Standard Solution, 25 to 30-mg/L (25,000 to
30,000 g/L) Cl2.
5. Prepare three sample spikes. Use the TenSette Pipet to add 0.1, 0.2 and 0.3 mL of standard to
three 50-mL samples, respectively. Swirl gently to mix.
Chlorine, Total
Page 369
Chlorine, Total
Method performance
Program
86
Standard
295 g/L Cl2
Precision
95% Confidence
Limits of Distribution
290300 g/L Cl2
Sensitivity
Point of curve
Concentration
Entire range
17 g/L Cl2
Summary of method
This method is designed for clean water, low in color and turbidity. The main applications include
monitoring for trace chlorine break-through of activated carbon beds and feedwater to reverse
osmosis membranes or ion-exchange resins.
Several modifications to the normal DPD chlorine method are necessary to measure trace levels
of chlorine. The Pour-Thru Cell must be used in the spectrophotometer. Liquid reagents are also
required. The reproducible optics of the Pour-Thru Cell give more stable readings than is possible
with movable sample cells, resulting in more stable measurements.
The reagents are packaged in ampules and sealed under argon gas to ensure stability. Use of
liquid reagents eliminates any slight turbidity that might be caused by using powdered reagents.
Due to the possible oxidation of the reagents (which could give a positive chlorine reading in the
blank), a reagent blank must be determined at least once a day for each lot of reagent used. This
reagent blank value is subtracted from the sample result and the corrected value is the actual
Chlorine, Total
Page 370
Chlorine, Total
Consumables and Replacement Items

Required Reagents
Description
Quantity/Test
Unit
1 mL
20/pkg
2493120
1 mL
20/pkg
2493220
1 mL
29 mL
2493023
ULR Chlorine Reagent Set (approximately 20 tests), includes:
Catalog number
2563000
Required Apparatus
Description
Quantity
Unit
Catalog number
PourRite Ampule Breaker
each
2484600
Beaker, 250-mL
each
50046H
each
189641
each
1970001
50/pkg
2185696
Unit
Catalog number
20/pkg
2630020
Recommended Standards
Description
PourRite

Description
Unit
100 mL
34332
100 mL
104732
127032
Sulfuric Acid, 1 N
100 mL
1000 mL
244953
100/pkg
2601300
1000/pkg
2185628
each
2196800
20/pkg
1426820
16/pkg
1426810

1
Catalog number
Voluette
ampules, 5075 mg/L
other sizes are available
Chlorine, Total
Page 371

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Free and Total, DT, 8210
Chlorine, Free and Total

DPD-FEAS Method
0 to 3.00 mg/L Cl2
DOC316.53.01172
Method 8210
Digital Titrator
Test preparation
It is best to use separate, dedicated flasks for free and total chlorine determinations.
mg/L combined chlorine = mg/L total chlorine mg/L free chlorine
1

Description
Quantity
DPD Free Chlorine Powder Pillow
1 pillow
DPD Total Chlorine Powder Pillow
1 pillow
Ferrous Ethylenediammonium Sulfate (FEAS) Titration Cartridge
1 cartridge
Digital titrator
Pipet, volumetric, 25-mL, Class A and pipet filler

Page 373



titrant until the solution is
colorless.
counter.
18. Hold the Digital

wipe the tip.
19. Use a pipet to

measure 25.0 mL of the
sample into a 50-mL
Erlenmeyer flask.
22. Calculate:
To measure total chlorine,

follow the procedure with
the following modification:
digits x 0.01 =
mg/L Free Chlorine as Cl2
Example: 25.0 mL of a
sample was titrated and
= 2.50 mg/L Cl2

one DPD Free Chlorine
mix.
of powder does not
dissolve.
Substitute a DPD Total

Chlorine Powder Pillow in
step 20. Wait three
minutes before proceeding
to step 21. The result in
step 22 will be expressed
as mg/L total chlorine.
Accuracy check
Use the standard additions method to find if the sample has an interference and to confirm the
Chlorine Standard Solution, PourRite Ampule, 5075 mg/L Cl2 (the exact concentration will
be shown on a certificate enclosed with the ampule)
Ampule breaker

Page 374

2. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to three
25-mL samples. Swirl to mix.
3. Follow the test procedure and titrate each of the spiked samples and a sample with no
standard added.
4. Each 0.1 mL of standard that was added will use 2030 digits to reach the endpoint. To find
the exact number of digits that should be used for each 0.1-mL addition, multiply the exact
concentration by 4 and by the spike volume.
(Example: 50 mg/L x 0.1 mL x 4 = 20 digits)
Interferences
Acidity
Greater than 150 mg/L CaCO3. May not develop full color or color may fade
instantly. Neutralize to pH 67 with 1 N Sodium Hydroxide. Determine amount to be
added on separate sample aliquot, then add the same amount to the sample being
tested.
Alkalinity
Greater than 250 mg/L CaCO3. May not develop full color or color may fade
instantly. Neutralize to pH 6 7 with 1 N Sulfuric Acid. Determine amount to be added
on separate sample aliquot, then add the same amount to the sample being tested.
Bromine, Br2
May interfere
Iodine, I2
Manganese, Oxidized
(Mn4+, Mn7+) or Chromium, Oxidized
(Cr6+)
1.
2.
3.
Add 3 drops Potassium Iodide (30-g/L) to a 10-mL sample.
4.
5.
6.
Add 3 drops Sodium Arsenite 1 (5-g/L) and mix.

Subtract the result from this test from the original analysis to obtain the correct
Ozone
Peroxides
May interfere
Extreme sample pH or Highly buffered

samples
Adjust to pH 67 using acid (Sulfuric Acid, 1.000 N) or base (Sodium Hydroxide,

1.00 N).
Temperature
Higher room temperatures tend to give higher free chlorine values due to reaction of
chloramines. Higher room temperatures also result in increased color fading.
Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). See the
current MSDS for proper disposal of hazardous material.

Page 375
in water.
container overflow with the sample several times,
Cap the sample containers so there is no headspace (air) above the sample.
Summary of method
The DPD-FEAS method provides a titrimetric procedure for determining free available chlorine
and for estimating free and combined chlorine fractions that are present together. The magenta
species, resulting from the oxidation of DPD by chlorine, is destroyed quantitatively by titration with
ferrous ethylenediammonium sulfate. The volume of titrant required to reach a colorless end point
is proportional to the chlorine concentration. Total residual chlorine may also be determined by
this test.

Required reagents
Description
Quantity/Test
Unit
Catalog number
(1) DPD Free Chlorine Powder Pillows
1 pillow
100/pkg
1407099
(1) DPD Total Chlorine Powder Pillows
1 pillow
100/pkg
1406499
(1) FEAS Titration Cartridge, 0.00564 N
varies
each
2292301
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50541
each
1451540
Free and Total Chlorine Reagent Set (approximately 100 tests):
2445300
Required apparatus
Description
each
1465100
each
1720500
each
4157800
Unit
Catalog number
20/pkg
1426820
each
2484600
Description
Chlorine Standard Solution, PourRite Ampule, 5075 mg/L Cl2, 2-mL
PourRite Breaker

Page 376

Description
Unit
Catalog number
Potassium Iodide Solution, 30-g/L
100 mL MDB
34332
Sodium Arsenite Solution, 5-g/L
100 mL MDB
104732
100 mL MDB
104532
each
2095352
100 mL MDB
127032
each
1970001
each
1940000
each
1940010
Water, deionized
500 mL
27249
100/pkg
2601300
Pipet tips
100/pkg
2185628
Pipet tips
50/pkg
2185696
Chlorine Standard, 5075 mg/L, 10 mL
16/pkg
1426810
Chlorine Standard, 2530 mg/L, 2 mL
16/pkg
2630020
each
2196800
Voluette breaker

Page 377

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Free, AFT, DT, 10024

P
Chlorine, Free
DOC316.53.01220
Amperometric Forward Titration using

0.00564 N PAO1
Method 10024
(0 to 1000 g/L Chlorine as Cl2)
Digital Titrator
Scope and Application: For drinking water; USEPA Accepted for reporting
1
Procedure is equivalent to Standard Method (18th ed.) 4500 Cl D for drinking water.
Test preparation

Make sure that the proper stir bar is used. The wrong size can cause the loss of chlorine, unstable readings and loss of
method sensitivity, especially when measuring low level chlorine concentrations.

Description
Quantity
Amperometric Titrator assembly
each
Digital Titrator
each
Beaker, low-form, 250-mL
each
Stir bar, octagonal, Teflon-coated, 50.8 x 7.9 mm
each
each
TitraStir mixer/stand assembly, 115 VAC
each
Probe Assembly, Amperometric Titrator
each
Delivery Tubes, 90 with hook
5/pkg
Phenylarsine Oxide Solution, 0.00564 N Digital Titrator Cartridge

each
1 bottle
Chlorine, Free
Page 379
Chlorine, Free
Amperometric forward titration using 0.00564 N PAO
1. Install the 0.00564 N

Phenylarsine Oxide (PAO)
cartridge. Flush the Digital
Titrator delivery tube by
turning the delivery knob
to eject a few drops of
titrant. Reset the counter
to zero and wipe the tip.
2. Assemble the
Amperometric Digital
Titrator System according
to the instructions in the
Amperometric Titrator
Instruction Manual.
3. Without excessive
agitation, measure 200 mL
of sample with a clean
graduated cylinder.
Transfer the sample to a
clean, 250-mL beaker
containing the 50-mm
stirring bar supplied with
the system.
4. If the pH is less
than 6 or greater than
7.5, add 1.0 mL of pH 7
Phosphate Buffer Solution
to make the prepared
sample.
5. Place the beaker on

the TitraStir stand and
immerse the tips of both
the probe and the delivery
tube in the solution. The
probes platinum wires
must be submerged. Turn
on the stirring motor.
6. Note the LED reading

on the Amperometric
Titrator. Unlock the BIAS
control and adjust the
BIAS control knob until a
reading between 0.50 and
0.06 is obtained. Lock the
bias control.
7. Using the Digital

Titrator delivery knob,
dispense the PAO Titrant
Solution in 5 to 10 digit
increments while noting
the downward reading.
8. As the end point of the

titration is approached,
record the LED readings
along with the
corresponding digits
displayed on the Digital
Titrator counter. Near the
titration end point, add 2 to
5 digits of titrant; wait a
few seconds for a stable
reading and record.
The BIAS adjustment

instrument reading is not
important; the relative
readings as the titration
proceeds are important. A
precise adjustment is not
required.
Chlorine, Free
Page 380
If the chlorine content of

the sample is high, add
titrant at a faster rate.
Chlorine, Free
Amperometric forward titration using 0.00564 N PAO
9. Continue the titration,

recording at least three
point is reached. The latter
points will cause little
change in the LED
readings.
10. Use linear graph

paper to plot the recorded
readings from the
Amperometric Titrator on
the vertical axis and the
corresponding Digital
Titrator digits on the
horizontal axis.

the points plotted as
shown above. Determine
the number of digits at the
intersection of the two
lines. This is the end point.
12. Determine the mg/L

free chlorine
concentration:
Digits at end point x 1.25 =
g/L free chlorine as Cl2
Interferences
Interference
Silver ions
Silver ions poison the electrode.
Copper ions
Interfere by plating on the electrode
High turbid water
Turbid water containing surface active agents
Oxidized manganese and

other oxidizing reagents
Positive interference
Samples containing organic

content
Some uncertainty in the endpoint may be observed on samples with high organic content.
Samples containing reducing

agents
Excess reducing agents, such as sulfur dioxide, sulfite and bisulfite, will cause either static,
or increasing LED readings because no free chlorine is present; these samples cannot be
titrated under the conditions of the test.
Buffered samples or extreme

sample pH
Highly buffered samples or extreme sample pH may exceed the buffering capacity of the
buffer reagent. If necessary, add additional buffer and check pH of sample prior to titration.
Chlorine, Free
Page 381
Chlorine, Free

Chlorine is rapidly lost from water.
Avoid exposure to sunlight or other strong light.
Avoid excessive agitation.
Analyze samples immediately.
Accuracy check
Chlorine Standard Solution Ampule
Fresh sample
1. Snap the top off a Chlorine Standard Solution Ampule. Note the certificate concentration of the
standard in mg/L.
2. Split a fresh sample into two 200-mL portions.
3. Use a TenSette Pipet to add 0.1 to 0.5 mL of the standard to one portion and swirl to mix. This
is the spiked sample.
4. Analyze both the sample and spiked sample and record the chlorine concentration of each.
5. Calculate the theoretical concentration of the spiked sample:
( Cu Vu ) + ( Cs Vs )
Theoretical concentration = ------------------------------------------------------Vu + Vs
Where:
Cu = measured concentration of sample, in mg/L (g/L divided by 1000)
Vu = volume of sample
Cs = concentration of chlorine standard (mg/L, certificate value)
Vs = volume of standard added
6. Calculate the percent spiked recovery:
Spiked sample result, in mg/L
% Spike recovery = ----------------------------------------------------------------------------------------------------------------------Theoretical concentration calculated, in mg/L
Chlorine, Free
Page 382
Chlorine, Free
Example:
Sample result (Cu) = 120 g/L or 0.120 mg/L
Spiked sample result = 185 g/L or 0.185 mg/L
Volume Sample (Vu) = 200 mL
Volume Standard (Vs) = 0.2 mL
Chlorine Standard (Cs) = 68.1 mg/L
( 0.120 200 ) + ( 68.1 0.2 )
Theoretical concentration = ------------------------------------------------------------------------- = 0.188 mg/L
200 + 0.2
0.185 mg/L
% Spike recovery = ------------------------------ 100 = 98%
0.188 mg/L
Ideally, the percent recovery should be 100%. Generally, results from 80120% recovery are
considered acceptable.
Method performance
Precision
In a single laboratory, a single operator used a standard solution of 338 g/L chlorine to obtain a
standard deviation of 5.2 g/L chlorine.
Detection Limit
With good operator technique, the estimated detectable concentration is approximately 15 g/L
chlorine using 0.00564 N PAO.
Summary of method
In the amperometric forward titration procedure for free chlorine, a small electrical current is
applied across two identical platinum electrodes. No current can flow between the electrodes
unless a substance that can be oxidized at the anode and a substance that can be reduced at the
cathode are both present. In the case of the free chlorine titration with phenylarsine oxide (PAO),
chlorine is reduced to chloride at the cathode due to the addition of PAO, and PAO is oxidized from
the +3 oxidation state to the +5 oxidation state at the anode. Prior to the end point of the titration,
both free chlorine and chloride are present in solution; allowing current to flow, even with a very
small applied potential. At the end point, no free chlorine remains and the solution cannot conduct
even if excess PAO titrant is added. The end point is defined when no change in current occurs,
signaling all free chlorine has been reacted.
Chlorine, Free
Page 383
Chlorine, Free

Required reagents
Description
Unit
each
199901
100 mL MDB
2155332
Description
Unit
Catalog number
Amperometric Titrator Assembly
each
1929900
Digital Titrator
each
1690001
each
50046H
each
50846
5/pkg
4157800
Catalog number
Required apparatus
each
1939000
Stir Bar, octagonal, Teflon-coated, 50.8 x 7.9 mm
each
2095355
TitraStir Mixer/Stand Assembly, 115 VAC

OR
each
1940000
1940010
Description
Chlorine Standard Solution Ampule, 5075 mg/L
Unit
Catalog number
20/pkg
1426820
4L
27256
Water, demineralized, each

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Hypochlorite, 10100
Chlorine, Hypochlorite
DOC316.53.01219
Iodometric Method1
Method 10100
HR (50 to 150 g/L) as Cl2 (5 to 15%)
Digital Titrator
Scope and Application: For testing concentrated liquid bleach (sodium hypochlorite, soda bleach) used as a
disinfectant in drinking water or wastewater treatment
1
Adapted from ASTM method D2022.
Test preparation

Description
Quantity
Digital Titrator Reagent Set, HR Hypochlorite (Bleach), 515% as Cl2
Potassium Iodide-Iodate Standard Solution, 0.0125 N
1L
Clippers, large
Delivery Tubes, 180
Digital Titrator
TenSette
Tips, for
Pipet 1970001
Iodometric method

tube into the 2.26 N
Sodium Thiosulfate Titrant
Solution cartridge. Attach
the cartridge to the titrator
body.
14. Flush the delivery tube

by turning the delivery
knob to eject a few drops
off the tip.
15. Fill the Erlenmeyer

flask to about the 75-mL
mark with deionized water
or tap water.

one Potassium Iodide
Powder Pillow to the flask
and swirl to mix.
The level of residual

chlorine found in tap water
will not interfere with the
test.
Page 385
Iodometric method

one Acid Reagent Powder
Pillow to the flask and swirl
to mix.
18. Attach a clean tip to

the TenSette Pipet.
21. Add one dropperful of

Starch Indicator Solution
to the flask and swirl to
mix. A dark blue or green
color will develop.

until the solution becomes
colorless. Record the
number of digits required.
Use the pipet to dispense

0.2 mL of bleach sample
below the solution level in
the flask.
19. Swirl to mix. The

solution will turn dark
brown.

tip into the solution and
swirl the flask while
titrating with the sodium
thiosulfate titrant until the
solution is pale yellow.
23. Calculate the g/L

chlorine:
Digits required x 0.5 =
g/L chlorine
g/L chlorine x 0.10 =
% chlorine by volume
(trade percent)
Interferences
The iodometric method is relatively free of interferences. The test will determine chlorite ion
(ClO2 ) in addition to the hypochlorite ion (ClO). However, the amount of chlorite in commercial
bleach is insignificant (typically less than 0.2%).

Interference level
Caustic agent
A large excess of caustic may cause low results.

1. After adding the Acid Reagent Powder Pillow (step 17), check the pH of the solution with
pH Paper. The pH should be less than 3.
2. If the pH is not less than 3, add additional Acid Reagent, one pillow at a time, until the
pH drops below 3.
Temperature
For most accurate results, the temperature of the dilution water should be less than 20 C
(68 F).
Page 386

Soda bleach solutions are relatively unstable.
Avoid exposing the sample to heat or light.
Collect samples in glass bottles and store in a cool, dark place until analyzed.
Analyze as soon as practical.
Accuracy check
For optimum test results, the manufacturer strongly recommends that reagent accuracy be
checked with each new lot of reagents. The strength of the Sodium Thiosulfate Standard Solution
can be checked using Potassium Iodide-Iodate Standard Solution:
Class A pipet, 50-mL
Pipet filler
Potassium Iodide Powder Pillow
50.00 mL of 0.0125 N Potassium Iodide-Iodate Standard Solution
7. Use a Class A pipet to transfer 50.00 mL of 0.0125 N Potassium Iodide-Iodate Standard

Solution to a clean 125-mL Erlenmeyer flask.
8. Add the contents of one Potassium Iodide Powder Pillow to the flask and swirl to mix.
9. Add the contents of three Acid Reagent Powder Pillows to the flask and swirl to mix. Swirl until
all powder is dissolved.
10. Continue the titration starting at step 20 of the procedure. It should take 217227 digits of
2.26 N Thiosulfate Standard Solution to reach the end point.
Precision
In a single laboratory, using a commercial bleach sample of 91.2 g/L (9.12%) Cl2, a single operator
obtained a standard deviation of 1.5 g/L ( 0.15%) Cl2.
Summary of method
Under acidic conditions, hypochlorite reacts with iodide to produce an equivalent amount of
triiodide (I3). The released I3 is titrated with standard sodium thiosulfate solution to a colorless
end point. The number of digits of sodium thiosulfate required is proportional to the hypochlorite
concentration in the original bleach sample.
Page 387

Required reagents
Description
Quantity/Test
Unit
Catalog number
Digital Titrator Reagent Set, HR Hypochlorite (Bleach), 515% as Cl2. Includes:

Acid Reagent Powder Pillows
1 pillow
100/pkg
104299
Potassium Iodide Powder Pillows
1 pillow
50/pkg
2059996
varies
each
2686901
100 mL MDB
34932
Quantity/Test
Unit
Catalog number
Sodium Thiosulfate Standard Solution Cartridge, 2.26 N

Required apparatus
Description
Clippers, large
each
96800
Delivery Tubes, 180
5/pkg
1720500
1690001
Digital Titrator
each
each
50543
each
1970001
Tips, for TenSette Pipet 1970001
50/pkg
2185696
Description
Unit
Catalog number
1L
1400153
Unit
Catalog number
100/pkg
2601300

Description
pH Test Strips, 014 pH
Bottle, sample, glass, 4 oz, with cap
3/pkg
2161303
Pipet, volumetric Class A, 50-mL
each
1451541
each
1465100

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total, ABT, DT, 10025
Chlorine, Total
DOC316.53.01221
Amperometric Back Titration1
Method 10025
Digital Titrator
Scope and Application: For drinking water and wastewater; USEPA accepted for reporting
1
Procedure is equivalent to Standard Method (18th ed.) 4500-Cl C for wastewater.
Test preparation
Before starting the test

When a new probe is used or the probe has not been used recently, prepare it according to the Probe Stabilization
instructions in the Amperometric Titrator Instruction Manual.
Use the proper stir bar (Catalog number 2095355). The wrong size can cause the loss of chlorine, unstable readings, and
loss of method sensitivity, especially when measuring low level chlorine concentrations.
To preserve the strength of the iodine titrant solution, always remove the delivery tube from the Digital Titrator cartridge and
replace the cap when not in use. Protect the iodine titrant solution from direct sunlight.
The sample may be fixed at the sample site for brief transportation delays but not for sample storage. (This fixing
technique is not acceptable for USEPA compliance monitoring.). See Sample collection, preservation and storage for
more information.

Description
Quantity
Standard Iodine Titrant Solution, Cartridge, 0.028 N
each
Digital Titrator
each
each
each
each
each
Acetate Buffer Solution, pH 4.0

TitraStir
Mixer/Stand Assembly, 115 or 230 VAC
100 mL MDB
100/pkg
each
Pipet, Volumetric, Class A, 1-mL
each
Pipet Filler
each
100 mL
each
5/pkg
Chlorine, Total
Page 389
Chlorine, Total
Part 1Adjusting the electrode response slope
1. Install the Standard

Iodine Titrant Cartridge,
0.028 N. Flush the Digital
2. Assemble the
Instruction Manual.
3. Use a graduated
cylinder to measure 200
mL of deionized water into
a clean 250-mL beaker.
Place the 50-mm stirring
bar into the beaker.
4. Add 1 mL of pH 4
Acetate Buffer and the
contents of one Potassium
Iodide Pillow.

6. Use the Digital Titrator

delivery knob to add
50 digits of Standard
Iodine Titrant Solution.

on the Amperometric
control knob until a stable
reading between 0.50 and
0.60 is obtained. Lock the
bias control.
8. Remove the probe

arm from the beaker and
rinse the platinum wires
Discard the sample.
Chlorine, Total
Page 390
The adjustment of the

electrode response slope
is complete.
Chlorine, Total
Part 2Standardization of the Iodine Titrant
1. Flush the Digital

2. Assemble the
Instruction Manual.
3. Use a graduated
cylinder to measure
200 mL of deionized water
into a clean 250-mL
beaker. Place the 50-mm
stirring bar into the beaker.
4. Use a Class A pipet to

transfer 1.00 mL of
0.00564 N Sodium
Thiosulfate Solution to the
beaker. Swirl to mix.
5. Add 1 mL of pH 4
Acetate Buffer Solution
and the contents of one
Potassium Iodide Powder
Pillow.


on the Amperometric
Titrator. It should read 0.00
0.05. DO NOT adjust the
bias control.

dispense 100 digits of
Standard Iodine Titrant
Solution and note the
reading.
Alternatively, use
Oxide (PAO) (Catalog
No. 199942) instead of
sodium thiosulfate.
Chlorine, Total
Page 391
Chlorine, Total
Part 2Standardization of the Iodine Titrant (continued)
titrant in five to ten digit
the reading.
13. Use a linear graph

paper to plot the recorded
readings from the
horizontal axis.
10. Record at least three

points (the null current
values) before the end
point is reached.

the plotted points as
shown above.
11. After the end point of

the titration (nominal 160
digits), record the
increasing LED readings
along with the
Titrator counter.
12. Add five to ten digits of

titrant and wait a few
seconds for a stable
reading. Record it.
15. Determine the number

of digits at the intersection
of the lines. That is the
standard end point.
16. Record the standard

end point digits value. Find
the multiplier from the Digit
multipliers table. This
multiplier will be used to
calculate the sample
Discard the sample.
Table 121 Digit multipliers
Chlorine, Total
Page 392
Stop adding titrant when

the LED readings exceed
0.60. LED readings above
0.60 will be excessively
noisy.
Digits (standard end point)
Multiplier
160
6.25
165
6.06
170
5.88
175
5.71
180
5.56
185
5.40
190
5.26
195
5.13
200
5.00
Chlorine, Total
Part 3Titration of sample for total residual Chlorine
1. Flush the Digital


2. Assemble the
Instruction Manual.

Reagent Power Pillow to
the beaker and allow the
powder to dissolve.
3. Place a clean, 50-mm

stir bar into a clean
250-mL beaker. Use a
Class A pipet to transfer
1.00 mL of 0.00564 N
Sodium Thiosulfate
Solution to the beaker.
4. Add 1 mL of pH 4
Acetate Buffer Solution to
the beaker. Then add
200 mL of the sample to
the beaker.
Alternatively, use
Oxide (PAO) (Catalog No.
199942) instead of sodium
thiosulfate.
Swirl to mix.

on the Amperometric
Titrator. It should read 0.00
0.05. DO NOT adjust the
bias control.

dispense Standard Iodine
Titrant Solution in five to
ten digit increments while
noting the reading.
Minimize agitation when

adding the sample.
Chlorine, Total
Page 393
Chlorine, Total
Part 3Titration of sample for total residual Chlorine (continued)
9. Record at least three

points (the null current
values) before the end
point is reached.

the plotted points as
shown above.
12. Using linear graph

paper, plot the recorded
readings from the
horizontal axis.
10. After the end point of

the titration is reached,
record the increasing LED
readings along with the
Titrator counter.
11. Add five to ten digits of

titrant and wait a few
seconds for a stable
reading. Record it.
14. Determine the number

of digits at the intersection
of the lines. That is the
sample end point.
15. Calculate the g/L total chlorine:
Stop adding titrant when

the LED readings exceed
0.60. LED readings above
0.60 will be excessively
noisy.
[Digits (Standard End Point) Digits (Sample End

Point)] x Multiplier = g/L Cl2
Use the multiplier from Part 2Standardization of the
Iodine Titrant, step 16.
Interpolation between values in the table is not
necessary.
Example:
Standard EP = 160 digits
Sample EP = 150 digits
Multiplier = 6.25, so:
g/L total chlorine [160 150] x 6.25 = 62.5, or 63 (round up)
Chlorine, Total
Page 394
Chlorine, Total
Interference
Silver ions
Silver ions poison the electrode
Copper ions
Interfere by plating on the electrode.
Turbid water
Interferences are sometimes found in highly turbid water and those containing surface active
agents
Oxidized manganese
Oxidized manganese and other oxidizing reagents give positive interferences.
Samples containing high

organic content.
Some uncertainty in the end point may be observed with samples containing high organic
content.
Iron and nitrite
Iron and nitrite interference are minimized by buffering to pH 4 before adding potassium
iodide.
Buffered samples or sample

pH
Dechlorinating agents
In samples that contain excess dechlorinating agents, such as sulfur dioxide, sulfite or
bisulfite, the titration end point (number of digits) will be greater than the number of digits
obtained during the standardization. It is not necessary to continue the titrant addition if the
number of digits used in the sample titration exceeds that calculated for the standardization
end point. This indicates that no free or combined chlorine is present in the sample.

Chlorine is rapidly lost from water. Avoid exposure to sunlight or other strong light. Avoid excessive
agitation. Analyze samples immediately or fix the sample by pre-addition of standard thiosulfate
and buffer. To fix the sample:
1. Pipet 1.00 mL of 0.00564 N Sodium Thiosulfate and add 1.0 mL of Acetate Buffer into a clean,
dry glass sampling bottle (e.g. BOD bottle).
2. At the sample site, measure 200 mL of sample with a graduated cylinder and transfer to the
sampling bottle. Swirl to mix.
3. Before analysis, quantitatively transfer the entire contents of the sampling bottle to the 250-mL
beaker. Minimize delay between sampling and analysis (1 hour maximum) to prevent
decomposition of thiosulfate in the sample. (This fixing technique is not acceptable for USEPA
compliance monitoring and should be used for brief transportation delaysnot for sample
storage.)
4. Start the analysis in Part 3Titration of sample for total residual Chlorine at step 5.
Accuracy check
Use the bias control prior to performing the analysis to adjust the electrode sensitivity. Set the bias
adjustment by adding a known amount of standard iodine titrant to deionized water and adjusting
the bias control to a given value on the display. The electrode sensitivity will vary depending on the
probe conditioning. Adjustment should be made at least daily or before each series of samples.
The iodine titrant concentration is approximately 0.0282 N, which relates to 160 digits needed to
titrate 1.00 mL of 0.00564 N Thiosulfate. If the calculated end point is greater than 160 digits, this
indicates that the Standard Iodine Titrant is weaker than when packaged. Discard the Standard
Iodine Titrant cartridge if the calculated standard end point in Part 2Standardization of the Iodine
Titrant is greater than 200 digits.
To preserve the strength of the iodine titrant solution, always remove the delivery tube from the
Digital Titrator cartridge and replace the cap when not in use. Protect the iodine titrant solution
from direct sunlight.
Chlorine, Total
Page 395
Chlorine, Total
Note: Standard additions is not applicable for samples containing excess reducing agents such as sulfur
dioxide, sulfite, or bisulfite.
Chlorine Standard Solution Ampule
1. Snap the top off a Chlorine Standard Solution Ampule. Note the certificate concentration of the
standard in mg/L.
3. Using a TenSette Pipet (Catalog number 1970001), add 0.1 to 0.5 mL of the standard to one
portion and swirl to mix. This is the spiked sample.
( Cu Vu ) + ( Cs Vs )
Where:
6. Calculate the percent spiked recovery:
Example:
( 0.120 200 ) + ( 68.1 0.2 )
200 + 0.2
0.185 mg/L
% Spike recovery = ------------------------------ 100 = 98%
0.188 mg/L
Chlorine, Total
Page 396
Chlorine, Total
Method performance
Precision
In a single laboratory, using a standard solution of 120 g/L chlorine, a single operator obtained a
standard deviation of 19 g/L chlorine.
Detection limit
The estimated detectable concentration is equivalent to one digit of 0.0282 N Standard Iodine
Titrant Solution, or approximately 6 g/L chlorine.
Summary of method
The back titration procedure minimizes errors caused by liberating the full concentration of iodine
in the sample and is the preferred method for amperometric measurement for total chlorine in
wastewaters. In this procedure, the end point signal is reversed because the remaining thiosulfate
(or phenylarsine oxide) added to the sample is titrated with standard iodine. The end point of the
back titration is reached just when free iodine exists in the sample resulting in a measurable
polarization current. The end point is estimated by continued addition of titrant, recording of the
current at each titrant addition, and graphing the data points. Where the best line between the data
points intersects the null current, the number of digits (from the Digital Titrator) at the end point can
be determined and the chlorine concentration calculated.

Required reagents
Description
Acetate buffer solution, pH 4.0
Potassium Iodide powder pillows
Unit
Catalog number
100 mL MDB
1490932
100/pkg
107799
Standard Iodine titrant solution, cartridge, 0.028 N
each
2333301
Sodium Thiosulfate standard solution, 0.00564 N
100 mL
2408842
Required apparatus
Description
Unit
Catalog number
each
1929900
each
50046H
each
50846
Delivery tubes, 90 with hook
5/pkg
4157800
Amperometric titrator assembly
Digital titrator
each
1690001
each
1451535
1939000
Probe assembly, Amperometric titrator
each
Stir bar, octagonal, Teflon-coated, 50.8 x 7.9 mm
each
2095355

OR
each
1940000
1940010
Description
Chlorine standard solution Ampule, 5075 mg/L
Water, demineralized, each
Unit
Catalog number
20/pkg
1426820
4L
27256
Chlorine, Total
Page 397
Chlorine, Total

Description

Unit
Catalog number
each
1970001
50/pkg
2185696
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total, AFT, DT, 10026
Chlorine, Total
DOC316.53.01222
Amperometric Forward Titration1
Method 10026
Digital Titrator
Scope and Application: For drinking water and wastewater; USEPA accepted for reporting
1
Procedure is equivalent to Standard Method (18th ed.) 4500-Cl D for drinking water and Standard Method (17th ed.) 4500-Cl D for wastewater
Test preparation

When a new probe is used or the probe has not been used recently, prepare it according to the Probe Stabilization
instructions in the Amperometric Titrator Instruction Manual.
Use the proper stir bar. The wrong size can cause the loss of chlorine, unstable readings and loss of method sensitivity,

Description
Quantity
each
5/pkg
each
each
each
each

Acetate Buffer Solution, pH 4
TitraStir
Mixer/Stand Assembly, 115 or 230 VAC
100/pkg
100 mL MBD
each
Digital Titrator
each
Graph paper
varies
each
Chlorine, Total
Page 399
Chlorine, Total
Amperometric forward titration
1. Install the
Phenylarsine Oxide (PAO)
Cartridge, 0.00564 N.
Flush the Digital Titrator
delivery tube by turning
the delivery knob to eject a
wipe the tip.
2. Assemble the
Instruction Manual.
3. Without excessive
agitation, measure 200 mL
of sample with a clean
graduated cylinder.
Transfer the sample to a
clean, 250-mL beaker
containing the 50-mm
stirring bar supplied with
the system.

Powder Pillow and swirl to
dissolve.
5. Add 1 mL of pH 4
Acetate Buffer solution.


on the Amperometric
control and adjust the
BIAS control knob until a
stable reading between
0.50 and 0.60 is obtained.
Lock the BIAS control.

dispense the PAO Titrant
Solution in 5 to 10 digit
the downward reading.
The BIAS adjustment

instrument reading is not
important; the relative
readings as the titration
proceeds are important. A
precise adjustment is not
required.
Chlorine, Total
Page 400
If the chlorine content of

the sample is high, add
titrant at a faster rate.
Chlorine, Total
Amperometric forward titration (continued)
9. As the end point of the

titration is approached,
record the LED readings
along with the
Titrator counter. Near the
titration end point, add 2 to
5 digits of titrant; wait a
few seconds for a stable
reading and record.
10. Continue the titration,

recording at least three
point is reached. The latter
points will cause little
change in the LED
readings.
11. Using linear graph

paper, plot the recorded
readings from the
horizontal axis.

the points plotted as
shown above. Determine
the number of digits at the
intersection of the two
lines. This is the end point.
13. Determine the g/L

total chlorine
concentration:
Digits at end point x 1.25 =
g/L total chlorine as Cl2
Chlorine, Total
Page 401
Chlorine, Total
Interferences
Interference
Silver ions
Silver ions poison the electrode.
Copper ions
Interfere by plating on the electrode.
Turbid water and water

containing surface active
agents
Interferences are sometimes found in highly turbid water and those containing surface active
agents.
Oxidized manganese and

other oxidizing reagents
Oxidized manganese and other oxidizing reagents give positive interferences.
Samples containing organic

content.
Some uncertainty in the endpoint may be observed on samples with high organic content.
Samples containing excess

reducing agents
Samples containing excess reducing agents, such as sulfur dioxide, sulfite and bisulfite do
not contain free chlorine or chloramines and can not be titrated under the conditions of the
test.
Buffered samples or sample

pH

Chlorine is rapidly lost from water.
Avoid exposure to sunlight or other strong light.
Avoid excessive agitation.
Accuracy check
Standard additions method* (sample spike)
Chlorine Standard Solution Ampule, 5075 mg/L Cl2
1. Snap the top off a Chlorine Standard Solution Ampule, 5075 mg/L Cl2. Note the specific
certificate concentration of the standard in mg/L.
3. Use the TenSette Pipet (Catalog. Number. 1970001) to add 0.1 to 0.5 mL of the standard to
one portion and swirl to mix. This is the spiked sample.
( Cu Vu ) + ( Cs Vs )
* The Standard Additions technique is not applicable to samples that contain excess reducing agents such as sulfur dioxide,
sulfite, or bisulfite.
Chlorine, Total
Page 402
Chlorine, Total
Where:
Calculate the percent spiked recovery:
Example:
( 0.120 200 ) + ( 68.1 0.2 )
200 + 0.2
0.185 mg/L
% Spike recovery = ------------------------------ 100 = 98%
0.188 mg/L
Method performance
Precision
In a single laboratory, using a standard solution of 347 g/L chlorine, a single operator obtained a
standard deviation of 3.2 g/L chlorine.
Detection limit
The estimated detectable concentration is approximately 15 g/L chlorine using 0.00564 N PAO.
Summary of method
In the amperometric forward titration procedure for total chlorine, a small electrical current is
applied across two identical platinum electrodes. No current can flow between the electrodes
unless a substance that can be oxidized at the anode and a substance that can be reduced at the
cathode are both present. In the case of the total chlorine, an equivalent amount of iodine forms
from the reaction of excess iodide with chlorine and combined chlorine at pH 4. During the titration
with phenylarsine oxide (PAO), the free iodine is reduced to iodide at the cathode and PAO is
oxidized from the +3 oxidation state to the +5 oxidation state at the anode. Prior to the end point of
the titration, both iodine and iodide are present in solution; therefore current can flow, even with a
very small applied potential. At the end point, no free iodine remains and the solution cannot
conduct even if excess PAO titrant is added. The end point is defined when no change in current
occurs, signaling all total chlorine has been reacted.
Chlorine, Total
Page 403
Chlorine, Total

Required reagents
Description
Unit
Phenylarsine Oxide Solution, 0.00564 N Digital Titrator cartridge
each
199901
100 mL MBD
1490932
100/pkg
107799
Description
Unit
Catalog number
each
1929900
each
50046H
each
50846
5/pkg
4157800
Digital Titrator
each
1690001
each
1939000
each
2095355
each
1940000

Catalog number
Required apparatus
Stir Bar, octagonal,
Teflon-coated,
50.8 x 7.9 mm

OR
1940010
Description
Chlorine Standard Solution Ampule, 5075 mg/L
Water, deionized, each
Unit
Catalog number
20/pkg
1426820
4L
27256
Unit
Catalog number
each
1970001
50/pkg
2185696

Description

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, total, BT, 8161
Chlorine, Total
DOC316.53.01154
Iodometric Method using Sodium Thiosulfate1

0 to 40,000 mg/L
Method 8161
Buret Titration

1
Adapted from Standard Methods for the Examination of Water and Wastewater (4500 Cl- D).
Test preparation


Description
Quantity
Dissolved Oxygen 3 Powder Pillow
1 bottle
Sodium Thiosulfate Standard Solution (see the Range-specific information table)
1 bottle
Graduated cylinder
Buret titration
See
Table 1

volume and corresponding
sodium thiosulfate solution
from the Range-specific
information table.

Sodium Thiosulfate
Standard Solution.
3. Use a graduated
measure the sample

Chlorine, Total
Page 405
Chlorine, Total

one Dissolved Oxygen 3
to mix.

to mix.

until the color changes to a
very light yellow.
The addition of the powder

pillow will lower the pH to 4
or less. If the sample is
highly alkaline, make sure
that the solution pH is 4 or
less with a pH meter or pH
paper before proceeding.

from blue to colorless.
8. Add one full dropper of

Starch Indicator Solution.
Swirl to mix. The sample
will change to a dark blue
color.
10. Calculate:
mL titrant used x multiplier = mg/L total chlorine as Cl2
Example: 100 mL of sample was titrated with the 0.10 N
sodium thiosulfate solution and 15 mL of titrant was
used to reach the endpoint.
The chlorine concentration is: 15 x 35.5 = 532 mg/L Cl2

Range (mg/L as Cl2)
Sample volume (mL)
Sodium thiosulfate concentration
Multiplier
0200
100
0.025 N
8.87
100400
50
0.025 N
17.7
200800
100
0.10 N
35.5
4001,600
50
0.10 N
70.9
1,0004,000
20
0.10 N
177
2,0008,000
10
0.10 N
355
5,00020,000
0.10 N
887
10,00040,000
0.10 N
1773
Chlorine, Total
Page 406
Chlorine, Total
Interferences
Oxidized forms of manganese, oxidizing agents and reducing agents such as organic sulfides can
interfere.

sunlight, pH, temperature and salinity can cause the decomposition of chlorine in water.
Summary of method
When potassium iodide is added to a sample containing chlorine at a pH less than 4, free iodine is
liberated in direct proportion to the amount of total chlorine present. The iodine is then titrated with
sodium thiosulfate. Starch indicator is added to enhance the end point. This method measures
both free chlorine and combined chlorine.
Chlorine, Total
Page 407
Chlorine, Total

Required reagents
Description
Quantity/Test
Unit
Dissolved Oxygen 3 Powder Pillows
1 pillow
100/pkg
98799
1 pillow
100/pkg
107799
1 mL
100 mL MDB
34932
varies
1L
35253
varies
1L
32353
varies
4L
27256
Catalog number
Titrantselect one or more based on range:
Water, deionized
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
2636540
Buret Clamp, double
each
32800
each
96800
100/pkg
2601300
each
50546
50837

each
each
50838
each
50840
each
50841
each
50842
each
56300
Support Stand

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, total, BT, 8168
Chlorine, Total
USEPA1 Amperometric Buret Titration Method2
(0.5 mg/L and above)
DOC316.53.01156
Method 8168
Buret Titration

1
USEPA accepted (4500 Cl- D).
Test preparation

Use only a 50-mm stir bar. The wrong size can cause the loss of chlorine, unstable readings and loss of method sensitivity,
For added convenience when stirring, use the TitraStir apparatus.

Description
Acetate Buffer Solution
Quantity
1 bottle
1 mL
Beaker, 250-mL
Chlorine, Total
Page 409
Chlorine, Total
Buret titration
1. Fill the 5-mL automatic

buret to the zero mark with
Oxide (PAO) Solution.
2. Put a 50-mm stir bar

into a 250-mL beaker.
5. Place the beaker of

prepared sample on the
TitraStir titration stand and
turn on the stirring motor.
Put the tip of the probe
fully into the prepared
sample. The platinum
wires must be submerged.
6. Turn the BIAS control

knob to adjust the value on
the display to
approximately 1.00.
If a stir plate other than the

TitraStir is used, set the
speed for moderate
mixing. Do not adjust the
speed after this point.
Chlorine, Total
Page 410

to measure 200 mL of
sample. Add the sample to
the beaker.
The BIAS adjustment

value is not important.
Only the relative value as
the titration continues is
important. A precise
adjustment is not
necessary.

mix.
4. Add 1.0 mL of pH 4
Acetate Buffer Solution to
make the prepared
sample.
7. Dispense the titrant

into the beaker in small
increments while
monitoring the values on
the Amperometric Titrator.
The values will decrease.
slowly. Near the end point
of the titration, write down
the value on the display
total volume of titrant that
was added. Read the
volume to the nearest
0.01 mL. Add a small
amount of titrant and wait
several seconds for a
stable value. Write down
the value.
Chlorine, Total

by recording at least three
point has been reached.
The value on the display
will not change
significantly after the
end point.
10. Make a graph of the

titration. Plot the values
from the amperometric
titrator on the vertical axis
volume of titrant on the
horizontal axis.

the points as shown
above. Find the volume of
titrant to the nearest
0.01 mL at the intersection
of the two lines. This is the
mL titrant end point. This
volume is equivalent to the
total chlorine
concentration in mg/L.
mL titrant =
mg/L total chlorine as Cl2
Interferences
Refer to the Amperometric Titrator Instruction Manual for a discussion of sources of errors and
interferences using the amperometric methods.

sunlight, pH, temperature, and salinity can cause the decomposition of chlorine in water.
A common error in testing for chlorine is introduced when a representative sample is not obtained.
If sampling from a tap, let the water flow for at least 5 minutes before sample collection. Let the
sample container overflow with the sample several times, then cap the container so that there is no
headspace (air) above the sample. Start the chlorine analysis immediately.
Summary of method
Total chlorine is measured after the addition of potassium iodide and acetate buffer by a titration at
pH 4 with PAO solution to the amperometric end point. The amperometric titration method has
greater sensitivity and accuracy when compared to colorimetric methods. Refer to the
Amperometric Titrator Instruction Manual for more information.
Chlorine, Total
Page 411
Chlorine, Total

Required reagents
Description
Quantity/Test
Unit
Catalog number
(2) Acetate Buffer Solution, pH 4
1 mL
100 mL MDB
(1) Phenylarsine Oxide Solution, 0.00564 N
varies
1L
199953
100/pkg
107799
Total Chlorine Reagent Set (approximately 200 tests), includes:
(2) Potassium Iodide Powder Pillows
2460700
1490932
Required apparatus
Description
Unit
Catalog number
each
1930010
each
1930012
Beaker, 250-mL
each
50046H
each
50846
Stir bar, 50 mm
each
2095355
each
1940000
TitraStir
apparatus, 230 VAC
pH paper, 014 pH range
each
1940010
100/pkg
2601300
Unit
Catalog number

Description
Chlorine Standard Solution, 10 mL Voluette Ampules, 5075 mg/L
16/pkg
1426810
Chlorine Standard Solution, 2 mL PourRite Ampule, 5075 mg/L
20/pkg
1426820
each
2196800
PourRite Ampule breaker, 2 mL
(each)
2484600
20/pkg
2630020
Water, deionized
500 mL
27249

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chlorine, Total, DT, 8209
Chlorine, Total
Iodometric Method Using Sodium Thiosulfate
1 to 400 mg/L or 20 to 70,000 mg/L Cl2
DOC316.53.01173
Method 8209
Digital Titrator
Test preparation

To convert mg/L chlorine to percent chlorine, divide the result in mg/L by 10,000.
These procedures can be used to measure iodine or bromine concentration if chlorine is not present. Multiply the test result
(in mg/L chlorine) by 3.58 or 2.25, respectively, to accurately express the iodine or bromine content of the sample.
For higher concentrations, see the hypochlorite procedure Method 10100.

Description
Quantity
Acetate Buffer Solution, pH 4 (for 1 to 400 mg/L Cl2 range)
1 bottle
Dissolved Oxygen 3 Powder Pillow (for 20 to 70,000 mg/L Cl2 range)
1 pillow

Sodium Thiosulfate Ttitration cartridge (see Range-specific information20 to 70,000 mg/L)
1 pillow
1 cartridge
1 bottle
Digital titrator
Graduated cylinder
Chlorine, Total
Page 413
Chlorine, Total
1 to 400 mg/L chlorine
See
Table 1
1. Select a sample
cartridge from the Rangespecific information1 to
400 mg/L table.


4. Use a clean graduated

measure the sample
volume from the Rangespecific information1 to
400 mg/L table in a
125 mL Erlenmeyer flask.

deionized water.
6. Add two full droppers

(2 mL) of Acetate Buffer
Solution, pH 4. Swirl to
mix.

mix.

titrant until the solution is a
pale yellow.
Chlorine, Total
Page 414
Chlorine, Total
1 to 400 mg/L chlorine

Swirl to mix.
A dark blue color will
develop.

until the solution changes
from dark blue to
colorless.
counter.

the Range-specific
information1 to 400 mg/
L table to calculate the
concentration:
mg/L Total Chlorine (Cl2)
Example: 100 mL of
sample was titrated and
= 2.5 mg/L chlorine
Table 125 Range-specific information1 to 400 mg/L

Range (mg/L as Cl2)
Sample volume (mL)
Titration cartridge (N)
Multiplier
14
100
0.02256
0.01
28
50
0.02256
0.02
520
20
0.02256
0.05
100400
0.02256
1.0
Chlorine, Total
Page 415
Chlorine, Total
20 to 70,000 mg/L chlorine
See
Table 2
1. Select a sample
cartridge from the Rangespecific information20 to
70,000 mg/L table.


4. Use a clean graduated

measure the sample
volume from the Rangespecific information20 to
70,000 mg/L table in a
125 mL Erlenmeyer flask.
5. Dilute to
deionized water.

mix.

mix.

pale yellow.
Normally, the addition of

the powder pillow will
lower the pH to 4 or less. If
the sample is large or
highly alkaline, make sure
that the solution is pH 4 or
less before proceeding.
Chlorine, Total
Page 416
Chlorine, Total
20 to 70,000 mg/L chlorine (continued)

Swirl to mix.
develop.

from dark blue to
colorless.
counter.

the Range-specific
information20 to 70,000
mg/L table to calculate the
concentration:
mg/L Total Chlorine (Cl2)
was titrated with the
0.113 N titration cartridge
and 250 digits were used
to reach the endpoint. The
= 125 mg/L chlorine
Table 126 Range-specific information20 to 70,000 mg/L

Range (mg/L as Cl2)
Sample volume (mL)
Titration cartridge (N)
Multiplier
2080
25
0.113
0.2
50200
10
0.113
0.5
100400
0.113
1.0
2501000
0.113
2.5
5002000
0.113
20009000 (0.20.9%)
2.00
22.2
500018,000 (0.51.8%)
2.00
44.3
10,00035,000 (1.03.5%)
2.00
88.7
20,00070,000 (2.07.0%)
0.5
2.00
177
Analyze samples for chlorine immediately after collection.
no headspace (air) above the sample.
Chlorine, Total
Page 417
Chlorine, Total
Accuracy check
Use the standard additions method to find if the sample has an interference and to confirm
Chlorine Standard Voluette Ampule, 5075 mg/L Cl2 (the exact concentration will be shown
on a certificate enclosed with the ampule)
Ampule breaker

samples of the same volume as used in the procedure. Swirl to mix.
standard added.
4. Each 0.2 mL of standard that was added will use approximately 10 digits to reach the
endpoint. To find the exact number of digits that should be used for each 0.2-mL addition,
multiply the exact concentration by the spike volume.
(Example: 50 mg/L x 0.2 mL = 10 digits)
If more or less titrant was used, the problem can be due to user technique, an interference or
a problem with reagents or apparatus.
samples of the same volume as used in the procedure. Swirl to mix.
standard added.
4. Each 1.0 mL of standard that was added will use approximately 1015 digits to reach the
endpoint. To find the exact number of digits that should be used for each 1.0-mL addition,
multiply the exact concentration by the spike volume and divide by 5.
(Example: 50 mg/L x 0.2 mL / 5 = 10 digits)
Summary of method
Total chlorine concentration equals the concentration of the free and the combined forms of
chlorine. Free chlorine reacts readily with ammonia to form combined chlorine such as
monochloramines. When potassium iodide is added to a sample containing chlorine at a pH less
than 8, free iodine is liberated in direct proportion to the amount of total chlorine present. The
iodine is then titrated with sodium thiosulfate.
Chlorine, Total
Page 418
Chlorine, Total

Required reagents
Description
Quantity/Test
Unit
Catalog number
1490932
1400 mg/L range:

2 mL
100 mL MDB
1 pillow
100/pkg
107799
Sodium Thiosulfate Titration Cartridge, 0.02256 N
varies
each
2409101
1 mL
100 mL MDB
202000 mg/L rangeReagent set (approximately 100 tests):
34932
2272500
(1) Dissolved Oxygen 3 Powder Pillows
1 pillow
100/pkg
98799
1 pillow
100/pkg
107799
2267301
(1) Sodium Thiosulfate Titration Cartridge, 0.113 N
varies
each
(1) Starch Indicator Solution
1 mL
100 mL MDB
200070,000 mg/L rangeReagent set (approximately 100 tests):
34932
2444800
(1) Dissolved Oxygen 3 Powder Pillows
1 pillow
100/pkg
98799
1 pillow
50/pkg
2059996
varies
each
1440101
1 mL
100 mL MDB
34932
Required apparatus
Description
Quantity/Test
Digital Titrator
Unit
Catalog number
each
1690001
each
50543

each
50837
each
50838
each
50840
each
50841
each
50842
each
1720500
each
4157800
Description
Chlorine Standard Voluette Ampule, 5075 mg/L, 10-mL
Voluette Breaker
Unit
Catalog number
16/pkg
1426810
each
2196800
Chlorine, Total
Page 419
Chlorine, Total
Description
Unit
Catalog number
each
2095352
each
1970001
each
1970010
each
1940000
each
1940010
Water, deionized
500 mL
27249
100/pkg
2601300
Pipet tips
100/pkg
2185628
Pipet tips
50/pkg
2185696
each
2484600
PourRite breaker

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chromate, DT, 8211
Chromate
DOC316.53.01174
Titration Method using Sodium Thiosulfate
Method 8211
20 to > 400 mg/L as CrO42
Digital Titrator
Scope and Application: Closed system cooling towers.
Test preparation

mg/L chromium = mg/L chromate x 0.448
mg/L sodium chromate = mg/L chromate x 1.4

Description
Quantity
1 pillow
Dissolved Oxygen 3 Reagent Powder Pillow
1 pillow
Sodium Thiosulfate titration cartridge (see Range-specific information)

1 cartridge
1 mL
Digital titrator
Graduated cylinder
Chromate
See
Table 1
1. Select a sample

3. Hold the Digital

wipe the tip.
4. Use a graduated
measure the sample
flask.
Chromate
Page 421
Chromate
Chromate

deionized water.

mix.

mix.
8. Wait 3 minutes.

pale yellow.

Swirl to mix.

from dark blue to
colorless.

the Range-specific
calculate the
concentration:

develop.

counter.
Do not wait more than

10 minutes.
mg/L chromate (CrO42)
endpoint. The
= 50 mg/L CrO42

Range (mg/L as CrO42)
Sample volume (mL)
Titration cartridge (N Na2S2O3)
2080
50
0.2068
0.2
50200
20
0.2068
0.5
100400
10
0.2068
1.0
> 400
0.2068
2.0
Chromate
Page 422
Multiplier
Chromate
Interferences

Interference level
Copper
Interfere to give high results. The effects of iron and copper can be masked by adding a
Magnesium CDTA Powder Pillow, followed by two 1.0-gram measuring spoons of Sodium
Acetate to the sample in step 7.
Iron, ferric
(Fe3+)
Substances capable of oxidizing iodide to iodine under acidic conditions (such as ferric iron
and copper) will interfere to give high results.
Other oxidants
Collect samples in an acid-washed plastic or glass bottles.
If sample can not be analyzed immediately, add 1 mL of concentrated sulfuric acid and swirl to
mix.
Accuracy check
Use the standard additions method to determine whether the sample has an interference and to
confirm the analytical technique.
Hexavalent Chromium Standard Solution, 1000-mg/L Cr6+
5. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of the standard to three samples.
Use the same sample volume that was used for the analysis. Swirl to mix.
6. Follow the test procedure and titrate the spiked samples to the end point. Write down the
amount of titrant that was used to reach the end point.
7. Each 0.1 mL of standard that was added will use approximately 22 digits of the titration
cartridge to reach the endpoint.
Complete the following test to make sure that the reagents and user technique are accurate.
Hexavalent Chromium Standard Solution, 1000-mg/L Cr6+
100-mL volumetric flask, Class A
1. Use a pipet to add 3.0 mL of the standard solution, 1000-mg/L as Cr6+, to a volumetric flask.
Dilute to 100 mL with deionized water and mix fully. The diluted standard is equivalent to
67 mg/L chromate.
2. Use 20 mL or 50 mL of the standard as the sample volume and follow the test procedure.
3. Titrate the standard to the end point and calculate the result.
Chromate
Page 423
Chromate
Summary of method
Chromate in the sample reacts with iodide under acidic conditions to form iodine as triiodide. The
addition of starch indicator produces a blue color complex with the iodine. This complex is titrated
with sodium thiosulfate to a colorless end point. The volume of titrant used is proportional to the
chromate concentration.

Required reagents
Description
Quantity/Test
Unit
(1) Dissolved Oxygen 3 Reagent Powder Pillows
1 pillow
100/pkg
98799
1 pillow
50/pkg
2059996
Chromate Reagent Set (approximately 100 tests):
Catalog number
2272400
varies
each
2267601
1 mL
each
34932
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50543
Required apparatus
Description

each
50838
each
50840
each
50841
each
1720500
each
4157800
Unit
Catalog number
100 mL
1466442
Description
Chromium, Hexavalent, Standard Solution, 1000-mg/L as
Chromate
Page 424
Cr6+
Chromate

Description
Magnesium CDTA Powder Pillows
Unit
Catalog number
100/pkg
1408099
Sodium Acetate, trihydrate, ACS
454 g
17801H
each
2095352
each
1970001
each
1940000
each
1940010

Water, deionized
500 mL
27249
Bottle, sampling
250 mL
2087076
Sulfuric Acid, ACS
500 mL
97949
Measuring spoon
1g
51000
Chromium standard, 50 mg/L
100 mL
81042H
Pipet tips
100/pkg
2185628
Pipet tips
50/pkg
2185696
Volumetric flask
100 mL
1457442
Volumetric Pipet
3 mL
1457442
Safety bulb
Tensette Pipet
Clippers
each
1465100
110 mL
1970010
each
96800
Chromate
Page 425

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chromium, Hexavalent, 8023
Chromium, Hexavalent
DOC316.53.01033
USEPA1 1,5-Diphenylcarbohydrazide
Method2
Method 8023
(0.010 to 0.700 mg/L Cr6+)
Scope and Application: For water and wastewater; USEPA accepted for reporting for wastewater analysis.3
1
Accepted USEPA and Standard Method 3500 Cr B.
Procedure is equivalent to USGS method 1-1230-85 for wastewater.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

At high chromium levels, a precipitate will form. Sample dilution may be necessary.
The final samples are highly acidic. Refer to reagent MSDS sheets for disposal information.

Description
Quantity
Powder Pillow Test:

ChromaVer 3 Chromium Reagent Powder Pillows
Sample cells, 10-mL
AccuVac Test:
ChromaVer 3 AccuVac Ampuls
Beaker, 50-mL
Page 427
Description
Quantity
Sample cell, 10-mL round, with cap
1,5-Diphenylcarbohydrazide for powder pillows
Stored Programs
90 Chromium, Hex.
Start
1. Select the test.


10 mL of sample.

for orientation.
mL of sample.
A purple color will form if

hexavalent chromium is
present.
6. When the timer

expires, insert the blank
display will show:

0.000 mg/L Cr6+
Page 428
3. Prepared Sample:
ChromaVer 3 Reagent
sample cell. Swirl to mix.

READ the results in
mg/L Cr6+.

timer.
period will begin.
1,5-Diphenylcarbohydrazide for AccuVac Ampuls
Stored Programs
95 Chromium, Hex. AV
Start
1. Select the test.

3. Prepared Sample:
Fill a ChromaVer 3
with sample from the
beaker. Keep the tip
fills completely.
6. When the timer

holder.
7. Wipe the Ampul and


for orientation.

timer.
period will begin.

Ampul several times
to mix.
READ the results in

mg/L Cr6+.

0.000 mg/L Cr6+
Interferences
Iron
May interfere above 1 mg/L
Mercurous & Mercuric Ions
Interfere slightly
pH
reagents and require sample pretreatment.
Vanadium
May interfere above 1 mg/L. Allow 10 minutes for the reaction period before reading.
Turbidity
For turbid samples, treat the blank with the contents of one Acid Reagent Powder Pillow1.
This will ensure that any turbidity dissolved by the acid in the ChromaVer 3 Chromium
Reagent will also be dissolved in the blank.
Page 429

Collect samples in a cleaned glass or plastic container. Store at 4 C (39 F) up to 24 hours.
Samples must be analyzed within 24 hours.
Accuracy check
Chromium Voluette Ampule Standard, 12.5 mg/L Cr6+
Ampule Breaker

or edited. After values are accepted, the unspiked sample reading will appear in the top row.
See the user manual for more information.
4. Open a Chromium Voluette Ampule Standard, 12.5 mg/L Cr6+.
5. For analysis using powder pillows, use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL
of standard, respectively to three 25-mL samples and mix thoroughly. Transfer 10 mL of each
solution into a 10-mL sample cell and analyze as described above.
Note: For AccuVac Ampuls, fill three mixing cylinders* with 50-mL of sample and spike with 0.2 mL, 0.4 mL
and 0.6 mL of standard. Transfer 40 mL from each of the three mixing cylinders to three 50-mL beakers.
Analyze each standard addition sample as described in the procedure above.
6. Accept each standard additions reading. Each addition should reflect approximately 100%
recovery.
Prepare a 0.50-mg/L Cr6+ standard solution daily, as follows:
1. Using a 5.00 mL pipet transfer 5.00 mL of Hexavalent Chromium Standard Solution, 50 mg/L,
into a Class A 500-mL volumetric flask.
2. Dilute to the mark with deionized water. Perform the test procedure as described above.
3. To adjust the calibration curve using the reading obtained with the 0.50-mg/L Standard
Solution, select Options>More>Standard Adjust from the instrument menu.
Note: Refer to the instrument user manual for specific software navigation instructions.
Page 430
Method performance
Standard
Precision
Distribution
Sensitivity
90
0.500 mg/L Cr6+
0.4970.503 mg/L Cr6+
0.005 mg/L Cr6+
95
0.500 mg/L Cr6+
0.4960.504 mg/L Cr6+
0.006 mg/L Cr6+
Program
Summary of method
Hexavalent chromium is determined by the 1,5-Diphenylcarbohydrazide method using a single dry
powder formulation called ChromaVer 3 Chromium Reagent. This reagent contains an acidic
buffer combined with 1,5-Diphenylcarbohydrazide, which reacts to give a purple color when
hexavalent chromium is present. Test results are measured at 540 nm.
Page 431

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
1271099
25/pkg
2505025
varies
4L
27256
Quantity
Unit
Catalog number
50041H

OR
ChromaVer 3 AccuVac Ampuls
Deionized Water
Required apparatus
Description
Beaker, 50-mL
each
Stopper for 18 mm Tube
6/pkg
173106
AccuVac Snapper
each
2405200
Description
Unit
Catalog number
Chromium, Hexavalent Standard Solution, 10-mL Voluette Ampules, 12.5-mg/L Cr6+
16/pkg
1425610
Chromium, Hexavalent Standard Solution, 50.0-mg/L Cr6+
100 mL
81042H
Unit
Catalog number

Description
100/pkg
212699
Ampule Breaker
Acid Reagent Powder Pillow
each
2196800
each
1457449
Pipet, Volumetric 5.00 mL
each
1451537
Pipet FIller, safety bulb
each
1465100
50 mL SCDB
245026

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chromium, Total, 8024
Chromium, Total
DOC316.53.01034
Alkaline Hypobromite Oxidation Method1, 2

0.01 to 0.70 mg/L Cr
Method 8024
Powder Pillows

1
This procedure is equivalent to Standard Method 3500-Cr D for wastewater.
Test preparation


Instrument
Sample cell
Orientation
DR 6000
2495402 (2) and 2401906 (1)
DR 5000
2495402 (2) and 2401906 (1)
DR 3900
2495402 (2) and 2401906 (1)
DR 3800, DR 2800, DR 2700
2495402 (2) and 2401906 (1)

deionized water instead of the sample. Subtract the reagent blank value from the final results or perform a reagent
blank adjust.
Prepare a boiling water bath for step 4. Use finger cots to handle hot sample cells.

Description
Quantity
ChromaVer
3 Chromium Reagent Powder Pillows
Chromium 1 Reagent Powder Pillows
Hot Plate
Water Bath and Rack
Finger Cots
1 pair
Sample Cells (Instrument-specific information)
varies
Chromium, Total
Page 433
Chromium, Total
Alkaline Hypobromite Oxidation method for powder pillows
Stored Programs
100 Chromium, Total
Start
1. Select the test.

2. Fill a 25-mL sample

period will begin.

one ChromaVer 3
Chromium Reagent
mix.
Chromium, Total
Page 434
4. Remove the cap.

Insert the prepared
sample into a boiling water
bath.
Swirl to mix.

for orientation.

timer.
3. Prepared Sample:
Chromium 1 Reagent
Powder Pillow.
6. When the timer

expires, remove the
prepared sample. Cap the
cell and use running water
to cool the cell to 25 C.
7. Remove cap and add

the contents of one
Chromium 2 Reagent
Powder Pillow. Cap and
invert to mix.

Pillow. Swirl to mix.

timer.
11. While the sample is

reacting, pour 10 mL from
the mixing bottle into a
second sample cell
information). This is the
prepared sample.
period will begin.
When the timer expires, fill

another sample cell with
10 mL of original sample.
Chromium, Total
Alkaline Hypobromite Oxidation method for powder pillows (continued)
Zero


0.00 mg/L Cr
Read

the cell holder.

mg/L Cr.
Interferences

extreme sample pH
Organic material
May inhibit complete oxidation of trivalent chromium. If high levels of organic material are
present, digestion may be required. Perform the analysis as described in this procedure on the
digested sample.
Turbidity
For turbid samples, treat a 25-mL blank and the sample the same during steps 38. Use this
solution as the blank.
Collect samples in acid-washed glass or plastic containers.
To preserve samples, adjust the pH to 2 or less with nitric acid. This requires approximately 2
mL per liter of the acid.
Store preserved samples at room temperature up to six months.
Adjust the pH to about 4 with 5.0 N Sodium Hydroxide before analysis.
Accuracy check
Trivalent Chromium Voluette Ampule Standard, 50-mg/L as Cr3+.
Ampule Breaker
Mixing cylinders

5. Prepare a 12.5 mg/L standard by pipetting 5.00 mL of Trivalent Chromium Standard Solution,
50 mg/L as Cr3+ into a mixing cylinder. Pipet 15.00 mL of demineralized water into the
cylinder. Stopper the cylinder and mix thoroughly.
Chromium, Total
Page 435
Chromium, Total
TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
10. Analyze each sample spike as described in the procedure above, starting with the smallest
sample spike. Accept each standard additions reading. Each addition should reflect
Prepare a 0.50-mg/L trivalent chromium standard as follows:

1. Dilute 5.00 mL of Trivalent Chromium Standard Solution, 50-mg/L as Cr3+, to 500 mL with
deionized water. Prepare this solution daily.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
90
0.500 mg/L Cr
0.470.53 mg/L Cr
0.005 mg/L Cr
Summary of method
Trivalent chromium in the sample is oxidized to the hexavalent form by hypobromite ion under
alkaline conditions. The sample is acidified. The total chromium content is determined by the 1,5Diphenylcarbohydrazide method. Determine trivalent chromium by subtracting the results of a
separate hexavalent chromium test from the results of the total chromium test. Test results are
measured at 540 nm.
Chromium, Total
Page 436
Chromium, Total

Required reagents
Description
Quantity/Test
Unit
Catalog number
2242500
100/pkg
212699
100/pkg
1206699
100/pkg
204399
100/pkg
204499
Quantity
Unit
Catalog number
each
1206701
Hot Plate, stirrer, 220 240 VAC
each
2881602
Water Bath and Rack
each
195555
Unit
Catalog number
100 mL
1415142
Description
Unit
Catalog number
Finger Cots
2/pkg
1464702
each
1457449
Pipette, volumetric, Class A, 5.00 mL
each
1451537
Tensette Pipet 0.11.0 mL
each
1970001
50/pkg
2185696
Total Chromium Reagent Set (100 tests), includes:
Required apparatus
Description
Hot plate, 3-inch diameter, 120 VAC, 50/60 Hz
OR
Description
Chromium, Trivalent, Standard Solution, 50-mg/L
Cr3+
Pipet Tips, Tensette

Pipet, Volumetric Class A, 15 mL
each
1451539
each
1465100
Cylinder, Mixing, 25 mL
each
189640
Chromium, Total
Page 437
Chromium, Total

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Cobalt, 8078
Cobalt
DOC316.53.01036
1-(2-Pyridylazo)-2-Naphthol (PAN) Method1
Method 8078
0.01 to 2.00 mg/L
Powder Pillows
Scope and Application: For water and wastewater; digestion is required for determining total recoverable cobalt;
if EDTA is present, use the vigorous digestion.
1
Adapted from Watanbe, H., Talanta, 21 295 (1974).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

adjust.
If the sample is less than 10 C (50 F), warm to room temperature prior to analysis.
Nickel can be determined with the same sample prepared with this method. Use program Number 340. A reagent blank is
necessary for the nickel procedure.
The Pour-Thru Cell can be used with 25-mL reagents only.
Total recoverable cobalt requires a prior digestion. See the Digestion section in the Water Analysis Guide.
For samples containing iron (Fe3+), all of the powder must dissolve before mixing the sample in step 6.
Cobalt
Page 439
Cobalt
Description
Quantity
EDTA reagent powder pillows
Phthalate-Phosphate reagent powder pillows
PAN indicator solution
1 mL
Water, deionized
25 mL
Sample cells (Instrument-specific information)
Stopper for sample cells
PAN method for powder pillows
Stored Programs
110 Cobalt
Start
1. Select the test.
2. Prepared Sample:
Fill a sample cell to the

10-mL mark with roomtemperature sample.

for orientation.
Cobalt
Page 440
room-temperature
deionized water.

one Phthalate-Phosphate
Reagent powder pillow to
each cell. Swirl to
completely dissolve.
Cobalt
PAN method for powder pillows (continued)
5. Use the plastic

dropper provided to add
0.5 mL of 0.3% PAN
Indicator Solution to each
cell.
6. Use a stopper to close

each cell.
Invert several times to mix.

timer. A three-minute
reaction time will begin.
During the reaction period,
the sample solution color
may vary from green to
dark red, depending on
the chemical makeup of
the sample. The deionized
water blank should be
yellow.
8. When the timer

expires, add the contents
of one EDTA Reagent
powder pillow to each cell.
Use a stopper to close
each cell.
Shake each cell to
dissolve.
Zero


0.00 mg/L Co
Read

the cell holder.

mg/L Co.
Interferences
Al3+
32 mg/L
Ca2+
1000 mg/L as CaCO3
Cd2+
20 mg/L
Cl
8000 mg/L
Cr3+
20 mg/L
Cr6+
40 mg/L
Cu2+
15 mg/L
Cobalt
Page 441
Cobalt
20 mg/L
Fe2+
Interferes directly and must not be present
Fe3+
10 mg/L
K+
500 mg/L
Mg2+
400 mg/L
Mn2+
25 mg/L
Mo6+
60 mg/L
Na+
5000 mg/L
Pb2+
20 mg/L
Zn2+
30 mg/L

require sample pretreatment.
Collect samples in acid-washed plastic bottles.
Adjust the sample pH to 2 or less with nitric acid* (about 5 mL per liter).
Preserved samples can be stored up to six months at room temperature.
Adjust the sample pH between 3 and 8 with 5.0 N Sodium Hydroxide Standard Solution* just
before analysis. Do not exceed pH 8 as this may cause some loss of cobalt as a precipitate.
Correct test results for volume additions.
Accuracy check
Prepare a 1.00 mg/L cobalt standard solution as follows:

1. Dilute 10.0 mL of a 10-mg/L working stock solution to 100 mL in a volumetric flask. Prepare
the 10-mg/L working stock solution daily by diluting 10.00 mL of Cobalt Standard Solution,
1000-mg/L as Co, to 1000 mL with deionized water.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
110
1.00 mg/L Co
0.991.01 mg/L Co
0.01 mg/L Co
Cobalt
Page 442
Cobalt
Summary of method
After buffering the sample and masking any Fe3+ with pyrophosphate, the cobalt is reacted with 1(2-Pyridylazo)-2-Naphthol indicator. The indicator forms complexes with most metals present.
After color development, EDTA is added to destroy all metal-PAN complexes except nickel and
cobalt, which can both be determined using the same sample.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2651600
(2) EDTA Reagent Powder Pillows
100/pkg
700599
(2) Phthalate-Phosphate Reagent Powder Pillows
100/pkg
2615199
Cobalt Reagent Set, 10-mL (100 tests), includes:
(1) PAN Indicator Solution, 0.3%
1 mL
100 mL
2150232
25 mL
4L
27256
Quantity
Unit
Catalog number
Stopper, rubber
6/pkg
173106
2/pkg
2495402
Unit
Catalog number
100 mL
2150342
Unit
Catalog number
Nitric Acid, 1:1
500 mL
254049
100 mL
245032
each
1451538
Water, deionized
Required apparatus
Description
Description
Cobalt Standard Solution, 1000-mg/L Co

Description
each
1457442
each
1457453
Pipet Filter, Safety bulb
each
1465100
100/pkg
2601300
pH paper
Cobalt
Page 443

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Color, True and Apparent, 8025
Color, True and Apparent

Platinum-Cobalt Standard Method1, 2, 3
DOC316.53.01037
Method 8025
15 to 500 units
Scope and Application: For water, wastewater and seawater; equivalent to NCASI method 253 and NCASI
Method Color 71.01 for pulp and paper effluent using 465 nm (requires pH adjustment).
1
Adapted from Standard Methods for the Examination of Water and Wastewater and National Council for Air and Stream Improvement
(NCASI) Methods Manual.
Adapted from Wat. Res. Vol. 30, No. 11, pp. 27712775, 1996.
NCASI Method 253 approved at 40 CFR part 136.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

The NCASI procedure requires pH adjustment. Adjust the pH to 7.6 with 1.0 N HCl or 1.0 N NaOH. When adjusting the pH, if
the overall volume change is greater than 1%, start over and use a stronger acid or base. Use program 125 when
performing the NCASI procedure.
One powder pillow of pH 8 Buffer (sodium phosphate/potassium phosphate) may be added to 50 ml of sample prior to final
pH adjustment to reduce sample dilution effects. Mix thoroughly to dissolve before making final pH adjustments.
To test for apparent color, omit steps 35 and step 7. Use unfiltered deionized water in step 6 and unfiltered sample in step 8.
For low level samples, use of a Pour-Thru Cell is recommended.

Description
Buffer, pH 8.0 (Program 125)
Hydrochloric Acid Solution, 1.0 N (Program 125)
Sodium Hydroxide, 1.00 N (Program 125)
Water, deionized
Quantity
1
varies
varies
100 mL
FIlter Apparatus: membrane filter, filter holder, filter flask and aspirator

Page 445

Description
Quantity
Stopper, rubber, one hole, No. 7
Tubing, rubber
Plantinum-Cobalt method
Stored Programs
120 Color, 455 nm
OR
125 Color, 465 nm
Start
NCASI: Use Program 125

for the NCASI test.
2. Collect 200 mL of
sample in a 400-mL
beaker.
NCASI: Adjust the pH as

described in Test
Preparation.
1. Select the test.

for orientation.
5. Filter another 50 mL of
deionized water through
the filter.
10-mL of filtered deionized
water from step 5.
Discard the excess water
in the flask.

Page 446
3. Assemble the filter

apparatus (0.45 micron
membrane filter, filter
holder, filter flask and
aspirator).
4. Filter about 50 mL of
deionized water to rinse
the filter. Discard the rinse
water.
NCASI: The NCASI test

prescribes a 0.8-micron
filter. A 1.0 micron prefilter
can be first used for
difficult to filter samples.
7. Filter about 50 mL of
sample through the filter.

a second sample cell with
10 mL of filtered sample.

Plantinum-Cobalt method (continued)
Zero

Read

0 units Pt-Co

the cell holder.

mg/L Pt-Co.
Collect samples in clean plastic or glass bottles. Most reliable results are obtained when
samples are analyzed as soon as possible after collection. If prompt analysis is impossible, fill
bottles completely and cap tightly.
Avoid excessive agitation or prolonged contact with air.
Samples can be stored for 24 hours by cooling to 4 C (39 F).
Warm to room temperature before analysis.
Accuracy check
Prepare a 250 platinum-cobalt units standard as follows:

1. Using Class A glassware, pipet 50.00 mL of a 500 Platinum-Cobalt Units Standard Solution
into a 100-mL volumetric flask. Dilute to the 100 mL mark with deionized water.
2. To adjust the calibration curve using the reading obtained with the 250 unit Standard Solution,
Select Options>More>Standard Adjust from the instrument menu.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
120
250 units Pt-Co
245255 units Pt-Co
16 units Pt-Co
125
250 units Pt-Co
245255 units Pt-Co
16 units Pt-Co

Page 447
Summary of method
Color may be expressed as apparent or true color. The apparent color includes that from
dissolved materials plus that from suspended matter. By filtering or centrifuging out the suspended
materials, the true color can be determined. The procedure describes true color analysis. If
apparent color is desired, it can be determined by measuring an unfiltered water sample. The
same stored program is used for both forms of color.
The stored program is calibrated in color units based on the APHA-recommended standard of 1
color unit being equal to 1 mg/L platinum as chloroplatinate ion. Test results for Programs 120 and
125 are measured at 455 and 465 nm, respectively.

Required reagents
Description
Quantity/Test
Unit
Catalog number
15/pK
1407995
Hydrochloric Acid Solution, 1.0 N
varies
1L
2321353
varies
1L
104553
100 mL
4L
27256
Catalog number
Buffer, pH 8.0
Water, deionized
Required apparatus
Description
Quantity
Unit
Aspirator, Nalgene vacuum pump
each
213100
Filter, membrane, 47-mm, 0.8-microns, Program 125
100/pkg
2640800
Filter, membrane, 47-mm, 0.45-microns, Program 120
100/pkg
1353000
each
54649
6/pkg
211907
Tubing, rubber, 7.9 x 2.4 mm

Filter Holder, 47 mm, 300 mL graduated
varies
12 ft
56019
each
1352900
Beaker, 400 mL
each
50048
2/pkg
2495402
Description
Unit
Catalog number
Color Standard Solution, 500 platinum-cobalt units
1L
141453
1L
2602853
Color Standard Solution, 500 platinum-cobalt units, 10-mL Voluette Ampules
16/pkg
141410
Filter, Glass Microfiber, 47-mm, 1.0 micron
100/pk
2551400
each
1457442
Pipet, volumetric, Class A, 50.00 mL
each
1451541
each
1465100

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Color, ADMI, 10048
Color, ADMI
DOC316.53.01122
ADMI Weighted Ordinate Method1
Method 10048
(5 to 250 units Pt-Co)

Scope and Application: For water and wastewater where color characteristics are significantly different from
platinum-cobalt standards, or for water and wastewater with color characteristics similar in hue to the standards.
Turbid samples must be filtered prior to analysis.
1
Adapted from Allen, et. al., 1973. Determination of color of water and wastewater by means of ADMI Color Values. Proc. 28th Ind. Waste
Conf., Purdue Univ., Eng. Ext. Ser. No. 142:661
Test preparation
The ADMI color method is available on the DR 5000 and DR 6000 spectrophotometer. One of
three sample cell sizes can be used (refer to Sample cell options). Select the test that corresponds
to the sample cells used (refer to step 1 of the procedure).
Table 136 Sample cell options

Program
Sample cell
Cell orientation
97
2495402
96
2095100
Clear side toward user
98
2629250
Clear side toward user

If the sample cannot be analyzed immediately, see Sample collection, preservation and storage.
Make pH adjustments with a minimum volume of acid or base. Make major adjustments with strong acid or base, then finetune with the 0.1 N solution.
The sample cells shown in the procedure are a generic representation. Each of the three stored programs uses a cell of a
different shape.

Description
Quantity
Sodium Hydroxide or Sulfuric Acid Solution, 10 N
varies
Sodium Hydroxide or Sulfuric Acid Solution, 0.100 N
varies
Water, deionized
50 mL
Beaker, 250 mL polypropylene
Cylinder, graduated, 100 mL polypropylene
Filter Apparatus: membrane filter, filter holder, filter flasks (2), tubing, stopper and aspirator
pH meter with electrode
Sample Cells, 1-inch square, 1 cm (10 mm) or 5 cm (50 mm)
Color, ADMI
Page 449
Color, ADMI
ADMI weighted ordinate method
Stored Programs
97 Color ADMI 1 inch
96 Color ADMI 10 mm
98 Color ADMI 50 mm
Start
1. Select the test.

Insert the multi-cell
adapter. Refer to Sample
cell options for cell
orientation.
2. If the sample is not

turbid, omit steps 36.
Pour two 100-mL aliquots
of sample into separate
beakers. Adjust the pH of
one of the aliquots to 7.6;
leave the other aliquot
as is.
3. Assemble the filtering

apparatus (0.45 micron
membrane filter, filter
holder, filter flask, and
aspirator).
4. Rinse the filter by

pouring about 50 mL of the
original sample aliquot
through the filter. Discard
the rinse water.
7. Prepared Sample:
Fill a sample cell with the
pH-adjusted sample.
Discard the excess.
Discard the excess water
in the flask.
Use 10 N sodium
hydroxide or concentrated
sulfuric acid to adjust the
pH. Use 0.1 N acid or base
near the end point.
5. Pour another 50 mL of
the original sample aliquot
through the filter.
Label the flask original.
Color, ADMI
Page 450
6. Repeat steps 35 for

the pH adjusted sample
and label it pH adjusted.
Color, ADMI
ADMI weighted ordinate method (continued)
Zero

Close the light shield.

The instrument will read
the percent transmittance
(%T) at 10-nm intervals
from 700 nm to 400 nm.
Read

sample (pH adjusted) and
Close the light shield.
12. READ the results.

the percent transmittance
(%T) at 10-nm intervals
from 700 nm to 400 nm.
When finished, the
instrument will display the
ADMI color value of the
pH-adjusted sample.
Repeat steps 712 for the
original sample.
For USEPA reporting,
report both results.
Interferences
Turbidity interferes directly and must be removed using filtration.
samples are analyzed as soon as possible after collection.
If prompt analysis is impossible, fill bottles completely and cap tightly.
Avoid excessive agitation or prolonged contact with air.
Samples can be stored for 24 hours by cooling to 4 C (39 F).
Color, ADMI
Page 451
Color, ADMI
Accuracy check
Deionized water
20-mL Class A volumetric pipet and pipet bulb
1. Prepare a 100 platinum-cobalt units standard solution as follows:

a. Pipet 20 mL of Color standard, 500 platinum-cobalt units, into a 100-mL volumetric flask.
b. Dilute to the mark with deionized water. Mix well.
2. Use this solution in place of the sample. Follow the ADMI weighted ordinate method test
procedure. Omit filtration steps 36.
3. To adjust the calibration curve using the reading obtained with the standard solution, press
OPTIONS>MORE>STANDARD ADJUST.
Summary of method
Three properties describe color: hue, chroma and value. Hue is color, whether it be blue, red,
green, yellow, etc. Chroma is color intensity (bright or dull). Value is the amount of color (light or
dark). This method measures only the amount of color, or color value. It is independent of the hue
and chroma.
This method determines the color value in a sample. Transmittance is measured from 400 to 700
nm and converted to a set of abstract numbers. These numbers describe the color as seen by an
average human eye. They are converted to a single number that indicates the color value. This
number is expressed on a scale used by the American Dye Manufacturers Institute (ADMI) to
measure color value. The ADMI has adopted the Platinum-Cobalt standard of the American Public
Health Association (APHA) as the standard for color value. Although this standard is yellow, the
ADMI method works for all hues. Three calibrations are stored in the instrument. Each calibration
corresponds to one of the three sample cell pathlengths.
Color, ADMI
Page 452
Color, ADMI

Required reagents
Description
Quantity/Test
Unit
Catalog number
Sodium Hydroxide Solution, 10 N
varies
500 mL
2545049
varies
1000 mL
19153
Sulfuric Acid, 10 N
varies
1000 mL
93153
varies
100 mL MDB
20232H
Water, deionized
10 mL
4 liters
27256
Catalog number
Required apparatus
Description
Quantity
Unit
Beaker, 250-mL polypropylene
each
108046
Cylinder, graduated, 100-mL, polypropylene
each
108142
pH meter, with electrode
each
HQ11d
Sample cells, 10 mL square, matched pair
2/pkg
2495402
Description
Unit
Catalog number
1L
141453
Description
Unit
Catalog number
each
213100
Filter Holder, 47-mm, 300-mL graduated
each
1352900
Filter, membrane, 47-mm, 0.45-microns
100/pkg
1353000
each
54649
each
1457442
each
1465100
each
1451520
6/pkg
211907
Tubing, rubber
12 ft
56019
Color, ADMI
Page 453

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Copper, Bicinchoninate, 8506 and 8026
Copper
DOC316.53.01039
Method 8506
Method 8026
USEPA1 Bicinchoninate Method2
(0.04 to 5.00 mg/L)
Scope and Application: For water, wastewater and seawater3; Method 8506 USEPA approved for reporting
wastewater analysis (digestion required).4
1
Approved, USEPA and Standard Method 3500 Cu C or E
Adapted from Nakano, S., Yakugaku Zasshi, 82 486-491 (1962) [Chemical Abstracts, 58 3390e (1963)].
Pretreatment required; see Interferences (Using Powder Pillows).
Federal Register, 45 (105) 36166 (May 29, 1980).
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Digestion is required for determining total copper.
Adjust the pH of acid-preserved samples to 46 with 8 N KOH before analysis.
adjust.
If copper is present, the sample will turn purple when it mixes with the reagent powder.
Accuracy is not affected by undissolved reagent.
Copper
Page 455
Copper

Description
Quantity
Powder Pillow Test:

CuVer 1 Copper Reagent powder pillow
Sample Cells, powder pillow test (Instrument-specific information)
AccuVac Test:
CuVer 2 Copper Reagent AccuVac Ampul
Beaker, 50-mL
Sample Cell, AccuVac test (Instrument-specific information)
Bicinchoninate method for powder pillows (Method 8506)
Stored Programs
135 Copper, Bicin.
Start
1. Select the test.

for orientation.
Copper
Page 456

a cell with 10 mL of
sample.

one CuVer 1 Copper
Reagent powder pillow to
the prepared sample cell.
Swirl sample cell to mix.
Use a CuVer 2 Copper
Reagent powder pillow for
samples containing high
levels of aluminum, iron
and hardness. A 25-mL
sample cell is required
(Interfering substances
and suggested treatments
for powder pillows).

timer. A two-minute
Copper
Bicinchoninate method for powder pillows (Method 8506) (continued)
Zero
10 mL of sample.

the cell holder.

0.00 mg/L Cu
8. Within 30 minutes
Cu.
Bicinchoninate method for AccuVac Ampuls (Method 8026)
Stored Programs
140 Copper, Bicin. AV
Start
1. Select the test.

for orientation.
3. Prepared Sample:
Fill a CuVer 2 AccuVac
Ampul with sample from
the beaker. Keep the tip
fills completely.
4. Place the stopper and

quickly invert the Ampul
several times to mix. Wipe
off all liquid and
fingerprints with a cloth or
soft paper towel.
Copper
Page 457
Copper
Bicinchoninate method for AccuVac Ampuls (Method 8026) (continued)
Zero

timer.
period will begin.
6. When the timer

holder.

0.00 mg/L Cu
8. Within 30 minutes
READ the results in
mg/L Cu.
Interferences
The Interfering substances and suggested treatments for powder pillows table suggests
treatments for powder pillows. The Interfering substances and suggested treatments for AccuVac
Ampuls tables suggests treatments for AccuVac Ampuls. To differentiate free copper from that
complexed to EDTA or other complexing agents, use a 25-mL sample cell and Free Copper
Reagent Powder Pillow instead of the CuVer 1 Powder Pillow in step 3. Add a Hydrosulfite
Reagent Powder Pillow to the same sample and re-read the result. This result will include the total
dissolved copper (free and complexed). Unlike CuVer 1 Reagent, CuVer 2 Reagent Powder
Pillows and AccuVac Ampuls react directly with copper, which is complexed by chelants such as
EDTA.
Table 138 Interfering substances and suggested treatments for powder pillows
Acidity
If the sample is extremely acidic (pH 2 or less) a precipitate may form. Add 8 N Potassium
Hydroxide Standard Solution drop-wise until sample pH is above 4. Continue with step 3.
Aluminum, Al3+
Follow the powder pillow procedure above, but substitute a CuVer 2 Copper Reagent Powder
Pillow for the CuVer 1 Pillow used in step 3. Results obtained will include total dissolved
copper (free and complexed). Requires a 25-mL sample volume.
Cyanide, CN
Prevents full color development. Before adding the CuVer 1 Powder Pillow Reagent, add 0.2
mL of formaldehyde to the 10-mL sample. Wait 4 minutes before taking the reading. Multiply
the test results by 1.02 to correct for sample dilution by the formaldehyde.
Hardness
Iron, Fe3+
Silver, Ag+
If a turbidity remains and turns black, silver interference is likely. Add 10 drops of saturated
Potassium Chloride Solution to 75 mL of sample, followed by filtering through a fine or highly
retentive filter. Use the filtered sample in the procedure.
Copper
Page 458
Copper
Table 139 Interfering substances and suggested treatments for AccuVac Ampuls
Acidity
If the sample is extremely acidic (pH 2 or less) a precipitate may form. Add 8 N Potassium
Hydroxide Standard Solution drop-wise until sample pH is above 4. Continue with step 3.
Aluminum, Al3+
Reagents accommodate high levels.
Cyanide, CN
Prevents full color development. Add 0.5 mL of formaldehyde per 25-mL of sample before
using CuVer 2 Reagent AccuVac Ampul. Wait 4 minutes before taking the reading. Multiply the
test results by 1.02 to correct for sample dilution by the formaldehyde.
Hardness
Iron, Fe3+
Silver, Ag+
If a turbidity remains and turns black, silver interference is likely. Add 10 drops of saturated
Potassium Chloride Solution to 75 mL of sample, followed by filtering through a fine or highly
retentive filter. Use the filtered sample in the procedure.
Collect samples in acid-cleaned glass or plastic containers.
Adjust the pH to 2 or less with concentrated nitric acid (about 2 mL per liter).
Store preserved samples up to six months at room temperature.
Before analysis, adjust the pH to 46 with 8 N Potassium Hydroxide. Do not exceed pH 6, as

copper may precipitate.
If only dissolved copper is to be determined, filter the sample before acid addition.
Accuracy check
Copper Standard Solution, 100 mg/L Cu
Mixing cylinders (3)
Beakers (3)

1. Prepare a 12.5 mg/L Copper standard by pipetting 5.0 mL of Copper standard solution,
100mg/L into a 50 mL mixing cylinder. Dilute the solution to 40 mL, stopper and mix.
5. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to the unspiked sample. Press
the timer icon. After the timer expires, read the result.
Press the timer icon. After the timer expires, read the result.
Copper
Page 459
Copper
Press the timer icon. After the timer expires, read the result. Each addition should reflect
Note: For AccuVac Ampuls, fill three mixing cylinders with 50-mL of sample and spike with 0.2 mL, 0.4 mL
and 0.6 mL of Copper Voluette Ampule Standard, 75-mg/L Cu. Transfer 40 mL from each of the three
mixing cylinders to three 50-mL beakers. Analyze each standard addition sample as described in the
procedure above. Accept each standard additions reading by pressing READ. Each addition should
Prepare a 4.00-mg/L Standard as follows:

1. Using Class A glassware, pipet 4.00 mL of Copper Standard Solution, 100-mg/L as Cu, into a
100-mL volumetric flask. Dilute to volume with deionized water, stopper and invert to mix.
Perform the procedure as described above.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
135
1.00 mg/L Cu
0.971.03 mg/L Cu
0.04 mg/L Cu
140
1.00 mg/L Cu
0.971.03 mg/L Cu
0.03 mg/L Cu
Summary of method
Copper in the sample reacts with a salt of bicinchoninic acid contained in CuVer 1 or CuVer 2
Copper Reagent to form a purple colored complex in proportion to the copper concentration. Test

Required reagents
Description
CuVer
1 Copper Reagent Powder Pillows
Quantity/Test
Unit
Catalog number
100/pkg
2105869
25/pkg
2504025
OR
CuVer 2 Copper Reagent AccuVac Ampuls
Copper
Page 460
Copper
Required apparatus
Description
Quantity
Unit
Catalog number
Beaker, 50-mL
each
50041H
6/pkg
173106
Unit
Catalog number
Description
Copper Standard Solution, 100-mg/L as Cu
100 mL
12842
Copper Voluette Ampule Standard, 75-mg/L as Cu, 10-mL
16/pkg
1424710
Metals Drinking Water Standard, LR for Cu, Fe, Mn
500 mL
2833749
Metals Drinking Water Standard, HR for Cu, Fe, Mn
500 mL
2833649

Description
Unit
Beaker, 50-mL
each
500-41H
100/pkg
2188299
each
189641
100 mL MDB
205932
CuVer 2 Copper Reagent Powder Pillow

Cylinder, mixing,. 50 mL
Formaldehyde, ACS
Catalog number
Nitric Acid, concentrated
500 mL
15249
Potassium Chloride Solution
100 mL
76542
Potassium Hydroxide Standard Solution, 8 N

Reagent Set for Free and Total Copper, includes:
100 mL MDB
28232H
each
2439200
Hydrosulfite Reagent Powder Pillows
100/pkg
2118869
Free Copper Reagent Powder Pillows
100/pkg
2182369
2612602
Sample Cells, 25 mLmatched, 1 square
2/pkg
AccuVac Snapper
each
2405200
Ampule Breaker
each
2196800
Sample Cells, 25 mm round
6/pkg
2401906
Copper
Page 461
Copper

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Copper, LR, 8143
Copper
DOC316.53.01038
Porphyrin Method1
Method 8143
LR (1 to 210 g/L)
Powder Pillows
Scope and Application: For water, wastewater and sea water

1
Adapted from Ishii and Koh, Bunseki Kagaku, 28 (473), 1979
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Digestion is required for determining total copper.
adjust.
Wash all glassware with detergent. Rinse with tap water. Rinse again with 1:1 Nitric Acid Solution. Rinse a third time with
copper-free, deionized water.
If samples contain high levels of metals, a slight metallic deposit or yellow buildup may form in the sample cell. Wash the cell
as described above.

Description
Quantity
Copper Masking Reagent Powder Pillows
Porphyrin 1 Reagent Powder Pillows
Porphyrin 2 Reagent Powder Pillows

2
varies
2
Copper
Page 463
Copper
Porphyrin method
Stored Programs
145 Copper, Porphyrin
Start
1. Select the test.

2. FIll two sample cells


for orientation.

one Porphyrin 2 Reagent
powder pillow to each
sample cell.
6. Swirl to dissolve.
If copper is present the
sample will briefly turn
blue, then return to yellow.
Zero

0 g/L Cu
Copper
Page 464
Copper Masking Reagent
powder pillow to one of the
sample cells to create the
blank. Swirl to dissolve.

one Porphyrin 1 Reagent
powder pillow to each
sample cell.

timer. A three-minute
8. When the timer

expires insert the blank
Read


g/L Cu.
Swirl to dissolve.
Copper
Interferences
Al3+
60 mg/L
Cadmium, Cd2+
10 mg/L
Calcium, Ca2+
1500 mg/L
Chelating agents
Interfere at all levels unless either the Digesdahl or vigorous digestion is

performed
Chloride, Cl
90,000 mg/L
Aluminum,
Chromium,
Cobalt,
Cr6+
110 mg/L
Co2+
100 mg/L
Fluoride, F
30,000 mg/L
Iron, Fe2+
6 mg/L
Lead, Pb2+
3 mg/L
Magnesium
10,000 mg/L
Manganese
140 mg/L
Mercury, Hg2+
3 mg/L
Molybdenum
11 mg/L
Nickel,
Ni2+
60 mg/L
Potassium, K+
60,000 mg/L
Sodium, Na+
90,000 mg/L
Zinc, Zn2+
9 mg/L
May exceed the buffering capacity of the reagents and require sample
pretreatment.
To preserve, adjust the pH to 2 or less with nitric acid (about 5 mL per liter).
Before testing, adjust the pH of the preserved sample to between 2 and 6. If the sample is too
acidic, adjust the pH with 5.0 N Sodium Hydroxide Standard Solution*.
Accuracy check
Copper Standard Solution, 4 mg/L Cu Pour-Rite Ampules
Copper
Page 465
Copper
7. Fill eight sample cells with 10 mL of sample. Use the TenSette Pipet to add 0.1 mL from a
4-mg/L Pour-Rite Ampule, to two of the sample cells. Then pipet 0.2 mL of the standard
solution into two more cells. Finally, pipet 0.3 mL of the standard solution into two more cells.
8. Analyze each standard addition sample as described in the procedure above, using one of the
two spiked samples in each set as the blank. Accept each standard additions reading by
pressing READ. The copper concentration reading should reflect approximately 100%
recovery.
relationship between the sample spikes and the "Ideal Line" of 100% recovery.
1. To assure the accuracy of the test, prepare a 150-g/L copper standard by pipetting 15.00 mL
of Copper Standard Solution, 10.0-mg/L Cu, into a 1000-mL volumetric flask.
2. Dilute to the mark with copper-free, reagent-grade water. Prepare this solution daily. Perform
the copper procedure as described above.
3. To adjust the calibration curve using the reading obtained with the 150-g/L Standard Solution,
Method performance
Program
Standard
Precision
Distribution
Sensitivity
145
50 g/L Cu
4753 g/L Cu
1 g/L Cu
Summary of method
The porphyrin method is very sensitive to trace amounts of free copper. The method is free from
most interferences and does not require any sample extraction or concentration before analysis.
Interferences from other metals are eliminated by the copper masking reagent. The porphyrin
indicator forms an intense, yellow-colored complex with any free copper present in sample. Test
Copper
Page 466
Copper

Description
Quantity/Test
Unit
2603300
(1) Copper Masking Reagent Powder Pillows
100/pkg
2603449
(2) Porphyin 1 Reagent Powder Pillows
100/pkg
2603549
(2) Porphyrin 2 Reagent Powder Pillows
100/pkg
2603649
varies
500 mL
254049
2/pkg
2495402
Copper Reagent Set (100 tests), includes:

Catalog number
Description
Unit
Catalog number
Copper Standard Solution, 4 mg/L, 2 mL Pour-Rite Ampules
20/pkg
2605720
Copper Standard Solution, 10-mg/L Cu
100 mL
12932
4L
27256
Unit
Catalog number
Water, deionized

Description
Sodium Hydroxide Standard Solution, 5.0 N MDB
100 mL
245032
Tensette Pipet, 0.11.0
each
1970001
50/pkg
2185696
Pipet, Volumetric, Class A, 15 mL
each
1451539
each
1457453
each
1465100
Sample Cells, 1" square matched set
8/pkg
2495408
100/pkg
2601300
pH paper
Copper
Page 467

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Cyanide, 8027
Cyanide
DOC316.53.01040
Pyridine-Pyrazalone Method1
Method 8027
(0.002 to 0.240 mg/L CN )
Powder Pillows

1
Adapted from Epstein, Joseph, Anal. Chem. 19(4), 272 (1947).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402
Before starting the test

The sample cell shown is a generic representation. Refer to Instrument-specific information for the correct sample cell and
adapter configuration.
Use a water bath to maintain the optimum temperature for the reaction in this test (25 C). Samples at less than 23 C require
longer reaction times and samples at greater than 25 C yield low results.
blank adjust.
The timing for steps 310 is critical. You may find it useful to open the necessary reagents before starting this sequence.
All samples to be analyzed for cyanide should be treated by acid distillation except when experience has shown that there is
no difference in results obtained with or without distillation. See Acid distillation.
See Pollution prevention and waste management for proper disposal of solutions containing cyanide.

Description
Quantity
CyaniVer Cyanide 3 Reagent Powder Pillow
Sample Cells, 1-inch square glass
Cyanide
Page 469
Cyanide
Description
Quantity
Pyridine-Pyrazalone
Stored Programs
160 Cyanide
Start
1. Select the test.

Insert the adapter, if
5. Leave the sample cell

undisturbed for an
additional 30 seconds.
cylinder, fill a sample cell
3. Prepared Sample:
CyaniVer 3 Cyanide
Reagent Powder Pillow.
Cap the cell.
4. Shake the sample cell

for 30 seconds.

one CyaniVer 4 Cyanide
7. Shake the sample for

10 seconds. Immediately
proceed to step 8.
(Delaying the addition of
the CyaniVer 5 for more
than 30 seconds will
produce low test results.)

one CyaniVer 5 Cyanide
Cap the sample cell.

Undissolved reagent will
not affect accuracy.
Cyanide
Page 470
Cap the sample cell.
Cyanide
9. Shake the cell

vigorously.
If cyanide is present, a
pink color will develop.
10. Set the timer.

period will begin.
The solution will turn from
pink to blue.

10 mL of sample.

Samples less than 25 C

require a longer reaction
time. Samples greater
than 25 C give low test
results.
Zero

0.000 mg/L CN .

the cell holder. Review the
user manual for cell
orientation.
Results are in mg/L CN.
Cyanide
Page 471
Cyanide

Special considerations for materials containing cyanide
Samples analyzed by this procedure may contain cyanide, which is regulated as reactive (D003)
waste by the federal RCRA. It is imperative these materials be handled safely to prevent the
release of hydrogen cyanide gas (an extremely toxic material with the smell of almonds). Most
cyanide compounds are stable and can be safely stored for disposal in highly alkaline solutions
(pH >11) such as 2 N sodium hydroxide. Never mix these wastes with other laboratory wastes
which may contain lower pH materials such as acids or even water.
In the event of a spill or release, special precautions must be taken to prevent exposure to
hydrogen cyanide gas. The following steps may be taken to destroy the cyanide compounds in the
event of an emergency:
Use a fume hood or supplied air or self contained breathing apparatus.
While stirring, add the waste to a beaker containing a strong solution of sodium hydroxide and
calcium hypochlorite or sodium hypochlorite (household bleach).
Maintain a strong excess of hydroxide and hypochlorite. Let the solution stand for 24 hours.
Neutralize and flush the solution down the drain with a large excess of water.
Note: If the solution contains other regulated materials such as chloroform or heavy metals, it may still need
to be collected for hazardous waste disposal. Never flush hazardous wastes down the drain.
Interferences
Interfering
substance
Chlorine
Large amounts of chlorine in the sample will cause a milky white precipitate after the addition of the
CyaniVer 5 Reagent. If chlorine or other oxidizing agents are known to be present, pretreat the sample
before testing using the procedure in this table for oxidizing agents.
Metals
Nickel or cobalt in concentrations up to 1 mg/L do not interfere. Eliminate the interference from up to
20 mg/L copper and 5 mg/L iron by adding the contents of one HexaVer Chelating Reagent Powder
Pillow to the sample and then mixing before adding the CyaniVer 3 Cyanide Reagent Powder Pillow in
step 3. Prepare a reagent blank of deionized water and reagents to zero the instrument in step 12.
1.
2.
Oxidizing agents
3.
4.
5.
1.
2.
Reducing agents
3.
4.
5.
6.
Cyanide
Page 472
Adjust a 25-mL portion of the alkaline sample to pH 79 with 2.5 N Hydrochloric Acid Standard
Solution. Count the number of drops of acid added.
Add two drops of Potassium Iodide Solution and two drops of Starch Indicator Solution to the
sample. Swirl to mix. The sample will turn blue if oxidizing agents are present.
Add Sodium Arsenite Solution drop-wise until the sample turns colorless. Swirl the sample
thoroughly after each drop. Count the number of drops.
Take another 25-mL sample and add the total number of drops of Hydrochloric Acid Standard
Solution counted in step 1.
Subtract one drop from the amount of Sodium Arsenite Solution added in step 3. Add this amount to
the sample and mix thoroughly. Continue with step 2 of the cyanide procedure.
Adjust a 25-mL portion of the alkaline sample to pH 79 with 2.5 N Hydrochloric Acid Standard
Solution. Count the number of drops added.
Add four drops of Potassium Iodide Solution and four drops of Starch Indicator Solution to the
sample. Swirl to mix. The sample should be colorless.
Add Bromine Water drop-wise until a blue color appears. Swirl the sample thoroughly after each
addition. Count the number of drops.
Take another 25-mL sample and add the total number of drops of Hydrochloric Acid Standard
Solution counted in step 1.
Add the total number of drops of Bromine Water counted in step 3 to the sample and mix thoroughly.
Continue with step 2 of the cyanide procedure.
Cyanide
Interfering
substance
Turbidity
Large amounts of turbidity will cause high readings. Use filter paper and a funnel to filter highly turbid
water samples before use in steps 1 and 11. The test results should then be recorded as soluble cyanide.
Collect samples in glass or plastic bottles and analyze as quickly as possible.
The presence of oxidizing agents, sulfides and fatty acids can cause the loss of cyanide during
sample storage. Samples containing these substances must be pretreated as described below
before preservation with sodium hydroxide. If the sample contains sulfide and is not
pretreated, it must be analyzed within 24 hours.
Preserve the sample by adding 4.0 mL of 5.0 N Sodium Hydroxide Standard Solution to each
liter (or quart) of sample, using a glass serological pipet and pipet filler.
Check the sample pH; 4-mL of sodium hydroxide is usually enough to raise the pH of most
water and wastewater samples to 12. Add more 5.0 N Sodium Hydroxide if necessary.
Store the samples at 4 C (39 F) or less. Samples preserved in this manner can be stored for
14 days.
Before testing, samples preserved with 5.0 N Sodium Hydroxide or samples that are highly
alkaline due to chlorination treatment processes or sample distillation procedures should be
adjusted to approximately pH 7 with 2.5 N Hydrochloric Acid Standard Solution.
Correct the volume when significant amounts of preservative are used.
Oxidizing agents
Oxidizing agents such as chlorine decompose cyanides during storage. To test for their presence
and to eliminate their effect, pretreat the sample as follows:
1. Take a 25-mL portion of the sample and add one drop of 10-g/L m-Nitrophenol Indicator
Solution. Swirl to mix.
2. Add 2.5 N Hydrochloric Acid Standard Solution drop-wise until the color changes from yellow
to colorless. Swirl the sample thoroughly after the addition of each drop.
3. Add two drops of Potassium Iodide Solution, 30-g/L and two drops of Starch Indicator Solution,
to the sample. Swirl to mix. The solution will turn blue if oxidizing agents are present.
4. If step 3 suggests the presence of oxidizing agents, add two level, 1-g measuring spoonfuls of
Ascorbic Acid per liter of sample.
5. Withdraw a 25-mL portion of sample treated with ascorbic acid and repeat steps 1 to 3. If the
sample turns blue, repeat steps 4 and 5.
6. If the 25-mL sample remains colorless, preserve the remaining sample to pH 12 for storage
with 5 N Sodium Hydroxide Standard Solution.
7. Perform the procedure given under Interfering Substances and Levels, Reducing Agents, to
eliminate the effect of excess ascorbic acid, before following the cyanide procedure.
Cyanide
Page 473
Cyanide
Sulfides
Sulfides will quickly convert cyanide to thiocyanate (SCN ). To test for the presence of sulfide and
eliminate its effect, pretreat the sample as follows:
1. Place a drop of sample on a disc of Hydrogen Sulfide Test Paper that has been wetted with pH
4 Buffer Solution.
2. If the test paper darkens, add a 1-g measuring spoon of Lead Acetate to the sample. Repeat
step 1.
3. If the test paper continues to turn dark, keep adding Lead Acetate until the sample tests
negative for sulfide.
4. Filter the lead sulfide precipitate through Filter Paper and a Funnel. Preserve the sample for
storage with 5 N Sodium Hydroxide Standard Solution or neutralize to a pH of 7 for analysis.
Fatty acids
CAUTION
Perform this operation under a ventilation hood and complete as quickly as possible.
When distilled, fatty acids will pass over with cyanide and under the alkaline conditions of the
absorber, will form soaps. If the presence of fatty acid is suspected, use the following pretreatment
before preserving samples with sodium hydroxide.
1. Acidify 500 mL of sample to pH 6 or 7 with a 4:1 dilution of glacial Acetic Acid.
2. Pour the sample into a 1000-mL separation funnel and add 50 mL of Hexane.
3. Stopper the funnel and shake for one minute. Allow the layers to separate.
4. Drain off the lower, sample layer into a 600-mL beaker. If the sample is to be stored, add
enough 5 N Sodium Hydroxide Standard Solution to raise the pH above 12.
Acid distillation
All samples to be analyzed for cyanide should be treated by acid distillation except when
experience has shown that there is no difference in results obtained with or without distillation.
With most compounds, a one-hour reflux is adequate.
If thiocyanate is present in the original sample, a distillation step is absolutely necessary as
thiocyanate causes a positive interference. High concentrations of thiocyanate can yield a
substantial quantity of sulfide in the distillate. The rotten egg smell of hydrogen sulfide will
accompany the distillate when sulfide is present. The sulfide must be removed from the distillate
prior to testing.
If cyanide is not present, the amount of thiocyanate can be determined. The sample is not distilled
and the final reading is multiplied by 2.2. The result is mg/L SCN .
The distillate can be tested and treated for sulfide after the last step of the distillation procedure by
using the following lead acetate treatment procedure.
1. Place a drop of the distillate (already diluted to 250 mL) on a disc of Hydrogen Sulfide Test
Paper that has been wetted with pH 4 Buffer Solution.
2. If the test paper darkens, add 2.5 N Hydrochloric Acid Standard Solution by drops to the
distillate until a neutral pH is obtained.
3. Add a 1-g measuring spoon of Lead Acetate to the distillate and mix. Repeat step 1.
4. If the test paper continues to turn dark, keep adding lead acetate until the distillate tests
negative for sulfide. Filter the black lead sulfide precipitate through filter paper and a funnel.
Neutralize the liquid filtrate to pH 7 and immediately analyze for cyanide.
Cyanide
Page 474
Cyanide
Distillation procedure
The following steps describe the distillation process using distillation apparatus and cyanide
glassware offered by the manufacturer:
1. Set up the distillation apparatus for cyanide recovery, leaving off the thistle tube. Refer to the
Distillation Apparatus Manual. Turn on the water and make certain it is flowing steadily through
the condenser.
2. Fill the distillation apparatus cylinder to the 50-mL mark with 0.25 N Sodium Hydroxide
Standard Solution.
3. Fill a clean 250-mL graduated cylinder to the 250-mL mark with sample and pour it into the
distillation flask. Place a stirring bar into the flask and attach the thistle tube.
4. Arrange the vacuum system as shown in the Distillation Apparatus Manual, but do not connect
the vacuum tubing to the gas bubbler. Turn on the water to the aspirator to full flow and adjust
the flow meter to 0.5 SCFH.
5. Connect the vacuum tubing to the gas bubbler, making certain that air flow is maintained
(check the flow meter) and that air is bubbling from the thistle tube and the gas bubbler.
6. Turn the power switch on and set the stir control to 5. Using a 50-mL graduated cylinder, pour
50 mL of 19.2 N Sulfuric Acid Standard Solution* through the thistle tube and into the
distillation flask.
7. Using a water bottle, rinse the thistle tube with a small amount of deionized water.
8. Allow the solution to mix for three minutes; then add 20 mL Magnesium Chloride Reagent*
through the thistle tube and rinse again. Allow the solution to mix for 3 more minutes.
9. Verify that there is a constant flow of water through the condenser.
10. Turn the heat control to 10.
11. Carefully monitor the distillation flask at this point in the procedure. Once the sample begins to
boil, slowly lower the air flow to 0.3 SCFH. If the contents of the distillation flask begin to back
up through the thistle tube, increase the air flow by adjusting the flow meter until the contents
do not back up through the thistle tube. Boil the sample for one hour.
12. After one hour, turn off the still, but maintain the air flow for 15 minutes more.
13. After 15 minutes, remove the rubber stopper on the 500-mL vacuum flask to break the vacuum
and turn off the water to the aspirator. Turn off the water to the condenser.
14. Remove the gas bubbler/cylinder assembly from the distillation apparatus. Separate the gas
bubbler from the cylinder and pour the contents of the cylinder into a 250-mL, Class A
volumetric flask. Rinse the gas bubbler, cylinder and J-tube connector with deionized water
and add the washings to the volumetric flask.
15. Fill the flask to the mark with deionized water and mix thoroughly. Neutralize the contents of
the flask and analyze for cyanide.
Cyanide
Page 475
Cyanide
Accuracy check
Standard solutions method
CAUTION
Cyanides and their solutions and the hydrogen cyanide liberated by acids, are very
poisonous. Both the solutions and the gas can be absorbed through the skin.
Potassium Cyanide
Deionized water
Prepare a 100-mg/L cyanide stock solution weekly as follows:

1. Dissolve 0.2503 grams of Potassium Cyanide in deionized water and dilute to 1000 mL.
2. Immediately before use, prepare a 0.200 mg/L cyanide working solution by diluting 2.00 mL of
the 100 mg/L stock solution to 1000 mL using deionized water.
3. To adjust the calibration curve using the reading obtained with the 0.200 mg/L standard
solution, select Options>More>Standard Adjust from the instrument menu.
Method performance
Program
160
Standard
0.100 mg/L
CN
Precision
Distribution
0.0900.110 mg/L
CN
Sensitivity
Point of Curve
Concentration
Entire Range
0.002 mg/L CN
Summary of method
The Pyridine-Pyrazalone method used for measuring cyanide gives an intense blue color with free
cyanide. A sample distillation is required to determine cyanide from transition and heavy metal
cyanide complexes. Test results are measured at 612 nm.
Cyanide
Page 476
Cyanide

Required reagents
Description
Cyanide Reagent Set, includes:
(1) CyaniVer 3 Cyanide Reagent Powder Pillow
Quantity/Test
Unit
Catalog number
2430200
100/pkg
2106869
100/pkg
2106969
100/pkg
2107069
Catalog number
Required apparatus
Description
Quantity
Unit
each
50838
2/pkg
2495402
Stopper, poly, hollow
6/pkg
1448000
Description
Unit
Cat. No.
Potassium Cyanide, ACS
125 g
76714
4L
27256
Water, deionized

Description
Acetic Acid, ACS
Ascorbic Acid
Unit
Catalog number
500 mL
10049
100 g
613826
Bromine Water, 30 g/L
29 mL
221120
Buffer Solution, pH 4
500 mL
1222349
Filter Paper, 12.5 cm
100/pkg
189457
each
108367
Hexane Solution, ACS
500 mL
1447849
HexaVer Chelating Reagent Powder Pillow
100/pkg
24399
Hydrochloric Acid Standard Solution, 2.5 N
100 mL MDB
141832
100/pkg
2537733
Funnel, 65 mm
Hydrogen Sulfide Test Paper

m-Nitrophenol Indicator Solution, 10 g/L
Magnesium Chloride Reagent
Potassium Iodide Solution, 30 g/L
100 mL MDB
247632
1L
1476253
100 mL MDB
34332
Sodium Arsenite Solution, 5 g/L
100 mL
104732
1000 mL
1476353
1L
245053
100 mL MDB
34932
500 mL
203849
Cyanide Glassware
each
2265800
Distillation Apparatus, 115 VAC
each
2274400
Cyanide
Page 477
Cyanide
Description
Unit
Catalog number
Distillation Apparatus, 230 VAC
each
2274402
Distillation Apparatus Set
each
2265300
Pipet, seriological, 5 mL
each
53237
each
1465100
100/pkg
2601300
pH paper
Spoon, measuring, 1 g
each
51000
Thermometer, -10225 C
each
2635700

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Cyanuric Acid, 8139
Cyanuric Acid
DOC316.53.01183
Turbidimetric Method
Method 8139
5 to 50 mg/L
Powder Pillows
Scope and Application: For water.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Filter highly turbid samples with filter paper and a funnel.
Clean sample cells with soap, water, and a brush soon after each test to avoid a build-up of film on the sample cell.
Do not use the Pour-Thru Cell with this procedure.

Description
Quantity
Bottle, mixing, square glass
Cyanuric Acid 2 Reagent Powder Pillow
Sample cells (See Instrument-specific information)
Cyanuric Acid
Page 479
Cyanuric Acid
Turbidimetric method
Stored Programs
170 Cyanuric Acid
Start
1. Select the test.

2. Fill a mixing bottle with

25 mL of sample.

timer.
period will begin.
After adding the reagent a

white turbidity will form if
cyanuric acid is present.

for orientation.
10 mL of sample from the
mixing bottle.
3. Prepared Sample:
Cyanuric Acid 2 Reagent
powder pillow. Swirl to mix.

blank sample cell into the
cell holder.
7. When the timer

expires, fill a second
sample cell with 10 mL of
prepared sample from the
mixing bottle.
0 mg/L Cyan Acid
8. Within seven minutes

Cyanuric Acid.

Collect samples in clean plastic or glass bottles. Samples must be analyzed within 24 hours.
Accuracy check
1. Dissolve 1.000 gram of cyanuric acid in 1 liter of deionized water to make a 1000-mg/L
solution. Cyanuric acid is difficult to dissolve; it may take several hours to completely dissolve.
This solution is stable for several weeks.
2. Dilute 3.00 mL of the 1000-mg/L solution to 100 mL with deionized water to make a 30-mg/L
solution. Prepare fresh daily. Testing the 30-mg/L solution should give test results of about
30 mg/L cyanuric acid.
Cyanuric Acid
Page 480
Cyanuric Acid
Method performance
Program
170
Standard
10 mg/L cyanuric acid
Sensitivity
Precision
95% Confidence Limits
of Distribution
Portion of curve
Concentration change per

0.010 Abs change
713 mg/L cyanuric acid
10 mg/L
0.3 mg/L cyanuric acid
30 mg/L
50 mg/L
Summary of method
The test for Cyanuric Acid uses the turbidimetric method. Cyanuric Acid 2 Reagent precipitates
any Cyanuric Acid present and holds it in suspension. The amount of turbidity caused by the
suspended particles is directly proportional to the amount of cyanuric acid present. Test results are
measured at 480 nm.
Cyanuric Acid
Page 481
Cyanuric Acid

Required reagents
Description
Quantity/Test
Unit
Catalog number
50/pkg
246066
Cyanuric Acid 2 Reagent Powder Pillow
Required apparatus
Description
Quantity
Unit
Catalog number
Bottle, mixing, square, with 25 mL mark
each
1704200
2/pkg
2495402
Description
Unit
Catalog number
Cyanuric Acid
25 g
712924
Water, deionized
4L
27256
Unit
Catalog number
Balance, 600 g x 0.01 g, 100240 VAC
each
2937201
Brush, test tube
each
69000
10/pkg
189457
Flask, volumetric, 100 mL
each
2636642
each
2636653
Funnel, filter
each
108367
1L
2088153

Description
Filter, paper, for funnel filter
Liqui-nox Solution
each
1970010
50/pkg
2199796
each
1451503
each
1465100
Tips for Pipet, TenSette

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Fluoride, 8029
Fluoride
DOC316.53.01041
USEPA SPADNS Method1

0.02 to 2.00 mg/L
Method 8029
Reagent Solution or AccuVac Ampuls
Scope and Application: For water, wastewater and seawater; USEPA accepted for reporting for drinking and
wastewater analyses (distillation required; see Distillation in this procedure).2
1
Adapted from Standard Methods for the Examination of Water and Wastewater, 4500-F B & D.
Procedure is equivalent to USEPA method 340.1 for drinking water and wastewater.
Test preparation


Reagent solution
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

The sample and deionized water should be at the same temperature (1 C). Temperature adjustments may be made before
or after reagent addition.
SPADNS Reagent is toxic and corrosive. Use care while handling the reagent.
SPADNS Reagent contains sodium arsenite. Final solutions will contain arsenic (D004) in sufficient concentration to be
regulated as a hazardous waste for Federal RCRA. Refer to the MSDS for disposal instructions.
For best results, measure the volume of SPADNS Reagent as accurately as possible.
Do not use the Pour-Thru Cell with this test.

Description
Quantity
Solution Test:
SPADNS Reagent Solution
4 mL
Deionized Water
10 mL
Pipet, volumetric, 2-mL
Fluoride
Page 483
Fluoride
Description
Quantity
Pipet Filler Bulb
Sample cells (Instrument-specific information)
AccuVac Test:
SPADNS Fluoride Reagent AccuVac Ampuls
Deionized Water
40 mL
Beaker, 50-mL
Stopper
SPADNS reagent solution method
Stored Programs
190 Fluoride
Start
1. Select the test.

2. Prepared Sample:
Pipet 10.0 mL of sample
into a dry sample cell.
Pipet 10.0 mL of deionized
water into a second dry
sample cell.
4. Carefully pipet 2.0 mL

of SPADNS Reagent into
each cell. Swirl to mix.

for orientation.
Zero

timer.
period will begin.
Fluoride
Page 484
6. When the timer


0.00 mg/L F

READ the result in
mg/L F.
Fluoride
SPADNS AccuVac Ampuls
Stored Programs
195 Fluoride AV
Start
1. Select the test.

for orientation.

timer.
period will begin.
2. Prepared Sample:
Fill one SPADNS Fluoride
fills completely.
6. When the timer

holder.
Pour at least 40 mL of
second beaker.
4. Close with stopper

and quickly invert both
Ampuls several times
to mix.
Fill a second Ampul with

deionized water. Keep the
tip immersed while the
Ampul fills completely.
7. Insert the Ampul that

contains the prepared
READ the results in
mg/L F.

0.00 mg/L F
Fluoride
Page 485
Fluoride
Interferences
This test is sensitive to small amounts of interference. Glassware must be very clean (acid rinse
before each use). Repeat the test with the same glassware to ensure that results are accurate.
Interference level
Alkalinity (as CaCO3)
At 5000 mg/L it causes a 0.1 mg/L F error.
Aluminum
At 0.1 mg/L it causes a 0.1 mg/L F error.

To check for interferences from aluminum, read the
concentration one minute after reagent addition, then again
after 15 minutes. An appreciable increase in concentration
suggests aluminum interference. Waiting 2 hours before
making the final reading will eliminate the effect of up to
3.0 mg/L aluminum.
Chloride
At 7000 mg/L causes a +0.1 mg/L F error.
Chlorine
SPADNS Reagent contains enough arsenite to eliminate

interference up to 5 mg/L chlorine. For higher chlorine levels,
add one drop of Sodium Arsenite Solution1 to 25 mL of
sample for each 2 mg/L of Chlorine.
Iron, ferric
At 10 mg/L it causes a 0.1 mg/L F error.
Phosphate, ortho
At 16 mg/L it causes a +0.1 mg/L F error.
Sodium Hexametaphosphate
At 1.0 mg/L it causes a +0.1 mg/L F error.
Sulfate
At 200 mg/L it causes a +0.1 mg/L F error.
Distillation
Distillation Solution Preparation:
1. Measure 60 mL of deionized water into a 250 mL glass Erlenmeyer flask.
2. With constant stirring, add 120 mL of concentrated Sulfuric Acid. Caution: The mixture will
become very hot. Allow the solution to cool before handling.
To eliminate most interferences, dilute the sample from the acid solution as described
below:
1. Set up the distillation apparatus for general purpose distillation. Refer to the Distillation
Apparatus manual for proper assembly. Use a 125-mL Erlenmeyer flask to collect the distillate.
2. Turn on the water and maintain a steady flow through the condenser.
3. Measure 100 mL of sample into the distillation flask using a 100-mL graduated cylinder. Add a
magnetic stir bar and 5 glass beads.
4. Turn the stirrer power switch on. Turn the stir control to 5.
5. Using a 250-mL graduated cylinder, carefully add 150 mL of Distillation Solution into the flask.
Note: When distilling samples with high amounts of chloride, add 5 mg of Silver Sulfate* to the sample for
every mg/L of chloride in the sample.
Fluoride
Page 486
Fluoride
6. With the thermometer in place, turn the heat control to 10. The yellow pilot lamp indicates the
heater is on.
7. When the temperature reaches 180 C or when 100 mL of distillate has been collected, turn
the still off (requires about 1 hour).
8. Dilute the distillate to a volume of 100 mL, if necessary. The distillate may now be analyzed by
the SPADNS or the fluoride ion-selective electrode method.
Samples may be stored in glass or plastic bottles for up to 28 days when cooled to 4 C
(39 F) or lower.
Warm samples to room temperature before analysis.
Accuracy check
A variety of standard solutions covering the entire range of the test is available. Use these instead
of sample to verify technique.
Minor variations between lots of reagent become measurable above 1.5 mg/L. While results in this
region are usable for most purposes, better accuracy may be obtained by diluting a fresh sample
1:1 with deionized water and retesting. Multiply the result by 2.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
190
1.00 mg/L F
0.971.03 mg/L F
0.024 mg/L F at 1 mg/L
195
1.00 mg/L F
0.921.08 mg/L F
Summary of method
The SPADNS Method for fluoride determination involves the reaction of fluoride with a red
zirconium-dye solution. The fluoride combines with part of the zirconium to form a colorless
complex, thus bleaching the red color in an amount proportional to the fluoride concentration. This
method is accepted by the EPA for NPDES and NPDWR reporting purposes when the samples
have been distilled. Seawater and wastewater samples require distillation. Test results are
measured at 580 nm.
Fluoride
Page 487
Fluoride

Required reagents
Description
Quantity/Test
Unit
Catalog number
4 mL
500 mL
44449
25/pkg
2506025
1040 mL
4L
27256
SPADNS Reagent Solution

OR
SPADNS Fluoride Reagent AccuVac Ampuls
Water, deionized
Required apparatus (solution)

Description
Quantity
Unit
Catalog number
each
1465100
each
1451536
Pipet, volumetric, Class A, 10.00-mL.
each
1451538
2/pkg
2495402
each
187701
Catalog number
Required apparatus (AccuVac)

Description
Quantity
Unit
Beaker, 50-mL
each
50041H
each
2122800
6/pkg
2427606
Description
Unit
Catalog number
500 mL
40502
Fluoride Standard Solution, 0.5-mg/L F
500 mL
40505
500 mL
40508
1000 mL
29153
500 mL
29149
500 mL
40512
500 mL
40515
500 mL
40520
500 mL
23249
500 mL
2833049
Fluoride Standard Solution, 0.2-mg/L
Fluoride Standard Solution, 100-mg/L
Standard, Drinking Water, Mixed Parameter, Inorganic for F, NO3, PO4, SO4
Distillation reagents and apparatus

Description
Quantity
Unit
Catalog number
each
50842
each
50846
Distillation Heater and Support Apparatus Set,115 VAC, 50/60 Hz
each
2274400
OR
Fluoride
Page 488
Fluoride
Description
Distillation Heater and Support Apparatus Set, 230 VAC, 50/60 Hz
Quantity
Unit
Catalog number
each
2274402
AND
Distillation Apparatus Set, General Purpose
each
2265300
each
2089743
Glass Beads
100/pkg
259600
Stir Bar, magnetic
each
1076416
Sulfuric Acid, concentrated, ACS
500 mL
97949
Unit
Catalog number

Description
Silver Sulfate
113 g
33414
100 mL
104732
AccuVac Snapper,
each
2405200
Wipes, disposable
280/pkg
2097000
Sodium Arsenite Solution, 5.0 g/L
Fluoride
Page 489

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Fluoride SPADNS 2, 10225
Fluoride
DOC316.53.01184
USEPA1 SPADNS 22
(0.02 to 2.00 mg/L
Method 10225
F )
Scope and Application: For water, wastewater and seawater; USEPA accepted for reporting for drinking and
wastewater analyses (distillation required; see Distillation in this procedure).
1
Procedure is equivalent to USEPA method 340.1 for drinking water and wastewater analysis
Adapted from Standard Methods for the Examination of Water and Wastewater, 4500-F B & D.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

The sample and deionized water should be at the same temperature ( 1 C). Temperature adjustments may be made before
or after reagent addition.
SPADNS 2 Reagent is corrosive. Use care while handling the reagent.
For best results, measure the volume of SPADNS 2 Reagent as accurately as possible.
If the instrument displays Over Measure Range!, dilute a fresh sample with an equal volume of deionized water and repeat
the test, using this solution in step 2. Multiply the result by 2.
SPADNS 2 Reagent contains a non-toxic reducing agent to prevent chlorine interference. SPADNS 2 does not contain
sodium arsenite.
Fluoride
Page 491
Fluoride

Description
Quantity
Solution test
SPADNS 2 Reagent Solution
4 mL
Deionized Water
10 mL
Pipet Filler Bulb
Thermometer
AccuVac test
SPADNS 2 Fluoride Reagent AccuVac Ampuls
Deionized Water
40 mL
Beaker, 50-mL
SPADNS 2 reagent solution
Stored Programs
190 Fluoride
Start
1. Select the test.

Fluoride
Page 492
2. Prepared Sample:
Pipet 10.0 mL of sample
water into a second dry
sample cell.
4. Carefully pipet 2.0 mL

of SPADNS 2 Reagent into
Fluoride
SPADNS 2 reagent solution (continued)
Zero

timer.
6. When the timer

expires, insert the blank.
period will begin.
7.

0.00 mg/L F

sample cell.
READ the results in
mg/L F.
SPADNS 2 AccuVac Ampuls
Stored Programs
195 Fluoride AV
Start
1. Select the test.

2. Prepared Sample:
Fill one SPADNS 2
Fluoride Reagent AccuVac
Ampul with sample. Keep
the tip immersed while the
Pour at least 40 mL of
second beaker.
4. Quickly invert both

Ampuls several times
to mix.

Fluoride
Page 493
Fluoride
SPADNS 2 AccuVac Ampuls (continued)

timer.
period will begin.
6. When the timer


sample into the cell holder
READ the results in
mg/L F.

0.00 mg/L F
Interferences
This test is sensitive to small amounts of interference. Glassware must be very clean (acid rinse
before each use). Repeat the test with the same glassware to make sure that the results are
accurate.

Interference level
Alkalinity (as CaCO3)
At 5000 mg/L causes a 0.1 mg/L F error
Aluminum
At 0.1 mg/L causes a 0.1 mg/L F error. To check for interferences from aluminum,
read the concentration one minute after reagent addition, then again after 15 minutes.
An appreciable increase in concentration suggests aluminum interference. Waiting 2
hours before making the final reading will eliminate the effect of up to 3.0 mg/L
aluminum.
Chloride
At 7000 mg/L causes a +0.1 mg/L F error
Chlorine
SPADNS 2 Reagent contains enough non-toxic reductant to eliminate interference up

to 5 mg/L chlorine.
For higher chlorine levels:
1. Dilute sample with deionized water by a factor that will lower chlorine
concentration to below 5 mg/L.
2. Perform the SPADNS 2 reagent solution or AccuVac procedure.
3. Multiply results by the dilution factor to obtain
mg/L Fluoride.
Iron, ferric
At 10 mg/L causes a 0.1 mg/L F error
Phosphate, ortho
Sodium Hexametaphosphate
At 1.0 mg/L causes a +0.1 mg/L F error
Sulfate
Fluoride
Page 494
Fluoride
Distillation
Distillation Solution Preparation:
1. Measure 60 mL of deionized water into a 250 mL glass Erlenmeyer flask.
2. With constant stirring, add 120 mL of concentrated Sulfuric Acid. Caution: The mixture will
become very hot. Allow the solution to cool before handling.
To eliminate most interferences, dilute the sample from the acid solution as described
below:
1. Set up the distillation apparatus for general purpose distillation. Refer to the Distillation
Apparatus manual for proper assembly. Use a 125-mL Erlenmeyer flask to collect the distillate.
2. Turn on the water and maintain a steady flow through the condenser.
3. Measure 100 mL of sample into the distillation flask using a 100-mL graduated cylinder. Add a
magnetic stir bar and 5 glass beads.
4. Turn the stirrer power switch on. Turn the stir control to 5.
5. Using a 250-mL graduated cylinder, carefully add 150 mL of Distillation Solution into the flask.
Note: When distilling samples with high amounts of chloride, add 5 mg of Silver Sulfate to the sample for
every mg/L of chloride in the sample.
6. With the thermometer in place, turn the heat control to 10. The yellow pilot lamp indicates the
heater is on.
7. When the temperature reaches 180 C or when 100 mL of distillate has been collected, turn
the still off (requires about 1 hour).
8. Dilute the distillate to a volume of 100 mL, if necessary. The distillate may now be analyzed by
the SPADNS, SPADNS 2 or the fluoride ion-selective electrode method.
Samples may be stored in glass or plastic bottles for at least seven days when cooled to 4 C
(39 F) or lower.
Accuracy check
A variety of standard solutions for the entire range of the test is available. Use standard solutions
instead of sample to verify the technique.
Minor variations between lots of reagent become measurable above 1.5 mg/L. While results in this
region are usable for most purposes, better accuracy may be obtained with steps 13.
1. Dilute a fresh sample 1:1 with deionized water.
2. Perform the test again
3. Multiply the result by 2.
4. To adjust the calibration curve with the reading obtained with the standard solution, select
Fluoride
Page 495
Fluoride
Method performance
Program
Standard
Precision
Distribution
Sensitivity
190
1.00 mg/L F
0.971.03 mg/L F
195
1.00 mg/L F
0.921.08 mg/L F
Safety
Follow good safety habits and laboratory techniques throughout the procedure. Consult the
Material Safety Data Sheet for information specific to the reagents used.

SPADNS 2 Reagent does not contain sodium arsenite. Instead, it contains a non-toxic species to
prevent chlorine interference. Dispose of all waste safely in accordance with local and
federal guidelines.
Summary of method
The SPADNS 2 Method for fluoride determination involves the reaction of fluoride with a red
zirconium-dye solution. The fluoride combines with part of the zirconium to form a colorless
complex that bleaches the red color in an amount proportional to the fluoride concentration. This
method is equivalent to the EPA method for NPDES and NPDWR reporting purposes when the
samples have been distilled. Seawater and wastewater samples require distillation. Test results
Fluoride
Page 496
Fluoride

Required reagents
Description
Quantity/Test
Unit
Catalog number
4 mL
500 mL
2947549
SPADNS 2 Reagent Solution

OR
25/pkg
2527025
10 mL
4L
27256
Quantity
Unit
Catalog number
each
1465100
each
1451536
Pipet, volumetric, Class A, 10.00-mL.
each
1451538
2/pkg
2495402
Thermometer
each
2635700
Quantity
Unit
Catalog number
SPADNS 2 Fluoride Reagent AccuVac Ampuls

Water, deionized
Required apparatus (solution)

Description

Description
Beaker, 50-mL
each
50041H
each
2122800
6/pkg
2427606
Description
Unit
Catalog number
500 mL
40502
500 mL
40505
500 mL
40508
1000 mL
29153
500 mL
29149
500 mL
40512
500 mL
40515
500 mL
40520
Fluoride Standard Solution, 100-mg/L F
500 mL
23249
Standard, Drinking Water, Mixed Parameter, Inorganic for F, NO3, PO4, SO4
500 mL
2833049
Fluoride
Page 497
Fluoride

Description
Quantity
Unit
Catalog number
50842
each
each
50846
each
2274400
each
2274402
AND
OR
Distillation Apparatus Set, General Purpose
each
2265300
each
2089743
Flask, Erlenmeyer, 250 mL, Glass
each
50546
Glass Beads
100/pkg
259600
Stir Bar, magnetic
each
1076416
Sulfuric Acid, ACS
500 mL
97949
Description
Unit
Catalog number
Silver Sulfate
113 g
33414
Balance Analytical 80 g x 0.1 mg 100240 V
each
2936701
500/pkg
1473800
Weighing papers

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Formaldehyde, 8110
Formaldehyde
DOC316.53.01042
MBTH Method1
Method 8110
3 to 500 g/L CH2O
Powder Pillows
Scope and Application: For water.

1
Adapted from Matthews, T.G. and Howell, T.C., Journal of the Air Pollution Control Association, 31 (11) 1181-1184 (1981).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Wash glassware with Chromic Acid Cleaning Solution1 to remove trace contaminants.
Time and temperature are very important to this test. The sample should be 25 1 C and the times specified in the
procedure steps must be followed precisely. A temperature-controlled water bath is recommended for best accuracy.
Obtain formaldehyde-free water by distilling water from alkaline permanganate (4 g sodium hydroxide1, 2 g potassium
permanganate per 500 mL of water). Discard the first 50100 mL of distillate.
The Pour-thru cell cannot be used for this test.
1

Description
Developing Solution for LR Formaldehyde
Quantity
5 mL
MBTH Powder Pillows
Pipet Filler
Formaldehyde
Page 499
Formaldehyde
MBTH method for powder pillows
Stored Programs
200 Formaldehyde
Start
1. Select the test.

2. Prepared Sample:
Accurately measure 25 mL
of sample in a 50-mL
mixing cylinder.
Accurately measure 25 mL
of formaldehyde-free
water in a second 50-mL
mixing cylinder.

one MBTH powder pillow
to the blank. Insert the
cylinder stopper.
6. Immediately after the

reaction period starts,
shake the blank sample
vigorously for 20 seconds.

one MBTH powder pillow
to the prepared sample
when the timer shows
15:00. Insert the cylinder
stopper.
8. Shake vigorously for

20 seconds.
11. Add 2.5 mL of

Developing Solution for
Low Range Formaldehyde
to the prepared sample
when the timer shows
10:00.
12. Insert the cylinder

stopper and invert to mix.

for orientation.

timer. A 17-minute
Proceed with step 6
immediately after the timer
starts.
9. Add 2.5 mL of
Developing Solution for
Low Range Formaldehyde
to the blank when the
timer shows 12:00.
Formaldehyde
Page 500
Do not wait for the timer

to expire.
10. Insert the cylinder

stopper and invert to mix.
Formaldehyde
MBTH method for powder pillows (continued)
Zero
13. Just before the timer

shows 2:00, pour at least
10 mL of the blank into the
sample cell. Pour the
solution slowly to avoid
bubble formation on the
cell walls. If bubbles form,
swirl to dislodge them.

blank and insert it into the
cell holder.
15. When the timer shows

2:00, ZERO the
instrument.

the prepared sample into a
sample cell.

0 g/L CH2O
Read

18. When the timer

expires, READ the results
in g/L CH2O.
Interferences
Interference level
Interference level
Acetate
Iron (Fe3+)
Aldehydes (other)
Positive interference at all levels
Lead
Ammonium (as N)
Manganese
Aniline
Mercury
Bicarbonate
Morpholine
Calcium
Nitrate
Carbonate
Nitrite
Greater than 8 mg/L
Chloride
Phenol
Copper
Phosphate
Cyclohexylamine
Silica
Formaldehyde
Page 501
Formaldehyde
Interference level
Interference level
Ethanolamine
Sulfate
Greater than 10,000 mg/L
Ethylenediamine
Urea
Glucose
Glycine
Zinc
Accuracy check
Formaldehyde Voluette Ampule Standard, 4000-mg/L CH2O
Flask, 100-mL volumetric Class A
Mixing cylinders, 50-mL (3)

4. Open a Formaldehyde Voluette Ampule Standard, 4000-mg/L CH2O.
5. Use a TenSette Pipet to add 0.2 mL of the standard to a 100-mL volumetric Class A flask.
Dilute to volume with formaldehyde-free water and mix well. Prepare daily. This is an
8000-g/L (8-mg/L) formaldehyde standard.
6. Prepare three sample spikes. Fill three 50-mL mixing cylinders* with 25 mL of sample. Use the
TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of 8000-g/L standard, respectively, to each
sample and mix thoroughly.
sample spike. Accept each standard addition. Each addition should reflect approximately
100% recovery.
relationship between the sample spikes and the Ideal Line of 100% recovery
Prepare a 320-g/L Formaldehyde Standard Solution by pipetting 1.0 mL of the 8000-g/L solution
into a 50-mL mixing cylinder. Dilute to 25.0 mL with formaldehyde-free water. Run the test directly
on this sample.
Formaldehyde
Page 502
Formaldehyde
Method performance
Program
Standard
Precision
Distribution
Sensitivity
200
320 g/L CH2O
312328 g/L CH2O
3 g/L CH2O
Summary of method
Formaldehyde reacts with MBTH (3-methyl-2-benzothiazoline hydrazone) and a developing
solution to form a blue color in proportion to the formaldehyde concentration. Test results are
measured at 630 nm.

Required reagents
Description
Formaldehyde Reagent Set (100 tests), includes:
Developing Solution for LR Formaldehyde
MBTH Powder Pillows
Quantity/Test
Unit
Catalog number
2257700
5 mL
500 mL
2257249
100/pkg
2257169
Quantity
Unit
Catalog number
189641
Required apparatus
Description
each
each
53237
each
1465100
2/pkg
2495402
Description
Formaldehyde Standard Solution, 10-mL Voluette Ampule, 4000-mg/L
Unit
Catalog number
16/pkg
2257310
Unit
Catalog number

Description
Chromic Acid Cleaning Solution
500 mL
123349
Potassium Permanganate
454 g
16801H
Sodium Hydroxide ACS
500 g
18734
each
1457442

Pipet Tips, Tensette
Ampule Breaker
Wipes, disposable
each
1970001
50/pkg
2185696
each
2196800
280/pkg
2097000
Formaldehyde
Page 503

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, 8030
Hardness
DOC316.53.01043
Calcium and Magnesium;

Calmagite Colorimetric Method
Method 8030
0.05 to 4.00 mg/L Ca and Mg as CaCO3

Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

For the most accurate magnesium test results, keep the sample temperature between 2129 C (7084 F).
The test will detect any calcium or magnesium contamination in the mixing cylinder, measuring droppers or sample cells. To
test cleanliness, repeat the test until results are consistent.
Total hardness in mg/L equals mg/L Ca as CaCO3 plus mg/L Mg as CaCO3.
Remaining traces of EDTA or EGTA from previous tests will give erroneous results. Rinse sample cells thoroughly
before using.

Description
Quantity
Alkali Solution for Calcium and Magnesium test
1 mL
Calcium and Magnesium Indicator Solution
1 mL
EDTA Solution, 1 M
1 drop
EGTA Solution
1 drop
Cylinder, 100-mL, graduated mixing
Dropper, measuring, 0.5 and 1.0 mL
Hardness
Page 505
Hardness
Description
Quantity
Calmagite
Stored Programs
225 Hardness, Mg
Start
1. Select the test.

2. Pour 100 mL of
sample into a 100-mL
3. Use a 1.0 mL
measuring dropper to add
1.0 mL of Calcium and
Magnesium Indicator
solution.

and invert it several times.

and invert it several times.
7. Pour 10 mL of the
solution into each of three
sample cells.
Add one drop of 1 M EDTA
Solution to the first cell
(blank).

for orientation.
5. Add 1.0 mL of Alkali

Solution for Calcium and
Magnesium Test using a
1.0 mL measuring
dropper.
Hardness
Page 506
Hardness
Calmagite (continued)
9. Swirl to mix.
10. Magnesium Sample:

Add one drop of EGTA
Solution to the second
cell.
Zero

0.00 mg/L Mg CaCO3
11. Swirl to mix.
12. Insert the blank (first

cell) into the cell holder.
Read
14. Insert the magnesium

sample (second cell) into
the cell holder.

mg/L magnesium as
calcium carbonate.
This value is the amount of
magnesium in the sample
expressed as CaCO3.
16. Do not remove the cell

from the instrument.
Record or store the
magnesium results before
proceeding with step 17.
Exit
220 Hardness, Ca
Zero
Read
Start
17. EXIT the magnesium

program
Select the calcium test.

0.00 mg/L Ca CaCO3
Remove the second cell.
19. Calcium Sample:

Insert the third cell into
the cell holder.

mg/L calcium as calcium
carbonate.
This value is the amount of
calcium in the sample
expressed as CaCO3.
Hardness
Page 507
Hardness
Interferences
Chromium (Cr 3+)
Above 0.25 mg/L
Copper (Cu 2+)
Above 0.75 mg/L
EDTA
Above 0.2 mg/L as CaCO3
EDTA or EGTA
Traces remaining in sample cells from previous tests will give erroneous results. Rinse cells
thoroughly before using.
Iron (Fe 2+)
Above 1.4 mg/L
Iron (Fe
3+)
Above 2.0 mg/L
Manganese (Mn 2+)
Above 0.20 mg/L
Zinc (Zn 2+)
Above 0.050 mg/L
Ca >1.0 mg/L;
Mg >0.25 mg/L
For the most accurate calcium test result, rerun the test on a diluted sample if the calcium is over
1.0 and the magnesium is over 0.25 mg/L as CaCO3. No retesting is needed if either is below
those respective concentrations.
Adjust the sample pH to 2 or less with Nitric Acid (about 5 mL per liter).
Cool samples to 4 C. Preserved samples can be stored up to six months.
Before analysis, adjust the sample pH to between 3 and 8 with 5.0 N Sodium Hydroxide
Standard Solution*.
Correct the test results for volume additions.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
220
2.00 mg/L Ca
1.902.10 mg/L Ca
0.05 mg/L Ca
225
2.00 mg/L Mg
1.922.08 mg/L Mg
0.02 mg/L Mg
Summary of method
The colorimetric method for measuring hardness supplements the conventional titrimetric method
because the colorimetric method can measure very low levels of calcium and magnesium. Also,
some metals (those listed in Table 152) that interfere in the titrimetric method may be
inconsequential when diluting the sample to bring it within the range of this test. The indicator dye
is calmagite, which forms a purplish-blue color in a strongly alkaline solution and changes to red
when it reacts with free calcium or magnesium. Calcium and magnesium determinations are made
by chelating calcium with EGTA to destroy any red color due to calcium and then chelating the
calcium and magnesium with EDTA to destroy the red color due to both calcium and magnesium.
* See Optional Reagents and Apparatus on page 4
Hardness
Page 508
Hardness
By measuring the red color in the different states, calcium and magnesium concentrations are
determined. Test results are measured at 522 nm.
Hardness
Page 509

Required reagents
Description
Quantity/Test
Unit
Catalog number
2319900
Alkali Solution for Calcium and Magnesium test
1 mL
100 mL MDB
2241732
Calcium and Magnesium Indicator Solution
1 mL
100 mL MDB
2241832
Hardness Reagent Set (100 tests), includes:
EDTA Solution, 1 M
1 drop
50 mL SCDB
2241926
EGTA Solution
1 drop
50 mL SCDB
2229726
Catalog number
Required apparatus
Description
Quantity
Unit
Cylinder, 100 mL, graduated mixing
each
189642
Dropper, measuring, 0.5 and 1.0 mL
20/pkg
2124720
2/pkg
2495402
Unit
Catalog number

Description
Nitric Acid, ACS
pH Paper, 014

500 mL
15249
10 mL MDB
245032
100/pkg
2601300
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, 8374
Hardness
DOC316.53.01044

Chlorophosphonazo Colorimetric Method
(8 to 1000 g/L Ca and Mg as CaCO3)
Method 8374
Solution Pillow
Scope and Application: For boiler and ultra-pure water.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

The test will detect any calcium or magnesium contamination in the measuring droppers or sample cells. To test cleanliness,
repeat the test until results are consistent.
If the sample concentration is greater than 750 g/L, a 1:1 dilution of the sample is recommended for greatest accuracy. Use
ultra-pure (aldehyde-free) water for the dilution. Repeat the analysis on the diluted sample and multiply the resulting
concentration by two.Alternate forms should only be used when the sample is known to contain only Mg or Ca. This method
does not distinguish between the two forms.
Alternate forms should only be used when the sample is known to contain only Mg or Ca. This method does not distinguish
between the two forms.
One mL of Chlorphosphonazo Solution may be used instead of the solution pillow in step 4.
Use dedicated plasticware for this analysis.
Use a bottle-top dispenser to dispense the reagent

Description
ULR Hardness Reagent Set
Quantity
1
Hardness
Page 511
Hardness
Description
Quantity
Chlorophosphonazo Indicator Solution Pillow
CDTA Solution
1 drop
Shears for opening powder pillow
Sample Cells (2410212)
Chlorophosphonazo method
Stored Programs
228 Hardness Tot ULR
Start
1. Select the test.
2. Rinse a plastic sample

cell and the cap three
times with the water to be
tested. Do not allow the
underside of the cap to
come in contact with
surfaces that may
contaminate it.
3. Fill the plastic sample

sample.

one Chlorophosphonazo
Solution Pillow to the
sample cell.
A small amount of solution
may remain in the pillow.
This will not affect results.
Zero
5. Cap the cell and swirl

to mix.
6. Insert the cell into the

cell holder.

0 g/L CaCO3
8. Remove the cell from

the holder. Add one drop
of CDTA Reagent for Ultra
Low Range Hardness.
Complete steps 10-11
within 1-2 minutes.
Hardness
Page 512
Hardness
Chlorophosphonazo method (continued)
Read
9. Cap the cell and swirl

to mix.
10. Insert the cell into the

holder.

g/L CaCO3 .
Interferences
Interference studies were conducted at various hardness levels between 0 and 500 g/L as
CaCO3 (Interfering substances and levels). Various cations and anions were evaluated at levels in
the range appropriate for ultra pure water applications. An ion is said to interfere when the
resulting concentration is changed by 10%.

Aluminum
Negative interference above 150 g/L
Ammonium
No interference at or below 1000 g/L
Copper
Positive interference above 250 g/L
Formaldehyde
No interference at or below 47,000 g/L
Nitrate
Potassium
Silicon
Sodium
Negative interference above 79,000 g/L
Do not use glass containers.
Collect samples in clean plastic containers, preferably with screw-type closures.
Rinse containers several times with the water to be analyzed before collecting the final
sample.
Seal to avoid contamination during transport.
Analyze as soon as possible.
Accuracy check
Calcium Chloride Standard Solution, 50 mg/L (50,000 g/L) as CaCO3
Hardness
Page 513
Hardness
1. Prepare a 20,000 g/L (20 mg/L) CaCO3 standard by pipetting 20 mL of 50 mg/L CaCO3
standard solution into a 50 mL plastic volumetric flask. Dilute the solution with 50 mL of
ultra-pure water.
4. Accept the default sample volume (25 mL).
5. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of the prepared 20,000 g/L
standard, respectively to three 25-mL samples and mix each thoroughly.
6. Follow the test procedure for each of the spiked samples.
1. Use a 0.50 mg/L CaCO3 (500-g/L as CaCO3) Calcium Chloride Standard Solution in place of
the sample.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
278
500 g/L
478522 mg/L
8 g/L Ca
Summary of method
Calcium and magnesium combine equivalently with the Chlorophosphonazo III indicator to form a
colored complex which absorbs light very strongly at 669 nm. One drop of the CDTA reagent
breaks up this complex and the resultant decrease in color is proportional to the amount of calcium
and magnesium in the sample (as CaCO3). Test results are measured at 669 nm.

Required reagents
Description
Unit
Catalog number
2603100
100/pkg
2589599
1 drop
10 mL SCDB
2589636
(1) Sample cell, 1-inch square, molded
each
2410201
(1) Sample cell cap
each
2410202
ULR Hardness Reagent Set (100 tests), includes:

(1) Chlorophosphonazo Indicator Solution Pillows
(1) CDTA Solution
Hardness
Page 514
Quantity/Test
Hardness
Required reagents
Description
Quantity/Test
Unit
Catalog number
2603101
OR
ULR Hardness Reagent Set (500 tests), includes:
(1) Chlorophosphonazo Indicator Solution
1 mL
500 mL
2589549
(2) CDTA Solution
1 drop
10 mL SCDB
2589636
(1) Sample cell, 1-inch square, molded
each
2410201
(1) Sample cell cap
each
2410202
100/pkg
2589599
1 drop
10 mL SCDB
2589636
Quantity
Unit
Catalog number
each
2369400
Chlorophosphonazo Indicator Solution Pillows

CDTA Solution
Required apparatus
Description
Shears

Description
Unit
Catalog number
Calcium Chloride Standard Solution, 50 mg/L as CaCO3
946 mL
2127716
Calcium Chloride Standard Solution, 500 g/L as CaCO3
946 mL
2058016
Chlorophosphonazo Indicator Solution
500 mL
2589549
Description
Unit
Catalog number
Dispenser, 1.0-mL, Repipet Jr.
each
2111302
each
1970001
50/pkg
2185696
each
1451520
Flask, volumetric, polypropylene, 50 mL
each
1406041
Pipet filler, Safety Bulb
each
1465100
12/pkg
Sample cell, 25-mL plastic with cap
Hardness
Page 515
Hardness

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Total, 8374
Hardness, Total
DOC316.53.01045

Chlorophosphonazo Rapid Liquid Method
Method 8374
ULR (4 to 1000 g/L Ca and Mg as CaCO3)
Pour-Thru Cell
Scope and Application: For boiler and ultra pure water.
Test preparation


Instrument
Pour-thru Kit
DR 6000
LQV175.99.20002
DR 5000
LZV479
DR 3900
DR 3800, DR 2800, DR 2700
Cell orientation
Adapter
Arrow faces right
LQV157.99.10002
5940400
LZV585 (B)

Pre-clean the Pour-Thru Cell and all labware as specified in Treating analysis labware.
The test will detect any calcium or magnesium contamination in the mixing cylinder, measuring droppers, or sample cells. To
test cleanliness, repeat the test until results are consistent.
If the sample concentration is greater than 750 g/L, a 1:1 dilution of the sample is recommended for greatest accuracy. Use
ultra-pure (aldehyde-free) water for the dilution. Repeat the analysis on the diluted sample and multiply the resulting
concentration by two.
Alternate forms should only be used when the sample is known to contain only Mg or Ca. This method does not distinguish
between the two forms.
Use dedicated plasticware for this analysis.
In bright light conditions (e.g. direct sunlight) it may be necessary to close the DR 2800 or DR 2700 cell compartment with the
protective cover during measurements.
Ensure the pour-thru cell is completely seated in the sample cell compartment.
Hardness, Total
Page 517
Hardness, Total

Description
Quantity
2 mL
CDTA Reagent for Ultra Low Range Hardness
1 drop
Water, Ultra-pure
Varies
Cylinder, graduated, 50-mL, poly
Flask, Erlenmeyer, PMP w/cap, 125-mL
Pour-Thru Cell Module (see Instrument-specific information table)
Chlorophosphonazo rapid liquid method
Stored Programs
227 Hardness Tot RL ULR
Start
1. Select the test.

5. Fill the rinsed cylinder

sample from the flask.
Discard remaining
contents of the flask.
Hardness, Total
Page 518

Cell with 50 mL of ultrapure water.
3. Fill a clean, 125-mL

plastic Erlenmeyer flask to
overflowing with sample.
4. Rinse a clean, 50-mL

plastic graduated cylinder
three times with sample.
Collect sample directly in

the flask if possible.

the 50-mL cylinder back
into the flask.
7. Add 2.0 mL of
Chlorophosphonazo
Reagent to the sample
with the Dispenser. Swirl
to mix.
half (25 mL) of the sample
into the Pour-Thru Cell.
Use a clean, dry, plastic
25-mL graduated cylinder
to measure the sample.
Hardness, Total
Chlorophosphonazo rapid liquid method (continued)
Zero


0 g/L CaCO3
Read
10. Add one drop of CDTA

Reagent for Ultra Low
Range Hardness to the
remaining sample in the
flask. Swirl to mix.
11. Pour the remaining

sample into the
Pour-Thru Cell.

g/L CaCO3.
Complete steps 11 and 12

within one to two minutes.
13. Using a wash bottle,

rinse the Pour-Thru Cell
with at least 75 mL of ultrapure water immediately
after use.
Rinse the flask with ultrapure water. Cap when
finished.
Hardness, Total
Page 519
Hardness, Total
Interferences
Interference level
Aluminum
Negative interference above 150 g/L
Ammonium
Copper
Formaldehyde
No interference at or below 47,000 g/L
Potassium
Silicon
Sodium
Negative interference above 79,000 g/L

Clean all containers used in this test thoroughly to remove any traces of calcium or magnesium. If
possible, use plastic containers for all analysis and storage. Clean containers by normal means,
then rinse with ultra-pure (aldehyde-free) water. Fill and soak for 10 minutes with a 1:25 dilution of
Chlorophosphonazo Reagent in ultra-pure water. Rinse well with ultra-pure water. Keep containers
tightly closed and dedicate them for ULR Hardness only. If containers are rinsed and capped after
each use, only occasional soaking is necessary. Fill the Pour-Thru cell with this same mixture of
chlorophosphonazo and water and let stand for several minutes. Rinse with ultra-pure water.
Avoid contamination of the Chlorophosphonazo Reagent bottle when placing the Repipet
dispenser on the bottle. Rinse the inlet tubing and inside of the dispenser cap with copious
amounts of ultra-pure water using a wash bottle. Place the inlet tubing into a beaker of ultra-pure
water and depress the plunger 1015 times to rinse the inside of the dispenser. (For best results,
pour a small amount of reagent into the beaker of rinse water.) Remove the dispenser from the
water and depress the plunger until all of the water has been expelled.
Shake off any excess water on the dispenser, place the dispenser on the bottle and tighten.
Do not use glass containers.
Collect samples in clean plastic containers, preferably with screw-type closures.
Rinse containers several times with the water to be analyzed before collecting the final
sample.
Seal to avoid contamination during transport.
Analyze as soon as possible.
Accuracy check
Calcium Chloride Standard Solution, 50 mg/L (50,000 g/L) as CaCO3

1. Prepare a 20,000 g/L (20 mg/L) CaCO3 standard by pipetting 20 mL of 50 mg/L CaCO3
standard solution into a 50 mL plastic volumetric flask. Dilute the solution with 50 mL of ultrapure water.
Hardness, Total
Page 520
Hardness, Total
or edited. Accept the default values then read the unspiked sample measurement. After
values are accepted, the unspiked sample reading will appear in the top row. See the user
manual for more information.
5. Use the TenSette Pipet to prepare spiked samples: add 0.2 mL, 0.4 mL, and 0.6 mL of the
prepared 20,000 g/L standard to three 50 mL portions of fresh sample.
6. Follow the test procedure for each of the spiked samples, starting with the 0.2 mL sample
spike. Measure each of the spiked samples in the instrument.
1. Using a 0.50 mg/L (500 g/L as CaCO3) Calcium Chloride Standard Solution, perform the
procedure using the standard in place of the sample.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
227
0.500 mg/L Cr6+
495505 g/L
8g/L Ca
Summary of method
Calcium and magnesium combine equivalently with the Chlorophosphonazo Indicator to form a
colored complex which absorbs light very strongly at 669 nm. One drop of the CDTA reagent
breaks up this complex, and the resultant decrease in color is proportional to the amount of
calcium and magnesium (as CaCO3) in the sample. Test results are measured at 669 nm.

Required reagents
Description
CDTA Reagent for Ultra Low Range Hardness
Quantity/Test
Unit
Catalog number
2 mL
500 mL
2589549
1 drop
10 mL SCDB
2589636
Quantity
Unit
Catalog number
Required apparatus
Description
Cylinder, graduated, 50 mL, poly
each
108141
Dispenser, variable-volume
each
2563137
Flask, Erlenmeyer, PMP w/cap, 125 mL
each
2089843
Hardness, Total
Page 521
Hardness, Total
Description
Unit
Catalog number
2127716
Calcium Standard Solution, 50 mg/L as CaCO3
946 mL
Calcium Standard Solution, 0.50 mg/L as CaCO3
946 mL
2058016
Water, ultra-pure (aldehyde-free)
500 mL
2594649
Unit
Catalog number

Description
each
1970001
50/pkg
2185696
each
1451520
each
1465100
each
1406041
Pipet,
TenSette
0.11.0 mL

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Calcium, DT, 8204
Hardness, Calcium
Titration Method using EDTA
DOC316.53.01175
Method 8204
Digital Titrator
Test preparation

Magnesium is not included in the results but must be present for a sharp end point. If magnesium is not present, add one to
two drops of Magnesium Standard Solution, 10-g/L as CaCO3 to the sample before the test is started.
One German degree of hardness (G.d.h.) = 17.9 mg/L hardness as CaCO3
mg/L Ca = Ca hardness in mg/L as CaCO3 x 0.40
A 0.1-g scoop of CalVer 2 Calcium Indicator Powder can be used in place of the CalVer 2 Calcium Indicator Powder Pillow.
1

Description
Quantity
CalVer 2 Calcium Indicator Powder Pillow
1 pillow
1 bottle
EDTA titration cartridge (see Range-specific informationmg/L (mg/L) or Range-specific

informationG.d.h. (German degrees of hardness))
1 cartridge
Digital titrator
Graduated cylinder
Hardness, Calcium
Page 523
Hardness, Calcium
Hardness, Calcium
See
Table 1
or
Table 2
1. Select a sample
cartridge from the Rangespecific informationmg/L
table or the Range-specific
informationG.d.h. table.

3. Hold the Digital

delivery knob to eject air
and a few drops of titrant.
Reset the counter to zero
and wipe the tip.
4. Use a graduated
measure the sample
volume from the Rangespecific informationmg/L
or the Range-specific
Transfer the sample into a

is 100 mL, add 2 mL of
8 N Potassium Hydroxide
Standard Solution. If the
sample volume is 50 mL or
less, add 1 mL of
Standard Solution. Swirl to
mix.

one CalVer 2 Calcium
Swirl to mix.

changes from red to pure
blue.
counter.

the Range-specific
informationmg/L table
(or the Range-specific
informationG.d.h. table)
to calculate the
concentration:
mg/L (or G.d.h.) Ca as
CaCO3
Example:
50 mL of sample titrated with the 0.800 M EDTA titration cartridge, and 250 digits to reach the
endpoint yields a calcium concentration of 250 x 2.0 = 500 mg/L as CaCO3 (or with the 0.714 M
EDTA titration cartridge, 250 x 0.1 = 25 mg/L G.d.h.)
Hardness, Calcium
Page 524
Hardness, Calcium
Table 157 Range-specific informationmg/L

Sample volume (mL)
Titration cartridge (M EDTA)
Multiplier
1040
100
0.0800
0.1
0.4
40160
25
0.0800
100400
100
0.800
1.0
200800
50
0.800
2.0
5002000
20
0.800
5.0
10004000
10
0.800
10.0
Table 158 Range-specific informationG.d.h.

Range (G.d.h as CaCO3)
Sample volume (mL)
14
100
0.1428
0.01
416
25
0.1428
0.04
Multiplier
1040
50
0.714
0.1
25100
20
0.714
0.25
> 100
10
0.714
0.5
Interferences
WARNING
Chemical hazard. Potassium cyanide is toxic. Always add it after the potassium hydroxide.
Follow local hazardous waste regulations for disposal of all cyanide-containing waste.
An interfering substance can prevent the color change at the titration end point. A dilution can
often reduce the interference to a level at which the substance does not interfere. If an interference
is suspected, decrease the sample volume, dilute to 100 mL and repeat the test.

Interference level
Acidity
The test can tolerate 10,000 mg/L acidity.
Alkalinity
The test can tolerate 10,000 mg/L alkalinity .
Aluminum
Interferes by causing a slow end point but up to 200 mg/L aluminum can be tolerated by
allowing sufficient time for the color change.
Barium
Barium is titrated along with calcium but is seldom found in natural waters in significant
amounts.
Chloride
Saturated solutions do not give a distinct end point. The test can be run directly in sea water
Cobalt
Interferes at all levels. 0.5 grams of potassium cyanide can be added after the potassium
hydroxide solution to remove interference from up to 20 mg/L cobalt.
Copper
Interferes at 0.1 mg/L copper. 0.5 grams of potassium cyanide can be added after the
potassium hydroxide solution to remove interference from up to 100 mg/L copper.
Iron
Interferes above 8 mg/L by causing an orange-red to green end point. Accurate results can
still be obtained up to approximately 20 mg/L iron with this end point.
Magnesium
Interference from magnesium is prevented up to 200 mg/L by the formation of magnesium

hydroxide at the high test pH but higher levels prevent a distinct end point.
Manganese
Interferes above 5 mg/L.
Hardness, Calcium
Page 525
Hardness, Calcium
Interference level
Nickel
Interferes at 0.5 mg/L nickel. 0.5 grams of potassium cyanide can be added after the
potassium hydroxide solution to remove interference from up to 200 mg/L nickel.
Orthophosphate
Causes a slow end point but does not interfere if the calcium phosphate that forms is allowed
to redissolve during the titration.
Polyphosphates
Interfere directly and must be absent.
Strontium
Strontium is titrated along with calcium but is seldom found in natural waters in significant
amounts.
Temperature
Samples at about 20 C (68 F) or colder should be titrated slowly near the end point to allow
sufficient time for the color change.
Zinc
Interferes at 5 mg/L zinc. 0.5 grams of potassium cyanide can be added after the potassium
hydroxide solution to remove interference from up to 100 mg/L zinc.

extreme sample pH
May exceed the buffering capacity of the reagents. Adjust the pH before starting the test
(see Sample collection, preservation and storage).
Collect samples in plastic or glass bottles that have been washed with a detergent and rinsed
with tap water.
Rinse the bottles in 1:1 nitric acid solution and deionized water.
The following storage instructions are necessary only when immediate analysis is not
possible. To preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix.
Measure the sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL
increments if necessary. Mix thoroughly and check the pH after each addition until the pH is 2
or less.
Preserved samples can be stored at least six months at room temperature.
Before running the test, adjust the sample to pH 7 by adding potassium hydroxide standard
solution and mixing thoroughly.
Accuracy check
Use the standard additions method to find if the sample has an interference and to make sure that
the user has followed the procedure correctly.
Hardness Voluette Ampule Standard Solution, 10,000-mg/L as CaCO3
Ampule breaker

4. Repeat steps 2 and 3.
Hardness, Calcium
Page 526
Hardness, Calcium
5. Each 0.1 mL of standard that was added will use approximately 10 digits of the 0.800 M
titration cartridge or 100 digits of the 0.0800 M titration cartridge to reach the endpoint
(11 digits of 0.714 M or 56 digits of 0.1428 M titrant).
Summary of method
The sample is made alkaline (pH 12 to 13) with potassium hydroxide to precipitate magnesium as
magnesium hydroxide. CalVer 2 Calcium Indicator is added as an indicator and combines with any
calcium to form a red color. As the EDTA is added, it will react with all the free calcium ions
present. At the end point of the titration, when no free calcium ions are present, the EDTA will
remove the calcium complexed with the indicator. The indicator will then change from red to blue.

Required reagents
Description
Quantity/Test
Unit
Catalog number
(1) CalVer 2 Calcium Indicator Powder Pillows
1 pillow
100/pkg
85299
(1) Potassium Hydroxide Standard Solution, 8 N
12 mL
100 mL MDB
28232H
varies
each
1436401
1 pillow
100/pkg
85299
12 mL
100 mL MDB
28232H
varies
each
1439901
1 pillow
100/pkg
85299
12 mL
100 mL MDB
28232H
varies
each
1496001
1 pillow
100/pkg
85299
12 mL
100 mL MDB
28232H
varies
each
1495901
(1) EDTA Titration Cartridge, 0.0800 M
2447200
2447500
116 G.d.h. rangeReagent set (approximately 100 tests):
2447300
10100+ G.d.h. rangeReagent set (approximately 100 tests):
2447400
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50546

each
50838
each
50840
each
50841
each
50842
Hardness, Calcium
Page 527
Hardness, Calcium
Description
Unit
Catalog number
16/pkg
218710
Description
Unit
Catalog number
CalVer 2 Calcium Indicator Powder
113 g
28114H
Magnesium Standard Solution, 10-g/L as CaCO3
29 mL
102233
Hardness Standard Solution, Voluette ampule, 10,000-mg/L as CaCO3, 10-mL
Nitric Acid Solution, ACS
500 mL
15249
500 mL
254049
CDTA Magnesium Salt Powder Pillow
100/pkg
1408099
29 mL
102233
Hardness 2 Indicator Solution
100 mL
42532
HexaVer Hardness Titrant, 0.020 N

1L
74053
113 g
28014
500 mL
15249
500 mL
254049
Sodium Hydroxide Standard Solution, 5 N
50 mL
245026
Potassium cyanide
125 g
76714
each
2095352
each
1970001
each
1940000
each
1940010
Water, deionized
500 mL
27249
Tensette Pipet tips
50/pkg
218596
Potassium hydroxide, 8 N
500 mL
28249
each
1451538
each
1451520
each
1465100
each
2087079
Bromphenol Green-Methyl Red indicator solution
100 mL MDB
2329232
100 mL MDB
16232
pH meter
each
Pipet tips
50/pkg
2185696
Delivery Tube, 180 Hook
5/pkg
1720500
each
1970001
each
51000
each
90700
each
51100
each
2196800
12/pkg
2087076

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Calcium, BT, 8222
Hardness, Calcium
USEPA1 Buret Titration Method2
0 to 25,000 mg/L as CaCO3
DOC316.53.01157
Method 8222
Buret Titration

1
USEPA accepted when 0.020 N titrant is used.
Test preparation

Magnesium is not included in the results but must be present for a sharp end point. If magnesium is not present, add one to
two drops of Magnesium Standard Solution, 10-g/L as CaCO3 to the sample before the test is started.

Description
TitraVer Hardness Titrant (see Range-specific information)
Quantity
1
1 mL
1 bottle
Graduated cylinder
Hardness, Calcium
Page 529
Hardness, Calcium
Buret titration
See
Table 1

volume and titrant
concentration from the
table.

TitraVer Hardness Titrant.
3. Use a graduated
measure the sample

with deionized water. If
magnesium is known to be
absent, add one or two
drops of Magnesium
Standard Solution,
10-g/L as CaCO3.
5. Add 1 mL of
Standard Solution using
the 1-mL dropper. Swirl to
mix.

Swirl to mix.

from red to pure blue.

to calculate the
concentration:
Magnesium must be
present (and usually is in
natural waters) for a sharp
end point, although it is not
measured in the titration.
mL titrant used x multiplier

= mg/L calcium as CaCO3
Hardness, Calcium
Page 530
0.020 N titrant solution
and 15 mL of titrant was
used to reach the
endpoint.
The calcium concentration
is: 15 x 20 = 300 mg/L as
CaCO3
Hardness, Calcium

Sample volume (mL)
Hardness titrant concentration
Multiplier
0500
50
0.020 N
20
4001000
25
0.020 N
40
10002500
10
0.020 N
100
20005000
0.020 N
200
10005000
50
0.200 N
200
400010,000
25
0.200 N
400
10,00025,000
10
0.200 N
1000
Table 161 Hardness conversions

To convert from
To
Multiply by
mg/L as Ca
0.400
grains per gallon (gpg)
0.058
German degrees hardness (Gdh)
0.056
mg/L as CaCO3
Interferences
WARNING
Dispose of all cyanide containing wastes according to local regulations.
reduce the interference to a level at which the substance does not interfere. If an interference is
suspected, decrease the sample volume, dilute to 50 mL and repeat the test.

Interference level
Acidity
Alkalinity
The test can tolerate 10,000 mg/L alkalinity.
Aluminum
Interferes by causing a slow end point but up to 200 mg/L aluminum can be tolerated by
allowing sufficient time for the color change.
Barium
Barium is titrated along with calcium but is seldom found in natural waters in significant
amounts.
Chloride
Saturated solutions do not give a distinct end point, but the test can be run directly under sea
water.
Cobalt
Copper
Iron
Magnesium
Interference from magnesium is prevented up to 200 mg/L by the formation of magnesium

hydroxide at the high test pH but higher levels prevent a distinct end point.
Manganese
Nickel
Hardness, Calcium
Page 531
Hardness, Calcium
Interference level
Orthophosphate
Polyphosphates
Strontium
Strontium is titrated along with calcium but is seldom found in natural waters in significant
amounts.
Temperature
Samples at about 20 C (68 F) or colder should be titrated slowly near the end point to allow
sufficient time for the color change.
Zinc

extreme sample pH

Collect samples in plastic or glass bottles that have been washed with a detergent and rinsed with
tap water. Then rinse the bottles in 1:1 nitric acid solution and deionized water.
The following storage instructions are necessary only when immediate analysis is not possible. To
preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix. Measure the
sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL increments if
necessary. Mix thoroughly and check the pH after each addition until the pH is 2 or less. Preserved
samples can be stored at least six months at room temperature.
Before running the test, adjust the sample to pH 7 by adding potassium hydroxide standard
solution and mixing thoroughly. Correct the test result for volume additions.
Accuracy check
solution method to confirm anaytical technique and reagent performance.
Ampule breaker
TenSette Pipet, 0.11.0 mL or 1.010.0 mL and Pipet Tips.

Hardness, Calcium
Page 532
Hardness, Calcium
concentration of the titrant has changed (see Accuracy check).
Complete the following test to confirm analytical technique and reagent performance.
1. Add 25.0 mL of a calcium chloride standard solution, 1000-mg/L as CaCO3, to an Erlenmeyer
flask. Dilute to 50 mL with deionized water and mix fully.
2. Add the potassium hydroxide and CalVer 2 indicator. Swirl to mix.
3. Titrate the standard to the end point with the 0.020 N hardness titrant and calculate the result.
The titration should use 25 mL of titrant.
1. Add 10.0 mL of a Hardness Voluette Ampule Standard Solution, 10,000-mg/L as CaCO3, to an
Erlenmeyer flask. Dilute to 50 mL with deionized water and mix fully.
2. Add the potassium hydroxide and CalVer 2 indicator. Swirl to mix.
The titration should use 10 mL of titrant.
Summary of method
The sample is made alkaline (pH 12 to 13) with potassium hydroxide to precipitate magnesium as
magnesium hydroxide. CalVer 2 Calcium Indicator is added as an indicator and combines with any
calcium to form a red color. As the TitraVer (EDTA) is added, it will react with all the free calcium
ions present. At the end point of the titration, when no free calcium ions are present, the EDTA will
remove the calcium complexed with the indicator. The indicator will then change from red to blue.
Hardness, Calcium
Page 533
Hardness, Calcium

Required reagents
Description
Quantity/Test
Unit
100/pkg
85299
1 mL
100 mL MDB
28232H
Hardness (Calcium) Reagent Set (approximately 100 tests), includes:
2447000
CalVer 2 Calcium Indicator Powder Pillows

TitraVer Hardness Titrant, 0.020 N
Catalog number
varies
1L
20553
varies
500 mL
102149
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
2636540
Buret Clamp, double
each
32800
each
50546
50837

each
each
50838
each
50840

Support Stand
each
50841
each
56300
Unit
Catalog number

Description
Calcium Chloride Standard Solution, 1000-mg/L as CaCO3
1L
12153
16/pkg
218710
Pipet, Class A volumetric, 10 mL
each
1451538
each
1451540
Safety bulb
each
1465100
Hardness, Calcium
Page 534
Hardness, Calcium

Description
Catalog number
CalVer 2 Calcium Indicator Powder
113 g
28114H
29 mL
102233
500 mL
15249
500 mL
254049
Potassium cyanide
Water, deionized
125 g
76714
500 mL
28249
each
1970001
500 mL
27249
each
51000
each
90700
each
51100
50/pkg
2185696
each
2196800
each
1970010
50/pkg
2199796
12/pkg
2087076
Unit
Hardness, Calcium
Page 535

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Total, Sequential 8329
Hardness, Total, Sequential
DOC316.53.01230

Method 8329
Digital Titrator
Test preparation

The first titration gives calcium hardness and the second titration gives total hardness. The difference between the values is
magnesium hardness. All of these concentrations are in mg/L as CaCO3. See Hardness conversions for conversions to
other units.
Four drops of Hardness 2 Indicator Solution or a 0.1-g scoop of ManVer 2 Hardness Indicator Powder can be added in place
of the ManVer 2 Hardness Indicator Powder Pillow.
1

Description
Quantity
1
1 mL
1 mL

1
1 mL
1 cartridge
Digital titrator
Graduated cylinder

Page 537

Hardness, total, sequential
See
Table 1
or
Table 2
1. Select a sample
cartridge from Rangespecific informationmg/L
or Range-specific
informationG.d.h..

3. Hold the Digital

wipe the tip.
4. Use a graduated
measure the sample
volume from Rangespecific informationmg/L
or Range-specific
informationG.d.h..

is 100 mL, add 2 mL of
Standard Solution. If the
sample volume is 50 mL or
less, add 1 mL of
Standard Solution. Swirl to
mix.

Swirl to mix.

blue.

Page 538

Range-specific
informationmg/L (or
Range-specific
informationG.d.h.) to
calculate the
concentration:
Total digits from step 7 and
step 12 x multiplier =
mg/L (or G.d.h.) Calcium
as CaCO3

Hardness, total, sequential (continued)
9. Add 1 mL of 5.25 N
Sulfuric Acid Standard
Solution. Continue to add
the acid by drops while
swirling until the solution
changes from pure blue to
purple and finally to red.
Swirl the flask to make
sure that all the
precipitated magnesium
hydroxide has dissolved.
10. Add 2 mL of
Hardness 1 Buffer

Swirl to mix.

blue.

Range-specific
informationmg/L (or
Range-specific
informationG.d.h.) to
calculate the
concentration:
mg/L (or G.d.h.) total
hardness as CaCO3

Page 539

Sample volume (mL)
Multiplier
1040
100
0.0800
0.1
40160
25
0.0800
0.4
100400
100
0.800
1.0
200800
50
0.800
2.0
5002000
20
0.800
5.0
10004000
10
0.800
10.0

Sample volume (mL)
14
100
0.1428
0.01
416
25
0.1428
0.04
Multiplier
1040
50
0.714
0.1
25100
20
0.714
0.25
> 100
10
0.714
0.5

To convert from
To
mg/L as CaCO3
mg/L as Ca
Multiply by
0.400
mg/L as Mg
0.243
0.0584
0.0559
Hardness relationships
mg/L Mg Hardness as CaCO 3
= mg/L Total Hardness as CaCO 3 mg/L Ca Hardness as CaCO 3
mg/L MgCO 3 = mg/L Mg Hardness as CaCO 3 0.842
mg/L Mg = mg/L MgCO 0.29
3
Interferences
WARNING:
Do not use potassium cyanide to eliminate interferences because it will generate deadly
hydrogen cyanide gas when the sulfuric acid solution is added.
Although less common than calcium and magnesium, other polyvalent metal ions cause the
same hardness effects and will be included in the results.
Some transition and heavy metals complex the indicator and prevent the color change at the
end point.

Page 540
Iron does not interfere up to 15 mg/L. Above this level it causes a red-orange to green end
point which is sharp and usable up to 30 mg/L iron. Substitute a 0.0800 M CDTA or 0.800 M
CDTA titration cartridge for the 0.0800 M EDTA or 0.800 M EDTA titration cartridges,
respectively, if iron interference is probable. For results in G.d.h., divide the mg/L result by
17.9.
Manganese titrates directly up to 20 mg/L but masks the end point above this level. Adding a
0.1-gram scoop of hydroxylamine hydrochloride raises this level to 200 mg/L manganese.
Copper interferes at levels of 0.10 and 0.20 mg/L. Cobalt and nickel interfere at all levels and
must be absent or masked.
Orthophosphate causes a slow end point and polyphosphate must be absent for accurate
results.
Acidity and alkalinity at 10,000 mg/L (as CaCO3) do not interfere.
Saturated sodium chloride solutions do not give a distinct end point, but the titration can be run
directly on sea water.
Adding the contents of one CDTA Magnesium Salt Powder Pillow removes metal interferences
at or below the levels shown in Metal interferences.
If more than one metal is present at or above the concentrations shown in Metal interferences,
an additional CDTA Magnesium Salt Powder Pillow may be required.
Results obtained by this procedure include the hardness contributed by polyvalent metal ions.
If the concentration of each metal is known, a correction can be applied to obtain the calcium
and magnesium hardness concentration. The hardness (in mg/L as CaCO3) contributed by
each mg/L of metal is listed in Hardness contributed by each mg/L of metal. Hardness
contributed by metals can be subtracted from the total hardness value obtained to determine
the calcium and magnesium hardness concentration.
Barium, strontium and zinc titrate directly.
Table 166 Metal interferences

Metal
CDTA Removes Interference Below this

Level
Aluminum
50 mg/L
Cobalt
200 mg/L
Copper
100 mg/L
Iron
100 mg/L
Manganese
200 mg/L
Nickel
400 mg/L
Zinc
300 mg/L

Page 541

Table 167 Hardness contributed by each mg/L of metal
Metal
Hardness as CaCO3 Contributed

by Each mg/L of Metal
Aluminum
3.710
Barium
0.729
Cobalt
1.698
Copper
1.575
Iron
1.792
Manganese
1.822
Nickel
1.705
Strontium
1.142
Zinc
1.531
with tap water. Then rinse the bottles in 1:1 nitric acid solution and deionized water.
increments if necessary.
Mix thoroughly and check the pH after each addition until the pH is 2 or less.
Store samples at 4 C (39 F) or below. Preserved samples can be stored for at least six
months.
Before starting the test, warm the sample to room temperature and adjust the pH to
approximately pH 7 with potassium hydroxide solution. Mix thoroughly.
If a significant amount of nitric acid was added, make a volume correction for the extra acid
and hydroxide.
Divide the total volume (sample + acid + hydroxide) by the volume of the sample and multiply
the result from the test by this value.
Summary of method
This test procedure is a combination of the calcium and total hardness procedures. Refer to each
method for a detailed description of the methods.

Page 542

Required reagents
Description
Quantity/Test
Unit
1 mL
100 mL MDB
42432
100/pkg
94799
Calcium and Total Hardness Reagent Set (approximately 100 tests)

(1) Buffer Solution, Hardness 1
Catalog number
2272100

(1) ManVer 2 Hardness Indicator Powder Pillows
100/pkg
85199
1 mL
100 mL MDB
28232H
varies
each
1436401
varies
each
1439901
varies
100 mL MDB
244932
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50546

each
50838
each
50840
each
50841
each
50842
Delivery tube for Digital Titrator, 180 hook
5/pkg
1720500
Delivery tube for Digital Titrator, 90 hook
5/pkg
4157800
Description
Unit
Catalog number
1L
12153

16/pkg
218710
each
2196800

Page 543

Description
Unit
Catalog number
CalVer
113 g
28114H
113 g
28014
2 Calcium Indicator Powder

500 mL
15249
each
2095352
each
1970001
each
1940000
each
1940010
Water, deionized
500 mL
27249
pH paper, 014
100/pkg
2601300
50/pkg
2185696
1000/pkg
2185628
Weighing papers
500/pkg
1473800
Spoon, measuring, 0.1 g capacity
each
51100
each
90700
Hydroxylamine Hydrochloride
113 g
24614
100/pkg
1408099
CDTA cartridge for digital titrator, 0.08 M
each
1440201
CDTA cartridge for digital titrator, 0.80 M
each
1440301
Magnesium CDTA powder pillows

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Total, DT, 8213
Hardness, Total
DOC316.53.01176
Method 8213
Digital Titrator
Test preparation

Hardness conversions: grains per gallon (gpg) as CaCO3 = mg/L x 0.058; German degrees hardness (Gdh) = mg/L x 0.056;
mg/L Total Hardness as Ca = mg/L Total Hardness as CaCO3 x 0.40
mg/L Total Hardness as CaCO3 = mg/L Ca as CaCO3 + mg/L Mg as CaCO3.
1

Description
Quantity
1
2 mL
1 cartridge
Digital titrator
Graduated cylinder
Hardness, Total
Page 545
Hardness, Total
Hardness, Total
See
Table 1
or
Table 2
1. Select a sample
cartridge from the Rangespecific informationmg/L

3. Hold the Digital

delivery knob to eject air
and a few drops of titrant.
Reset the counter to zero
and wipe the tip.
4. Use a graduated
measure the sample
volume from the Rangespecific informationmg/L
Hardness, Total
Page 546
Hardness, Total
Hardness, Total
5. Add 2 mL of Hardness
1 Buffer Solution and swirl
to mix.

Swirl to mix.

blue.

the Range-specific
informationmg/L table
(or the Range-specific
informationG.d.h. table)
to calculate the
concentration:
mg/L (or G.d.h.) total
hardness as CaCO3
0.800 M EDTA titration
cartridge and 250 digits
endpoint. The total
hardness concentration is:
250 x 2.0 = 500 mg/L as
CaCO3 (or with the
0.714 M EDTA titration
cartridge, 250 x 0.1 = 25
mg/L G.d.h.)

Sample volume (mL)
Multiplier
1040
100
0.0800
0.1
40160
25
0.0800
0.4
100400
100
0.800
1.0
200800
50
0.800
2.0
5002000
20
0.800
5.0
10004000
10
0.800
10.0
Hardness, Total
Page 547
Hardness, Total

Sample volume (mL)
Multiplier
14
100
0.1428
0.01
0.04
416
25
0.1428
1040
50
0.714
0.1
25100
20
0.714
0.25
> 100
10
0.714
0.5
Interferences
WARNING
Follow local hazardous waste regulations for disposal of all cyanide-containing waste.
often reduce the interference to a level at which the substance does not interfere. If an interference
is suspected, decrease the sample volume, dilute to 100 mL and repeat the test.

Interference level
Acidity
Alkalinity
The test can tolerate 10,000 mg/L alkalinity and can be run directly in sea water.
Aluminum
Aluminum interferes above 0.20 mg/L aluminum. 0.5 grams of potassium cyanide can be
added after the buffer solution to remove interference from up to 1 mg/L aluminum.
Barium
Barium is included in the results but is seldom found in natural waters in significant amounts.
Cobalt
Copper
Iron
Manganese
Nickel
Orthophosphate
Polyphosphates
Polyvalent metal ions
Sodium chloride
Saturated solutions do not give a distinct end point.
Strontium
Strontium is included in the results but is seldom found in natural waters in significant
amounts.
Zinc

extreme sample pH
Hardness, Total
Page 548
Hardness, Total
The addition of one CDTA Magnesium Salt Powder Pillow will remove metals interferences at or
below the levels shown in Interference level with CDTA pillow. If more than one metal is present at
or above the concentrations in Interference level with CDTA pillow, an additional CDTA
Magnesium Salt Powder Pillow may be necessary.
Table 171 Interference level with CDTA pillow

Interference level, mg/L
Aluminum
50
Cobalt
200
Copper
100
Iron
100
Manganese
200
Nickel
400
Zinc
300
The results obtained with CDTA Magnesium Salt will include the hardness contributed by the
metals. If the concentration of each metal is known, a correction can be made to obtain the
hardness contributed by calcium and magnesium only. The hardness contributed per mg/L metal
ion is listed in Hardness equivalence factors. The mg/L of metal in the sample multiplied by its
calcium carbonate hardness equivalent factor should be subtracted from the total hardness
determined in step 3 of the procedure to obtain the hardness contributed by calcium and
magnesium only.
Table 172 Hardness equivalence factors

Hardness equivalence factors, mg/L as CaCO3
Aluminum
3.710
Barium
0.729
Cobalt
1.698
Copper
1.575
Iron
1.792
Manganese
1.822
Nickel
1.705
Strontium
1.142
Zinc
1.531
Hardness, Total
Page 549
Hardness, Total
with tap water.
Rinse the bottles in 1:1 nitric acid solution and deionized water.
increments if necessary. Mix thoroughly and check the pH after each addition until the pH is 2
or less.
Store samples at 4 C (39 F) or below. Preserved samples can be stored for at least seven
days.
approximately pH 7 with 5.0 N sodium hydroxide.
Mix thoroughly. If a significant amount of nitric acid was added, make a volume correction for
the extra acid and hydroxide. Divide the total volume (sample + acid + hydroxide) by the
volume of the sample and multiply the result from the test by this value.
Accuracy check
Ampule breaker

titration cartridge or 100 digits of the 0.0800 M titration cartridge to reach the endpoint
(11 digits of 0.714 M or 56 digits of 0.1428 M titrant).
Hardness, Total
Page 550
Hardness, Total
Complete the following test to make sure that the reagents are user technique are accurate.
1. Add 20.0 mL of the standard solution to an Erlenmeyer flask. Dilute to about 100 mL with
deionized water and mix fully.
2. Add the Hardness 1 Buffer Solution and ManVer 2 indicator. Swirl to mix.
3. Titrate the standard to the end point with the titration cartridge and calculate the result. The
result should be close to 1000 mg/L or 55.9 G.d.h. as CaCO3.
Summary of method
In the total hardness test, the water sample is first buffered (using an organic amine and one of its
salts) to a pH of 10.1. An organic dye, calmagite, is added as the indicator for the test. This dye
reacts with calcium and magnesium ions to give a red-colored complex.
EDTA (ethylenediaminetetraacetic acid) is added as a titrant. The EDTA will react with all free
calcium and magnesium ions in the sample. At the end point of the titration, when free magnesium
ions are no longer available, EDTA will remove magnesium ions from the indicator, causing a color
change from red to blue.

Required reagents
Description
Quantity/Test
Unit
100/pkg
85199
1 mL
100 mL MDB
42432
varies
each
1436401
100/pkg
85199
1 mL
100 mL MDB
42432
100/pkg
1 mL
100 mL MDB
42432
varies
each
1496001
100/pkg
1 mL
100 mL MDB
42432
varies
each
1495901
varies
each
1439901

2448000

2448100
116 G.d.h. rangeReagent set (approximately 100 tests):

2447800
10100+ G.d.h. rangeReagent set (approximately 100 tests):

Catalog number
85199
2447900
85199
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50546
each
50838

Hardness, Total
Page 551
Hardness, Total
Description
Quantity/Test
Unit
Catalog number
50840
each
each
50841
each
50842
Unit
Catalog number
Description
1L
12153
16/pkg
218710
Unit
Catalog number
100/pkg
1408099
113 g
28014
29 mL
102233
100 mL
42532
1L
74053
50 mL
245026
500 mL
15249
500 mL
254049

Description
Potassium cyanide
125 g
76714
each
2095352
each
1970001
each
1940000
each
1940010
Water, deionized
500 mL
27249
Pipet tips
50/pkg
2185696
Pipet, Volumetric, 10 mL Class A
each
1451538
Pipet, Volumetric, 20 mL Class A
each
1451520
Pipet filler safety bulb
each
1465100
Delivery Tube, 180 Hook
5/pkg
1720500
each
1970001
each
51000
each
90700
each
51100
50/pkg
2199796
12/pkg
2087076

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Total, BT, 8226
Hardness, Total
DOC316.53.01158
USEPA1 ManVer 2 Buret Titration Method2
Method 8226
Buret Titration

1
USEPA accepted when 0.020 N titrant is used.
Test preparation

Hardness conversions: grains per gallon (gpg) as CaCO3 = mg/L x 0.058; German degrees hardness (Gdh) = mg/L x 0.056

Description
Quantity
1 mL
1 bottle
Graduated cylinder
Buret titration
See
Table 1

volume and titrant
concentration from Rangespecific information.

3. Use a graduated
measure the sample

Hardness, Total
Page 553
Hardness, Total
5. Add 1 mL of
Hardness 1 Buffer
Solution using the 1-mL
dropper. Swirl to mix.

Swirl to mix.


the Rangespecific information to
calculate the
concentration:
= mg/L total hardness as
CaCO3
0.020 N titrant and 15 mL
of titrant was used to
reach the endpoint.
The hardness
concentration is: 15 x 20 =
300 mg/L as CaCO3

Sample volume (mL)
Multiplier
0500
50
0.020 N
20
4001000
25
0.020 N
40
10002500
10
0.020 N
100
10005000
50
0.200 N
200
400010,000
25
0.200 N
400
10,00025,000
10
0.200 N
1000
Interferences
WARNING
Chemical hazard. Potassium cyanide is toxic. Always add it after the buffer solution.
Dispose of all cyanide containing wastes according to local regulations
reduce the interference to a level at which the substance does not interfere. If an interference is
suspected, decrease the sample volume, dilute to 50 mL and repeat the test.
Hardness, Total
Page 554
Hardness, Total

Interference level
Acidity
Acidity at 10,000 mg/L as CaCO3 does not interfere.
Alkalinity
Alkalinity at 10,000 mg/L as CaCO3 does not interfere.
Aluminum
Aluminum interferes above 0.20 mg/L aluminum. 0.5 grams of potassium cyanide can be added
after the buffer solution to remove interference from up to 1 mg/L aluminum.
Barium
Barium is titrated directly but is seldom found in natural waters in significant amounts.
Chloride
Saturated solutions do not give a distinct end point. The test can be run directly in sea water.
Cobalt
Interferes at all levels and must be absent or masked. 0.5 grams of potassium cyanide can be
added after the buffer solution to remove interference from up to 100 mg/L cobalt.
Copper
Copper interferes above 0.10 mg/L copper. 0.5 grams of potassium cyanide can be added after
the buffer solution to remove interference from up to 100 mg/L copper.
Iron
still be obtained up to approximately 30 mg/L iron with this end point. For slightly sharper end
points in solutions containing higher levels of iron, HexaVer Hardness Titrant (CDTA) can be
used in place of the TitraVer Hardness Titrant (EDTA).
Manganese
Interferes above 20 mg/L. Adding a 0.10-gram scoop of Hydroxylamine Hydrochloride

Monohydrate raises this level to 200 mg/L manganese.
Nickel
Interferes at all levels and must be absent or masked. 0.5 grams of potassium cyanide can be
added after the buffer solution to remove interference from up to 100 mg/L nickel.
Orthophosphate
Causes a slow end point but does not interfere if the calcium phosphate that forms is allowed to
redissolve during the titration.
Polyphosphates
Polyvalent metal ions
Strontium
Strontium is titrated directly but is seldom found in natural waters in significant amounts.
Zinc
Titrates directly. 0.5 grams of potassium cyanide can be added after the buffer solution to
remove interference from up to 100 mg/L zinc.

extreme sample pH
The addition of one CDTA Magnesium Salt Powder Pillow will remove metals interferences at or
below the levels shown in the Interference level with CDTA pillow table. If more than one metal is
present at or above the concentrations in the Interference level with CDTA pillow table, an
additional CDTA Magnesium Salt Powder Pillow may be necessary.
Table 175 Interference level with CDTA pillow

Interference level, mg/L
Aluminum
50
Cobalt
200
Copper
100
Iron
100
Manganese
200
Nickel
400
Zinc
300
The results obtained with CDTA Magnesium Salt will include the hardness contributed by those
soluble metal ions present. If the concentration of soluble metal ion is known, a correction can be
made to obtain the hardness contributed by calcium and magnesium only. The hardness
Hardness, Total
Page 555
Hardness, Total
contributed per mg/L metal ion is listed in Hardness equivalence factors for the individual metals.
The mg/L of metal present multiplied by its calcium carbonate hardness equivalent factor should
be subtracted for each metal present from the total hardness determined in step 8 of the procedure
to obtain the hardness contributed by calcium and magnesium only.
Table 176 Hardness equivalence factors

Hardness equivalence factors, mg/L as CaCO3
Aluminum
3.710
Barium
0.729
Cobalt
1.698
Copper
1.575
Iron
1.792
Manganese
1.822
Nickel
1.705
Strontium
1.142
Zinc
1.531

Collect samples in plastic or glass bottles that have been washed with a detergent and rinsed with
tap water. Then rinse the bottles in 1:1 nitric acid solution and deionized water.
The following storage instructions are necessary only when immediate analysis is not possible. To
preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix. Measure the
sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL increments if
necessary. Mix thoroughly and check the pH after each addition until the pH is 2 or less. Store
samples at 4 C (39 F) or below. Preserved samples can be stored for at least seven days.
Before starting the test, warm the sample to room temperature and adjust the pH to approximately
pH 7 with 5.0 N sodium hydroxide. Mix thoroughly. If a significant amount of nitric acid was added,
make a volume correction for the extra acid and hydroxide. Divide the total volume (sample + acid
+ hydroxide) by the volume of the sample and multiply the result from the test by this value.
Accuracy check
solution method to confirm analytical technique and reagent performance.
Ampule breaker
TenSette Pipet, 0.11.0 mL or 1.010.0 mL and Pipet Tips

Hardness, Total
Page 556
Hardness, Total
Complete the following test to confirm the analytical technique and reagent performance.
The titration should use 25 ( 0.3) mL of titrant.
Hardness, Total
Page 557
Hardness, Total
Summary of method
In the total hardness test, the water sample is first buffered (using an organic amine and one of its
salts) to a pH of 10.1. An organic dye, calmagite, is added as the indicator for the test. This dye
reacts with calcium and magnesium ions to give a red-colored complex.
EDTA (ethylenediaminetetraacetic acid) is added as a titrant. The EDTA will react with all free
calcium and magnesium ions present in the sample. At the end point of the titration, when free
magnesium ions are no longer available, EDTA will remove magnesium ions from the indicator,
causing a color change from red to blue.

Required reagents
Description
Quantity/Test
Unit
100/pkg
85199
1 mL
100 mL MDB
42432
(1) TitraVer Hardness Titrant, 0.020 N
varies
1L
Hardness (Total) Reagent Set (approximately 100 tests), includes;
Catalog number
2447600
Hardness (Total) Reagent Set (approximately 100 tests), includes:
20553
2447700
100/pkg
85199
1 mL
100 mL MDB
42432
varies
500 mL
102149
Quantity/Test
Unit
Catalog number
each
2636540
Buret Clamp, double
each
32800
each
50546
50838
Required apparatus
Description

each
each
50840
each
50841
each
56300
Unit
Catalog number
Support Stand

Description
1L
12153
16/pkg
218710
each
1451538
each
1451540
Safety bulb
each
1465100
Hardness, Total
Page 558
Hardness, Total

Description
Unit
Catalog number
100/pkg
1408099
29 mL
102233
100 mL
42532

1L
74053
113 g
28014
500 mL
15249
500 mL
254049
Potassium cyanide
125 g
76714
50 mL
245026
each
1970001

Water, deionized
500 mL
27249
each
51000
each
90700
each
51100
50/pkg
2185696

each
2196800
each
1970010
50/pkg
2199796
12/pkg
2087076
Pipet,
TenSette,
Pipet, 1.010.0 mL
Hardness, Total
Page 559

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hardness, Total, Sequential, BT, 8338

DOC316.53.01159
Method 8338
Buret Titration

1
Test preparation

The first titration gives calcium hardness and the second titration gives total hardness. The difference between the values is
magnesium hardness. All of these concentrations are in mg/L as CaCO3. See Hardness conversions for conversions to
other units.
Two drops of Hardness 2 Indicator Solution can be added in place of the ManVer 2 Hardness Indicator Powder Pillow.

Description
Quantity
1
1 mL
1 mL

1
1 mL
1 bottle
Graduated cylinder

Page 561

Buret titration
See
Table 1

volume and titrant
concentration from Rangespecific information.

3. Use a graduated
measure the sample
volume from Rangespecific information.
If the sample volume is
less than 50 mL, dilute to
deionized water.
4. Add 1 mL of
Standard Solution using
the 1-mL dropper. Swirl to
mix.

Swirl to mix.


to calculate the
concentration:
Magnesium must be
present (and usually is in
natural waters) for a sharp
end point. Record the
volume of titrant used.

= mg/L calcium as CaCO3
8. Add 1 mL of 5.25 N
Solution. Continue to add
the acid by drops while
swirling until the solution
changes from pure blue to
purple and finally to red.
Swirl the flask to make
sure that all the
precipitated magnesium
hydroxide has dissolved.

Page 562

9. Add 1 mL of
Hardness 1 Buffer
Solution using the 1-mL
dropper. Swirl to mix.

Swirl to mix.


to calculate the
concentration:
Record the volume of

titrant used.
Total mL titrant used in

step 6 and step 11 x
multiplier = mg/L total
hardness as CaCO3
0.020 N titrant and 15 mL
of titrant was used to
reach the endpoint.
The hardness
concentration is: 15 x 20 =
300 mg/L as CaCO3

Sample volume (mL)
Multiplier
0500
50
0.020 N
20
4001000
25
0.020 N
40
10002500
10
0.020 N
100
10005000
50
0.200 N
200
400010,000
25
0.200 N
400
10,00025,000
10
0.200 N
1000

To convert from
mg/L as CaCO3
To
Multiply by
mg/L as Ca
0.400
mg/L as Mg
0.243
0.0584
0.0559

Page 563
Hardness relationships
mg/L Mg Hardness as CaCO 3
= mg/L Total Hardness as CaCO mg/L Ca Hardness as CaCO
3
3
mg/L MgCO = mg/L Mg Hardness as CaCO 0.842
3
3
mg/L Mg = mg/L MgCO 0.29
3
Interferences
WARNING:
Do not use potassium cyanide to eliminate interferences because it will generate deadly
hydrogen cyanide gas when the sulfuric acid solution is added.
Some transition and heavy metals complex the indicator and prevent the color change at the
end point.
Iron does not interfere up to 15 mg/L. Above this level it causes a red-orange to green end
point which is sharp and usable up to 30 mg/L iron. For results in G.d.h., divide the mg/L result
by 17.9.
Manganese titrates directly up to 20 mg/L but masks the end point above this level. Adding a
0.1-gram scoop of hydroxylamine hydrochloride raises this level to 200 mg/L manganese.
Copper interferes at levels of 0.10 and 0.20 mg/L. Cobalt and nickel interfere at all levels and
must be absent or masked.
Orthophosphate causes a slow end point and polyphosphate must be absent for accurate
results.
Acidity and alkalinity at 10,000 mg/L (as CaCO3) do not interfere.
Saturated sodium chloride solutions do not give a distinct end point, but the titration can be run
directly on sea water.
Adding the contents of one CDTA Magnesium Salt Powder Pillow removes metal interferences
at or below the levels shown in the Metal interferences table.
If more than one metal is present at or above the concentrations shown in the Metal
interferences table, an additional CDTA Magnesium Salt Powder Pillow may be required.

Page 564
Results obtained by this procedure include the hardness contributed by polyvalent metal ions.
If the concentration of each metal is known, a correction can be applied to obtain the calcium
and magnesium hardness concentration. The hardness (in mg/L as CaCO3) contributed by
each mg/L of metal is listed in the Hardness of each mg/L of metal table. Hardness contributed
by metals can be subtracted from the total hardness value to determine the calcium and
magnesium hardness concentration.
Barium, strontium and zinc titrate directly.
Table 179 Metal interferences

Metal
CDTA removes interference below this

level
Aluminum
50 mg/L
Cobalt
200 mg/L
Copper
100 mg/L
Iron
100 mg/L
Manganese
200 mg/L
Nickel
400 mg/L
Zinc
300 mg/L
Table 180 Hardness of each mg/L of metal

Metal
Hardness as CaCO3 contributed

by each mg/L of Metal
Aluminum
3.710
Barium
0.729
Cobalt
1.698
Copper
1.575
Iron
1.792
Manganese
1.822
Nickel
1.705
Strontium
1.142
Zinc
1.531
with tap water. Then, rinse the bottles in 1:1 nitric acid solution and deionized water.
The storage instructions are necessary only when immediate analysis is not possible. To
preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix.
increments if necessary.
Mix thoroughly and check the pH after each addition until the pH is 2 or less.
Store samples at 4 C (39 F) or below. Preserved samples can be stored for at least six
months.
approximately pH 7 with potassium hydroxide solution. Mix thoroughly.
If a significant amount of nitric acid was added, make a volume correction for the extra acid
and hydroxide.

Page 565
Divide the total volume (sample + acid + hydroxide) by the volume of the sample and multiply
the result from the test by this value.
Accuracy check
solution method to make sure that the user has followed the test correctly and that the reagents
are good.
Ampule breaker

2. Use the TenSette Pipet to add 0.1 mL of the standard to the titrated sample from step 7 or
step 12. Swirl to mix.
2. Use the TenSette Pipet to add 1.0 mL of the standard to the titrated sample from step 7 or
step 12. Swirl to mix.
Page 566

Complete the following test to make sure that the reagents are user technique are accurate.
2. Add the reagents as shown in the test procedure. Swirl to mix.
2. Add the reagents as shown in the test procedure. Swirl to mix.
Summary of method
This test procedure is a combination of the calcium and total hardness procedures. Refer to each
method for a detailed description of the methods.

Required reagents
Description
Quantity/Test
Unit
1 mL
100 mL MDB
42432
100/pkg
94799
Hardness (Total) Reagent Set (approximately 100 tests), includes;

Catalog number
2448200
100/pkg
92899
1 mL
100 mL MDB
28232H
(1) Sulfuric Acid Standard Solution, 5.25 N
varies
100 mL MDB
244932
varies
1L
20553
varies
500 mL
102149
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
2636540
Buret Clamp, double
each
32800
each
50546
50838

each
each
50840
each
50841
each
56300
Support Stand

Page 567

Description
Unit

Catalog number
1L
12153
16/pkg
218710
each
2196800
Unit
Catalog number

Description
500 mL
15249
each
1970001
Water, deionized
500 mL
27249
pH paper, 014
100/pkg
2601300
50/pkg
2185696
1000/pkg
2185628
each
51100
each
90700
Hydroxylamine Hydrochloride
Magnesium CDTA powder pillows
Hardness 2 indicator solution

113 g
24614
100/pkg
1408099
100 mL MDB
42532
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Hydrazine, 8141
Hydrazine
DOC316.53.01046
p-Dimethylaminobenzaldehyde Method1
Method 8141
4 to 600 g/L N2H4

Scope and Application: For boiler water/feedwater.
1
Adapted from ASTM Manual of Industrial Water, D1385-78, 376 (1979).
Test preparation


Reagent solution
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Adapter
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
LZV584 (C)

Samples cannot be preserved and must be analyzed immediately
Sample temperature should be 21 4 C (70 7 F).
After adding the HydraVer 2 Hydrazine Reagent, a yellow color will develop in the sample if hydrazine is present. The blank
may also have a faint yellow color.
Important Note: The final samples will have a pH less than 2, which is considered corrosive (0002) by the
Federal RCRA. Refer to the MSDS for disposal instructions.
Description
Quantity
Solution Test:
HydraVer 2 Reagent Solution
1 mL
Deionized Water
10 mL
Graduated Cylinder, 25 mL
AccuVac Test:
HydraVer 2 Reagent AccuVac Ampuls
Hydrazine
Page 569
Hydrazine
Collect the following items (continued):
Description
Quantity
Deionized Water
40 mL
Beaker, 50 mL
Stopper for Ampules
Reagent solution method
Stored Programs
231 Hydrazine
Start
1. Select the test.

to pour 10 mL of deionized
water into a sample cell.
3. Prepared Sample:
to pour 10 mL of sample
into a second sample cell.
4. Add 0.5 mL of
HydraVer 2 Hydrazine
Reagent to each sample
cell. Swirl to mix.

for orientation.
Zero

timer.
period will begin.
Complete steps 68
during this period.
Hydrazine
Page 570
6. Insert the blank cell.

0 g/L N2H4

sample cell.
Immediately after the
timer expires READ the
results in g/L N2H4.
Hydrazine
AccuVac Ampuls
Stored Programs
232 Hydrazine AV
Start
1. Select the test.

for orientation.
2. Prepared Sample:
Fill a HydraVer Hydrazine
AccuVac Ampul with
fills completely. Cap with a
stopper and mix.

timer.
period will begin.
Complete steps 57
during this period.
Pour at least 40-mL of
second beaker.
Cap with a stopper and
mix.
Zero

the cell holder.

0 g/L N2H4

sample in the cell holder.
Immediately after the

timer expires READ the
results in g/L N2H4.
Interferences
Interference level
Ammonia
No interference up to 10 mg/L. May cause a positive interference of up to 20% at 20

mg/L.
Highly colored or turbid samples
Prepare a blank by oxidizing the hydrazine in a portion of the sample with a 1:1
mixture of deionized water and household bleach. Add one drop of the mixture to 25
mL of sample in a graduated mixing cylinder and invert to mix. Use this solution in step
2, instead of deionized water, to prepare the blank.
Morpholine
No interference up to 10 mg/L.
Hydrazine
Page 571
Hydrazine
Samples collected in glass or plastic bottles should be filled completely and capped tightly.
Avoid excessive agitation or exposure to air.
Samples must be analyzed immediately after collection and cannot be preserved for later
analysis.
Accuracy check
1. Prepare a 25 mg/L stock solution. Dissolve 0.1016 g of hydrazine sulfate in 1000 mL of

oxygen-free deionized water. Prepare this stock solution daily.
2. Using Class A glassware, prepare a 0.25 mg/L (250 g/L) hydrazine working solution by
diluting 10.00 mL of the 25 mg/L stock solution to 1000 mL with deoxygenated
deionized water. Prepare just before analysis. Perform either hydrazine procedure.
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
231
DR 5000
250 g/L N2H4
247253 g/L N2H4
4 g/L N2H4
232
DR 5000
250 g/L N2H4
246254 g/L N2H4
4 g/L N2H4
Summary of method
Hydrazine in the sample reacts with the p-dimethylaminobenzaldehyde from the HydraVer 2
Reagent to form a yellow color which is proportional to the hydrazine concentration. Test results

Required reagents
Description
HydraVer 2 Hydrazine Reagent
Quantity/Test
Unit
Catalog number
1 mL
100 mL MDB
179032
25/pkg
2524025
1040 mL
4L
27256
OR
HydraVer 2 Hydrazine Reagent AccuVac Ampuls
Water, deionized
Hydrazine
Page 572
Hydrazine
Required apparatus (Reagent solution)

Description
Quantity
Unit
Catalog number
Cylinder, graduated, 25-mL.
each
50840
2/pkg
2495402

Description
Quantity
Unit
Catalog number
Beaker, 50 mL
each
50041H
Stopper
6/pkg
173106
Description
Unit
Catalog number
Hydrazine Sulfate, ACS
100 g
74226
Description
Unit
Catalog number
Cylinder, graduated mixing
each
189640
each
1457453
each
1451538
each
1465100
AccuVac Snapper
each
2405200
Hydrazine
Page 573

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iodine, 8031
Iodine
DOC316.53.01047
DPD Method 1
Method 8031
0.07 to 7.00 mg/L
Scope and Application: For testing dissolved iodine residual used as disinfectant in process water, treated water,
estuary water and seawater
1
Adapted from Palin, A.T., Inst. Water Eng., 21 (6), 537-547 (1967).
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Analyze samples immediately. Do not preserve for later analysis
blank adjust.
Use DPD Total Chlorine reagents. Do not use DPD Free Chlorine reagents for this test.
If the sample temporarily turns yellow after reagent addition, dilute a fresh sample. Repeat the test. A slight loss of iodine
may occur due to the dilution. Apply the appropriate dilution factor.
Iodine
Page 575
Iodine
Description
Quantity
Powder Pillow Test:

DPD Total Chlorine Reagent Powder Pillow
Sample cells, 1-inch square, 10-mL (see Instrument-specific information)
AccuVac Test:
DPD Total Chlorine Reagent AccuVac Ampul
Beaker, 50-mL
DPD for powder pillows
Stored Programs
245 Iodine
Start
1. Select the test.

for orientation.
Iodine
Page 576
2. Prepared sample: Fill

a sample cell with 10 mL
of sample.
DPD Total Chlorine
sample cell.
3. Swirl for about 20

second to mix.

timer.

iodine is present.
time will begin.
Iodine
DPD for powder pillows (continued)
Zero
5. Blank preparation:

(See Instrumentspecific information.)

0.00 mg/L I2
Close the cover.

holder.
(See Instrumentspecific information.)
Read

mg/L I2.
DPD for AccuVac Ampuls
Stored Programs
246 Iodine AV
Start
1. Select the test.

for orientation.
2. Prepared Sample:
fills completely.

Ampul several times
to mix.
iodine is present.

timer.
period will begin.
Iodine
Page 577
Iodine
DPD for AccuVac Ampuls (continued)
Zero


0.00 mg/L l2.

the cell holder.
READ the results in
mg/L I2.
Interferences
Interference level
Acidity
Neutralize to pH 67 with 1 N Sodium Hydroxide1. Determine amount to be added on

separate sample aliquot, then add the same amount to the sample being tested. Correct
for volume addition.
Greater than 250 mg/L CaCO3: May not develop full color or color may fade instantly.
1.
2.
Bromine
3.
Chlorine and chloramines
Causes a positive interference at all levels
Chlorine Dioxide
May interfere
Hardness
Manganese, Oxidized (Mn4+,

Mn7+) or
Chromium, Oxidized (Cr6+)
Neutralize to pH 67 with 1 N Sulfuric Acid1.

Determine amount to be added on separate sample aliquot. Add the same amount to
Correct for volume addition.
Alkalinity
1.
Adjust sample pH to 67.
2.
3.
Add 3 drops Potassium Iodide1 (30-g/L) to a 25-mL sample.

4.
5.
6.
Add 3 drops Sodium Arsenite1, 2 (5-g/L) and mix.

Subtract the result from this test from the original analysis to obtain the correct iodine
concentration.
Ozone
Peroxides
May interfere

buffered samples
Adjust to pH 67.
Iodine
Page 578
Iodine
2
for arsenic (D004). Refer to the current MSDS for disposal information.
Collect samples in clean, dry glass containers.
If sampling from a tap, allow the water to flow at least 5 minutes to ensure a representative
sample.
Avoid excessive agitation and exposure to sunlight when sampling.
Allow several volumes of water to overflow the container and cap the container so there is no
headspace above the sample.
If sampling with a sample cell, rinse the cell several times with the sample, then carefully fill to
the 10-mL mark.
Proceed with the analysis immediately.
Accuracy check
Standard additions method (sample spike) for powder pillows
LR Chlorine PourRite Ampule Standard, 2530 mg/L
Ampule breaker
1. Analyze the sample and record the result.

3. Use the TenSette Pipet to prepare a spiked sample: add 0.1 mL of standard to a 10-mL portion
of reacted sample. Swirl to mix.
4. Insert the spiked sample cell in the instrument.
6. Read the result.
7. Calculate the equivalent concentration of mg/L iodine added to the sample:
0.1 (vol. standard added) mg/L Chlorine (certificate value) 3.6
mg/L Iodine = ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------10.1 (sample + standard volume)
8. The spiked sample result from step 6 should reflect the analyzed sample result in step 1 plus
the added, calculated mg/L iodine in step 7. If the result does not reflect the increase, refer to
Standard Additions in the Water Analysis Guide.
Standard additions method (sample spike) for AccuVac Ampuls
LR Chlorine PourRite Ampule Standard, 2530 mg/L
DPD Total Chlorine AccuVac Ampul (2)
Graduated cylinder
Beakers
Ampule breaker
Iodine
Page 579
Iodine
TenSette Pipet

2. Use a graduated cylinder to measure 25 mL of sample into a beaker. Use the TenSette Pipet
to add 0.2 mL of standard to the beaker. Swirl to mix. This is the spiked sample.
3. Use a graduated cylinder to measure 25 mL of sample into a second beaker.
4. Fill an AccuVac Ampul from the spiked sample. Fill the second Ampul from the second beaker.
5. Follow the DPD for AccuVac Ampuls test procedure.
7. Read the result.
8. Calculate the equivalent concentration of mg/L iodine added to the sample:
0.2 (vol. standard added) mg/L Chlorine (certificate value) 3.6
mg/L Iodine = ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------25.2 (sample + standard volume)
9. The spiked sample result should reflect the analyzed sample result plus the added, calculated
mg/L iodine in step 8. If the result does not reflect the increase, refer to Standard Additions in
the Water Analysis Guide.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
240
4.47 mg/L I2
4.404.54 mg/L I2
0.07 mg/L I2
242
4.47 mg/L I2
4.334.61 mg/L I2
0.07 mg/L I2
Summary of method
Iodine reacts with DPD (N, N-diethyl-p-phenylenediamine) to form a pink color, the intensity of
which is proportional to the total iodine concentration. Test results are measured at 530 nm.
Iodine
Page 580
Iodine

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
2105669
25/pkg
2503025
Catalog number
OR
Required apparatus
Description
Quantity
Unit
Beaker, 50-mL
each
50041H
each
2122800
6/pkg
2427606
2/pkg
2495402
Unit
Catalog number
AccuVac Snapper Kit
each
2405200
Ampule Breaker Kit
each
2196800

Description
100 mL MDB
34332
100 mL
104732
100 mL MDB
104532
25 / pkg
173125
100 mL MDB
127032
20/pkg
2630020
Sulfuric Acid, 1 N
Chlorine Standard, 25-30 mg/L ampules
Deionized Water
4L
27256
each
1970001
Pipet tips for TenSette Pipet 1970001
50 / pkg
2185696
1000 / pkg
2185628
Iodine
Page 581

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron, FerroZine, 8147
Iron
DOC316.53.01048
Ferrozine Method1
Method 8147
0.009 to 1.400 mg/L
FerroZine Reagent Solution Pillows

1
Adapted from Stookey, L.L., Anal. Chem., 42(7), 779 (1970)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Digestion is required for total iron determination.
Rinse glassware with a 1:1 hydrochloric acid solution. Rinse again with deionized water. These two steps will remove iron
deposits that can cause slightly high results.
blank adjust.
Instead of solution pillows, 0.5 mL of FerroZine Iron Reagent Solution can be used.
If the sample contains rust, see Interferences.
Use clean clippers, free of rust and wipe with a dry towel. Do not allow clippers to contact contents of the pillow.
FerroZine Iron Reagent may crystallize or precipitate when exposed to cold temperatures during shipment. Reagent quality
is not affected. Place the reagent in warm water to redissolve.
Iron
Page 583
Iron

Description
Quantity
FerroZine Iron Reagent Solution Pillows
OR
FerroZine Iron Reagent Solution
0.5 mL
Cylinder, 25 mL graduated mixing, with stopper
Clippers for solution pillows
FerroZine solution pillows
Stored Programs
260 Iron, FerroZine
Start
1. Select the test.

2. Fill a clean 25 mL
to the 25 mL mark with
sample.

for orientation.
3. Prepared sample:
FerroZine Iron Reagent
mixing cylinder.
Stopper and invert to mix.

timer.
period will begin. A purple
color will develop if iron is
present.
Zero
10 mL of sample.
Iron
Page 584
6. When the timer

expires, pour 10 mL of the
prepared sample into a
second clean sample cell.

the cell holder.

0.000 mg/L Fe
Iron
FerroZine solution pillows (continued)
Read


mg/L Fe.
Interferences
Interference level
Strong chelants (EDTA)
Interfere at all levels. Use the FerroVer or TPTZ methods for these samples. Use the
TPTZ method for low iron concentrations.
Cobalt
May give slightly high results
Copper
Hydroxides
Boil the sample, with the FerroZine Iron Reagent added to it from step 3, for 1 minute in a
boiling water bath. Cool to 24 C (75 F) before proceeding with step 4. Return the sample
volume to 25 mL with deionized water.
1.
2.
Magnetite (black iron oxide) or

Ferrites
Rust
Fill a 25 mL graduated cylinder with 25 mL of sample.

Transfer this sample into a 125-mL Erlenmeyer flask.
3. Add the contents of one FerroZine Iron Reagent Solution Pillow and swirl to mix.
4. Place the flask on a hot plate or over a flame and bring to a boil.
5. Continue boiling gently for 20 to 30 minutes.
Note: Do not allow to boil dry. A purple color will develop if iron is present.
6. Return the boiled sample to the 25 mL graduated cylinder. Rinse the Erlenmeyer flask
with small amounts of deionized water and empty into the graduated cylinder.
7. Return the sample volume to the 25 mL mark with deionized water.
8. Pour this solution into a sample cell and swirl to mix.
9. Proceed with steps 510.
Iron
Page 585
Iron
Collect samples in acid-washed glass or plastic bottles.
To preserve samples, adjust the sample pH to 2 or less with concentrated Nitric Acid, ACS*
(about 2 mL per liter). Samples preserved in this manner can be stored up to six months at
room temperature.
If only reporting dissolved iron, filter the sample immediately after collection and before adding
nitric acid.
Before testing, adjust the sample pH to 35 with Ammonium Hydroxide 10%.
Do not exceed pH 5 or iron may precipitate.
Accuracy check
Iron Voluette Ampule Standard, 10 mg/L Fe
Ampule breaker
3. Accept the default values for standard concentration, sample volume and spike volumes. After
the values are accepted, the unspiked sample reading will appear in the top row. See the user
5. Follow the FerroZine solution pillows test procedure for each of the spiked samples:
a. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to the unspiked sample.
Start the instrument timer. After the timer expires, read the result.
b. Prepare a 0.2 mL sample spike by adding 0.1 mL of standard to the 0.1 mL sample spike.
c. Prepare a 0.3 mL sample spike by adding 0.1 mL of standard to the 0.2 mL sample spike.
Start the instrument timer. After the timer expires, read the result. Each addition should
Iron Standard Solution, 100-mg/L
500 mL Class A volumetric flask
Class A volumetric pipet, 5 mL and Pipet Filler
Iron
Page 586
Iron
1. Prepare a 1.0 mg/L Fe standard solution:

a. Pipet 5.00 mL of Iron Standard, 100-mg/L into a 500-mL volumetric flask.
c. Prepare this solution daily.
2. Follow the FerroZine solution pillows test procedure.
3. To adjust the calibration curve using the reading obtained with the 1.00 mg/L Standard
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
260
DR 5000
1.000 mg/L Fe
0.9851.015 mg/L Fe
0.009 mg/L Fe
Summary of method
The FerroZine Iron Reagent forms a purple-colored complex with trace amounts of iron in
samples that are buffered to a pH of 3.5. This method is applicable for determining trace levels of
iron in chemical reagents and glycols and with digestion can be used to analyze samples
containing magnetite (black iron oxide) or ferrites. Test results are measured at 562 nm.
Iron
Page 587
Iron

Required reagents
Description
Quantity/Test
Unit
Catalog number
0.5 mL
500 mL
230149
50/pkg
230166
Quantity
Unit
Catalog number

OR
FerroZine Iron Reagent Solution Pillows
Required apparatus
Description
Clippers for solution pillows
each
96800
Cylinder, graduated mixing, 25 mL with stopper
each
2088640
2/pkg
2495402
Unit
Catalog number

Description
Iron Standard Solution, 100 mg/L Fe
100 mL
1417542
Iron Standard Solution, 10 mL Voluette ampule, 25 mg/L Fe
16/pkg
1425310
500 mL
2833749
each
1457449
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451537
each
1465100
Unit
Catalog number
100 mL MDB
1473632

Description
Ammonium Hydroxide, 10%
Hydrochloric Acid, 1:1, 6N
500 mL
88449
Nitric Acid, ACS, concentrated
500 mL
15249

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron, Ferrous, 8146
Iron, Ferrous
DOC316.53.01049
1-10 Phenanthroline Method1
Method 8146
0.02 to 3.00 mg/L

1
Adapted from Standard Methods for the Examination of Water and Wastewater, 15th ed. 201 (1980)
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

adjust.
Analyze samples as soon as possible to prevent air oxidation of ferrous iron to ferric iron, which is not determined.
If ferrous iron is present, an orange color will form after adding the reagent.

Description
Quantity
Powder Pillow Test:

Ferrous Iron Reagent Powder Pillows
AccuVac Test:
Ferrous Iron Reagent AccuVac Ampuls
Beaker, 50 mL (AccuVac test)
Iron, Ferrous
Page 589
Iron, Ferrous
Description
Quantity
1-10 Phenanthroline method for powder pillows
Stored Programs
255 Iron, Ferrous
Start
1. Select the test.

2. Fill a clean graduated

mixing cylinder with 25 mL
of sample.
3. Prepared Sample:
Ferrous Iron Reagent
powder pillow to the
cylinder.
4. Insert a stopper and

invert to mix. Undissolved
powder does not affect
accuracy.
10 mL of sample.
7. Fill a second sample

cell with the prepared
sample from the mixing
cylinder in step 4.
8. When the timer


for orientation.

timer.
period will begin.
Zero

0.00 mg/L Fe2+
Iron, Ferrous
Page 590
Read


mg/L Fe2+.
Iron, Ferrous
1-10 Phenanthroline method for AccuVac Ampuls
Stored Programs
257 Iron, Ferrous AV
Start
1. Select the test.
3. Prepared Sample:
Fill a Ferrous Iron Reagent
AccuVac Ampul with
sample from the beaker.
Keep the tip immersed
while the Ampul fills
completely.

timer.


period will begin.
READ the results in

mg/L Fe2+.
for orientation.

Ampul several times
to mix.
0.00 mg/L Fe2+
Collect samples in plastic or glass bottles.
Analyze samples as soon as possible after collection.
Accuracy check
Ferrous Ammonium Sulfate, hexahydrate, 0.7022 g
1 L Class A volumetric flask
Deionized water
Iron, Ferrous
Page 591
Iron, Ferrous
Analytical balance
2 mL Class A volumetric pipet and pipet filler
1. Prepare a 100 mg/L Fe2+ ferrous iron stock solution as follows:

a. Dissolve 0.7022 grams of Ferrous Ammonium Sulfate, hexahydrate, in deionized water.
b. Dilute to one liter in a Class A volumetric flask.
c. In a 100 mL Class A volumetric flask, dilute 2.00 mL of this solution to 100 mL with
deionized water to make a 2.0 mg/L standard solution. Prepare this solution immediately
before use.
2. Follow the 1-10 Phenanthroline method for powder pillows or the 1-10 Phenanthroline method
for AccuVac Ampuls test procedure.
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
255
DR 5000
2.00 mg/L Fe2+
1.992.01 mg/L Fe2+
0.021 mg/L Fe2+
257
DR 2800
Fe2+
Fe2+
0.023 mg/L Fe2+
2.00 mg/L
1.982.02 mg/L
Summary of method
The 1-10 phenanthroline indicator in the Ferrous Iron Reagent reacts with ferrous iron (Fe2+) in the
sample to form an orange color in proportion to the iron concentration. Ferric iron (Fe3+)does not
react. The ferric iron concentration can be determined by subtracting the ferrous iron concentration
from the results of a total iron test. Test results are measured at 510 nm.
Iron, Ferrous
Page 592
Iron, Ferrous

Required reagents
Description
Ferrous Iron Reagent Powder Pillows
Quantity/Test
Unit
Catalog number
100/pkg
103769
25/pkg
2514025
Catalog number
OR
Ferrous Iron Reagent AccuVac Ampuls
Required apparatus
Description
Quantity/Test
Unit
Beaker, 50 mL
each
50041H
each
2122800
6/pkg
2427606
2/pkg
2495402
Unit
Catalog number
2936701

Description
Balance, analytical, 80 g x 0.1 mg 100240 VAC
each
Ferrous Ammonium Sulfate, hexahydrate, ACS
113 g
1125614
each
1457453
Pipet filler, safety bulb
each
1465100
each
1451535
Water, deionized
Wipers, disposable
4L
27256
280/pkg
2097000
Iron, Ferrous
Page 593

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron Total FerroVer, 8008
Iron, Total
DOC316.53.01053
USEPA1 FerroVer Method2
Method 8008
0.02 to 3.00 mg/L
Scope and Application: For water, wastewater and seawater; digestion is required for determining total iron
1
USEPA approved for reporting wastewater analysis, Federal Register, June 27, 1980; 45 (126:43459)
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Digestion is required for determining total iron for EPA reporting purposes. Use the mild or vigorous digestion. Refer to the
Water Analysis Guide for more information.
adjust. See the user manual for more information.
Adjust pH of stored samples before analysis.
For turbid samples, treat the blank with one 0.1-g scoop of RoVer Rust Remover. Swirl to mix.

Description
Quantity
Powder Pillow Test:

FerroVer Iron Reagent Powder Pillow
Iron, Total
Page 595
Iron, Total
Description
Quantity
AccuVac Ampul test:

FerroVer Iron Reagent AccuVac Ampul
Beaker, 50-mL
Stopper of 18 mm tubes
FerroVer method for powder pillows
Stored Programs
265 Iron, FerroVer
Start
1. Select the test.


a clean sample cell with
10 mL of sample

one FerroVer Iron Reagent
Accuracy is not affected by
undissolved powder.

for orientation.

timer.
period will begin. An
orange color will form, if
iron is present.
(Allow samples that
contain rust to react for at
least 5 minutes.)
Zero
Iron, Total
Page 596
6. When the timer


0.00 mg/L Fe

READ the results in
mg/L Fe.
Iron, Total
FerroVer method for AccuVac Ampuls
Stored Programs
267 Iron, FerroVer AV
Start
1. Select the test.


for orientation.
3. Prepared Sample:
Fill a FerroVer Iron
AccuVac Ampul with
completely.

Ampul several times
to mix.
Accuracy is not affected by
undissolved powder.
Zero

timer.

orange color will develop if
iron is present.

0.00 mg/L Fe

READ the results in
mg/L Fe.
Interferences
Calcium,
Ca2+
Interference level
No effect at less than 10,000 mg/L as CaCO3.
Chloride, Cl
No effect at less than 185,000 mg/L.
Copper, Cu2+
No effect. Masking agent is contained in FerroVer Reagent.
High Iron Levels
Inhibit color development. Dilute sample and re-test to verify results.
Iron Oxide
Requires mild, vigorous or Digesdahl digestion. After digestion, adjust sample to pH 35 with
sodium hydroxide, then analyze.
Magnesium
No effect at 100,000 mg/L as calcium carbonate.
Iron, Total
Page 597
Iron, Total
Interference level
Molybdate Molybdenum
No effect at 50 mg/L as Mo.
High Sulfide Levels, S2
1.
Treat in fume hood or well-ventilated area. Add 5 mL hydrochloric acid1, ACS to 100 mL
sample in a 250 mL Erlenmeyer flask. Boil 20 minutes.
2.
Cool. Adjust pH to 35 with Sodium Hydroxide1. Readjust volume to 100 mL with

deionized water.
Analyze using FerroVer method for powder pillows or FerroVer method for AccuVac
Ampuls.
3.
Turbidity
1.
2.
3.
Add 0.1 g scoop of RoVer Rust Remover to the blank. Swirl to mix.
Zero the instrument with this blank.
If sample remains turbid, add three 0.2 g scoops of RoVer to a 75 mL sample.
Let stand 5 minutes.
4.
5.
Filter through a Glass Membrane Filter and Filter Holder1.

Use the filtered sample as the prepared sample and the blank.
Extreme Sample pH
Adjust pH to 35.
Highly Buffered Samples
Adjust pH to 35.
Collect samples in acid-cleaned glass or plastic containers. No acid addition is necessary if

analyzing the sample immediately.
To preserve samples, adjust the pH to 2 or less with concentrated nitric acid (about 2 mL per
liter). Preserved samples may be stored up to six months at room temperature.
Before analysis, adjust the pH to between 3 and 5 with 5.0 N Sodium Hydroxide Standard
Solution.
If only dissolved iron is to be determined, filter the sample before acid addition.
Accuracy check
Iron Voluette Ampule Standard, 25 mg/L
Ampule breaker
5. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to 10 mL of unspiked sample.
6. Prepare a 0.2 mL sample spike by adding 0.1 mL of standard to the 0.1 mL sample spike. Start
the instrument timer. After the timer expires, read the result.
Iron, Total
Page 598
Iron, Total
7. Prepare a 0.3 mL sample spike by adding 0.1 mL of standard to the 0.2 mL sample spike. Start
the instrument timer. After the timer expires, read the result.
Standard additions method for AccuVac Ampuls (sample spike)
1. Fill three mixing cylinders each with 50 mL of sample and spike with 0.2 mL, 0.4 mL and
0.6 mL of standard. Stopper and invert to mix.
2. Transfer 40 mL from each of the three mixing cylinders to three 50 mL beakers.
3. Analyze each standard addition sample as described in the FerroVer method for AccuVac
Ampuls.
recovery.
Iron Standard Solution, 100 mg/L
100-mL volumetric flask
Class A volumetric pipet, 2 mL
Deionized water
Pipet filler
1. Prepare a 2.00-mg/L Fe standard solution as follows:

a. Pipet 2.00 mL of Iron Standard Solution, 100 mg/L, into a 100 mL volumetric flask.
b. Dilute to the mark with deionized water. Mix well. Prepare this solution daily.
2. Use the 2.00 mg/L Fe standard solution in place of the sample. Follow the FerroVer method for
powder pillows test procedure.
3. To adjust the calibration curve using the reading obtained with the Standard Solution, select
concentration is used, enter the concentration and adjust the curve to that value. Mixedparameter standards are also available to simulate various matrices.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
265
2.00 mg/L Fe
1.992.01 mg/L Fe
0.021 mg/L Fe
267
2.00 mg/L Fe
1.982.02 mg/L Fe
0.023 mg/L Fe
Iron, Total
Page 599
Iron, Total
Summary of method
FerroVer Iron Reagent converts all soluble iron and most insoluble forms of iron in the sample to
soluble ferrous iron. The ferrous iron reacts with the 1-10 phenanthroline indicator in the reagent to
form an orange color in proportion to the iron concentration. Test results are measured at 510 nm.

Required reagents
Description
FerroVer Iron Reagent Powder Pillows (for 10-mL sample)
Quantity/Test
Unit
Catalog number
100/pkg
2105769
25/pkg
2507025
Quantity
Unit
Catalog number
OR
FerroVer Iron Reagent AccuVac Ampuls
Required apparatus
Description
Beaker, 50 mL
each
50041H
each
2122800
6/pkg
2427606
2/pkg
2495402
6/pkg
173106
Unit
Catalog number
Description
100 mL
1417542
Iron Standard Solution, 10 mL Voluette Ampule, 25 mg/L as Fe
16/pkg
1425310
500 mL
2833749
500 mL
2833649
Water, deionized
4L
27256
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1457442
each
1451536
each
1465100
Description
Unit
Catalog number
Beaker, 50 mL
each
50041H
each
189641
Hydrochloric Acid, concentrated
500 mL
13449
500 mL
15249
Iron, Total
Page 600
Iron, Total
Description
Unit
Catalog number
245032
100 mL
Glass Membrane Filter, 47 mm
100/pkg
253000
Glass Membrane Filter Holder
each
234000
RoVer Rust Remover
454 g
30001
each
51100
Iron, Total
Page 601
Iron, Total

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron Total, 8112
Iron, Total
DOC316.53.01051
TPTZ Method1
Method 8112
0.012 to 1.800 mg/L

1
Adapted from G. Frederic Smith Chemical Co., The Iron Reagents, 3rd ed. (1980)
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Digestion is required for determining total iron.
adjust.
Rinse all glassware with a 1:1 Hydrochloric Acid Solution1. Rinse again with deionized water. This process will remove iron
After adding reagent, a blue color will develop if iron is present.
Adjust the pH of stored samples to 34. Do not exceed pH 5 or iron may precipitate.
1

Description
Quantity
Powder Pillow Test:

TPTZ Iron Reagent Powder Pillow
Iron, Total
Page 603
Iron, Total
Description
Quantity
AccuVac Test:
TPTZ Low Range Iron Reagent AccuVac
Beaker, 50 mL
TPTZ method for powder pillows
Stored Programs
270 Iron, TPTZ
Start
1. Select the test.

for orientation.

one clean sample cell with
10 mL of sample.
10-mL TPTZ Iron Reagent
prepared sample. Swirl at
least 30 seconds to
dissolve.

timer.
period will begin.
water.
Proceed with step 4 and 5

while the timer is running.
Zero

one 10-mL TPTZ Iron
the blank. Swirl at least 30
seconds to dissolve. This
will be the reagent blank.
Iron, Total
Page 604
6. When the timer


0.000 mg/L Fe

READ the results in
mg/L Fe.
Iron, Total
TPTZ method for AccuVac Ampuls
Stored Programs
272 Iron, TPTZ AV
Start
1. Select the test.

10 mL of sample.

for orientation.
3. Prepared Sample:
Fill a TPTZ Iron AccuVac

Ampul several times
to mix.
Zero

timer.
period will begin.


0.000 mg/L Fe

READ the results in
mg/L Fe
Iron, Total
Page 605
Iron, Total
Interferences
Interferences are tested (see the Interfering substances table) with an iron concentration of
0.5 mg/L. The following do not interfere with the method when present up to the levels given.
Interference level
Cadmium
4.0 mg/L
Chromium
3+
Chromium
6+
0.25 mg/L
1.2 mg/L
Cobalt
0.05 mg/L
Copper
0.6 mg/L
Cyanide
2.8 mg/L
Manganese
50.0 mg/L
Mercury
0.4 mg/L
Molybdenum
4.0 mg/L
Nickel
1.0 mg/L
Nitrite Ion
0.8 mg/L
Color or turbidity
In the powder pillow procedure, if the sample, without a TPTZ Iron Reagent Powder Pillow,
has a color or turbidity greater than the blank (deionized water plus TPTZ Iron Reagent),
then use the sample as the blank.
pH
A sample pH of less than 3 or greater than 4 after the addition of reagent may inhibit color
formation, cause the developed color to fade quickly or to result in turbidity. Adjust the
sample pH in the sample cell before the addition of reagent:
1. Measure the current pH by using a pH meter or pH paper.
2. Adjust the sample pH to between 3 and 4 by adding an appropriate amount of iron-free
acid or base such as 1.0 N Sulfuric Acid Standard Solution1 or 1.0 N Sodium Hydroxide
Standard Solution1.
3. Make a volume correction if significant volumes of acid or base are used. Refer to the
To preserve samples, adjust the sample pH to 2 or less with about 2 mL/L Nitric Acid, ACS*.
If reporting only dissolved iron, filter sample immediately after collection and before adding
nitric acid.
Before testing, adjust the pH of the stored sample to between 34 with 5.0 N Sodium
Hydroxide Standard Solution*. Do not exceed pH 5 as iron may precipitate.
Iron, Total
Page 606
Iron, Total
Accuracy check
Standard additions method for powder pillows (sample spike)
Beakers, 50 mL (3)
Graduated mixing cylinders, 50 mL (3)
TenSette Pipet
4. Open a fresh bottle of standard solution.
5. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of
6. Follow the TPTZ method for powder pillows test procedure for each of the spiked samples
using the powder pillows or AccuVac ampules, starting with the 0.1 mL sample spike. Measure
each of the spiked samples in the instrument.
1. Fill three mixing cylinders each with 50 mL of sample and spike with 0.5 mL, 1.0 mL and 1.5
mL of standard. Stopper and invert to mix.
2. Transfer 40 mL from each of the three mixing cylinders to three 50 mL beakers.
3. Analyze each standard addition sample as described in the TPTZ method for AccuVac
Ampuls.
4. Accept each standard additions reading. Each addition should reflect approximately
100% recovery.
Iron Standard Solution, 1 mg/L or
5 mL Class A volumetric pipet and pipet filler
Deionized water
1. Use a 1 mg/L Iron Standard Solution or prepare a 1.00 mg/L iron standard solution as follows:
Iron, Total
Page 607
Iron, Total
2. Use the 1.00 mg/L iron standard solution in place of the sample. Follow the TPTZ method for
powder pillows or Accuvac Ampuls.
3. To adjust the calibration curve using the reading obtained with the 1.00 mg/L Standard
concentration is used, enter the concentration and adjust the curve to that value. Mixedparameter standards are also available to simulate various matrices.
Method performance
Precision
Distribution
Sensitivity
Program
Standard
270
1.000 mg/L Fe
0.9891.011 mg/L Fe
0.011 mg/L Fe
272
1.000 mg/L Fe
0.9841.016 mg/L Fe
0.012 mg/L Fe
Summary of method
The TPTZ Iron Reagent forms a deep blue-purple color with ferrous iron (Fe2+). The indicator is
combined with a reducing agent that converts precipitated or suspended iron, such as rust, to the
ferrous state. The amount of ferric iron (Fe3+) present can be determined as the difference
between the results of a ferrous iron test and the concentration of total iron. Test results are
measured at 590 nm.
Iron, Total
Page 608
Iron, Total

Required reagents
Description
TPTZ Iron Reagent Powder Pillows (for 10-mL sample)
Quantity/Test
Unit
Catalog number
100/pkg
2608799
25/pkg
2510025
Quantity
Unit
Catalog number
OR
TPTZ Low Range Iron Reagent AccuVac Ampuls
Required apparatus
Description
Beaker, 50 mL
each
50041H
each
2122800
6/pkg
2427606
2/pkg
2495402
Unit
Catalog number
Description
100 mL
1417542
500 mL
14049
500 mL
13949
500 mL
2833749
500 mL
2833649
4L
27256
Unit
Catalog
number
each
189641
Water, deionized

Description
Nitric Acid, ACS concentrated
500 mL
15249
50 mL SCDB
245026
100 mL SCDB
127032
100 mL MDB
104532
6/pkg
173106
Tensette Pipet, 0.11.0 mL
each
1970001
50/pkg
2185696
each
1457449
Pipet, volumetric, 5 mL Class A
each
1451537
Pipet Filler, Safety bulb
each
1465100
Iron, Total
Page 609

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron, FerroZine, 8147
Iron
DOC316.53.01050
FerroZine Rapid Liquid Method1
Method 8147
0.009 to 1.400 mg/L Fe
Pour-Thru Cell
Scope and Application: For boiler, cooling and natural waters

1
Adapted from Stookey, L.L., Anal.Chem., 42 (7) 779 1970.
Test preparation


Instrument
Pour-thru Kit
Cell orientation
Adapter
DR 6000
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
LQV157.99.10002
5940400
LZV585 (B)
DR 3800, DR 2800, DR 2700

If sample contains rust, see Interferences.
adjust.
If iron is present, a purple color will form after adding the reagent
Rinse glassware with a 1:1 Hydrochloric acid1 solution. Rinse again with deionized water. This will remove residual iron that
may interfere
Prepare the Pour-Thru cell by preparing a solution of 1 mL Ferrozine Reagent per 50 mL of deionized water. Pour this into
the cell and allow to stand for approximately 5 minutes to react with any trace iron in the cell and cell tubing. Flush with
deionized water.
FerroZine Iron Reagent may crystallize or precipitate when exposed to cold temperatures during shipment; reagent quality is
not affected. Place the reagent bottle in warm water to dissolve.
1
Iron
Page 611
Iron

Description
Quantity
1.0 mL
Water, deionized
varies
Cylinder, graduated, 50 mL poly
Dispenser, fixed volume, 1.0 mL, with bottle
Flask, Erlenmeyer, PMP w/cap, 125 mL
Pour-Thru Cell Module (see Instrument-specific information)
FerroZine Rapid Liquid Method for Pour-Thru Cell
Stored Programs
261 Iron, FerroZine RL
Start
1. Select the test.


Cell with 50 mL of
deionized water.
3. Rinse two clean

125 mL Erlenmeyer flasks
with the sample three
times.
4. Rinse a clean 50-mL

three times with the
sample.

the 50 mL cylinder into
one of the flasks.
7. Prepared sample:
Add 1.0 mL of FerroZine
Iron Reagent Solution to
flask using the Dispenser.
Swirl to mix.

timer.

for orientation.

to the 50 mL mark with
sample.
Iron
Page 612
period will begin.
Iron
FerroZine Rapid Liquid Method for Pour-Thru Cell (continued)
Zero
Measure a second 50 mL
portion of sample into the
graduated cylinder and
pour the contents into the
second flask.
10. When the timer

expires the display will
show mg/L Fe.
Pour the contents of the
flask containing the blank
into the Pour-Thru Cell


0.000 mg/L Fe

the flask containing the
Pour-Thru Cell.
Read

READ the results in
mg/L Fe.

Cell with at least 50 mL of
deionized water
Iron
Page 613
Iron
Interferences
Interference level
Strong Chelants (EDTA)
Interfere at all levels. Use the FerroVer or TPTZ methods for these samples. Use the
TPTZ method for low iron concentrations.
Cobalt
Copper
Hydroxides
Boil the sample with the FerroZine Iron Reagent added to it from step 7 of the test procedure
for 1 minute in a boiling water bath. Cool to 24 C (75 F) before proceeding with step 9.
Return the sample volume to 50 mL with deionized water.
Magnetite (black iron oxide) or

Ferrites
1.
2.
Fill a 50 mL graduated cylinder with 50 mL of sample.

Transfer the sample into a clean glass 125 mL Erlenmeyer flask.
3.
4.
5.
6.
Add 1.0 mL of FerroZine Iron Reagent Solution1 and swirl to mix.

Place the flask on a hot plate or over a flame and bring to a boil.
Continue boiling gently for 20 to 30 minutes. Do not allow to boil dry.
Return the boiled sample to the graduated cylinder. Rinse the Erlenmeyer flask with
small amounts of deionized water and empty into the graduated cylinder. A purple color
will develop if iron is present.
Return the sample volume to the 50 mL mark with deionized water.
Pour the solution into a 125 mL Erlenmeyer flask and swirl to mix.
Proceed with steps 914 of the test procedure.
7.
8.
9.
Rust
1
To preserve samples, adjust the sample pH to 2 or less with concentrated Nitric Acid, ACS*
(about 2 mL per liter). Samples preserved in this manner can be stored up to six months at
room temperature.
If only reporting dissolved iron, filter the sample immediately after collection and before adding
nitric acid.
Before testing, adjust the sample pH to 35 with Ammonium Hydroxide, 10%. Do not exceed
pH 5 or iron may precipitate.
Correct test results for volume additions. Refer to the Water Analysis Guide for more
information.
Iron
Page 614
Iron
Labware
All containers used in this test must be cleaned thoroughly to remove any traces of iron.
Rinse labware and the Pour-Thru Cell with a 1:1 HCl solution* or with a 1:50 dilution of
FerroZine Reagent. Rinse several times with deionized water.
Keep flasks tightly closed when not in use. Dedicate these containers for iron analysis only. If
containers are rinsed and capped after each use, only occasional treatment with HCl or
FerroZine is necessary.

The Pour-Thru Cell may accumulate a buildup of color products, especially if the reacted solutions
are allowed to stand in the cell for long periods after measurement. Remove the color by rinsing
with a 1:5 dilution of Ammonium Hydroxide*, followed by several rinses with deionized water.
Cover the Pour-Thru Cell after use.
Accuracy check
Ampule breaker
Graduated mixing cylinder, 50 mL (3)
standard to three 50 mL portions of fresh sample.
6. Follow the FerroZine Rapid Liquid Method for Pour-Thru Cell test procedure for each of the
spiked samples starting with the 0.2 mL sample spike. Measure each of the spiked samples in
the instrument.
addition results to the theoretical 100% recovery. Each addition should reflect approximately
100% recovery.
1.0 mg/L Iron Standard Solution or
100 mg/L Iron Standard Solution
Deionized water
Iron
Page 615
Iron
5 mL Class A volumetric pipet and filler
1. To check the accuracy, use a 1.0 mg/L Iron Standard Solution or prepare a 1.0 mg/L iron
working solution as follows:
a. Pipet 5.00 mL of iron standard solution, 100 mg/L Fe, into a 500 mL volumetric flask.
b. Dilute to volume with deionized water. Prepare this solution daily.
2. Follow the FerroZine Rapid Liquid Method for Pour-Thru Cell test procedure.
concentration is used, enter the concentration and adjust the curve to that value. Mixedparameter standards are also available to simulate various test matrices.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
261
1.000 mg/L Fe
0.9971.003 mg/L Fe
0.009 mg/L Fe
Summary of method
The FerroZine Iron Reagent forms a purple colored complex with trace amounts of iron in
samples that are buffered to a pH of 3.5. This method is applicable for determining trace levels of
iron in chemical reagents and glycols and with digestion can be used to analyze samples
containing magnetite (black iron oxide) or ferrites. The test results are measured at 562 nm.
Iron
Page 616
Iron

Required reagents
Description
Quantity/Test
Unit
Catalog number
1 mL
500 mL
230149
Water, deionized
varies
4L
27256
Quantity
Unit
Catalog number
Required apparatus
Description
each
108141
Dispenser, fixed volume, 1.0-mL
each
2111302
Flask, Erlenmeyer, PMP w/cap, 125-mL
each
2089843
Unit
Catalog number
each
1457449

Description
Iron Standard Solution, 100-mg/L Fe
100 mL
1417542
Iron Standard Solution, Voluette ampule, 25-mg/L Fe, 10-mL
16/pkg
1425310
500 mL
13949
500 mL
2833749
Pipet, TenSette 0.11.0 mL
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451537
each
1465100

Description
Unit
Catalog number
Ammonium Hydroxide, ACS, 58%
500 mL
10649
Ammonium Hydroxide, ACS, 10%
100 mL MDB
1473632
Hydrochloric Acid, 1:1, 6N
500 mL
88449
Nitric Acid ACS, concentrated
500 mL
15249
Iron
Page 617

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron Total FerroMo, 8365
Iron, Total
DOC316.53.01052
FerroMo Method1
Method 8365
0.01 to 1.80 mg/L
Powder Pillows
Scope and Application: For cooling water containing molybdate-based treatment

1
Adapted from G. Frederick Smith Chemical Co., The Iron Reagents, 3rd ed. (1980)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

adjust.
Rinse glassware with a 1:1 hydrochloric acid solution. Rinse again with deionized water. These two steps will remove iron
After the addition of the reagent, the sample pH should be between 35.
If the sample contains high levels of molybdate (100 mg/L MoO42 or greater), read the sample immediately after zeroing
the blank.

Description
Quantity
FerroMo
Reagent 1 Powder Pillow
FerroMo
Cylinder, graduated mixing, 25 mL, with stopper
Iron, Total
Page 619
Iron, Total
Description
Quantity
FerroMo method for powder pillows
Stored Programs
275 Iron, FerroMo
Start
1. Select the test.


a 50-mL graduated mixing
cylinder with 50 mL of
sample.

one FerroMo Iron
to the graduated mixing
cylinder. Insert the
stopper.

dissolve the reagents.
6. Developed sample:
FerroMo Iron Reagent 2
sample in the 25-mL
mixing cylinder.
7. Insert the stopper and

invert to dissolve the
reagents. A blue color will
develop if iron is present.

timer.

for orientation.
5. Fill a clean, 25-mL

25-mL mark with prepared
sample. Save the
remaining prepared
sample for step 10.
Iron, Total
Page 620
A small amount of
undissolved reagent will
not affect the results.
time will begin.
Iron, Total
FerroMo method for powder pillows (continued)
Zero
9. When the timer

expires, pour 10 mL of the
developed sample from
step 7 into a sample cell.
10. Blank preparation: Fill

10 mL of the remaining
prepared sample from
step 5.

the cell holder.

0.00 mg/L Fe
Read
13. Insert the developed


mg/L Fe.
Interferences
Interference level
pH
A sample pH of less than 3 or greater than 4 after the addition of reagent may inhibit color
formation, cause the developed color to fade quickly or result in turbidity. Adjust the sample
pH to between 3 and 8 in the graduated cylinder before the addition of reagent:
1. Add by drops an appropriate amount of iron-free acid or base such as 1.0 N Sulfuric
Acid Standard Solution1 or 1.0 N Sodium Hydroxide Standard Solution1.
2. Make a volume correction if significant volumes of acid or base are used. Refer to the
To preserve samples, adjust the sample pH to 2 or less with hydrochloric acid (about 2 mL per
liter)*. Samples preserved in this manner can be stored up to six months at room temperature.
Iron, Total
Page 621
Iron, Total
If only dissolved iron is to be reported, filter sample immediately after collection through a 0.45
micron filter or equivalent medium before adding hydrochloric acid.
Before testing, adjust the sample pH to 35 with 5.0 N Sodium Hydroxide Standard Solution*.
Do not exceed pH 5 as iron may precipitate.
Accuracy check
Mixing cylinders, 50 mL (3)
Ampule breaker
6. Follow the FerroMo method for powder pillows test procedure for each of the spiked samples,
starting with the 0.1 mL sample spike. Measure each of the spiked samples in the instrument.
Accept each standard additions reading. Each addition should reflect approximately 100%
recovery.
Iron Standard Solution, 1 mg/L or
1 mL Class A volumetric pipet
Deionized water
1. Use a 1.0 mg/L standard or prepare a 1.00 mg/L iron standard solution as follows:
2. Use the 1.00 mg/L iron standard solution in place of the sample. Follow the FerroMo method
for powder pillows test procedure.
Iron, Total
Page 622
Iron, Total
Method performance
Program
Standard
Precision
Distribution
Sensitivity
275
1.00 mg/L Fe
0.981.02 mg/L Fe
0.01 mg/L Fe
Summary of method
FerroMo Iron Reagent 1 contains a reducing agent combined with a masking agent. The masking
agent eliminates interference from high levels of molybdate. The reducing agent converts
precipitated or suspended iron, such as rust, to the ferrous state. FerroMo Iron Reagent 2 contains
the indicator combined with a buffering agent. The indicator reacts with ferrous iron in the sample,
buffered between pH 3 and 5, resulting in a deep blue-purple color. Test results are measured at
590 nm.

Required reagents
Description
Quantity/Test
Unit
FerroMo Iron Reagent Set (100 tests), includes:
Catalog number
2544800
(4) FerroMo Reagent 1 Powder Pillows
25/pkg
2543768
(2) FerroMo Reagent 2 Powder Pillows
50/pkg
2543866
Quantity
Unit
Catalog number
Required apparatus
Description
each
2088640
each
2088641
2/pkg
2495402
Unit
Catalog number
1417542
Description
100 mL
500 mL
13949
Iron Standard Solution, 10 mL Voluette ampule, 50 mg/L Fe.
16/pkg
1425410
4L
27256
Water, deionized
Iron, Total
Page 623

Description
Unit
Catalog number
each
1457442
each
1451535
each
1465100

100 mL MDB
104532
100 mL MDB
245032
100 mL MDB
127032
100 mL MDB
245032
500 mL
88449
Hydrochloric Acid, 1:1

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Iron, DT, 8214
Iron
DOC316.53.01177
TitraVer Titration Method
Method 8214
10 to 1000 mg/L as Fe
Digital Titrator
Test preparation


Description
Quantity
Citrate Buffer Powder Pillow
1 pillow
Sodium Periodate Powder Pillow
1 pillow
Sulfosalicylic Acid Powder Pillow
1 pillow
TitraVer Standard Solution titration cartridge (see Range-specific information)
1 cartridge
Digital titrator
Graduated cylinder
Iron
See
Table 1
1. Select a sample

3. Hold the Digital

wipe the tip.
4. Use a graduated
measure the sample
flask.
Iron
Page 625
Iron
Iron


changes from red to the
original yellow.
counter.

one Citrate Buffer Powder
Pillow. Swirl to mix.

one Sodium Periodate
mix.

one Sulfosalicylic Acid
mix.
A yellow color will develop

if iron is present.
A red color will develop if

iron is present.

the Range-specific
calculate the
concentration:
mg/L Fe
0.0716 N cartridge and
= 25 mg/L Fe

Range (mg/L as Fe)
Sample volume (mL)
Titration cartridge (M TitraVer)
Multiplier
1040
50
0.0716
0.1
25100
20
0.0716
0.25
100400
50
0.716
1.0
2501000
20
0.716
2.5
Iron
Page 626
Iron

Accuracy check
Iron Standard Solution, 1000-mg/L as Fe
titration cartridge or 100 digits of the 0.0716 M titration cartridge to reach the endpoint.
Summary of method
Ferrous iron (Fe2+) is oxidized by sodium periodate to ferric ion (Fe3+). The ferric ion forms a red
complex with sulfosalicylic acid. The red complex is destroyed by titration with EDTA. Citric acid is
used to buffer the solution and to stabilize the ferric ion in solution.

Required reagents
Description
Quantity/Test
Unit
(1) Citrate Buffer Powder Pillows
1 pillow
100/pkg
(1) Sodium Periodate Powder Pillows
1 pillow
100/pkg
98499
(1) Sulfosalicylic Acid Powder Pillows
1 pillow
100/pkg
2081669
varies
each
2081701
(1) Citrate Buffer Powder Pillows
1 pillow
100/pkg
2081599
(1) Sodium Periodate Powder Pillows
1 pillow
100/pkg
98499
(1) Sulfosalicylic Acid Powder Pillows
1 pillow
100/pkg
2081669
varies
each
2081801
10100 mg/L rangeReagent Set (approximately 100 tests):
(1) TitraVer Standard Solution Titration Cartridge, 0.0716 M
2449200
1001000 mg/L rangeReagent Set (approximately 100 tests):
(1) TitraVer Standard Solution Titration Cartridge, 0.716 M
Catalog number
2081599
2449300
Iron
Page 627
Iron
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50543

each
50840
each
50841
each
1720500
each
4157800
Unit
Catalog number
100 mL
227142
Description
Unit
Catalog number
each
2095352
each
1970001
each
1940000
each
1940010
Description
Iron Standard Solution, 1000-mg/L as Fe

Water, deionized
500 mL
27249
Bottle, sampling
250 mL
2087076
Iron standard, 10 mg/L
500 mL
14049
10 mL/16
1425310
10 mL/16
1425410
100 mL
1417542
Pipet tips
100/pkg
2185628
Pipet tips
50/pkg
2185696
each
2196800
Voluette breaker

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Lead, 8033
Lead
DOC316.53.01055
USEPA1 Dithizone Method2
Method 8033
3 to 300 g/L
Powder Pillows

1
USEPA accepted for reporting for wastewater analysis (digestion is required)
Procedure is equivalent to Standard Method 3500-Pb D for wastewater analysis.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2612602
DR 5000
2612602
DR 3900
2612602
DR 3800, DR 2800, DR 2700
2612602

Clean all glassware with a 1:1 Nitric Acid Solution. Rinse with deionized water.
Cloudy and turbid samples may require filtering before running the test. Report results as g/L soluble lead. Use glass
membrane type filter to avoid loss of lead by adsorption onto the filter paper.
If samples cannot be analyzed immediately, see Sample collection, preservation and storage. Adjust the pH of preserved
For more accurate results, adjust the sample to pH 11.011.5 using a pH meter in step 11. Omit the five additional drops of
Sodium Hydroxide Standard Solution in step 12
The DithiVer powder will not completely dissolve in the chloroform. For further notes see DithiVer solution preparation,
storage and reagent blank.
Read the MSDS before testing. Spilled reagent will affect test accuracy and is hazardous to skin and other materials.
Digestion is required to for determine the total lead for EPA reporting purposes. Use mild or vigorous digestion.
Lead
Page 629
Lead

Description
Quantity
Citrate Buffer Powder Pillows
Chloroform
50 mL
Potassium Cyanide
2g
varies
Cotton Balls
Clippers
Cylinder, 50 mL graduated mixing
Cylinder, 5 mL graduated
Funnel, 500 mL separatory
Support Ring (4 inch) and Stand (5 x 8 inch base)
Dithizone method for powder pillows
Stored Programs
280 Lead Dithizone
Start
1. Select the test.

for orientation.
Lead
Page 630
2. Fill a 250 mL
250 mL mark with sample.

into 500 mL separatory
funnel.

one Buffer Powder Pillow,
citrate type.
Lead
Dithizone method for powder pillows (continued)
5. Insert the stopper into

the funnel and shake to
dissolve.
9. Add 5 mL of 5.0 N
Sodium Hydroxide
Standard Solution.
6. DithiVer Solution
preparation:
7. Stopper the cylinder.

Invert several times to mix.
Add 50 mL of chloroform
to a 50-mL mixing
graduated cylinder. Add
the contents of one
DithiVer Metals Reagent
Powder Pillow.
10. Stopper. Invert. Open

stopcock to vent. Close
the stopcock and shake
the funnel once or twice
and vent again.
If the solution turns orange
after shaking, the pH is too
high. Add a few drops of
5.25 N Sulfuric Acid to the
solution to decrease the
pH.
The blue-green color will
reappear (alternatively, to
avoid higher blanks,
repeat on new sample and
use less sodium hydroxide
in step 9).
8. Measure 30 mL of the
prepared dithizone
solution with a second
graduated cylinder and
add to the separatory
funnel.
Insert the stopper and
invert to mix. Open
stopcock to vent. Close
the stopcock.
11. Continue adding 5.0 N

Sodium Hydroxide
Standard Solution
dropwise and shaking the
funnel after every few
drops until the color of the
solution being shaken
changes from blue-green
to orange.
Large amounts of zinc
cause the color transition
at the end point to be
indistinct.
12. Add 5 more drops of

5.0 N Sodium Hydroxide
Standard Solution.
A pink color in the bottom
(chloroform) layer at this
point does not necessarily
indicate lead is present.
Only after adding the
potassium cyanide in the
next step will the presence
of lead be confirmed by a
pink color.
Lead
Page 631
Lead
Dithizone method for powder pillows (continued)
13. Add 2 heaping 1.0 g

scoops of potassium
cyanide to the funnel.
Stopper.
Shake vigorously until the
potassium cyanide is all
dissolved (about 15
seconds).
14. Wait one minute for

the layers to separate. The
will be pink if lead is
present.
15. Prepared sample:

Insert a cotton plug
the size of a pea into the
delivery tube of the funnel
and slowly drain the
Insert the stopper.

Measure 10 mL of
chloroform into another
sample cell.
Insert the stopper.
The lead-dithizone
complex is stable for at
least thirty minutes if the
sample cell is kept tightly
capped and out of direct
sunlight.
Zero

the cell holder.

0 g/L Pb2+
Lead
Page 632
Read


g/L Pb2+.
Lead
Interferences
Table 198 Substances that do not interfere
Non-interfering substance
Non-interfering substance
Aluminum
Lead
Antimony
Magnesium
Arsenic
Manganese
Calcium
Nickel
Chromium
Tin
Cobalt
Zinc
Iron
Interference from the metals in the Interfering substances table can be eliminated by inserting the
Interference treatment for metals procedure after step 6 of the Dithizone method for powder
pillows procedure.

Interference level

extreme sample pH
All levels. See Interference treatment for metals.
Bismuth
Copper
Mercury
Silver
Tin
Interference treatment for metals

1. Measure about 5 mL of the DithiVer solution into the separatory funnel. Stopper the funnel,
invert and open the stopcock to vent. Close the stopcock and shake the solution vigorously for
15 seconds. Allow the funnel to stand undisturbed until the layers separate (about 30
seconds). A yellow, red or bronze color in the bottom (chloroform) layer confirms the presence
of interfering metals. Draw off and collect the bottom (chloroform) layer for proper disposal.
2. Repeat extraction with fresh 5 mL portions of prepared dithizone solution (collecting the
bottom layer each time in appropriate waste collection vessel) until the bottom layer shows a
pure dark green color for three successive extracts. Extractions can be repeated a number of
times without appreciably affecting the amount of lead in the sample.
3. Extract the solution with several 2 or 3 mL portions of pure chloroform to remove any
remaining dithizone, again collecting the bottom layer each time for proper disposal.
4. Continue the procedure, substituting 28.5 mL of prepared dithizone solution for the 30 mL in
step 8.
Lead
Page 633
Lead
DithiVer solution preparation, storage and reagent blank
Store DithiVer Powder Pillows away from light and heat.
A convenient way to prepare this solution is to add the contents of 10 DithiVer Metals Reagent
Powder Pillows to a 500 mL bottle of chloroform.
Invert several times until well mixed (carrier powder may not dissolve).
Store dithizone solution in an amber glass bottle. This solution is stable for 24 hours.
Carry out a reagent blank using deionized water through the entire method to obtain the most
accurate results.
Collect samples in an acid-washed glass or plastic containers.
Adjust the pH to 2 or less with nitric acid (about 2 mL per liter).
Adjust the pH to 2.5 with 5.0 N sodium hydroxide before analysis.
Accuracy check
Lead Voluette Ampule Standard, 50 mg/L Pb
Ampule breaker
1. After reading test results, leave the sample cell (unspiked sample) in the instrument.Verify that
units are in g/L.
6. Follow the Dithizone method for powder pillows test procedure for each of the spiked samples
Lead
Page 634
Lead Standard Solution, 100 mg/L
Deionized water
Lead
Pipet filler
1. Prepare a 10 mg/L lead standard solution as follows:

a. Pipet 10.00 mL of Lead Standard, 100 mg/L, into a 100 mL volumetric flask.
2. Prepare a 200 g/L lead standard solution as follows:
Use a graduated cylinder to measure 245 mL of deionized water into the 500 mL separatory
funnel (step 3 of the Dithizone method for powder pillows test). Pipet 5.00 mL of the 10.0 mg/L
Lead standard into the funnel.
3. Follow the Dithizone method for powder pillows test procedure.
4. To adjust the calibration curve using the reading obtained with the 200 g/L Standard Solution,
Method performance
Program
Standard
Precision
Distribution
Sensitivity
280
150 g/L Pb
140160 g/L Pb
2.3 g/L
Summary of method
The dithizone method is designed for the determination of lead in water and wastewater. The
DithiVer Metals Reagent is a stable powder form of dithizone. Lead ions in basic solution react with
dithizone to form a pink to red lead-dithizonate complex, which is extracted with chloroform. Test

Required reagents
Description
Lead Reagent Set (100 Tests)
Quantity/Test
Unit
Catalog number
2243100
1420299
Includes: (1) 1420299, (2) 1445817, (1) 1261699, (2) 76714, (1) 245053, (2) 245026
Buffer Powder Pillows, citrate
Chloroform, ACS
100/pkg
30 mL
4L
1445817
100/pkg
1261699
Potassium Cyanide
0.1 g
125 g
76714
Sodium Hydroxide Solution, 5.0 N
5 mL
1000 mL
245053
varies
59 mL DB
245026
Lead
Page 635
Lead
Required apparatus
Description
Quantity
Unit
each
Catalog number
96800
100/pkg
257201
each
50837
each
50841
each
50846
Cylinder, graduated, mixing, 50 mL
each
189641
Funnel, separatory, 500 mL
each
52049
pH Meter, sension1, portable, with electrode
each
5170010
Spoon, measuring,1 g
each
51000
Support Ring, 4"
each
58001
Support Ring Stand, 5" x 8" base
each
56300
Sample Cell, 1-inch square, w/stopper, matched pair
2/pkg
2612602
Unit
Catalog number
Description
Lead Standard Solution, 100 mg/L Pb
100 mL
1261742
Lead Standard Solution, 10 mL Voluette Ampules, 50 mg/L Pb
16/pkg
1426210
Unit
Catalog number

Description
Ampule Breaker Kit
each
2196800
Chloroform, ACS
500 mL
1445849
Filter Discs, glass, 47 mm
100/pkg
253000
Filter Holder, glass, for 47-mm filter
each
234000
Flask, Erlenmeyer, 500 mL
each
50549
Flask, filtering, 500 mL
each
54649
each
1457442
500 mL
254049
Nitric Acid, ACS
500 mL
15249
5 rolls/pkg
39133
Pipet, serological, 2 mL
each
53236

each
1970001
50/pkg
2185696
each
1451537
each
1451538

Water, deionized

each
1465100
100 mL MDB
244932
4L
27256
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Lead, LeadTrak, 8317
Lead
DOC316.53.01054
LeadTrak Fast Column Extraction Method
Method 8317
5 to 150 g/L
Scope and Application: For drinking water
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

adjust.
The sampling requirements for first-draw analysis are detailed in Sample collection, preservation and storage.
Reagents will stain the sample cells, rinse the cells with 1:1 nitric acid, LeadTrak followed by deionized water.

Description
Quantity
LeadTrak Reagent Set
Beaker, polypropylene, 150 mL
Clamp, 2-prong extension, with clamp holder
Cylinder, graduated polypropylene, 25 mL
Dropper, 0.5 and 1.0 mL marks
Support for Ring Stand
Lead
Page 637
Lead
LeadTrak Fast Column Extraction
Stored Programs
283 Lead, LeadTrak
Start
1. Select the test.

for orientation.
2. Fill a 100 mL plastic

graduated cylinder with
100 mL of the sample.
Pour the measured
sample into a 250 mL
plastic beaker.
3. Using a plastic 1mL

dropper, add 1.0 mL of
pPb-1 Acid Preservative
Solution to the sample and
swirl to mix.

timer.
period will begin.
If the sample has been

preserved previously with
pPb-1 Acid Preservative at
a ratio of 1.0 mL per 100
mL sample, omit steps 3
and 4.
Samples preserved with
Nitric Acid require steps 3
and 4.
5. When the timer

expires, use a second
1 mL plastic dropper to
add 2.0 mL of pPb-2 Fixer
6. Mount a new Fast

Column Extractor in a ring
stand with a clamp. Place
a 150-mL plastic beaker
under the Extractor.
Field samples that have

been preserved with nitric
acid or samples that have
been digested may
exceed the buffer capacity
of the Fixer Solution. After
step 5, check the pH of
these samples and adjust
with 5 N Sodium
Hydroxide to a pH of 6.7
7.1 before proceeding with
step 6.
A Fast Column Extractor is

included in the LeadTrak
Reagent Set. A new
extractor is required for
each test.
Lead
Page 638
7. Soak the cotton plug

with deionized water and
compress it with the
plunger. Remove the
plunger. If the cotton plug
moves up the column,
push it back to the bottom
with a clean, blunt rod.
The cotton plug should fit
snugly against the inner
wall of the column.
8. Pour the prepared

sample slowly into the
center of the Column
Extractor. Wait for the
sample to flow through.
The sample solution
should flow relatively
slowly (2 drops per
second) through the
column.
Keep the level of the
sample solution just above
the cotton plug.
Lead
LeadTrak Fast Column Extraction (continued)
9. After the flow has

stopped, fully compress
the absorbent pad in the
Extractor with the plunger.
Discard the contents of the
beaker. Slowly withdraw
the plunger from the
Extractor.
The absorbent pad should
remain at the bottom of the
Extractor when the
plunger is removed. If the
cotton plug moves up the
column, push it back to the
bottom with a clean,
blunt rod.

one pPb-5 Indicator
beaker and swirl
thoroughly to mix.
10. Place a clean, dry 150

mL beaker under the
Extractor. Using a 25 mL
plastic graduated cylinder,
add 25 mL of pPb-3 Eluant
Solution to the Extractor.
11. Allow the Eluant

Solution to drip slowly from
the Extractor.
After the flow has stopped,
fully compress the
absorbent pad.
Keep the level of the

eluent solution just above
the absorbent pad.
14. Pour 10 mL of solution

into a sample cell.

timer.
period will begin.
12. Using a 1 mL plastic

dropper, add 1.0 mL of
pPb-4 Neutralizer Solution
to the beaker. Swirl
thoroughly to mix and
proceed immediately to
step 13.
16. When the timer

expires, insert the sample
cell.
The solution will turn

brown.
Lead
Page 639
Lead
LeadTrak Fast Column Extraction (continued)
Zero
Read

0 g/L Pb
18. Remove the sample

cell and add 3 drops of
pPb-6 Decolorizer Solution
to the cell. Swirl vigorously
to mix
19. Insert the sample cell


g/L Pb.
Interferences
Interference studies were conducted by preparing a known lead solution of 25 g/L as well as the
potential interfering ion. The ion was said to interfere when the resulting lead concentration
changed by 10%. Samples containing levels exceeding these concentration values may be
diluted 1:1 and analyzed. Multiply the value obtained by a factor of 2 to determine the lead present
in the original sample.
To avoid contamination, do not use black rubber stoppers, black dropper bulbs and droppers with
inked graduations. Use the plastic droppers provided in the reagent set.
Acid-wash all glassware and plasticware to prevent sample contamination, especially if the
previous sample had a high lead level (see Apparatus and sample preparation).
The Extractor plunger may be reused for more than one test but should be rinsed with lead-free
water between uses.

Aluminum,
Al3+
Interference level
0.5 mg/L
Ammonium, NH4+
500 mg/L
Barium, Ba2+
6 mg/L
Calcium, Ca2+
500 mg/L
Chloride,
Cl
1000 mg/L
Copper, Cu2+
2 mg/L
Fluoride, F
10 mg/L
Iron, Fe2+
2 mg/L
Magnesium, Mg2+
500 mg/L
Manganese, Mn2+
0.5 mg/L
Nitrate,
NO3
Sulfate, SO42
Zinc,
Zn2+
Lead
Page 640
1000 mg/L
1000 mg/L
1 mg/L
Lead
Apparatus and sample preparation

Because lead is very common to our environment, care must be taken to prevent sample
contamination. Follow these steps for greatest test accuracy:
Lead-free water is necessary to minimize sample contamination when rinsing apparatus or

diluting sample. The water may be either distilled or deionized. If the water is obtained from a
grocery store, verify the lead concentration is zero from the label. If the lead concentration is
uncertain, determine the lead concentration with the LeadTrak test.
Plastic or glass sample containers and lids may be checked for contamination by rinsing with 1
mL of pPb-1 Acid Preservative Reagent*. Add 100 mL of lead-free water. After 24 hours,
analyze this solution using the LeadTrak test to confirm the absence of lead.
Rinse glassware used in this test with a small amount of dilute lead-free 0.1 N nitric acid or
pPb-1 Acid Preservative Reagent followed by rinsing with lead-free water.
pPb-5 Indicator may be rinsed from the glass sample cells with a few drops of pPb-1 Acid
Preservative Reagent or a small amount of dilute lead-free nitric acid.
Acidify solutions containing lead with Nitric Acid or pPb-1 to below pH 2 to prevent adsorption
of lead onto the container walls. See Sample collection, preservation and storage.
Samples may be collected either from household pipes (point-of-use) or from water sources.
Preserved samples may be stored up to six months.
Each sample type typically requires different sampling procedures. Consult with the
appropriate regulatory agency for more information about specific sampling requirements.
Sampling for lead contamination in household pipes for point-of-use drinking water
The sample should be collected after sitting in pipes with no flow for a minimum of six hours.
Add 10 mL of pPb-1 Acid Preservative* to a one-liter bottle.
Turn on tap and collect exactly the first liter of water in the bottle containing acid preservative.
Cap and invert several times to mix.
After two minutes the sample is ready for analysis. Steps 3 and 4 are skipped in the analysis
procedure. Use 100 mL of this preserved sample directly in step 5.
Sampling for lead contamination from drinking water sources such as well water or water from main
supply lines
Add 10 mL of pPb-1 Acid Preservative* to a one-liter bottle.
Turn on the tap for 35 minutes or until the water temperature has been stable for 3 minutes.
Collect exactly one liter of water into the bottle containing the acid preservative.
Cap and invert several times to mix.
After two minutes the sample is ready for analysis. Steps 3 and 4 are skipped in the analysis
procedure. Use 100 mL of this preserved sample directly in step 5.
At least one liter should be collected to obtain a representative sample. If less than one liter is
collected, use 1 mL of pPb-1 Acid Preservative per 100 mL of sample.
If nitric acid is to be substituted for pPb-1 as a preservative or the sample is digested, the
buffering capacity of the pPb-2 Fixer Solution* may be exceeded. Adjust the sample pH to
6.77.1 pH with 5 N Sodium Hydroxide* after step 6.
Lead
Page 641
Lead
Accuracy check
10 mg/L (10,000 g/L) Lead Standard Solution
TenSette Pipet and pipet tips
6. Follow the LeadTrak Fast Column Extraction test procedure for each of the spiked samples
Lead Standard Solution, 1000 mg/L or Lead Voluette Ampule Standard Solution, 50-mg/L
as Pb
Lead-free water or Deionized water
100 mL Class A volumetric flask or 100-mL plastic volumetric flask
1.0 mL Class A volumetric pipet
1. Prepare a 100 g/L Lead standard solution as follows:

a. Pipet 1.0 mL of Lead Standard, 1000 mg/L, into a 100 mL volumetric flask.
b. Use a Tensette Pipet to add 0.2 mL of concentrated nitric acid to the flask
c. Dilute to the mark with lead-free deionized water.
d. Pipet 10.00 mL of this prepared solution into a 1 liter plastic volumetric flask.
e. Add 2.0 mL of nitric acid to the flask.
f.
Dilute to the mark with lead-free water.
g. Prepare this solution immediately before use.

OR
1. Prepare a 100 g/L Lead Standard Solution as follows:
a. Use a Tensette Pipet to add 0.2 mL from a Lead Voluette Ampule Standard Solution, 50
mg/L as Pb into a 100 mL plastic volumetric flask.
b. DIlute to volume with deionized water. Prepare solution immediately before use
Lead
Page 642
Lead
2. Use the standard solution in place of the sample. Follow the LeadTrak Fast Column Extraction
test procedure.
3. To adjust the calibration curve using the reading obtained with the 100-mg/L Standard
Method performance
Program
Standard
283
50 g/L
Sensitivity
Precision
Distribution
Pb2+
4555 g/L
Point of Curve
Concentration
Entire curve
4 g/L Pb2+
Pb2+
Summary of method
Acid soluble lead, as Pb2+, in a potable water sample is first concentrated on a Fast Column
Extractor. The lead is then eluted from the Extractor and determined colorimetrically with an
indicator. Test results are measured at 477 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
LeadTrak
20/pkg
2375000
Quantity
Unit
Catalog number
108044
Reagent Set
Required apparatus
Description
each
each
108046
Clamp, 2-prong extension
each
2114500
Clamp Holder
each
32600
each
108140
each
108142
20/pkg
2124720
2/pkg
2495402
Support for Ring Stand
each
56300
Unit
Catalog number

Description
each
2099553
each
2099542
Lead Standard Solution, 1000 mg/L as Pb
100 mL
1279642
Lead Standard Solution, 50-mg/L 10 mL Voluette Ampules
16/pkg
1426210
Lead Standard Solution, 10 mg/L
25 mL
2374820
Nitric Acid, ACS
500 mL
15249
Lead
Page 643
Lead
Recommended standards and apparatus (continued)
Description
Unit
Catalog number
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451535
Pipet,
TenSette,
0.1 to 1.0 mL

each
1465100
each
1451538
4L
27256
Water, deionized

Description
Unit
Catalog number
pPb-1 Acid Preservative Reagent
236 mL
2368531
pPb-2 Fixer Solution
43 mL
2368655
1L
245053

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Manganese, HR, 8034
Manganese
DOC316.53.01058
USEPA1 Periodate Oxidation Method2
Method 8034
HR (0.1 to 20.0 mg/L)
Powder Pillows
Scope and Application: For soluble manganese in water and wastewater

1
USEPA Approved for reporting wastewater analyses (digestion required). Federal Register, 44(116)34 193 (June 14, 1979)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Digestion is required for reporting wastewater analyses.
If only dissolved manganese is to be determined, filter the sample before acid addition.
blank adjust.

Description
Quantity
High Range Manganese Reagent Set
Manganese
Page 645
Manganese
Periodate Oxidation method for powder pillows
Stored Programs
295 Manganese, HR
Start
1. Select the test.


one Sodium Periodate
sample cell.
2. Prepared Sample:
mL of sample.

one Buffer Powder Pillow,
Citrate Type for
Manganese.
4. Stopper or cap and

invert to mix.

invert to mix.

timer.
A violet color will develop if

manganese is present.

will begin.
Zero
9. When the timer

Manganese
Page 646

0.0 mg/L Mn
Read
11. Within eight minutes

insert the sample into the
cell holder

mg/L Mn.
Manganese
Interferences
Interference level
Calcium
700 mg/L
Chloride
70,000 mg/L
Iron
5 mg/L
Magnesium
100,000 mg/L
pH
Collect samples in acid-washed plastic bottles. Do not use glass containers due to possible
adsorption of Mn to glass.
If samples are acidified, adjust the pH to 45 with 5.0 N Sodium Hydroxide before analysis.
Do not exceed pH 5, as manganese may precipitate.
Accuracy check
Manganese Voluette Ampule Standard, 250 mg/L Mn
Ampule breaker
TenSette Pipet
standard to three 10-mL portions of fresh sample. Mix thoroughly.
6. Follow the Periodate Oxidation method for powder pillows test procedure for each of the
spiked samples using the powder pillows, starting with the 0.1 mL sample spike. Measure
Manganese Standard Solution, 1000 mg/L
Deionized water
Manganese
Page 647
Manganese
1. Prepare a 10.0 mg/L manganese standard solution as follows:

a. Pipet 10.0 mL of Manganese Standard, 1000 mg/L, into a 1000 mL (1 liter) volumetric
flask.
2. Use this solution in place of the sample. Follow the Periodate Oxidation method for powder
pillows test procedure.
Method performance
Program
Standard
295
10.0 mg/L Mn
Sensitivity
Precision
95% Confidence
Portion of Curve
Concentration
Entire curve
0.1 mg/L Mn
9.610.4 mg/L Mn
Summary of method
Manganese in the sample is oxidized to the purple permanganate state by sodium periodate, after
buffering the sample with citrate. The purple color is directly proportional to the manganese
concentration. Test results are measured at 525 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Manganese Reagent Set, High Range (100 tests), includes:
2430000
Buffer Powder Pillows, citrate type for Manganese
100/pkg
2107669
Sodium Periodate Powder Pillows for Manganese
100/pkg
2107769
Quantity
Unit
Catalog number
2/pkg
2495402
Stopper, rubber
6/pkg
173106
Required apparatus
Description
Manganese
Page 648
Manganese
Description
Unit
Catalog number
Manganese Standard Solution, 1000 mg/L Mn
100 mL
1279142
Manganese Standard Solution, 250 mg/L Mn, 10-mL Voluette ampule
16/pkg
1425810
Water, deionized
Voluette Ampule breaker
4L
27256
each
2196800

Description
Unit
Manganese Standard Solution, 2 mL PourRite Ampule, 25 mg/L

Manganese Standard Solution, 2 mL
PourRite Ampule,
pH paper, 014
Pipe filler, safety bulb
10 mg/L
Catalog number
20/pkg
2112820
20/pkg
2605820
100/pkg
2601300
each
1465100
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1970010
250/pkg
2199725
50/pkg
2199796
each
2484600
PourRite Ampule breaker

100 mL
245032
Volumetric flask, Class A, 1000 mL
each
1457453
Volumetric pipet, Class A, 10 mL
each
1451538
Manganese
Page 649

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Manganese, LR, 8149
Manganese
DOC316.53.01057
1-(2-Pyridylazo)-2-Naphthol PAN Method1
Method 8149
LR (0.006 to 0.700 mg/L)
Powder Pillows
Scope and Application: For water and wastewater; digestion is required for determining total manganese
1
Adapted from Goto, K., et al., Talanta, 24, 652-3 (1977)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Rinse all glassware with 1:1 Nitric Acid Solution. Rinse again with deionized water.
The alkaline cyanide solution contains cyanide. Cyanide solutions should be collected for disposal as a reactive (D001)
waste. Be sure cyanide solutions are stored in a caustic solution with pH >11 to prevent release of hydrogen cyanide gas.
Refer to the current MSDS for safe handling and disposal instructions.
Total manganese determination requires a prior digestion. Refer to the Water Analysis Guide for more information.

Description
Quantity
Alkaline Cyanide Reagent
12 drops
PAN Indicator Solution, 0.1%
12 drops
Deionized Water
10 mL
Stoppers for 18 mm tube
Manganese
Page 651
Manganese
Stored Programs
290 Manganese, LR PAN
Start
1. Select the test.

5. Stopper and invert to

Pour 10.0 mL of deionized
water into a sample cell.
3. Prepared Sample:
Pour 10.0 mL of sample
into another sample cell.

one Ascorbic Acid Powder
Pillow to each cell.
6. Add 12 drops of
Alkaline-Cyanide Reagent
Solution to each cell. Swirl
gently to mix.
7. Add 12 drops of PAN

Indicator Solution, 0.1%,
to each sample cell. Swirl
gently to mix.

timer.
A cloudy solution may

form. The turbidity should
dissipate after step 7.
An orange color will

develop in the sample if
manganese is present.
Total manganese
determination requires
prior digestion.
Zero
9. When the timer

and place it in the cell
holder.
Manganese
Page 652

0.000 mg/L Mn
period will begin.
Read
11. Wipe the prepared cell

and place it in the holder.

mg/L Mn.
Manganese
Interferences
For samples that contain hardness greater than 300 mg/L CaCO3, add 4 drops of Rochelle Salt
Solution to the sample after adding the Ascorbic Acid Powder Pillow in step 4.

Interference level
Aluminum
20 mg/L
Cadmium
10 mg/L
Calcium
1000 mg/L as CaCO3
Cobalt
20 mg/L
Copper
50 mg/L
Iron
25 mg/L (If sample contains more than 5 mg/L iron, allow a 10-minute reaction period in
step 8.)
Lead
0.5 mg/L
Magnesium
300 mg/L as CaCO3
Nickel
40 mg/L
Zinc
15 mg/L
Collect samples in a clean plastic container.
Adjust the pH to 2 or less with Concentrated Nitric Acid* (about 2 mL per liter).
Preserved samples can be stored up to six months at room temperature.
Adjust the pH to between 45 with 5.0 N Sodium Hydroxide before analysis.
Accuracy check
Manganese PourRite Ampule Standard, 10-mg/L Mn
Ampule breaker, PourRite
TenSette Pipet
standard to three 10 mL portions of fresh sample. Mix thoroughly.
Manganese
Page 653
Manganese
6. Follow the PAN method for powder pillows test procedure for each of the spiked samples
using the powder pillows, starting with the 0.1 mL sample spike. Measure each of the spiked
samples in the instrument.
Manganese Voluette Standard Solution, 250 mg/L Mn
Deionized water
1. Prepare a 0.5 mg/L manganese standard solution as follows:

a. Pipet 2.0 mL of Manganese Standard, 250 mg/L as Mn, into a 1000 mL (1 liter)
volumetric flask.
2. Use this solution in place of the sample. Follow the PAN method for powder pillows test
procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
290
0.500 mg/L Mn
0.4910.509 mg/L Mn
0.006 mg/L Mn
Summary of method
The PAN method is a highly sensitive and rapid procedure for detecting low levels of manganese.
An ascorbic acid reagent is used initially to reduce all oxidized forms of manganese to Mn2+. An
alkaline-cyanide reagent is added to mask any potential interferences. PAN Indicator is then
added to combine with the Mn2+ to form an orange-colored complex. Test results are measured at
560 nm.
Manganese
Page 654
Manganese

Required reagents
Description
Manganese Reagent Set, 10 mL (50 tests), includes:
Alkaline Cyanide Reagent
Quantity/Test
Unit
Catalog number
2651700
12 drops
50 mL SCDB
2122326
2 pillows
100/pkg
1457799
12 drops
50 mL SCDB
2122426
10 mL
4L
27256
Water, deionized
Required apparatus
Description
Quantity
Unit
Catalog number
2/pkg
2495402
Stoppers for 18 mm Tube
6/pkg
173106
Description
Manganese Standard Solution, 10-mg/L Mn, 2 mL
PourRite ampule
Unit
Catalog number
20/pkg
2605820
16/pkg
1425810
each
2196800
PourRite Ampule breaker 2 mL
each
2484600
Description
Unit
Catalog number
each
2088640
500 mL
15249
pH paper, 0-14
100/pkg
2601300
Manganese Standard Solution, 250-mg/L Mn, 10-mL
Voluette
ampule
each
1465100
TenSette
each
1970001
Pipet,
0.11.0 mL
each
1970010
50/pkg
2185696
2185628
1000/pkg
50/pkg
2199796
250/pkg
2199725
172533
29 mL
100 mL
245032
25/pkg
173125
each
1457453
each
1451536
PourRite Ampule breaker 2 mL
each
2484600
20/pkg
2112820
Manganese Standard Solution, 2-mL PourRite Ampule, 25 mg/L
Manganese
Page 655

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Mercury, 10065
Mercury
DOC316.53.01059
Cold Vapor Mercury Concentration Method1
Method 10065
0.1 to 2.5 g/L Hg

1
Patent number 5,733,786
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Perform phase 1 of the procedure in a fume hood. Toxic chlorine or other gases may be produced.
Use dedicated digestion glassware and sample cells for this procedure.
Determine a reagent blank for each new lot of reagent by running the entire procedure, including the digestion, using one liter
of deionized water instead of sample. Add the same amount of potassium permanganate as required by the sample. Subtract
the reagent blank value from the final results or perform a reagent blank adjust.

Description
Cold Vapor Mercury Reagent Set (see Required apparatus for contents of reagent set)
Digestion Reagents and Apparatus (see Required digestion reagents and apparatus)
Quantity
1
varies
Cold Vapor Mercury Apparatus Set
Sample Cells, 1-inch square, 10-mL, matched pair (wherever applicable)
See Required apparatus for a complete list of required apparatus.

Mercury
Page 657
Mercury
Phase 1: Sample digestion
DANGER
Toxic gas hazard. Test must be performed under a fume hood.
1. Transfer one liter of

the sample to a 2000 mL
Erlenmeyer flask. Add a
50-mm magnetic stir bar to
the sample. Set the flask
on a magnetic stirring hot
plate and begin stirring.
2. Add 50 mL of
concentrated sulfuric acid
to the sample.
5. Add 7.5 g of
potassium permanganate
to the sample. Stir until
dissolved.
6. Cover the flask with a

watch glass. Begin heating
the sample to a
temperature of 90 C after
the reagents have
dissolved. Do not boil.
Alternatively, add a
10 gram measuring scoop
of potassium
permanganate to the
sample.
Mercury
Page 658
3. Add 25 mL of
concentrated nitric acid to
the sample.
4. Add 4.0 g of
potassium persulfate to
the sample. Stir until
dissolved.
Alternatively, add one
5 gram measuring scoop
of potassium persulfate to
the sample.
For a mercury standard or

reagent blank in distilled
water, the heating step is
not necessary.
7. Continue to stir and

heat the sample at 90 C
for two hours.
The solution must remain
dark purple throughout the
entire digestion. Some
samples, such as sea
waters, industrial effluents
or other samples high in
organic matter or chloride
concentration, require
additional permanganate.
It may be difficult to see a
dark purple color if the
sample contains black/
brown manganese dioxide
precipitate. Add more
potassium permanganate
if the solution is not dark
purple.
8. Cool the digested

sample to room
temperature. Turn the hot
plate off.
A brown/black precipitate
of manganese dioxide
may settle during cooling.
If the digested sample
does not have a purple
color, the digestion may be
incomplete. Add more
potassium permanganate.
Return the sample to the
magnetic stirring hot plate
and continue the digestion
until the purple color
persists.
Mercury
Phase 1: Sample digestion (continued)
DANGER
9. Return the cool,

digested sample to the
cool, magnetic stirring hot
plate. Turn on the stirrer.
10. Using a 0.5 g

measuring spoon, add
0.5 g additions of
hydroxylaminehydrochloride until the
purple color disappears.
Wait 30 seconds after
each addition to see if the
purple disappears. Add
hydroxylaminehydrochloride until all
manganese dioxide is
dissolved.
11. Remove the stir bar.
12. The digested sample

is now ready for
processing by cold vapor
separation and
preconcentration. Proceed
to Phase 2.
Phase 2: Cold vapor separation and preconcentration of mercury

DANGER
1. Transfer the digested

sample to the Cold Vapor
Gas Washing Bottle. (The
volume of the digested
sample should contain
0.1 to 2.5 g Hg.)
2. Set the Gas Washing

Bottle in the support ring.
Place the top on the Gas
Washing Bottle. Wait until
step 9 to connect the
mercury absorber column
to the Gas Washing Bottle.
3. Connect the 100 mL

mercury absorber column.
4. Pipet 8 mL of
HgEx Reagent B into the
Mercury Absorber column.
Mercury
Page 659
Mercury
Phase 2: Cold vapor separation and preconcentration of mercury (continued)
DANGER
5. Connect the power to

the vacuum pump and
apply vacuum to the
Mercury Absorber
Column. Draw most of the
HgEx Reagent B into the
Erlenmeyer flask.
6. Disconnect the
vacuum using the quick
disconnect when
HgEx Reagent B begins to
drip from the inner delivery
tube on the Mercury
Absorber Column (about
10 seconds after starting
the vacuum). Do not draw
enough air through the
column to begin drying the
packing.
7. Remove the 100 mL

Erlenmeyer flask from the
Mercury Absorber
Column. Replace it with
the 10 mL Distilling
Receiver.
8. Pipet 2 mL of HgEx
Reagent C into the
Mercury Absorber
Column.
9. Connect the Mercury

Absorber column to the
Gas Washing Bottle using
the glass elbow.
10. Shake an ampule of

HgEx Reagent A to
suspend undissolved
reagent.
11. Stopper the side neck

on the Glass Washing
Bottle.
12. Reconnect the

vacuum to the Mercury
Absorber Column using
the quick disconnect. The
vacuum will pull HgEx
Reagent C through the
Mercury Absorber Column
packing and into the
10 mL receiver. Air
bubbles should be
produced at the gas
dispersion tube in the Gas
Washing Bottle. Perform
steps 1314 immediately.
Open the ampule and

gently shake the contents
into the Gas Washing
Bottle through the side
neck.
Mercury
Page 660
Mercury
DANGER
Stored Programs
312 Mercury, Cold Vap
Start

timer.
period will begin. Let the
solution bubble for this
period.
Air flow rate through the
Gas Washing Bottle
should be between
15 L/min. Allow more
bubbling time for lower air
flow rates. For example, if
the air flow rate is 1 L/min.,
let the solution bubble for
10 minutes.

remove the glass elbow
from the top of the
Mercury Absorber
Column. Keep the vacuum
pump on.

Reagent B into the
to elute the captured
mercury.
Continue to apply vacuum
to pull the HgEx Reagent
B into the Distilling
Receiver.
Mercury
Page 661
Mercury
DANGER
17. Turn off or disconnect

power to the vacuum
pump when the volume in
the Distilling Receiver
reaches the 10 mL mark.
If necessary, the volume in
the Distilling Receiver may
be brought up to 10 mL
with HgEx Reagent B. To
avoid low volumes in the
future, disconnect the
vacuum a little sooner in
step 6. This leaves more
HgEx Reagent B in the
packing of the Mercury
Absorber Column.
18. Remove the distilling

Receiver from the Mercury
Absorber Column.
Reconnect the 100-mL
column.

Reagent B into the
without applying vacuum.
This keeps the absorber
packing wet between
tests.
The Mercury Absorber
Column eluate in the
Distilling Receiver is ready
for analysis.
Proceed to Phase 3.
Phase 3: Colorimetric analysis

DANGER
1. Using the funnel

provided, add the contents
of one HgEx Reagent 3 foil
pillow to the eluate in the
Distilling Receiver.
Stopper the receiver.
Invert to dissolve the
reagent.
Mercury
Page 662

one HgEx Reagent 4 foil
pillow to the Distilling
Receiver using the funnel
provided. Stopper the
receiver. Invert to dissolve
the reagent.
3. Add 8 drops of HgEx

Reagent 5 to the Distilling
Receiver. Stopper the
Receiver. Invert to mix.

timer.
period will begin.
Mercury
Phase 3: Colorimetric analysis (continued)
DANGER
Zero

period, transfer the
solution to a sample cell.
6. Wipe the sample cell.

After the timer expires,
insert the sample into the
cell holder.

0.1 g/L Hg (this
program uses a non-zero
intercept)
8. Remove the cell from

the cell holder. Add the
contents of one HgEx
Reagent 6 foil pillow to the
solution. Swirl the cell until
the reagent is completely
dissolved. Immediately go
to step 9.
Do not use the funnel to
add HgEx Reagent 6 to
the sample cell. Any HgEx
Reagent 6 in the funnel
will make mercury
undetectable in
subsequent tests.
Read
9. Return the sample cell

to the cell holder.

g/L Hg. This is the
concentration in the
original sample.
Interferences
Standards were used to prepare a single test solution with substances at the concentrations listed
in the Interfering substances and levels table. A second test solution containing only mercury at
the same concentration was prepared as the control. The two solutions were digested, then
analyzed concurrently. There was no interference from the matrix of the test solution at the
concentrations listed.
In addition, no interference occurred with a test solution containing 1000 mg/L Na+, 1000 mg/L K+,
1000 mg/L Mg2+ and 400 mg/L Ca2+.
Mercury
Page 663
Mercury

Interference level
Ag+
7 mg/L Ag+
Al+3
10 mg/L Al+3
Au+3
500 g/L Au+3
Cd+2
10 mg/ L Cd+2
Co+2
10 mg/L Co+2
Cr+6
10 mg/L Cr+6
Cu+2
10 mg/L Cu+2
1.0 mg/L F
Fe+2
100 mg/L Fe+2
Hg+2
1 g/L Hg+2
Mo+6
10 mg/L Mo+6
Ni+2
10 mg/L Ni+2
NO3
50 mg/L NO3N
Pb2+
10 mg/L Pb2+
SiO2
100 mg/L SiO2
Zn+2
10 mg/L Zn+2
Collect 1000 mL of sample in an analytically clean, glass or polyethylene terephthalate (PET)

container.
Add 10 mL of concentrated hydrochloric acid to preserve the sample before sample collection.
Fill the container completely full to minimize air space when closed.
Note: Close a glass container with a ground glass stopper. Close a PET container with a PET cap or a
polypropylene cap (no liner).
Store aqueous samples at 26 C. Acid-preserved samples are stable for at least 6 months.
Accuracy check
Standard additions method
1. Prepare a 10.0 mg/L Mercury Standard Solution as described under Standard solution
method, step 3a.
2. Use a TenSette Pipet to add 0.10 mL of the 10.0 mg/L Mercury Standard Solution to the
purged solution in the Gas Washing Bottle after an analysis has been performed. Immediately
stopper the Gas Washing Bottle.
3. Begin at step 3 of Phase 2. Follow the procedure steps.
4. Test the eluate as described in Phase 3. The displayed concentration should be 0.91.1 g/L
Hg.
1. Transfer 800 mL of deionized water into the Gas Washing Bottle.
Mercury
Page 664
Mercury
2. Add 50 mL of concentrated sulfuric acid and 25 mL of concentrated nitric acid to the water.
Swirl to mix.
3. Prepare a 0.1 mg/L mercury standard solution by serially diluting a 1000 mg/L Mercury
Standard Solution:
a. To make a 10.0 mg/L standard, add 1.0 mL of concentrated nitric acid to a 500 mL
volumetric flask. Dilute 5.00 mL of a 1000 mg/L standard to 500 mL with deionized water.
Mix well.
b. To make a 1.0 mg/L standard solution, add 0.2 mL of concentrated nitric acid to a 100 mL
volumetric flask. Dilute 10.0 mL of the 10.0 mg/L standard to 100 mL with deionized water.
Mix well.
c. To make a 0.1 mg/L standard solution, add 0.2 mL of concentrated nitric acid to a 100 mL
volumetric flask. Dilute 10.00 mL of the 1.0 mg/L solution to 100 mL with deionized water.
Mix well.
4. Pipet 10.0 mL of the 0.1 mg/L mercury standard solution into the Gas Washing Bottle. Swirl to
mix.
Hg.
System start up
For more accurate results, perform a few analyses on mercury standards and blanks for system
equilibration before beginning sample testing. This allows the system to stabilize before
processing samples.
Startup standard
1. Test a mercury standard solution by following the procedure under Accuracy check using the
Standard solution method. Continue with step 2 of the Startup Standard procedure if the value
is not within specified limits.
2. Pipet 10.0 mL of the 0.1 mg/L mercury standard solution into the purged solution in the Gas
Washing Bottle. Immediately stopper the Gas Washing Bottle.
Hg. Repeat steps 13 if the value is not within these limits.
Startup blank
Run a system blank by using the purged solution in the Gas Washing Bottle after a satisfactory test
of the Startup Standard has been completed.
1. Leave the purged solution in the Gas Washing Bottle. Do not add an aliquot of
mercury standard.
3. Test the eluate as described in Phase 3. The displayed concentration should be
0.2 g/L Hg. Repeat the Startup Blank procedure until a reproducible value is obtained.
Mercury
Page 665
Mercury
Method performance
Program
Standard
Precision
Distribution
Sensitivity
312
1.0 g/L Hg
0.91.1 g/L Hg
0.03 g/L Hg
Storage and maintenance of the cold vapor mercury apparatus

Storage
Store the apparatus as follows for fastest system stabilization and greatest sensitivity:
Store the Gas Washing Bottle filled with deionized water containing 15 mL of concentrated
sulfuric acid. Seal the bottle with the Gas Washing Bottle stopper and top.
Store the Mercury Absorber Column with the packing wetted with HgEx Reagent B. The
Erlenmeyer flask should be kept attached underneath the column. The top of the Mercury
Absorber column should be attached to the Gas Washing Bottle with the glass elbow as in the
procedure.
Glassware care
Use of dedicated glassware and sample cells is recommended because of the sensitivity of this
procedure. Thoroughly clean the glassware and sample cells between tests. After washing, rinse
with 1:1 hydrochloric acid solution, then rinse several times with deionized water.
Maintaining the system
With proper care and storage, the Mercury Absorber Column may be used an unlimited
number of times.
Replace the Mercury Scrubber in the air trap housing at least once for every reagent set used.
Moisture build up on the Gas Washing Bottle side of the Acro 50 Vent Filter will reduce the
purging air flow rate. If this occurs replace the filter or dry it in an oven at 110 C.
Summary of method
The sample is digested to convert all forms of mercury in the sample to mercuric (Hg2+) ions. The
mercuric ions in the digested sample are converted to mercury vapor in a semi-closed system. The
vapor is carried by ambient air into a chemically activated absorber column where the mercury
vapor is converted to mercuric chloride.
The mercuric chloride is eluted off the column and a sensitive indicator is added. The instrument is
zeroed using the absorbance peak of the unreacted indicator. A complexing agent is added to
break the mercury:indicator complex. The increase in unreacted indicator causes an increase in
absorbance proportional to the amount of mercury in the original sample. Test results are
measured at 412 nm.
Safety
Wear personal protective equipment such as safety glasses with side shields or a face shield to
protect your eyes. Use other protective equipment as necessary (such as a fume hood) to avoid
chemical exposure. Perform all steps exactly as prescribed in the procedure.

Proper management and disposal of waste is the responsibility of the waste generator. It is up to
the generator to arrange for proper disposal and comply with applicable local, state and federal
Mercury
Page 666
Mercury
regulations governing waste disposal. The manufacturer makes no guarantees or warranties,
express or implied, for the waste disposal information represented in this procedure.
1. Dispose of the solution in the Gas Washing Bottle by neutralizing the solution to a pH of 69
and flushing to the sanitary sewer with water for several minutes.
2. The mercury contained in one liter of sample is concentrated by a factor of 100 by the Mercury
Absorber Column. Mercury analysis within the range of the test may produce a solution in the
sample cell that is above the RCRA Toxicity Characteristic limit of 0.20 mg/L Hg or other
regulatory limits. The sample cell will contain 0.25 mg/L mercury if the original sample was at
2.5 g/L mercury (the upper limit of the test range). Dispose of the solution in the sample cell
according to applicable regulations.
3. The mercury scrubber will capture mercury vapor if the Mercury Absorber Column is not
properly activated using HgEx Reagent B and HgEx Reagent C. In addition, mercury is also
captured if the capacity of the Absorber Column is exceeded. If the Mercury Scrubber has
captured mercury vapor, it must be disposed of according to applicable regulations.

Required reagents
Description
Quantity/Test
Unit
Catalog number
25/pkg
2658825
HgEx Reagent B, Sulfuric Acid Solution
19 mL
500 mL
2658949
HgEx Reagent C, Sodium Hypochlorite Solution
2 mL
55 mL
2659059
1 pillow
25/pkg
2658448
Cold Vapor Mercury Reagent Set (25 tests), includes:

HgEx Reagent A, Stannous Sulfate Solution,
20 mL ampules
HgEx Reagent 3, Alkaline Reagent Powder Pillows
2658300
HgEx Reagent 4, Indicator Powder Pillows
1 pillow
25/pkg
2658548
HgEx Reagent 5, Sodium Hydroxide Solution
8 drops
10 mL SCDB
2658636
HgEx Reagent 6, Complexing Reagent Powder Pillows
1 pillow
25/pkg
2658748
2/reagent set
2/pkg
2655800
Quantity
Unit
Catalog number
Acro 50 Vent Filter
18/pkg
2683318
Air Trap Holder Assembly
each
2663900
Mercury Scrubber
Required apparatus
Description
Cold Vapor Mercury Apparatus Set, includes:
2674400
Ampule Breaker
each
2564000
Breaker/Capper Tool for Mercury Scrubber
each
2664000
C-flex Tubing, 0.25-inch ID, white

Clamp for Mercury Absorber Column
Clamp Holder
Sample cell
4 ft
25 ft
2327367
each
2656200
each
32600
10 mL Matched
2/pkg
2495402
4528200
Riser Cell, 1 Square DR2000/2010
each
Riser cell, 1, Square DR3000
each
4840300
Distilling Receiver, 10-mL
each
2655438
each
2655342
Mercury
Page 667
Mercury
Description
Quantity
Unit
Catalog number
Funnel, micro
each
2584335
Gas Washing Bottle, 1200-mL
each
2662200
Glass Elbow, 90-degree, with hose adapter
each
2655200
each
2655510
Support Ring for Gas Washing Bottle
each
2656300
Stopper, for Distilling Receiver
each
2655900
2662300
Stopper, for Gas Washing Bottle
each
Support, Base and Rod
each
32900
Tubing Quick Disconnect, HDPE
12/pkg
1481000
each
50841
each
1970001
each
1970010
varies
50/pkg
2185696
2199796
varies
50/pkg
2/pkg
2495402
Vacuum Pump, 115 VAC w/ North American Plug
each
2824800
Vacuum Pump, 230 VAC w/ North American Plug
each
2824801
Vacuum Pump, 230 V w/ European Plug
each
2824802
Required digestion reagents and apparatus

Description
Quantity
Unit
Catalog number
each
2489454
Hot Plate/Stirrer, 7 x 7, Cimarec Digital, 115 VAC
each
2881600
Digital Hot Plate 7 x 7, 230 VAC
each
2881502
Hydroxylamine Hydrochloride, ACS
varies
113 g
24614
Nitric Acid, ACS
25 mL
500 mL
15249
Potassium Permanganate, ACS
varies
454 g
16801H
Potassium Persulfate, ACS
4.0 g
454 g
2617501
50 mL
2.5 L
97909
each
90700

Stir Bar, Octagonal 50.8 x 7.9 mm
each
2095355
each
56601
Watch Glass, Pyrex, 65 mm
each
57867
Description
Mercury Standard Solution, 1000 mg/L Hg (NIST)
Water, deionized

Unit
Catalog number
100 mL
1419542
4L
27256
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Molybdenum, HR, 8036
Molybdenum
DOC316.53.01061
Mercaptoacetic Acid Method1
Method 8036
HR (0.2 to 40.0 mg/L Mo)
Scope and Application: For water, wastewater, boiler and cooling waters
1
Adapted from Analytical Chemistry. 25(9) 1363 (1953)
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

deionized water instead of the sample. Subtract the reagent blank from the result or adjust a reagent blank.
Filter turbid samples using filter paper and a funnel.
After all reagents have been added, the presence of molybdenum will cause a yellow color to form.
Adjust pH of acidified stored samples to pH 7 before analysis.

Description
Quantity
Powder Pillow Test:

MolyVer 1 Molybdenum Reagent Powder Pillows
MolyVer
3 Molybdenum Reagent Powder Pillows
Sample Cell for blank (see Instrument-specific information)
Molybdenum
Page 669
Molybdenum
Description
Quantity
AccuVac Test:
CDTA Solution, 0.4 M
4 drops
MolyVer 6 Reagent AccuVac Ampuls
Beaker, 50 mL
Mercaptoacetic Acid for powder pillows
Stored Programs
320 Molybdenum HR
Start
1. Select the test.


one MolyVer 3 Reagent
mix.
Molybdenum
Page 670

10-mL of sample.
3. Prepared Sample:
MolyVer 1 Reagent
to mix.

one MolyVer 2 Reagent
to mix.

timer.
10 mL of the original
sample.

the cell holder
period will begin.
Molybdenum
Mercaptoacetic Acid for powder pillows (continued)
Zero

Read


mg/L Mo6+.
0.0 mg/L Mo6+
Mercaptoacetic Acid for AccuVac Ampuls
Stored Programs
322 Molybdenum HR
Start
1. Select the test.

10 mL of sample.
3. Prepared Sample:
Collect 40 mL of sample in
a 50-mL beaker. Add four
drops of 0.4 M CDTA
Standard Solution to the
sample in the beaker. Swirl
to mix
Zero

Ampul several times
to mix.

timer.
period will begin.
7. When the timer

expired insert the blank
4. Fill a MolyVer 6
AccuVac Ampul with the
treated sample. Make sure
that the tip is immersed
when filling the Ampul.
Read

READ the results in
mg/L Mo6+

0.0 mg/L Mo6+
Molybdenum
Page 671
Molybdenum
Interferences
Interference level
Aluminum
Chromium
Copper
Samples containing 10 mg/L copper or more will exhibit an increasing positive interference
upon standing. Read these samples as soon as possible after the five minute reaction period
is complete.
Iron
Nickel
Nitrite
Interference from up to 2000 mg/L as NO2 can be eliminated by adding one Sulfamic Acid
Powder Pillow1 to the sample.

extreme sample pH
Adjust the pH to 2 or less with nitric acid (about 2 mL/L).
Preserved samples can be stored up to 6 months at room temperature.
Adjust the pH to 7 with 5.0 N Sodium Hydroxide before analysis.
Accuracy check
Standard additions method (sample spike) for powder pillows
Molybdenum Standard Solution, 1000-mg/L Mo6+
TenSette Pipet, 0.1 to 1.0 mL and Pipet tips
6. Follow the Mercaptoacetic Acid for powder pillows test procedure for each of the spiked
samples using the powder pillows, starting with the 0.2 mL sample spike. Measure each of the
spiked samples in the instrument.
Molybdenum
Page 672
Molybdenum
1. Fill three 100 mL mixing cylinders each with 60-mL of sample and spike with 0.4 mL, 0.8 mL
and 1.2 mL of standard.
2. Transfer 40 mL from each of the three mixing cylinders to three 50-mL beakers.
3. Analyze each standard addition sample as described in the Mercaptoacetic Acid for
AccuVac Ampuls.
recovery.
Molybdenum Standard Solution,10.0-mg/L Mo6+
1. Use the molybdenum standard solution in place of the sample. Follow the Mercaptoacetic Acid
for powder pillows or the Mercaptoacetic Acid for AccuVac Ampuls test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
320
10.0 mg/L Mo6+
9.710.3 mg/L Mo6+
0.2 mg/L Mo6+
322
Mo6+
Mo6+
0.2 mg/L Mo6+
10.0 mg/L
9.710.3 mg/L
Summary of method
MolyVer 1 and 2 Reagents are added to buffer and condition the sample. MolyVer 3 provides the
mercaptoacetic acid which reacts with molybdate molybdenum to form a yellow color proportional
to the molybdenum concentration. Test results are measured at 420 nm.

Required reagents
Description
Quantity/Test
Unit
Molybdenum Reagent Set, for 10-mL samples (100 tests):
Catalog number
2604100
100/pkg
2604299
100/pkg
2604399
100/pkg
2604499
25/pkg
2522025
4 drops
15 mL SCDB
2615436
OR
MolyVer 6 Reagent AccuVac Ampuls
CDTA Solution, 0.4 M
Molybdenum
Page 673
Molybdenum
Required apparatus
Description
Quantity
Unit
Catalog number
Beaker, 50-mL
each
50041H
each
2122800
6/pkg
2427606
2/pkg
2495402
Unit
Catalog number
Description
Molybdenum Standard Solution, 10-mg-L as Mo
100 mL
1418742
Molybdenum Standard Solution, 1000-mg-L as Mo
100 mL
1418642
4L
27256
Unit
Catalog number
Beakers, 50 mL
each
50041H
each
189641
100/pkg
189457
Water, deionized

Description
Filter Paper, folded, 12.5 cm

Funnel, poly, 65 mm
each
108367
each
1970001
50/pkg
2185696
Sulfamic Acid Powder Pillows
100/pkg
105599
each
2088642

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Molybdenum, LR, 8169
Molybdenum
DOC316.53.01062
Ternary Complex Method
Method 8169
LR (0.02 to 3.00 mg/L Mo)
Powder Pillows
Scope and Application: For boiler and cooling tower waters
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Analyze samples immediately after collection.
Filter turbid samples using filter paper1 and a funnel1.
1
Description
Quantity
Molybdenum Reagent Set for 20-mL sample

Molybdenum 1 Reagent (LR) Molybdate Powder Pillow
Molybdenum 2 Reagent Solution
1
0.5 mL
Molybdenum
Page 675
Molybdenum
Ternary Complex Method for powder pillows
Stored Programs
315 Molybdenum LR
Start
1. Select the test.

2. Fill a 25-mL graduated

of sample.

one Molybdenum 1
the graduated cylinder.
4. Prepared Sample:
Stopper the cylinder and
shake to completely
6. Developed Sample:
Add 0.5 mL of
Molybdenum 2 Reagent to
the sample cell.
7.

timer.

for orientation.
5. Pour 10-mL of the

prepared sample into a
sample cell.
Swirl to mix.

will begin.
Zero
of the remaining prepared
sample.
Molybdenum
Page 676


0.00 mg/L Mo6+
12. Wipe the developed

the cell holder.
READ the results in
mg/L Mo6+.
Molybdenum
Interferences
Interference studies were conducted by preparing a molybdenum standard solution
(2 mg/L Mo6+) containing the potential interfering ion. When the standard solution concentration
changed by 5% with a given ion concentration, the ion was considered an interference.
Interference results are summarized in Substances that cause a negative interference,
Substances that cause a positive interference and Non-interfering substances.
Highly buffered samples or extreme sample pH may exceed the buffering capacity of the reagent
and require sample pretreatment. Adjust the sample pH to between 35 by adding, by drops, an
appropriate amount of acid or base such as 1.0 N Sulfuric Acid Standard Solution* or 1.0 N
Sodium Hydroxide Standard Solution*. If significant volumes of acid or base are used, a volume
correction should be made by dividing the total volume (sample + acid + base) by the original
volume and multiplying the test result by this factor.
After a number of samples have been analyzed, the sample cells may exhibit a slight bluish
discoloration. Rinse the cells with 1:1 Hydrochloric Acid Solution* to eliminate this build-up.
Table 211 Substances that cause a negative interference

Interference level
Alum
Greater than 7 mg/L
Aluminum
Greater than 2 mg/L
AMP (Phosphonate)
Bicarbonate
Bisulfate
Borate
Chloride
Chromium
Greater than 4.5 mg/L1
Copper
Diethanoldithiocarbamate
EDTA
Ethylene Glycol
Greater than 2% (by volume)
Iron
Lignin Sulfonate
Nitrite
Orthophosphate
Phosphonohydroxyacetic Acid
Phosphonate HEDP
Positive interference of about 10% up to 30 mg/L. As the concentration increases above

30 mg/L, a decrease in the molybdenum concentration reading occurs (negative
interference).
Sulfite
Read the molybdenum concentration immediately after the 2-minute reaction period.
Table 212 Substances that cause a positive interference

Interference level
Benzotriazole
Carbonate
Morpholine
Greater than 6 mg/L
Molybdenum
Page 677
Molybdenum
Table 212 Substances that cause a positive interference (continued)
Interference level
Phosphonate HEDP
The presence of the phosphonate HEDP at concentrations up to 30 mg/L will increase the
apparent molybdenum concentration reading by approximately 10% (positive interference).
Multiply the value obtained in step 12 by 0.9 to obtain the actual Mo6+ concentration.
Silica

Interference level
Bisulfite
9600 mg/L
Calcium
720 mg/L
Chlorine
7.5 mg/L
Magnesium
8000 mg/L
Manganese
1600 mg/L
Nickel
250 mg/L
PBTC (phosphonate)
500 mg/L
Sulfate
12,800 mg/L
Zinc
400 mg/L
Collect samples in glass or plastic bottles.
Accuracy check
Graduated cylinder, 250 mL
Erlenmeyer flasks (3)
TenSette Pipet and Pipet tips
chemical form.
4. Open the standard solution bottle.
6. Follow the Ternary Complex Method for powder pillows test procedure for each of the spiked
Molybdenum
Page 678
Molybdenum
Molybdenum Standard Solution, 10.00-mg/L
Deionized water
10 mL, Class A volumetric pipet
Pipet filler
1. Prepare a 2.00-mg/L molybdenum standard solution as follows:

a. Pipet 10.00 mL of Molybdenum Standard Solution, 10.00-mg/L, into a 50-mL volumetric
flask.
2. Use this solution in place of the sample. Follow the Ternary Complex Method for powder
Method performance
Program
Standard
Precision
Distribution
Sensitivity
315
2.00 mg/L Mo6+
1.942.06 mg/L Mo6+
0.02 mg/L Mo6+
Summary of method
The ternary complex method for molybdenum determination is a method in which molybdate
molybdenum reacts with an indicator and a sensitizing agent to give a stable blue complex. Test
Molybdenum
Page 679
Molybdenum

Required reagents
Description
Quantity/Test
Molybdenum Reagent Set for 20-mL sample (100 tests), includes:
Unit
Catalog number
2449400
100/pkg
2352449
0.5 mL
50 mL MDB
2352512
Quantity
Unit
Catalog number
(1) Molybdenum 1 Reagent (LR) Molybdate Powder Pillows

(1) Molybdenum 2 Reagent Solution
Required apparatus
Description
each
189640
2/pkg
2495402
Unit
Catalog number
100 mL
1418742
100 mL
1418642
4L
27256
Unit
Catalog number
Cylinder graduated, 250 mL
each
108146
100/pkg
189457
each
108367
Description
Water, deionized

Description
Funnel, poly, 65 mm
each
1970001
50/pkg
2185696
each
50546
Pipet, Volumetric Class A, 10 mL
each
1451538
each
1465100

Hydrochloric Acid Solution 1:1
500 mL
88449
100 mL MDB
104532
100 mL MDB
127032

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nickel, 8037
Nickel
DOC316.53.01064
USEPA1 Heptoxime Method2
Method 8037
0.02 to 1.8 mg/L Ni
Powder Pillows
Scope and Application: For Water, Wastewater and Seawater

1
USEPA accepted for reporting wastewater analyses (digestion required). Procedure is equivalent to Standard Method 3500-Ni D for
wastewater
Adapted from Chemie Analytique, 36 43 (1954)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2612602
DR 3900
2612602
DR 3800, DR 2800, DR 2700
2612602

adjust.
Make the cotton plug pea-size. A larger plug will restrict the flow; a smaller plug may become dislodged from the delivery
tube of the funnel.
Chloroform (D022) solutions are regulated as hazardous waste. Do not pour these materials down the drain. Water saturated
with chloroform, chloroform solutions and the cotton plug used in the delivery tube of the separatory funnel should be
collected for proper disposal. Refer to a current MSDS for safe handling and disposal instructions.
In bright light conditions (e.g. direct sunlight) it may be necessary to close the DR 3900, DR 3800, DR 2800 and DR 2700 cell
compartment with the protective cover during measurements.

Description
Chloroform, ACS
Quantity
30 mL
Nickel 1 Reagent Powder Pillow
Nickel 2 Reagent Powder Pillow
Nickel
Page 681
Nickel
Description
Quantity
Clippers for Opening Pillows
Cotton Balls
varies
Cylinder, graduated. 10-mL
Funnel, separatory with stand and stopper
Heptoxime method for powder pillows
Stored Programs
335 Nickel Heptoxime
Start
1. Select the test.

5. When the timer

of one Nickel 2 Reagent
funnel. Stopper and invert
to mix.
Nickel
Page 682
2. Measure 300 mL of
sample in a 500-mL
graduated cylinder. Pour
into a 500-mL separatory
funnel.

one Nickel 1 Reagent
funnel. Stopper and invert
to mix.

timer.

timer.
7. When the timer

expires, add 10 mL of
chloroform. Insert the
stopper and invert gently.
With the funnel inverted
and the tip pointed away
from people, open the
stopcock to vent.
8. Close the stopcock

and invert for 30 seconds.
A second five-minute
A five-minute reaction time

will begin.
Nickel
Heptoxime method for powder pillows (continued)

timer.
A third five-minute reaction
period will begin.
Invert the funnel several
times over the five minute
period.

When the timer expires,
wait for the layers to
separate. Insert a
pea-sized cotton plug into
the delivery tube of the
funnel. Remove the
stopper and drain the
chloroform layer (bottom
layer) into a sample cell.
11. Repeat steps 7

through 10 two additional
times with 10-mL volumes
of chloroform. The
five-minute reaction period
is not necessary.
12. Cap the sample cell

and invert to mix the
extracts. The final volume
will be about 25 mL due to
the slight solubility of
chloroform in water.
Insert the stopper into the

funnel.
Zero

with 10 mL of chloroform.
Cap the cell.


0.00 mg/L Ni

the cell holder.
Ni.
Interferences
Cobalt, copper and iron interferences can be overcome by adding additional Nickel 1 Reagent
Powder Pillows in step 3 of the Heptoxime method for powder pillows. The tolerance limits of these
interferences are shown in the Interfering substances table.
A preliminary acid digestion is required to determine any suspended or precipitated nickel and to
eliminate interference by organic matter. To eliminate this interference or to determine total
recoverable nickel perform the USEPA approved digestion.
Nickel
Page 683
Nickel

Tolerance Limit (mg/L)
Pillows of Nickel 1
Reagent
Cobalt
Copper
Iron
10
20
16
65
13
22
110
18
28
155
25
35
200
Adjust the sample pH to 2 or less with Nitric Acid*, about 5 mL per liter. Preserved samples
can be stored up to six months at room temperature.
Before analysis, adjust the sample pH to between 38 with 5.0 N Sodium Hydroxide Standard
Solution*. Do not exceed pH 8 as this may cause some loss of nickel as a precipitate.
Accuracy check
Prepare a 300 mg/L nickel standard by pipetting 15 mL of 1000 mg/L Nickel standard into a 50 ml
volumetric flask. Dilute to volume and mix well.
Nickel Standard Solution, 1000-mg/L Ni
Volumetric Pipet, 15 mL
Volumetric Flask, 50 mL
Pipet Filler
300 mg/L standard to three 300-mL portions of fresh sample.
5. Follow the Heptoxime method for powder pillows test procedure for each of the spiked
Nickel
Page 684
Nickel
Nickel Standard Solution, 1000-mg/L
Deionized water
Volumetric flasks, 500 mL and 1000 mL
Volumetric pipets, 10 mL and 50 mL
Pipet filler
1. Prepare a 10.0 mg/L nickel working solution as follows:

a. Pipet 10.0 mL of a Nickel Standard Solution, 1000-mg/L, into a 1000-mL (1 liter)
volumetric flask.
2. Prepare a 1.0-mg/L nickel standard solution by diluting 50.0 mL of the 10-mg/L working
standard solution to 500 mL in a volumetric flask.
3. Use the standard solution in place of the sample. Follow the Heptoxime method for powder
Method performance
Program
Standard
Precision
Distribution
Sensitivity
335
1.00 mg/L Ni
0.931.07 mg/L Ni
0.02 mg/L Ni
Summary of method
Nickel ion reacts with heptoxime to form a yellow-colored complex which is then extracted into
chloroform to concentrate the color and enable a more sensitive determination. Chelating agents
are added to the sample to overcome the interferences caused by cobalt, copper and iron.
Readings are taken at 430 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2243500
30 mL
500 mL
1445849
(2) Nickel 1 Reagent Powder Pillows
25/pkg
212368
(2) Nickel 2 Reagent Powder Pillows
25/pkg
212468
Nickel Reagent Set (50 Tests), includes:

(3) Chloroform, ACS
Nickel
Page 685
Nickel
Required apparatus
Description
Quantity
Unit
Catalog number
Clippers
each
96800
100/pkg
257201
50838
each
each
50849
each
52049
Ring, support, 4-inch
each
58001
Sample Cell, 1-inch square, w/stopper, matched pair
2/pkg
2612602
Stand, support, 5 x 8-inch base
each
56300
Unit
Catalog number
100 mL
1417642
4L
27256
Description
Unit
Catalog number
each
189640
each
1451539
each
1451541
each
1457449
each
1457453
Description
Nickel Standard Solution, 1000-mg/L Ni (NIST)
Water, deionized
each
1457441
each
1465100
Nitric Acid 1:1
500 mL
254049
100 mL
245053
each
1451538
Pipet, volumetric, 10 mL

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nickel, 8150
Nickel
DOC316.53.01063
1-(2 Pyridylazo)-2-Napthol (PAN) Method1
Method 8150
0.006 to 1.000 mg/L Ni
Powder Pillows
Scope and Application: For water and wastewater; digestion is required for determining total nickel
1
Adapted from Watanabe, H., Talanta, 21 295 (1974)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Cobalt concentration can be determined with the same sample by using Program Number 110.
If the sample is less than 10 C (50 F), warm to room temperature before analysis. Adjust the pH of acidified stored
samples.

Description
Quantity
EDTA Powder Pillow
Phthalate-Phosphate Reagent Powder Pillow
1 mL
Deionized Water
25 mL
Stoppers
Nickel
Page 687
Nickel
Stored Programs
340 Nickel, PAN
Start
1. Select the test.

2. Prepared Sample:
deionized water.

one Phthalate-Phosphate
each cell.
6. Using the plastic

dropper provided, add
0.5 mL of 0.3% PAN
Indicator Solution to
each cell.
7. Insert stoppers into

the cells. Invert several
times to mix.

timer.

for orientation.
5. Stopper the cells.

Immediately shake to
dissolve.
If the sample contains iron,
make sure that all the
powder is dissolved before
proceeding to step 6.
Nickel
Page 688
period will begin.
During color development,
the sample solution color
may vary from yellowishorange to dark red,
depending on the
chemical makeup of the
sample. The blank should
be yellow.
Nickel
PAN method for powder pillows (continued)
Zero
9. When the timer

of one EDTA Reagent
10. Stopper the cells and

shake to dissolve.


0.00 mg/L Ni
The instrument will zero at
560 and 620 nm.
Read
13. Wipe the sample cell

holder.

mg/L Ni and Co.
Interferences
Interference level
Al3+
32 mg/L
Ca2+
1000 mg/L as (CaCO3)
Cd2+
20 mg/L
Cl
8000 mg/L
Chelating agents (e.g., EDTA)
Interfere at all levels. Use either the Digesdahl or vigorous digestion to eliminate
this interference.
Cr3+
20 mg/L
Cr6+
40 mg/L
Cu2+
15 mg/L
20 mg/L
Fe3+
10 mg/L
Nickel
Page 689
Nickel
Interference level
Fe2+
Interferes directly and must not be present.
K+
500 mg/L
Mg2+
400 mg/L
Mn2+
25 mg/L
Mo6+
60 mg/L
Na+
5000 mg/L
Pb2+
20 mg/L
Zn2+
30 mg/L

extreme sample pH
Adjust the sample pH to 2 or less with Nitric Acid*, about 5 mL per liter. Preserved samples
can be stored up to six months at room temperature.
Before analysis, adjust the sample pH to between 3 and 8 with 5.0 N Sodium Hydroxide
Standard Solution*. Do not exceed pH 8 as this may cause some loss of nickel as a
precipitate.
If the sample is less than 10 C, warm it to room temperature.
Accuracy check
Nickel Standard solution, 1000-mg/L Ni
5-mL Volumetric Pipet, Class A
100 mL Volumetric Flask
Pipet Filler
Deionized Water
1. Prepare a 50 mg/L Nickel standard by pipetting 5.00 mL of 1000 mg/L Ni standard solution into
a 100 mL volumetric flask. Dilute the solution to the required volume and mix well.
Nickel
Page 690
Nickel
standard to three 25-mL portions of fresh sample. Mix well.
6. Transfer 10-mL of each solution into sample cells. Follow the PAN method for powder pillows
test procedure for each of the spiked samples using the powder pillows, starting with the
Nickel Standard Solution, 1000-mg/L as Ni
Deionized water
1-L volumetric flask, Class A
100-mL Volumetric flask
Volumetric pipets, 5 mL and 10 mL
Pipet filler
1. Prepare a 5.00 mg/L nickel stock solution as follows:

a. Pipet 5.00 mL of Nickel Standard Solution, 1000-mg/L as Ni, into a 1000-mL (1 liter)
volumetric flask.
2. Prepare a 0.5 mg/L nickel working solution as follows:
a. 10.0 mL of the 5.00-mg/L nickel stock solution into a 100-mL volumetric flask.
3. Use the working solution in place of the sample. Follow the PAN method for powder pillows
test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
340
0.500 mg/L Ni
0.4920.508 mg.L Ni
0.006 mg/L Ni
Summary of method
After buffering the sample and masking any Fe3+ with pyrophosphate, the nickel is reacted with 1(2-Pyridylazo)-2-Naphthol indicator. The indicator forms complexes with most metals present.
After color development, EDTA is added to destroy all metal-PAN complexes except nickel and
cobalt. The instrument automatically adjusts for cobalt interference by measuring the absorbance
Nickel
Page 691
Nickel
of the sample at both 560 nm and 620 nm. This method is unique because both nickel and cobalt
can be determined on the same sample when using a spectrophotometer.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2651600
(2) EDTA Reagent Powder Pillows
100/pkg
700599
(2) Phthalate-Phosphate Reagent Powder Pillows
100/pkg
2615199
Nickel Reagent Set (100 Tests), includes:
(1) PAN Indicator Solution, 0.3%
1 mL
100 mL MDB
2150232
25 mL
4L
27256
Quantity
Unit
Catalog number
2/pkg
2495402
Stoppers
6/pkg
173106
Unit
Catalog number
100 mL
1417642
Description
Unit
Catalog number
Water, deionized
Required apparatus
Description
Description
Nickel Standard Solution, 1000-mg/L Ni (NIST)
each
189640
each
1457442
each
1451537
each
1465100
each
1451538
each
1457453
Water, deionized
Nitric Acid 1:1

4L
27256
500 mL
254049
100 mL MDB
245032
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate HR, TNT, 10020
Nitrate
DOC316.53.01068
Chromotropic Acid Method

HR (0.2 to 30.0 mg/L
Method 10020
NO3N)
Test N Tube Vials
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

For DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield before performing this test.
deionized water (nitrate-free) in place of the sample. Subtract the reagent blank value from the final results or perform a
reagent blank adjust.
This test is technique-sensitive. Invert the vials as described here to avoid low results: Hold the vial in a vertical position with
the cap pointing up. Turn the vial upside-down. Wait for all of the solution to flow down to the cap. Pause. Return the vial to
an upright position. Wait for all the solution to flow to the bottom of the vial. This process equals one inversion.

Description
Quantity

Test N Tube
NitraVer
X Reagent Set
Funnel, micro, poly

Pipet,
TenSette,
0.01 to 1.0 mL, plus tips
Test Tube Rack
1
1
1
1
1
Nitrate
Page 693
Nitrate
Chromotropic Acid method for TNT
Stored Programs
344 N, Nitrate HR, TNT
Start
1. Select the test.

for orientation.
Remove the cap from a
NitraVer X Reagent A Test
N Tube vial and add
1.00 mL of sample.
3. Cap the tube and

invert ten times to mix.

insert it into the 16-mm cell
holder.
6. Prepared Sample:
Remove the vial from the
instrument. Using a funnel,
NitraVer X Reagent B
Powder Pillow to the vial.
7. Cap and invert ten

times to mix.

timer.
Some solid matter will

not dissolve.

will begin. Do not invert
the vial again.
Zero

0.0 mg/L NO3N
Read

holder.
Nitrate
Page 694

mg/L NO3N.
To display other chemical
forms, refer to the
instrument user manual.

if nitrate is present.
Nitrate
Interferences
Interference level
Barium
A negative interference at concentrations greater than 1 mg/L.
Chloride
Does not interfere below 1000 mg/L.
Nitrite
A positive interference at concentrations greater than 12 mg/L. (Remove nitrite interference

up to 100 mg/L by adding 400 mg (one full 0.5 g Hach measuring spoon) of Urea to 10 mL of
sample. Swirl to dissolve. Proceed with the nitrate test as usual.)
Copper
Positive interference at all levels.
Store the samples at 4 C (39 F) or lower if the sample will be analyzed within 24 to 48 hours.
For longer storage periods (up to 14 days), adjust sample pH to 2 or less with concentrated
Sulfuric Acid, ACS* (about 2 mL per liter). Sample refrigeration is still required.
Before testing, warm the stored sample to room temperature and neutralize with 5.0 N Sodium
Hydroxide Standard Solution*. Do not use mercury compounds as preservatives.
Correct the test result for volume additions:

( acid volume + base volume + sample volume )
Corrected result = ---------------------------------------------------------------------------------------------------------------------------- test result
original sample volume
Accuracy check
High Range Nitrate Nitrogen Voluette Ampule Standard, 500 mg/L NO3N
Ampule breaker
standard to three mixing cylinders with 25-mL portions of fresh sample.
6. Add 1 mL of the 0.1 mL spiked sample to a TNT vial and follow the Chromotropic Acid method
for TNT test procedure. Repeat for each spiked sample. Measure each of the spiked samples
in the instrument.
Nitrate
Page 695
Nitrate
10.0-mg/L Nitrate Nitrogen Standard Solution
1. Use the 10.0-mg/L Nitrate Nitrogen Standard Solution solution in place of the sample. Follow
the Chromotropic Acid method for TNT test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
344
10.0 mg/L NO3N
9.510.5 mg/L NO3N
0.2 mg/L NO3N
Summary of method
Nitrate in the sample reacts with chromotropic acid under strongly acidic conditions to yield a
yellow product with a maximum absorbance at 410 nm.

Required reagents
Description
Test N Tube NitraVer X Nitrate Reagent Set
Quantity/Test
Unit
Catalog number
50/pkg
2605345
Quantity
Unit
Catalog number
each
2584335
each
1970001
varies
50/pkg
2185696
each
1864100
Unit
Catalog number

Description
Funnel, micro, poly
Pipet,
TenSette,
0.1 to 1.0 mL

Test Tube Rack
Description
Nitrate Nitrogen Standard Solution, 10-mg/L: N03N
500 mL
30749
Nitrate Nitrogen Standard Solution, Voluette Ampule, 500-mg/L N03N
16/pkg
1426010
Wastewater Influent Inorganics Standard for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833149
4L
27256
Water, deionized
Nitrate
Page 696
Nitrate

Description
Unit
Catalog number
Ampule breaker
each
2196800
each
2088640

Sulfuric Acid ACS, Concentrated
each
1000/pkg
50 mL SCDB
245026
500 mL
97949
each
90700
Urea, ACS grade
100 g
1123726
Nitrate
Page 697

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate, HR, 8039
Nitrate
DOC316.53.01066
Cadmium Reduction Method
Method 8039
NO3N)
Powder Pillow or AccuVac Ampuls
HR (0.3 to 30.0 mg/L
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

adjust.
A deposit of unoxidized metal will remain at the bottom of the cell after the NitraVer 5 dissolves. The deposit will not affect
results.
This method is technique-sensitive. Shaking time and technique influence color development. For most accurate results,
make successive tests on a 10-mg/L Nitrate Nitrogen Standard solution. Adjust shaking time and technique to obtain the
correct result. Use this technique for all the samples.
Rinse the sample cell immediately after use to remove all cadmium particles. Prepared samples will contain cadmium and
must be disposed of according to federal, state and local hazardous waste regulations. Refer to the current MSDS for safe
handling and disposal instructions.
Nitrate
Page 699
Nitrate

Description
Quantity
Powder Pillow Test:

NitraVer 5 Nitrate Reagent Powder Pillow
Sample Cells, 1-inch, 10-mL, with stopper
AccuVac Test:
NitraVer 5 Nitrate Reagent AccuVac Ampul
Beaker, 50-mL
Sample Cell, for blank
Cadmium Reduction Method for powder pillows

CAUTION
Hazardous waste exposure. Prepared samples contain cadmium. Refer to the current MSDS for safe
handling and disposal instructions. Follow all federal, state and local hazardous waste
disposal regulations.
Stored Programs
355 N, Nitrate HR PP
Start
1. Select the test.

for orientation.
Nitrate
Page 700

10 mL of sample.
3. Prepared Sample:
NitraVer 5 Nitrate Reagent
Powder Pillow. Stopper.

timer. A one-minute
Nitrate
Cadmium Reduction Method for powder pillows (continued)
CAUTION
5. Shake the cell

vigorously until the timer
expires.
Note: Some solid materials
may not dissolve.
6. When the timer

expires, start the timer
again. A five-minute

0.0 mg/L NO3N

An amber color will

develop if nitrate is
present.
Zero
10 mL of sample.
Read

holder.

mg/L NO3N.
To display other chemical
forms, refer to the user
manual.
Nitrate
Page 701
Nitrate
Cadmium Reduction Method for AccuVac Ampuls
CAUTION
Stored Programs
361 N, Nitrate HR AV
Start
1. Select the test.

2. Prepared Sample:
3. Tap the bottom of a

NitraVer 5 Nitrate
AccuVac Ampul on a
hard surface to dislodge
powder. Fill the Ampul with
fills completely. Insert a
cap over the Ampul tip.

timer.
6. When the timer

again. A five-minute
Do not agitate or disturb
the sample during this
time.
a round sample cell with
10 mL of sample.


for orientation.
5. Invert the Ampul

4852 times as the timer
counts down.
An amber color will

present.
Nitrate
Page 702
period will begin.
Nitrate
Cadmium Reduction Method for AccuVac Ampuls (continued)
CAUTION
Zero

0.0 mg/L NO3N
Read


mg/L NO3N.
Interferences
Interference level
Chloride
Chloride concentrations above 100 mg/L will cause low results. The test may be used at high
chloride concentrations (seawater), but a calibration must be done using standards spiked to
the same chloride concentration. Refer to Seawater calibration.
Ferric iron
Nitrite

Compensate for nitrite interference as follows:
Before performing step 3, add 30-g/L Bromine Water1 dropwise to the sample until a yellow
color remains.
Add one drop of 30-g/L Phenol Solution1 to destroy the color.
Proceed with step 3. Report the results as total nitrate and nitrite.
pH
Strong oxidizing and reducing

substances
Nitrate
Page 703
Nitrate
Seawater calibration
Chloride concentrations above 100 mg/L will cause low results. To perform this test in water with
high interference level, calibrate the water using standards spiked to the same chloride
concentrations as the required samples. To prepare calibration standards containing 5.0, 10.0,
20.0 and 30.0 mg/L nitrate as NO3N:
1. Prepare a 1 L volume of chloride water that matches the concentration of the samples, using
the following equation:
c. Add necessary Chloride concentration (g/L) x (1.6485) = g of ACS grade NaCl to 1 L of
deionized water. 18.8 g/L is a typical seawater chloride concentration.
d. Mix this solution thoroughly to get a homogeneous solution. Use this water as the dilution
water instead of the deionized water when preparing the nitrate standards.
2. Use Class A glassware or a Tensette Pipet to pipet 0.5, 1.0, 2.0, and 3.0 mL of the 1000 mg/L
Nitrogen-Nitrate as NO3N (NIST) Standard Solution (Catalog Number 1279249) into four
different 100 mL Class A volumetric flasks.
3. Dilute to the mark with the prepared chloride water. Mix thoroughly.
4. Use the prepared chloride water for the 0-mg/L nitrate as NO3N standard.
More reliable results are obtained when samples are analyzed as soon as possible after
collection. If prompt analysis is impossible, store samples in clean plastic or glass bottles for
up to 24 hours at 4 C. To preserve samples for longer periods, add 2 mL of Concentrated
Sulfuric Acid (H2SO4)* per liter and store at 4 C. The results are reported as total nitrate plus
nitrite.
Before analysis, warm the sample to room temperature and adjust the pH to 7 with 5.0 N
Sodium Hydroxide Standard Solution*. Do not use mercury compounds as preservatives.
Correct the test result for volume additions by dividing the total volume (acid + base + sample)
by the original sample volume and multiplying the test result by this factor.
Accuracy check
Standard additions method for powder pillows (sample spike)
Nitrate Nitrogen Standard solution, 1000-mg/L NO3N
Mixing cylinders
25-mL Volumetric pipet
Pipet filler
100 mL Volumetric Flask
1. Prepare a 250-mL nitrate nitrogen standard solution by pipetting 25 mL of a 1000 mg/L Nitrate
Nitrogen standard solution into a 100 mL volumetric flask. Dilute the solution to the required
volume with deionized water and mix thoroughly.
Nitrate
Page 704
Nitrate
6. Follow the Cadmium Reduction Method for powder pillows test procedure for each of the
1. Fill three mixing cylinders each with 50-mL of sample and spike with 0.4 mL, 0.8 mL and
1.2 mL of 250-mg/L standard.
3. Analyze each standard addition sample as described in the Cadmium Reduction Method for
AccuVac Ampuls.
recovery.
1. Use the 10.0-mg/L Nitrate Nitrogen Standard Solution in place of the sample. Follow the
Cadmium Reduction Method for powder pillows and Cadmium Reduction Method for
AccuVac Ampuls test procedures.
Method performance
Program
Standard
Precision
Distribution
355
10 mg/L NO3N
9.310.7 mg/L NO3N
361
10 mg/L
NO3N
9.310.7 mg/L NO3
Sensitivity
Point of curve
Concentration
0 ppm
0.3 mg/L NO3N
10 ppm
0.5 mg/L NO3N
30 ppm
0.8 mg/L NO3N
0 ppm
0.5 mg/L NO3N
10 ppm
0.6 mg/L NO3N
30 ppm
0.8 mg/L NO3N
Nitrate
Page 705
Nitrate
Summary of method
Cadmium metal reduces nitrates in the sample to nitrite. The nitrite ion reacts in an acidic medium
with sulfanilic acid to form an intermediate diazonium salt. The salt couples with gentisic acid to
form an amber colored solution. Test results are measured at 500 nm.

Required reagents
Description
NitraVer
5 Nitrate Reagent Powder Pillows (for 10-mL sample)
Quantity/Test
Unit
Catalog number
100/pkg
2106169
25/pkg
2511025
Quantity
Unit
Catalog number
OR

Description
2/pkg
2495402
Stopper, Neoprene, solid, size #1
12/pkg
1480801
6/pkg
173106
Quantity
Unit
Catalog number
or
Stopper

Description
Beaker, 50-mL
each
50041H
each
2122800
6/pkg
2427606
Unit
Catalog number
Description
Nitrate Nitrogen Standard Solution, 10.0-mg/L NO3N
500 mL
30749
NO3N
500 mL
1279249
500 mL
2833149
4L
27256
Unit
Catalog number
Nitrate Nitrogen Standard Solution 1000 mg/L
Wastewater Influent Standard, Mixed Parameter, for NH3N, NO3N, PO4, COD, SO4,
TOC
Water, deionized

Description
29 mL
221120
each
2088641
each
1970001
Pipet, Volumetric, Class A, 0.5 mL
each
1451534
each
1451535
each
1451536
each
1451503
50/pkg
2185696
Nitrate
Page 706
Nitrate
Description
Phenol Solution, 30 g/L
Unit
Catalog number
1000/pkg
2185628
29 mL
211220
each
1451540
each
1465100
AccuVac Snapper
each
2405200
50 mL SCDB
245026

500 mL
97949
each
1457442
Nitrate
Page 707

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate, LR, 8192
Nitrate
DOC316.53.01067

LR (0.01 to 0.50 mg/L
Method 8192
NO3N)
Powder Pillows
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

adjust.
results.
Shaking time and technique influence color development. Analyze a standard solution several times and adjust the shaking
time to obtain the correct result. Use this technique for analyzing samples. See the Standard solution method.
Rinse the sample cell and mixing cylinder immediately after use to remove all cadmium particles.
Properly dispose of the used sample. Prepared samples contain cadmium and must be disposed of according to Federal,
State and local hazardous waste regulations. Refer to the current MSDS for safe handling and disposal information.

Description
Quantity
NitraVer 6 Nitrate Reagent powder pillow
NitriVer
3 Nitrite Reagent powder pillow
Nitrate
Page 709
Nitrate
Cadmium Reduction Method for powder pillows

CAUTION
Stored Programs
351 N, Nitrate LR
Start

of sample.

one NitraVer 6 Reagent
cylinder. Insert the
stopper.

timer.
5. Shake the cylinder

vigorously during the
three-minute timer.
6. When the timer

expires, start the
instrument timer again.
Note: Some solid materials

may not dissolve.
period will begin.
7. When the timer

expires, carefully pour
10 mL of the sample into a
clean sample cell. Do not
transfer any cadmium
particles to the sample
cell.
8. Prepared Sample:
NitriVer 3 Nitrite Reagent
sample cell.
1. Select the test.

for orientation.
Nitrate
Page 710
time will begin.
Nitrate
Cadmium Reduction Method for powder pillows (continued)
CAUTION

timer. A 30-second

sample cell gently during
the 30-second timer.
nitrate is present.

timer.
period will begin.
Zero

blank into the cell holder.

0.00 mg/L NO3N

a second square sample
cell with 10 mL of original
sample.
Read

cell holder.

mg/L NO3N.
Nitrate
Page 711
Nitrate
Interferences
Interference level
Calcium
100 mg/L
Chloride
chloride concentrations (seawater) but a calibration must be done using standards spiked to
the same chloride concentration. (Refer to Sea Water Calibration.)
Ferric iron
All levels
All levels: This method measures both the nitrate and nitrite in the sample. If nitrite is
present, the nitrite nitrogen test (Program #371) should be done on the sample. Pretreat the
nitrate nitrogen sample with the following pretreatment. Then subtract the amount of nitrite
found from the results of the LR nitrate nitrogen test.
1. Add 30-g/L Bromine Water1 dropwise to the sample in step 3 until a yellow color
remains. Mix after each drop.
Nitrite
2.
3.

Proceed with the Cadmium Reduction Method for powder pillows.
pH

substances
high-interference level, calibrate the water using standards spiked to the same chloride
concentrations as the required samples. To prepare calibration standards containing 0.06, 0.1, 0.3
and 0.4 mg/L nitrate as NO3N:
e. Add necessary Chloride concentration (g/L) x (1.6485) = g of ACS grade NaCl to 1 L of
deionized water. (
Note: 18.8 g/L is a typical seawater chloride concentration.
f.
Mix this solution thoroughly to make sure that it is a homogeneous solution. Use this water
as the dilution water instead of the deionized water when preparing the nitrate standards.
2. Use Class A glassware or a Tensette Pipet to pipet 0.6, 1, 3 and 4 mL of the 10 mg/L
Nitrogen-Nitrate as NO3-N (NIST) Standard Solution (Catalog Number 30749) into four
Nitrate
Page 712
up to 48 hours at 4 C. The results will be in total nitrate and plus nitrate.
To preserve samples for longer periods, add 2 mL of Concentrated Sulfuric Acid* per liter and
store at 4 C.
Nitrate
Accuracy check
Nitrate Nitrogen Standard, Solution, 100-mg/L NO3N
TenSette Pipet 110 mL and tips
50-mL Volumetric Flask
Pipet filler
1. Prepare a 12-mL nitrate nitrogen standard solution by pipetting 6.0 mL of a 100 mg/L Nitrate
Nitrogen standard solution into a 50 mL volumetric flask. Dilute the solution to required
volume with deionized water and mix thoroughly.
5. Use the TenSette Pipet to prepare spiked samples in three mixing cylinders: add 0.1 mL, 0.2
mL and 0.3 mL of 12 mg/L standard to three 15-mL portions of fresh sample.
6. Follow the Cadmium Reduction Method for powder pillows test procedure for each of the
Nitrate Nitrogen Standard Solution, 10-mg/L
Deionized water
Volumetric flask, 100 mL
Volumetric pipet, 4 mL
1. Prepare a 0.40-mg/L NO3N standard solution as follows:

a. Pipet 4.00 mL of Nitrate Nitrogen Standard Solution, 10 mg/L, into a 100 mL volumetric
flask.
Nitrate
Page 713
Nitrate
2. Use this solution in place of the sample. Follow the Cadmium Reduction Method for powder
Method performance
Program
Standard
Precision
Distribution
Sensitivity
351
0.40 mg/L NO3N
0.350.45 mg/L NO3N
0.003 mg/L NO3N
Summary of method
with sulfanilic acid to form an intermediate diazonium salt. The salt couples with chromotropic acid
to form a pink-colored product. Test results are measured at 507 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Low Range Nitrate Reagent Set (100 tests), includes:
2429800
NitraVer 6 Nitrate Reagent Powder Pillows
100/pkg
2107249
NitriVer 3 Nitrite Reagent Powder Pillows
100/pkg
2107169
Quantity
Unit
Catalog number
Required apparatus
Description
each
2088640
2/pkg
2495402
Stopper
6/pkg
173106
Unit
Catalog number
each
1457442
Description
Nitrate Nitrogen Standard Solution, 10.0 mg/L NO3N
Nitrate Nitrogen Standard Solution, 100 mg/L NO3
Water, deionized
Nitrate
Page 714
500 mL
30749
500 mL
194749
4L
27256
Nitrate

Description
Unit

Catalog number
29 mL
221120
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451504
Flask, Volumetric, 50 mL
each
1457441
each
1465100
Phenol Solution, 30-g/L

Sulfuric Acid, concentrated
Sodium chloride, ACS
Pipet, TenSette 110 mL

29 mL
211220
1L
245053
500 mL
97949
454 g
18201H
each
1970010
50/pkg
2199796
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate
Nitrate
Page 716
Nitrate, MR, 8171
Nitrate
DOC316.53.01069

MR (0.1 to 10.0 mg/L NO3
Method 8171
N)
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

results.
This method is technique-sensitive. Shaking time and technique influence color development. For most accurate results,
make successive tests on a 10.0-mg/L Nitrate Nitrogen Standard solution. Adjust shaking times to obtain the correct result.
Rinse the sample cell immediately after use to remove all cadmium particles. Retain the used sample for proper hazardous
waste disposal for cadmium.
Prepared samples will contain cadmium and must be disposed of according to Federal, State and local hazardous waste
regulations. Refer to the current MSDS for safe handling and disposal instructions.
Nitrate
Page 717
Nitrate
Description
Quantity
Powder Pillow Test:

NitraVer 5 Nitrate Reagent Powder Pillow
Stopper, Neoprene, #1, solid
AccuVac Test:
Beaker, 50-mL
Sample Cell for blank (see Instrument-specific information)
Cadmium reduction method for powder pillows
Stored Programs
353 N, Nitrate MR PP
Start
1. Select the test.

for orientation.

10 mL of sample.
3. Prepared Sample:
NitraVer 5 Nitrate Reagent
Powder Pillow. Insert a
stopper into the cell.

timer.
period will begin.
Shake the cell vigorously
until the timer expires.
Note: Some solid material
will not dissolve.
Nitrate
Page 718
Nitrate
Cadmium reduction method for powder pillows (continued)
Zero
5. When the timer

again.
period will begin.
10 mL of sample.


0.0 mg/L NO3N
An amber color will

present.
Read
9. Within two minutes

wipe and insert the
cell holder.

mg/L NO3N.
to display other chemical
forms.
Nitrate
Page 719
Nitrate
Cadmium reduction method for AccuVac Ampuls
Stored Programs
359 N, Nitrate MR AV
Start
1. Select the test.

2. Prepared Sample:
3. Fill a NitraVer 5 Nitrate

AccuVac Ampul with
fills completely. Place a
stopper over the Ampul tip.

timer.
6. When the timer

again.
a round sample cell with
10 mL of sample.


for orientation.
5. Invert the Ampul 48

52 times as the timer
counts down.
amber color will develop if
nitrate is present.
Zero

0.0 mg/L NO3N
Nitrate
Page 720
Read
10. Within two minutes


mg/L NO3N.
period will begin.
Nitrate
Interferences
Interference level
Chloride
chloride concentrations (seawater) but a calibration must be done using standards spiked to
the same chloride concentration (see Seawater calibration).
Ferric iron
Nitrite

Compensate for nitrite interference as follows:
1. Add 30-g/L Bromine Water1 drop-wise to the sample until a yellow color remains.
2.
3.

Proceed with Step 2 of the test. Report the results as total nitrate and nitrite.
pH

substances
high interference level, calibrate the water using standards spiked to the same chloride
concentrations as the required samples. To prepare calibration standards containing 1.0, 3.0, 5.0
and 10.0 mg/L nitrate as NO3N:
c. Add necessary Chloride concentration (g/L) x (1.6485) = g of ACS grade NaCl to 1 L of
deionized water.
Note: 18.8 g/L is a typical seawater chloride concentration.
d. Mix this solution thoroughly to make sure that it is a homogeneous solution. Use this water
as the dilution water instead of the deionized water when preparing the nitrate standards.
2. Use Class A glassware or a Tensette Pipet to pipet 1.0, 3.0, 5.0, and 10.0 mL of the 100 mg/L
Nitrogen-Nitrate as NO3N (NIST) Standard Solution (Catalog Number 194749) into four
Most reliable results are obtained when samples are analyzed as soon as possible after
up to 24 hours at 4 C. To preserve samples for longer periods, add 2 mL of Concentrated
Sulfuric Acid (H2SO4)* per liter and store at 4 C. The results are reported as total nitrate and
nitrite.
Nitrate
Page 721
Nitrate
Accuracy check
Nitrate Nitrogen Standard,100-mg/L NO3N
4. Open the standard solution bottle.
6. Follow the Cadmium reduction method for powder pillows test procedure for each of the
spiked samples, starting with the 0.1 mL sample spike. Measure each of the spiked samples in
the instrument.
500 mg/L Nitrate Nitrogen Ampule Standard Solution
Ampule breaker
Mixing cylinder, 50-mL (3)
1. Fill three mixing cylinders each with 50-mL of sample and spike with 0.1 mL, 0.2 mL and 0.3
mL of 500 mg/L Nitrate Nitrogen Ampule Standard Solution.
3. Analyze each standard addition sample as described in the Cadmium reduction method for
AccuVac Ampuls.
recovery. Standard solution method

Nitrate
Page 722
5.0-mg/L Nitrate Nitrogen Standard Solution (prepared)
100-mg/L Nitrate Nitrogen Standard
Deionized water
Nitrate
5-mL Volumetric pipet and pipet filler

OR
1. Prepare a 5.0-mg/L nitrate nitrogen standard solution as follows:

a. Pipet 5.0 mL of 100-mg/L Nitrate Nitrogen Standard, into a 100-mL volumetric flask.
2. Use this 5.0 mg/L nitrate nitrogen standard solution in place of the sample. Follow the
Cadmium reduction method for powder pillows test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
353
5.0 mg/L NO3N
4.85.2 mg/L NO3N
0.04 mg/L NO3N
359
NO3N
0.05 mg/L NO3N
5.0 mg/L
4.65.4 mg/L NO3
Summary of method
with sulfanilic acid to form an intermediate diazonium salt. The salt couples with gentisic acid to
form an amber colored solution. Test results are measured at 400 nm.

Required reagents
Description
NitraVer
5 Nitrate Reagent Powder Pillows (for 10 mL sample)
Quantity/Test
Unit
Catalog number
100/pkg
2106169
25/pkg
2511025
Catalog number
OR

Description
Quantity
Unit
2/pkg
2495402
Stopper, Neoprene, solid, size #1
12/pkg
1480801
Nitrate
Page 723
Nitrate

Description
Quantity
Unit
Beaker, 50-mL
each
Catalog number
50041H
each
2122800
6/pkg
2427606
Description
Unit
Catalog number
500 mL
2833049
500 mL
194749
16/pkg
1426010
4L
27256
Description
Unit
Catalog number
Ampule breaker for 10 mL ampules
each
2196800
Mixed Parameter Drinking Water Standard, for F, NO3N, PO4, SO4

Nitrate Nitrogen Standard Solution, 100-mg/L
NO3N
Nitrate Nitrogen Standard Solution, 500 mg/L NO3N, 10-mL ampules

Water, deionized
Bromine Water, 30-mg/L
29 mL
221120
each
2088641
Flask, volumetric, 100-mL
each
1457442
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1451537

each
1451535
each
1451503
each
1451538
each
1465100
29 mL
211220
1L
245053
500 mL
97949
5.0 N Sodium Hydroxide Standard Solution


HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate Nitrogen, UV, 10049
Nitrate
DOC316.53.01072
UV Screening Method1
0.1 to 10.0 mg/L NO3
Method 10049
Scope and Application: For the screening of uncontaminated natural and potable water supplies containing low
concentrations of organic matter
1
Adapted from Standard Methods for the Examination of Water and Wastewater, part 4500-NO3B.
Test preparation

The instrument specific information table displays requirements that may vary between

Instrument
Sample cell
Adapter
DR 6000
4822800
A23618
DR 5000
4822800
A23618

Turbid samples must be filtered prior to analysis.
This method is very sensitive to small differences in the measurement wavelength. For best results, use the Standard Adjust
feature (as described in Accuracy check, Standard solution method) to optimize results on each instrument.

Description
Quantity
Hydrochloric Acid Standard Solution, 1.0 M
1 mL
Water, deionized
10 mL
Beaker, 100-mL
Sample Cells, 1-cm quartz
Nitrate
Page 725
Nitrate
UV screening method
Stored Programs
357 N Nitrate UV
Start
1. Select the test.

required.
for orientation.
2. Prepared Sample:
Collect 50 mL of clear
sample in a 100-mL
beaker.
3. Add 1 mL of 1.0 N
Hydrochloric Acid
beaker and swirl to mix.
4. Rinse and fill a 1-cm

quartz sample cell with
sample. Discard the
excess.
Zero
Fill another 1-cm quartz
sample cell with deionized
water.

the cell holder and close
the lid. For the DR 5000,
the transparent face of the
cell should face the user.
For the DR 6000, the
transparent face of the cell
should face right.

0.0 mg/L NO3N
Interferences
Interference level
Chlorate
May interfere
Chromium
All levels
Dissolved organic matter
All levels
Nitrite
All levels
Surfactants
All levels
Suspended particulate matter
Remove using filtration.
Nitrate
Page 726

the sample at 220 and 275
nm. When finished, the
display will show the
sample nitrate nitrogen
concentration.
Nitrate
collection.
If prompt analysis is impossible, store samples in clean plastic or glass bottles for up to 24
hours at 4 C.
To preserve samples for longer periods, add 2 mL of Concentrated Sulfuric Acid (H2SO4)* per
liter and store at 4 C.
Accuracy check
Deionized water
100 mL volumetric flask
5-mL Volumetric pipet and pipet filler
100 mg/L Nitrate Nitrogen Standard

OR
5 mg/L Nitrate Nitrogen Voluette Ampul Standard
1. Prepare a 5.0 mg/L Nitrate Nitrogen standard solution as follows:

a. Pipet 5.0 mL of 100 mg/L Nitrate Nitrogen Standard, into a 100-mL volumetric flask.
b. Dilute to volume with deionized water. Insert a stopper and invert to mix. Alternatively,
open a 5 mg/L Nitrate Nitrogen Voluette Ampul Standard.
2. Use the 5.0 mg/L nitrate nitrogen standard solution in place of the sample. Follow the UV
screening method test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
357
5.0 mg/L NO3N
4.95.1 mg/L NO3N
0.1 mg/L NO3N
Summary of method
The UV nitrate direct screening method offers rapid determination of nitrate. Because both nitrate
and organic constituents absorb at 220 nm and nitrate does not absorb at 275 nm, the second
reading at 275 nm is used to correct for the absorbance attributed to organic matter. Although this
Nitrate
Page 727
Nitrate
method is useful for monitoring nitrate, it is not recommended for samples containing high
concentrations of organics. Adding hydrochloric acid prevents interference from hydroxide or
carbonate concentrations up to 1000 mg/L CaCO3.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Hydrochloric Acid Standard Solution, 1.0 M
1 mL
1L
2321353
Water, deionized
10 mL
4L
27256
Catalog number
Required apparatus
Description
Quantity
Unit
Beaker, 100-mL
each
50042H
Sample cell with cap, 1-cm quartz, matched pair
2/pkg
4822800
Description
Unit
Catalog number
Nitrate Nitrogen Standard Solution, 100-mg/L NO3N
500 mL
194749
Nitrate Nitrogen Voluette Standard, 5 mg/L NO3-N, 10 mL ampuls
16/pkg
2557810
each
2196800
Description
Unit
Catalog number
each
213100
20/pkg
2124720
each
1352900
100/pkg
1353000
Ampule Breaker - Voluette
Dropper, 0.5 and 1.0 mL

FIlter holder, 47-mm
Filter membrane, 47-mm, 0.45 micron
each
54649
Flask, volumetric, 100-mL
each
1457442
each
1465100
each
53236
each
1451537
Stopper, No. 7, one hole

Tubing, rubber latex

6/pkg
211907
500 mL
97949
12 ft
56019
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrite, HR, 8153
Nitrite
DOC316.53.01075
Ferrous Sulfate Method1
Method 8153
Powder Pillows
HR (2 to 250 mg/L NO2
Scope and Application: For cooling systems

1
Adapted from McAlpine, R. and Soule, B., Qualitative Chemical Analysis, New York, 476, 575 (1933)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

blank adjust.
After adding the reagent, a greenish-brown color will develop if nitrite is present.

Description
Deionized water
Quantity
1
varies
Stopper, Neoprene, solid # 1
Nitrite
Page 729
Nitrite
Ferrous sulfate method for powder pillows
Stored Programs
373 N, Nitrite HR PP
Start
1. Select the test.


10 mL of sample.
3. Prepared Sample:
Powder Pillow.

shake to dissolve.

for orientation.
Zero

timer.
period will begin. To
prevent low results, leave
the sample on a flat
surface and do not
disturb it during the
reaction period.

Read

cap and gently invert the
prepared sample twice.
Avoid excessive mixing or
low results may occur.
Nitrite
Page 730

the cell holder.

mg/L NO2.

0 mg/L NO2
Nitrite
Interferences
This test does not measure nitrates nor is it applicable to glycol-based samples. Dilute glycolbased samples and follow the Low Range Nitrite procedure, Method 8507.

The following storage instructions are necessary when prompt analysis is impossible. Store at 4 C
(39 F) or lower if the sample is to be analyzed within 24 to 48 hours. Warm to room temperature
before running the test. Do not use acid preservatives.
Accuracy check
1. Preparing nitrite standards is difficult. Use the standard preparation instructions in Standard
Methods for the Examination of Water and Wastewater. Prepare a 200-mg/L standard using
Sodium Nitrite, ACS*, reagent grade.
2. Use the 200-mg/L solution in place of the sample. Follow the Ferrous sulfate method for
3. To adjust the calibration curve using the reading obtained with the standard solution, navigate
to Standard Adjust in the software OPTIONS>MORE>STANDARD ADJUST.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
373
Standard: 200 mg/L NO2
191209 mg/L NO2
1.4 mg/L NO2
Summary of method
The method uses ferrous sulfate in an acidic medium to reduce nitrite to nitrous oxide. Ferrous
ions combine with the nitrous oxide to form a greenish-brown complex in direct proportion to the
nitrite present. Test results are measured at 585 nm.

Required reagents
Description
NitriVer
2 Nitrite Reagent Powder Pillows
Quantity/Test
Unit
Catalog number
100/ pkg
2107569
Nitrite
Page 731
Nitrite
Description
Quantity
Unit
Catalog number
Stopper, Neoprene, solid #1
12/pkg
1480801
2/pkg
2495402
Description
Unit
Catalog number
Balance, Analytical, 80 g capacity
each
2936701
Handbook, Standard Methods for the Examination of Water and Wastewater
each
2270800
Water, deionized
Sodium Nitrite, ACS

4L
27256
454 g
245201
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrite, LR, 10019
Nitrite
DOC316.53.01073
Diazotization Method
Method 10019
LR (0.003 to 0.500 mg/L NO2
N)
Test N Tube Vials
Test preparation


Instrument
DR 6000
Light shield
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield in cell compartment #2 before performing this test.
blank adjust.

Description
Quantity
Test N Tube NitriVer 3 Nitrite Reagent Set
Pipet, TenSette, 1.0 to 10.0 mL, plus tips
Nitrite
Page 733
Nitrite
Diazotization method, TNT
Stored Programs
345 N, Nitrite LR TNT
Start
1. Select the test.

2. Fill a Test N Tube

NitriVer 3 Nitrite vial with
5 mL of sample.
3. Prepared Sample:
Cap and shake to dissolve
the powder.
nitrite-nitrogen is present.

for orientation.

timer.
period will begin.
Zero
an empty Test N Tube
vial with 5 mL of sample.

insert it into the 16-mm
round cell holder.

0.000 mg/L NO2N

prepared sample cell into
the 16-mm round cell
holder.
READ the results in
mg/L NO2N.
Interferences
Interference level
Antimonous ions
Interfere by causing precipitation
Auric ions
Bismuth ions
Chloroplatinate ions
Cupric ions
Cause low results
Ferric ions
Ferrous ions
Cause low results
Lead ions
Nitrite
Page 734
Nitrite
Interference level
Mercurous ions
Metavanadate ions
Nitrate
Very high levels of nitrate (>100 mg/L nitrate as N) appear to undergo a slight amount of
reduction to nitrite, either spontaneously or during the course of the test. A small amount of
nitrite will be found at these levels.
Silver ions

substances
Store the samples at 4 C (39 F) or lower if the sample is to be analyzed within 24 to 48

hours.
Warm the sample to room temperature before running the test.
Accuracy check
Methods for the Examination of Water and Wastewater, Method 4500-NO2 B. Prepare a
0.300-mg/L standard. Use this solution in place of the sample. Follow the Diazotization
method, TNT test procedure.
Method performance
Program
Standard

Distribution
SensitivityConcentration
per 0.010 Abs
345
0.300 mg/L NO2N
0.2940.306 mg/L NO2N
0.003 mg/L NO2N
Summary of method
Nitrite in the sample reacts with sulfanilic acid to form an intermediate diazonium salt. This couples
with chromotropic acid to produce a pink colored complex directly proportional to the amount of
Nitrite
Page 735
Nitrite

Required reagents
Description
Quantity/Test
Unit
Catalog number
50/pkg
2608345
Quantity
Unit
Catalog number
Test N Tube NitriVer 3 Nitrite Reagent Set
Required apparatus
Description
each
1970010
varies
50/pkg
2199796
Description
Unit
Catalog number
each
2270800
each
2936701
250/pkg
2199725
454 g
245201
4L
27256
Pipet,
TenSette,
1.0 to 10.0 mL
Recommended standards, reagents and apparatus

Sodium Nitrite, ACS
Water, deionized

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrite, LR, 8507
Nitrite
DOC316.53.01074
USEPA1 Diazotization
LR (0.002 to 0.300 mg/L
Method 8507
NO2N)

1
USEPA approved for wastewater analysis, Federal Register, 44(85), 25505 (May 1, 1979)
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)


Description
Quantity
Powder Pillow Test:

AccuVac Test:
NitriVer 3 Nitrite Reagent AccuVac Ampul.
Beaker, 50-mL
Nitrite
Page 737
Nitrite
Diazotization method for powder pillows
Stored Programs
371 N, Nitrite LR PP
Start
1. Select the test.


10 mL of sample.
3. Prepared Sample:
Powder Pillow.
4. Swirl to dissolve.
nitrite is present.

for orientation.
Zero

timer.
period will begin.
10 mL of sample.
Read

the cell holder.
Nitrite
Page 738

mg/L NO2N.


0.000 mg/L NO2N
Nitrite
Diazotization method for AccuVac Ampuls
Stored Programs
375 N, Nitrite LR AV
Start
1. Select the test.

2. Prepared Sample:
sample into a 50-mL
beaker.
3. Invert the Ampul

several times to mix. A
pink color will develop if
nitrite is present.

for orientation.
Fill a NitriVer 3 Nitrite

AccuVac Ampul with
fills.
of sample.


READ the results in

mg/L NO2N.

0.000 mg/L NO2N.

timer.
period will begin.
Nitrite
Page 739
Nitrite
Interferences
Interference level
Antimonous ions
Auric ions
Bismuth ions
Chloroplatinate ions
Cupric ions
Cause low results
Ferric ions
Ferrous ions
Cause low results
Lead ions
Mercurous ions
Metavanadate ions
Nitrate
Very high levels of nitrate (>100 mg/L nitrate as N) appear to undergo a slight amount of
reduction to nitrite, either spontaneously or during the course of the test. A small amount of
nitrite will be found at these levels.
Silver ions

substances
Store at 4 C (39 F) or lower if the sample is to be analyzed within 24 to 48 hours.
Warm to room temperature before running the test.
Do not use acid preservatives.
Accuracy check
Methods for the Examination of Water and Wastewater, Method 4500NO2-B. Prepare a
0.150-mg/L standard.
2. Use the 0.150 mg/L solution in place of the sample. Follow the Diazotization method for
Nitrite
Page 740
Nitrite
Method performance
Program
Standard
Precision
Distribution
Sensitivity
371
0.150 mg/L NO2N
0.1470.153 mg/L NO2N
0.002 mg/L NO2N
375
NO2N
NO2N
0.002 mg/L NO2N
0.150 mg/L
0.1470.153 mg/L
Summary of method
Nitrite in the sample reacts with sulfanilic acid to form an intermediate diazonium salt. This couples
with chromotropic acid to produce a pink colored complex directly proportional to the amount of

Required reagents
Description
NitriVer
Quantity/Test
Unit
Catalog number
100/pkg
2107169
25/pkg
2512025
Catalog number
3 Nitrite Reagent Powder Pillows
OR
NitriVer 3 Nitrite Reagent AccuVac Ampul
Required apparatus
Description
Quantity
Unit
Beaker, 50-mL
each
50041H
each
2122800
6/pkg
2427606
2/pkg
2495402
Description
Unit
Catalog number
each
2936701
Recommended standards, reagents and apparatus
AccuVac ampules, for blanks
25/pkg
2677925
AccuVac Snapper
each
2405200
AccuVac Drainer
each
4103600
each
2270800
Sodium Nitrite, ACS
454 g
245201
4L
27256
Water, deionized
Nitrite
Page 741
Nitrite

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrite, DT, 8351
Nitrite
DOC316.53.01178
Ceric Acid Titration Method
Method 8351
100 to 2500 mg/L as NaNO2
Digital Titrator
Scope and Application: For cooling tower waters.
Test preparation

A pipet is recommended for measuring the sample volume when the volume is less than 10 mL.

Description
Quantity
Ferroin Indicator Solution
1 bottle
1 bottle
Ceric Standard Solution Titration Cartridge, 0.5 N
1 cartridge
Digital titrator
Graduated cylinder
Nitrite
Page 743
Nitrite
Nitrite
See
Table 1
1. Select a sample

3. Hold the Digital

wipe the tip.
4. Use a graduated
measure the sample
flask.
5. Dilute to
deionized water.
6. Add five drops of

7. Add one drop of

to the flask. Swirl to mix.

changes from orange to
pale blue.
counter.
Nitrite
Page 744
Nitrite
Nitrite (continued)

the Range-specific
calculate the
concentration:
mg/L sodium nitrite
(NaNO2)
endpoint. The
= 215 mg/L as NaNO2
Nitrite
Page 745
Nitrite

Range (mg/L as NaNO2)
Sample volume (mL)
Multiplier
100400
25
0.86
400800
10
2.15
8001500
4.31
15002500
10.78

Collect samples in clean plastic or glass bottles. Prompt analysis is recommended.
If prompt analysis is not possible, store samples for 24 to 48 hours at 4 C (39 F) or lower. Warm
to room temperature before analysis. Do not use acid preservatives.
Accuracy check
Sodium Nitrite, ACS
1. Prepare a 1000-mg/L sodium nitrite standard solution as follows. Add 1.000 gram of sodium
nitrite to the volumetric flask. Add deionized water to the mark and mix fully.
2. Add 5.0 mL of the solution to an Erlenmeyer flask. Dilute to approximately 75 mL with
deionized water and mix fully.
3. Add the sulfuric acid and Ferroin indicator. Swirl to mix.
4. Titrate the standard to the end point with the titration cartridge and calculate the result. The
result should be close to 1000 mg/L as NaNO2.
Standardization of ceric standard solution
The normality of the ceric standard solution will sometimes decrease over time. Before use, verify
the normality with the following procedure. This standardization should be done monthly.
1. Use a graduated cylinder or pipet to measure 50 mL of deionized water into a 125-mL
Erlenmeyer flask.
2. Add 5 mL of 19.2 N Sulfuric Acid Standard Solution. Swirl to mix.
3. Insert a clean delivery tube into a Ceric Standard Titration Cartridge.
4. Hold the Digital Titrator with the cartridge tip pointing up. Turn the delivery knob until a few
drops of titrant are expelled. Reset the counter to zero and wipe the tip.
5. Place the delivery tube tip into the solution. While swirling the flask, add 200 digits of Ceric
Standard.
6. Insert a clean delivery tube into a 0.200 N Sodium Thiosulfate Titration Cartridge.
7. Hold the Digital Titrator with the cartridge tip pointing up. Turn the delivery knob until a few
drops of titrant are expelled. Reset the counter to zero and wipe the tip.
8. Place the delivery tube tip into the solution. While swirling the flask, titrate with the sodium
thiosulfate from an intense yellow color to a faint yellow color. Record the number of digits
required. This step should require about 400450 digits of titrant.
Nitrite
Page 746
Nitrite
9. Add one drop of Ferroin Indicator Solution. Swirl to mix. The solution will turn a faint blue.
10. Continue titrating with the Sodium Thiosulfate Standard Solution from a faint blue to orange
color. Record the number of digits required.
11. Divide the number of digits by 500 to calculate the correction factor (number of digits 500 =
correction factor).
12. Multiply the mg/L sodium nitrite from the titration procedure by the correction factor to obtain
the correct sodium nitrite concentration.
Summary of method
Sodium nitrite is titrated with tetravalent cerium ion, a strong oxidant, in the presence of ferroin
indicator. After the cerium oxidizes the nitrite, the indicator is oxidized and causes a color change
from orange to pale blue. The concentration of sodium nitrite is proportional to the amount
of titrant.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Ceric Standard Solution Titration Cartridge, 0.5 N
1 pillow
each
2270701
1 pillow
29 mL DB
181233
1 pillow
100 mL MDB
244932
Quantity/Test
Unit
Catalog number
Digital Titrator
each
1690001
each
50543
each
50840
Required apparatus
Description
each
1720500
each
4157800
Description
Unit
Catalog number
Sodium Nitrite, ACS
454 g
245201
each
2267501
100 mL
203832
Nitrite
Page 747
Nitrite

Description
Unit
Catalog number
each
2095352
each
1970010
each
1940000
each
1940010

Water, deionized
500 mL
27249
Thermometer -10 to 225 C
405 mm
2635700
each
1457453
Volumetric pipet, 5 mL
each
1451537
Sampling bottle, 250 mL
each
2087076
Analytical balance
each
2936701
Weighing papers
500/pkg
1473800

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Ammonia, 8038
Nitrogen, Ammonia
DOC316.53.01078
USEPA1 Nessler Method2
Method 8038
0.02 to 2.50 mg/L NH3N

Scope and Application: For water, wastewater and seawater; distillation is required for wastewater and
seawater; USEPA accepted for wastewater analysis (distillation required); see Distillation in this procedure.
1
USEPA accepted for wastewater analysis (distillation required)
Adapted from Standard Methods for the Examination of Water and Wastewater 4500-NH3 B & C.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Nessler Reagent contains mercuric iodide. Both the sample and the blank will contain mercury (D009) at a concentration
regulated as a hazardous waste. Do not pour these solutions down the drain. Refer to a current MSDS for safe disposal and
handling instructions.
When dispensing reagent from a dropper bottle, hold the bottle vertically. Do not hold the bottle at an angle.
If the Flow Cell is used, periodically clean the cells by pouring a few sodium thiosulfate pentahydrate crystals in to the cell
funnel. Flush with enough deionized water to dissolve. Rinse the cell thoroughly.
Nitrogen, Ammonia
Page 749
Nitrogen, Ammonia
Description
Quantity
Ammonia Nitrogen Reagent set
Deionized Water
25 mL
Graduated Mixing Cylinders
Sample Cells, 1-inch, 10-mL
Serological Pipet, 1-mL
Nessler method
Stored Programs
380 N, Ammonia, Ness
Start
1. Select the test.

2. Prepared Sample:
Fill a 25-mL mixing
Fill a 25-mL mixing
25-mL mark with
deionized water.
4. Add three drops

of Mineral Stabilizer to
each cylinder. Stopper and
invert several times to mix.
6. Pipet 1.0 mL of
Nessler Reagent into each
cylinder. Stopper and

timer.
8. Pour 10 mL of each
solution into a sample cell.

for orientation.
5. Add three drops of

Polyvinyl Alcohol
Dispersing Agent to each
Nitrogen, Ammonia
Page 750
period will begin.
Nitrogen, Ammonia
Nessler method (continued)
Zero
9. When the timer


0.00 mg/L NH3 N
Read

the cell holder.

mg/L NH3 N.
Interferences
Interference level
Chlorine
Remove residual chlorine from a 250 mL sample by adding 1 drop of sodium thiosulfate for
each mg/L chlorine (Cl2). Sodium arsenite can be used instead of sodium thiosulfate. See
Sample collection, preservation and storage.
Hardness
A solution containing a mixture of 500 mg/L CaCO3 and 500 mg/L Mg as CaCO3 does not
interfere. If the hardness concentration exceeds these concentrations, add extra
Mineral Stabilizer.
Iron
Interferes at all levels by causing turbidity with Nessler Reagent.
Seawater
May be analyzed by adding of 1.0 mL (27 drops) of Mineral Stabilizer to the sample before
analysis. This complexes the high magnesium concentrations found in sea water, but the
sensitivity of the test is reduced by 30 percent due to the high chloride concentration. For best
results, perform a calibration, using standards spiked to the equivalent chloride concentration
or distill the sample as described below.
Sulfide
Interferes at all levels by causing turbidity with Nessler Reagent.
Glycine, various aliphatic and

aromatic amines, organic
chloramines, acetone,
aldehydes and alcohols
May cause greenish or other off colors or turbidity. Distill the sample if these compounds
are present.
Collect samples in clean glass or plastic bottles.
If chlorine is present, add one drop of 0.1 N Sodium Thiosulfate* for each 0.3 mg/L Cl2 in a
1-liter sample.
Preserve the sample by reducing the pH to 2 or less with sulfuric acid (at least 2 mL/L). Store
at 4 C (39 F) or less.
Preserved samples may be stored up to 28 days.
Warm samples to room temperature and neutralize with 5 N Sodium Hydroxide* before
analysis.
Nitrogen, Ammonia
Page 751
Nitrogen, Ammonia
Accuracy check
Nitrogen Ammonia Voluette Ampule Standard, 50-mg/L NH3N
Ampule breaker
25-mL Mixing cylinders (3)
2. Select standard additions from the instrument menu: OPTIONS>MORE>STANDARD
ADDITIONS.
standard to three 25-mL portions of fresh sample in three mixing cylinders.
6. Follow the Nessler method test procedure for each of the spiked samples, starting with the 0.1
mL sample spike. Measure each of the spiked samples in the instrument.
Nitrogen Ammonia Standard solution 1-mg/L NH3N
1. Use a 1-mg/L Nitrogen Ammonia Standard solution in place of the sample. Follow the Nessler
method test procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST.
Distillation
1. Measure 250 mL of sample into a 250-mL graduated cylinder and pour into a 400-mL beaker.
Destroy chlorine, if necessary, by adding 1 drop of Sodium thiosulfate Solution 0.1 N per mg/L
Cl2.
2. Add 25 mL of Borate Buffer Solution and mix. Adjust the pH to about 9.5 with 1 N sodium
hydroxide solution. Use a pH meter.
3. Set up the General Purpose Distillation Apparatus as shown in the Distillation Apparatus
Manual. Pour the solution into the distillation flask. Add a stir bar.
Nitrogen, Ammonia
Page 752
Nitrogen, Ammonia
4. Use a graduated cylinder to measure 25 mL of deionized water into a 250-mL Erlenmeyer
flask. Add the contents of one Boric Acid Powder Pillow. Mix thoroughly. Set the flask under
the distillation apparatus drip tube. Elevate the flask so the end of the tube is immersed in the
solution.
5. Turn on the heater power switch. Set the stir control to 5 and the heat control to 10. Turn on
the water and adjust to maintain a constant flow through the condenser.
6. Turn off the heater after collecting 150 mL of distillate. Immediately remove the collection flask
to avoid sucking solution into the still. Measure the distillate to ensure 150 mL was collected
(total volume = 175 mL).
7. Adjust the pH of the distillate to about 7 with 1 N sodium hydroxide. Use a pH meter.
8. Pour the distillate into a 250-mL volumetric flask; rinse the Erlenmeyer with deionized water.
Add the rinsings to the volumetric flask. Dilute to the mark. Stopper. Mix thoroughly. Analyze
as described above.
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
380
DR 5000
1.00 mg/L NH3N
0.991.01 mg/L NH3 N
0.02 mg/L NH3 N2
Summary of method
The Mineral Stabilizer complexes hardness in the sample. The Polyvinyl Alcohol Dispersing Agent
aids the color formation in the reaction of Nessler Reagent with ammonia and certain other
amines. A yellow color is formed proportional to the ammonia concentration. Test results are
measured at 425 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2458200
Nessler Reagent
2 mL
500 mL
2119449
Mineral Stabilizer
6 drops
50 mL SCDB
2376626
Polyvinyl Alcohol Dispersing Agent
6 drops
50 mL SCDB
2376526
25 mL
4L
27256
Quantity
Unit
Catalog number
2088640
Ammonia Nitrogen Reagent Set, includes:
Water, deionized
Required apparatus
Description
each
each
919002
each
1465100
Nitrogen, Ammonia
Page 753
Nitrogen, Ammonia

Description
Unit
Catalog number
Nitrogen, Ammonia Standard Solution, 1-mg/L NH3N
500 mL
189149
Nitrogen, Ammonia Standard Solution, 10-mL Voluette Ampule, 50-mg/L NH3N
16/pkg
1479110
each
1970001
Pipet, TenSette 0.1 - 1.0 mL

50/pkg
2185696
1000/pkg
2185628
500 mL
2833249
Description
Unit
Catalog number
Distillation Apparatus, General
each
2265300
Heater and Support Apparatus, 115 VAC, 60 Hz
each
2274400
each
2274402
Pour-Thru Cell Kit for DR 2800
each
5940400
Pour-Thru Cell Module for DR 5000
each
LZV479
Ampule breaker, Voluette
each
2196800
Sodium Thiosulfate, 0.1 N
100 mL
32332
100 mL
245032
Wastewater, Effluent Inorganics, for NH3N, NO3N, PO4, COD, SO4, TOC

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Ammonia, 8155
Nitrogen, Ammonia
DOC316.53.01077
Salicylate Method1
Method 8155
0.01 to 0.50 mg/L NH3N
Powder Pillows

1
Adapted from Clin. Chim. Acta., 14, 403 (1966)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

A green color will develop if ammonia nitrogen is present.

Description
Quantity
Ammonia Cyanurate Reagent pillows
Ammonia Salicylate Reagent pillows
Stopper for sample cells
Nitrogen, Ammonia
Page 755
Nitrogen, Ammonia
Salicylate method for powder pillows
Stored Programs
385 N, Ammonia, Salic
Start
1. Select the test.

2. Prepared Sample:
Fill a sample cell to the 10mL mark with sample.
deionized water.

one Ammonia Salicylate

timer.
7. When the timer

of one Ammonia
Cyanurate Reagent

shake to dissolve.

for orientation.
5. Insert the stopper or

cap the cell and shake to
dissolve.
period will begin.
Zero

timer.
period will begin.
if ammonia-nitrogen
is present.
Nitrogen, Ammonia
Page 756
10. When the timer

holder

0.00 mg/L NH3N.
12. Wipe the sample and

NH3N.
Nitrogen, Ammonia
Interferences
Interference level
Calcium
Iron
All levels. Correct for iron interference as follows:

1. Determine the amount of iron present in the sample by following one of the Iron, Total,
procedures.
2. Add the same iron concentration to the ammonia-free water in step 2. The interference
will be successfully blanked out.
Magnesium
Monochloramine
Monochloramine present in chloraminated drinking water interferes directly at all levels,

giving high results. Use Method 10200, Free Ammonia and Monochloramine, to determine
free ammonia in these sample matrices.
Nitrate
Greater than 100 mg/L as NO3N
Nitrite
Greater than 12 mg/L as NO2N
Phosphate
Greater than 100 mg/L as PO43P
Sulfate
Greater than 300 mg/L as SO42

Sulfide will intensify the color. Eliminate sulfide interference as follows:
1. Measure about 350 mL of sample in a 500-mL Erlenmeyer flask1.
Sulfide
Other Substances
2.
Add the contents of one Sulfide Inhibitor Reagent1 Powder Pillow. Swirl to mix.
3.
4.
Filter the sample through a Folded Filter Paper1 and Filter Funnel1.
Use the filtered solution in step 3.
Less common interferences such as hydrazine and glycine will cause intensified colors in
the prepared sample. Turbidity and color will give erroneous high values. Samples with
severe interferences require distillation. Use the distillation procedure with the General
Purpose Distillation Set.
samples are analyzed as soon as possible after collection.
If chlorine is known to be present, add one drop of 0.1 N Sodium Thiosulfate* for each 0.3
mg/L Cl2 in a one-liter sample.
Adjust the pH to 2 or less with concentrated (about 2 mL per liter) Sulfuric Acid.
Store samples at 4 C or less. Samples preserved in this manner can be stored up to 28 days.
Just before testing the stored sample, warm to room temperature and neutralize with 5.0 N
Sodium Hydroxide Standard Solution.
Nitrogen, Ammonia
Page 757
Nitrogen, Ammonia
Accuracy check
Ammonia Nitrogen Standard Solution, 10-mg/L as NH3N
25-mL Mixing cylinders (3)
2. Select standard additions from the instrument menu OPTIONS>MORE>STANDARD
ADDITIONS.
6. Follow the Salicylate method for powder pillows test procedure for each of the spiked samples,

OR
Ammonia Nitrogen Voluette Standard Solution, 50-mg/L as NH3N
Deionized water
4-mL Class A volumetric pipet
1. Prepare a 0.40 mg/L ammonia nitrogen standard solution as follows:
Dilute 4.00 mL of Ammonia Nitrogen Standard Solution, 10-mg/L to 100 mL with

deionized water.
OR
Use the TenSette Pipet to dilute 0.8 mL of an Ammonia Nitrogen Voluette Standard
Solution, 50-mg/L as NH3N, to 100 mL with deionized water
2. Use the 0.40-mg/L solution in place of the sample. Follow the Salicylate method for powder
Nitrogen, Ammonia
Page 758
Nitrogen, Ammonia
Method performance
Program
Standard
Precision95% Confidence
per 0.010 Abs
385
0.40 mg/L NH3N
0.380.42 mg/L NH3N
0.004 mg/L NH3N
Summary of method
Ammonia compounds combine with chlorine to form monochloramine. Monochloramine reacts
with salicylate to form 5-aminosalicylate. The 5-aminosalicylate is oxidized in the presence of a
sodium nitroprusside catalyst to form a blue-colored compound. The blue color is masked by the
yellow color from the excess reagent present to give a final green-colored solution. Test results are
measured at 655 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2668000
Ammonia Nitrogen Reagent Set for 10-mL samples (100 tests),

includes:
Includes:
(2) Ammonia Cyanurate Reagent Powder Pillows
100/pkg
2653199
(2) Ammonia Salicylate Reagent Powder Pillows
100/pkg
2653299
Unit
Catalog number

Description
Nitrogen, Ammonia Standard Solution, 10-mg/L NH3N
500 mL
15349
Nitrogen, Ammonia Standard Solution, 10-mL Voluette Ampule, 50-mg/L NH3N
16/pkg
147910
Wastewater, Effluent Inorganics, for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833249
each
1970001
50/pkg
2185696
1000/pkg
2185628
Pipet, TenSette 0.1 - 1.0 mL
each
1457442
each
1451504
Stopper, 18 mm
each
6/pkg
Water, deionized
4L
27256
2/pkg
2495402

Description
Unit
Catalog number
each
2088640
Nitrogen, Ammonia
Page 759
Nitrogen, Ammonia
Description
Unit
Catalog number
Distillation Set, general purpose
each
2265300
Erlenmeyer Flask, 500 mL
each
50549
Filter Funnel, Analytical PP, 65 mm
each
108367
100/pkg
189457
Heater and support apparatus; 115 Vac, 60 Hz
each
2274400
2274402
Heater and support apparatus, 230 Vac, 50 Hz
each
Pipet, 2 mL Serological
each
53236
Ampule breaker, Voluette
each
2196800
50 mL SCDB
245026
100/pkg
241899
Sulfuric Acid, Conc
500 mL
97949

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Ammonia, HR, 10031
Nitrogen, Ammonia
DOC316.53.01079
Salicylate Method
Method 10031
HR (0.4 to 50.0 mg/L NH3N)
Test N Tube Vials
Test preparation


Instrument
DR 6000
Light shield
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

Small sample sizes (such as 0.1 mL) may not be representative of the entire sample. Mix the sample well before testing or
repeat the test, sampling from different portions of the sample.
Good safety habits and laboratory techniques should be used throughout the procedure. Consult the Material Safety Data
Sheet (MSDS) for information specific to the reagent used.
In some lab environments, airborne cross-contamination of the blank is possible. To avoid the transfer of ammonia, complete
the preparation of the blank before opening or handling samples and standards. If the sample or standard containers have
already been opened, move to a separate area of the lab to prepare the blank.

Description
Quantity
High Range Test N Tube AmVer Nitrogen Ammonia Reagent
Funnel, micro (for adding reagent)
varies
Nitrogen, Ammonia
Page 761
Nitrogen, Ammonia
Salicylate method, TNT
Stored Programs
343 N, Ammonia HR TNT
Start
1. Select the test.

shield if required (see
Instrument-specific
information).
for orientation.

one Ammonia Cyanurate
each vial.
2. Prepared Sample:
Add 0.1 mL of sample to
one AmVer Diluent
Reagent Test N Tube for
High Range Ammonia
Nitrogen.
Add 0.1 mL of ammoniafree water to one AmVer
Diluent Reagent Test N
Tube for High Range
Ammonia Nitrogen.

Reagent Powder Pillow for
5 mL sample to each vial.
6. Cap the vials tightly

and shake thoroughly to

timer.
8.
Zero

0.0 mg/L NH3N
Nitrogen, Ammonia
Page 762
period will begin.
Read
10.

mg/L NH3N.
Nitrogen, Ammonia
Interferences
Interference level
Calcium
Glycine, hydrazine
Will cause intensified colors in the prepared sample.
Magnesium
Monochloramine
Monochloramine present in chloraminated drinking water interferes directly at all levels

Iron
Eliminate iron interference as follows:

1. Determine the amount of iron present in the sample using one of the total iron
procedures.
2. Add the same iron concentration to the deionized water in step 3.
The interference will then be successfully blanked out.
Nitrite
600 mg/L as NO2N
Nitrate
5000 mg/L as NO3N
Orthophosphate
5000 mg/L as PO43P
pH
Acidic or basic samples should be adjusted to approximately pH 7. Use 1 N Sodium

Hydroxide Standard Solution1 for acidic samples and 1 N Hydrochloric Acid Standard
Solution1 for basic samples.
Sulfate
6000 mg/L as SO42

Sulfide will intensify the color. Eliminate sulfide interference as follows:
1. Measure about 350 mL of sample in a 500-mL Erlenmeyer flask.
Sulfide
Add the contents of one Sulfide Inhibitor Reagent Powder Pillow1. Swirl to mix.
3.
Filter the sample through folded filter paper1. Use the solution in step 2.
Other
2.
Collect samples in clean plastic or glass bottles. Best results are obtained with immediate
analysis.
Preserve samples by reducing the pH to 2 or less with at least 2 mL of Hydrochloric Acid.
Store at 4 C (39 F) or less.
Neutralize preserved samples to a pH of 7.0 with 5.0 N Sodium Hydroxide before analysis.
Nitrogen, Ammonia
Page 763
Nitrogen, Ammonia
Accuracy check
Nitrogen, Ammonia Ampule Standard, 150-mg/L NH3N
Ampule breaker
Mixing cylinders, 25-mL, (3)
ADDITIONS.
5. Use the TenSette Pipet to prepare spiked samples: add 0.2 mL, 0.4 mL, and 0.6 mL of
6. Follow the Salicylate method, TNT test procedure for each of the spiked samples, starting with
7. Select GRAPH to view the results. Select IDEAL LINE (or best-fit) to compare the standard.
100-mg/L Ammonia Nitrogen standard
Deionized water
20-mL Class A volumetric pipet and pipet filler

a. Pipet 20.00 mL of 100-mg/L Ammonia Nitrogen standard, into a 50-mL volumetric flask.
2. Use the 40.0 mg/L solution in place of the sample. Follow the Salicylate method, TNT test
procedure.
Nitrogen, Ammonia
Page 764
Nitrogen, Ammonia
Method performance
Program
Standard
Precision95%
Confidence Limits of
Distribution
Sensitivity
Concentration
per 0.010 Abs
343
40.00 mg/L NH3N
38.141.9 mg/L NH3N
0.312 mg/L NH3N
Summary of method
sodium nitroprusside catalyst to form a blue colored compound. The blue color is masked by the
yellow color from the excess reagent present to give a green-colored solution. Test results are
measured at 655 nm.

Required reagents
Description
Reagent Set, High Range Test N Tube AmVer Nitrogen Ammonia
Quantity/Test
Unit
Catalog number
25 tests
2606945
Quantity
Unit
Catalog number
Required apparatus
Description
each
2584335
each
1970001
varies
50/pkg
2185696
each
1864100

Tube Rack, 16 mm
Description
Unit
Catalog number
Nitrogen Ammonia Standard Solution, 10-mg/L NH3N
500 mL
15349
Nitrogen Ammonia Standard Solution, 100-mg/L NH3N
500 mL
2406549
Nitrogen Ammonia Standard Solution, 150-mg/L NH3N, 10-mL Voulette Ampules
16/pkg
2128410
Nitrogen Ammonia Standard Solution, 50-mg/L NH3N, 10-mL Voluette Ampules
16/pkg
1479110
Wastewater, Effluent Inorganics Standard, for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833249
4L
27256
Description
Unit
Catalog number
Cylinders, mixing, 25 mL tall form
each
2088640
2265300
100/pkg
69257
Water, deionized

Nitrogen, Ammonia
Page 765
Nitrogen, Ammonia
Description
Unit
Catalog number
1000/pkg
2185628
Ampule Breaker, Voluette
each
2196800
each
2274400
Heater and Support Apparatus, 230 VAC, 50 Hz;
each
2274402
Filter Funnel, poly, 65 mm
each
108367
Pipet, 2 mL serological
each
50549
each
1465100
Hydrochloric Acid Standard Solution, 1 N
500 mL
13449
100 mL
104532
Sulfide Inhibitor Reagent Powder Pillows
100/pkg
241899
Hydrochloric Acid, conc
500 mL
13449
50 mL SCDB
245026

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Ammonia, LR, 10023
Nitrogen, Ammonia
DOC316.53.01080
Salicylate Method1
Method 10023
LR (0.02 to 2.50 mg/L NH3N)
Test N Tube Vials

1
Adapted from Clin. Chim. Acta, 14, 403 (1966)
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

Good safety habits and laboratory techniques should be used throughout the procedure. Consult the Material Safety Data
Sheet (MSDS) for information specific to the reagent used.

Description
Quantity
Low Range Test N Tube AmVer Nitrogen Ammonia Reagent
varies
Nitrogen, Ammonia
Page 767
Nitrogen, Ammonia
Salicylate method, LR, TNT
Stored Programs
342, Ammonia LR TNT
Start
1. Select the test.

Instrument-specific
information).
for orientation.

one Ammonia Cyanurate
each vial.
2. Prepared Sample:
Add 2.0 mL of sample to
one AmVer Diluent
Reagent Test N Tube for
Low Range Ammonia
Nitrogen.
Add 2.0 mL of ammoniafree water to one AmVer
Tube for Low Range
Ammonia Nitrogen.

each vial.


timer.
8.
Zero

0.00 mg/L NH3N
Nitrogen, Ammonia
Page 768
period will begin.
Read
10.

mg/L NH3N.
Nitrogen, Ammonia
Interferences
Interference level
Calcium
2500 mg/L as CaCO3
Iron
Determine the amount of iron present in the sample by using one of the Iron, Total,
procedures.
Add the same iron concentration to the ammonia-free water in step 3. The interference will
then be successfully blanked out.
Magnesium
Monochloramine
Monochloramine present in chloraminated drinking water Interferes directly at all levels

Nitrate
250 mg/L as NO3N
Nitrite
30 mg/L as NO2N
Orthophosphate
250 mg/L as PO43P
pH
Acidic or basic samples should be adjusted to approximately pH 7. Use 1 N Sodium

Hydroxide Standard Solution1 for acidic samples and 1 N Hydrochloric Acid Standard
Solution1 for basic samples.
Sulfate
300 mg/L as SO42
Sulfide
Measure about 350 mL of sample in a 500-mL Erlenmeyer flask.
2.
3.
4.
Filter the sample through a folded filter paper1.

Use the filtered solution in step 3.
Other
1.
analysis.
Preserve samples by reducing the pH to 2 or less with at least 2 mL of Hydrochloric Acid.
Store at 4 C (39 F) or less.
Neutralize preserved samples to a pH of 7.0 with 5.0 N Sodium Hydroxide before analysis.
Nitrogen, Ammonia
Page 769
Nitrogen, Ammonia
Accuracy check
Nitrogen, Ammonia Ampule Standard, 50-mg/L NH3N
Ampule breaker
Mixing cylinders, 25-mL, (3)
ADDITIONS.
6. Follow the Salicylate method, LR, TNT test procedure for each of the spiked samples, starting
with the 0.2 mL sample spike. Measure each of the spiked samples in the instrument.
7. Select GRAPH to view the results. Select IDEAL LINE (or best-fit) to compare the standard.
1.0-mg/L Ammonia Nitrogen standard
1. Use a 1.0-mg/L Ammonia Nitrogen standard in place of the sample. Follow the Salicylate
method, LR, TNT test procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST. .
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
DConcentration
per 0.010 DAbs
342
1.00 mg/L NH3N
0.901.10 mg/L NH3N
0.014 mg/L NH3N
Nitrogen, Ammonia
Page 770
Nitrogen, Ammonia
Summary of method
sodium nitroprusside catalyst to form a blue colored compound. The blue color is masked by the
yellow color from the excess reagent present to give a green-colored solution. Test results are
measured at 655 nm.

Required reagents
Description
Reagent Set, Low Range Test N Tube AmVer Nitrogen Ammonia
Quantity/Test
Unit
Catalog number
25 tests
2604545
Catalog number
Required apparatus
Description
Quantity
Unit
each
2584335
each
1970010
13
each
1864100
Unit
Catalog number
500 mL
189149
Tube Rack, 16 mm
Description
Nitrogen Ammonia Standard Solution, 1.0-mg/L NH3N
Nitrogen Ammonia Standard Solution, 50-mg/L NH3N, 10-mL Voluette Ampules
16/pkg
1479110
250/pkg
2199725
Wastewater, Effluent Inorganics Standard, for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833249
4L
27256
Unit
Catalog number
Water, deionized

Description
Ampule Breaker, Voluette
each
2196800
Cylinders, mixing, 25 mL
each
2088640
each
2265300
Filter Funnel, poly, 65 mm
each
108367
100/pkg
189457
each
2274400
each
2274402
Hydrochloric Acid Standard Solution, 1 N
1000 mL
2321353
Hydrochloric Acid, concentrated ACS
500 mL
13449
50/pkg
2199796
Pipet, 2 mL serological
each
50549
each
1465100
100 mL
104532
Nitrogen, Ammonia
Page 771
Nitrogen, Ammonia
Description
Unit
Catalog number
100 mL
245026
100/pkg
241899

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Free Ammonia, 10201
DOC316.53.01084
Indophenol Method1
Method 10201
(0.01 to 0.50 mg/L NH3N)
Powder Pillows
Scope and Application: For controlling free ammonia levels during the production of chloramines, at booster
stations and for monitoring free ammonia levels in potable distribution system waters.
1
U.S. Patent 6,315,950
Test preparation


Instrument
Sample Cell
Cell Orientation
Adapter
DR 6000
4864302
Orientation key faces right
DR 5000
4864302
DR 3900
4864302
LZV846 (A)
DR 3800, DR 2800, DR 2700
5940506
1-cm (flat) path aligned with the arrow

on the adapter
LZV585 (B)
A23618

Use Method 10200, Nitrogen, Free Ammonia and Chloramine (Mono) to determine free ammonia and monochloramine
simultaneously on the same sample.
Description
Free Ammonia Reagent Set
Quantity
1 drop
2
2

Page 773

Indophenol method for powder pillows
Stored Programs
388 N, Ammonia Free
Start
1. Select the test.

2. Fill two cells to the

10-mL line with sample.
Label one cell sample
and one cell blank.
3. Prepared Sample:
Add one drop of Free
Ammonia Reagent
Solution to the sample.
4. Cap the reagent bottle

to maintain reagent
performance and stability.
7. When the timer

of one MonoChlor F
powder pillow to each cell.
8. Cap and shake both

cells about 20 seconds to

for orientation.
5. Cap and invert the

sample to mix.

timer.
If the sample becomes

cloudy by the end of the
reaction period, pretreat
the sample and retest. See
Interferences.

will begin.

Page 774

depends on sample
See Color development
based on sample
temperature.

if monochloramine is
present.

Indophenol method for powder pillows (continued)
Zero

timer.
will begin.
10. When the timer

vial into the cell

The display will show: 0.00
mg/L NH3N f
12. Insert the sample into

the cell
Remove the blank.

depends on sample
See Color development
based on sample
temperature.
Read

mg/L NH3N f.
Interferences
This method is intended for finished, chloraminated drinking water samples that have a
measurable combined (total) chlorine disinfectant residual. Samples where the disinfectant
residual has disappeared and samples which exhibit a chlorine demand may produce low
ammonia test results. Blanks and ammonia standards analyzed without a disinfectant residual
must be prepared using high quality, reagent grade water.
The following do not interfere in the free ammonia determination at or below the stated
concentration.

Page 775

Interference level
Aluminum
0.2 mg/L
Chloride
1200 mg/L Cl-
Copper
1 mg/L Cu
Iron
0.3 mg/L Fe
Manganese
0.05 mg/L Mn
Nitrate
10 mg/L NO3N
Nitrite
1 mg/L NO2N
Phosphate
2 mg/L oPO4
Silica
100 mg/L SiO2
Sulfate
1600 ppm as CaCO3
Zinc
5 ppm Zn
Samples containing high levels of both Total Hardness and Alkalinity may become cloudy after the
addition of the Free Ammonia Reagent Solution. If this occurs by the end of the first reaction
period, the sample for Free Ammonia measurement must be pretreated as follows:
Note: The blank does not need pretreatment.
1. Measure 10 mL of sample into the cell labeled for the sample.

2. Add the contents of one Hardness Treatment Reagent Powder Pillow to the sample.
3. Cap the cell and invert until the reagent is dissolved.
4. Remove the cap.
5. Continue with the analysis at step 2 using the pretreated sample as the sample.

Test results are strongly influenced by sample temperature. Both reaction periods in the
procedure are the same and depend on the temperature of the sample. The reaction periods
indicated in the procedure are for a sample temperature of 1820 C (6468 F). Adjust both
reaction periods (see Color development based on sample temperature). The samples can be
read up to 15 minutes after the development times listed in Table 244 as the developed color is
stable for an extended period of time.

Sample Temperature
Development Time (minutes)
C
41
10
45
47
10
50
12
54
14
57
16
61
18
64

Page 776

Sample Temperature
Development Time (minutes)
C
20
68
23
73
2.5
25
77
greater than 25
greater than 77

Collect samples in clean glass bottles. Most reliable results are obtained when samples are
analyzed as soon as possible after collection.
Accuracy check
Dilution water is required when testing a diluted sample and preparing standard solutions. Dilution
water must be free of ammonia, chlorine and chlorine demand. A convenient source is a
recirculating, deionizer system with carbon filtration which produces 18 megohm-cm water.
Ammonia Nitrogen Standard, 10 mg/L as NH3-N
Ampule breaker
Mixing cylinders, 50 mL (3)
ADDITIONS.
6. Follow the Indophenol method for powder pillows test procedure for each of the spiked
instrument.

Page 777

Ammonia Nitrogen Standard Solution, 10 mg/L NH3N
Deionized water
Volumetric Pipet, 2 mL or Tensette Pipet and Pipet tips
Dilute 2.00 mL of Ammonia Nitrogen Standard Solution, 10 mg/L to 100 mL with deionized
water.
Or, use the TenSette Pipet to dilute 0.4 mL of a Ammonia Nitrogen Voluette Standard
Solution, 50 mg/L as NH3N, to 100 mL with dilution water.
2. Use this solution in place of the sample. Follow the Indophenol method for powder pillows test
procedure.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
388
0.20mg/L NH3N
0.190.21 mg/L NH3N
0.01 NH3N
Summary of method
Monochloramine (NH2Cl) and free ammonia (NH3 and NH4+) can exist in the same water sample.
Added hypochlorite combines with free ammonia to form more monochloramine. In the presence
of a cyanoferrate catalyst, monochloramine in the sample reacts with a substituted phenol to form
an intermediate monoimine compound. The intermediate couples with excess substituted phenol
to form a green-colored indophenol, which is proportional to the amount of monochloramine
present in the sample. Free ammonia is determined by comparing the color intensities, with and
without added hypochlorite. Test results are measured at 655 nm.

Page 778

Required reagents
Description
Free Ammonia Reagent Set (50 tests), includes:
Quantity/Test
Unit
Catalog number
2879700
1 drop
4 mL SCDB
2877336
100/pkg
2802299
(1) 2802299, (1) 2877336

Description
Unit
Catalog number
Hardness Treatment Reagent Pillows (1 per test)
50/pkg
2882346
Nitrogen Ammonia Standard Solution, 10 mg/L as NH3N
500 mL
15349
Nitrogen Ammonia Standard Ampule, 50 mg/L as NH3N, 10 mL
16/pkg
1479110
Water, organic-free water
500-mL
2641549
Description
Unit
Catalog number
Ampule Breaker Kit
each
2196800
each
1457442
each
1465100
each
1970001
50/pkg
2185696

each
187701
Wipers, Disposable, 30 x 30 cm, 280/box
box
2097000
each
1451536
each
189641

Page 779

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Total, 10072
Nitrogen, Total
DOC316.53.01085
Persulfate Digestion Method
Method 10072
HR (2 to 150 mg/L N)
Test N Tube Vials
Test preparation


Instrument
DR 6000
Light shield
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

DR 3900, DR 3800, DR 2800, DR 2700: Install the light shield in Cell Compartment #2 before performing this test.
Digestion is required for determining total nitrogen.
If the test overranges, repeat the digestion and measurement with diluted sample. The digestion must be repeated for
accurate results.
Use the deionized water provided in the reagent set or Organic-free Water to prepare the standards and perform
the procedure.
Nitrogen, Total
Page 781
Nitrogen, Total

Description
Quantity
Test N Tube HR Total Nitrogen Reagent Set
DRB200 Reactor
Funnel, micro
Light Shield or Adapter (see Instrument-specific information)
Pipet,
TenSette,
0.1 to 1.0 mL plus tips
Pipet,
TenSette,
Test Tube Cooling Rack
Persulfate digestion method
1. Turn on the DRB200

Reactor and heat to
105 C.
Nitrogen, Total
Page 782
2. Using a funnel, add

the contents of one Total
Nitrogen Persulfate
each of two HR Total
Nitrogen Hydroxide
Digestion Reagent vials.
Wipe off any reagent that
may get on the lip or the
tube threads.
3. Prepared Sample:
Add 0.5 mL of sample to a
vial.
Blank Preparation: Add
0.5 mL of the deionized
water included in the kit to
a second vial. Use only
water that is free of all
nitrogen-containing
species as a substitute for
the deionized water
provided.
4. Cap both vials. Shake

vigorously for at least
30 seconds to mix. The
persulfate reagent may not
dissolve completely after
shaking. This will not affect
accuracy.
Nitrogen, Total
Persulfate digestion method (continued)
Stored Programs
394 N, Total HR TNT
Start
5. Insert the vials in the

reactor and close the lid.
Heat for exactly 30
minutes.
6. Using finger cots,

immediately remove the
hot vials from the reactor.
Cool the vials to room
temperature.
7. Select the test.
9. Cap the tubes and

shake for 15 seconds.

timer.

remove the caps from the
vials and add one TN
Reagent B Powder Pillow
to each vial.

shake for 15 seconds. The
reagent will not completely
dissolve. This will not
affect accuracy. The
solution will begin to turn
yellow.

pipet 2 mL of digested,
treated sample into one
TN Reagent C vial.
15. Cap the vials and

Use slow, deliberate
inversions for complete
recovery.

timer.
Blank: Add 2 mL of
digested, treated reagent
blank to the second TN
Reagent C vial.
The tubes will be warm to

the touch.
period will begin.

timer.
period will begin.
8. Remove the caps from

the digested vials and add
Nitrogen (TN) Reagent A
Powder Pillow to each vial.
period will begin.
The yellow color will
intensify.
Nitrogen, Total
Page 783
Nitrogen, Total
Zero
17.

0 mg/L N
Read
19. Wipe the reagent vial

and insert it into the 16mm round cell holder.

mg/L N.
Blanks for colorimetric measurement

The reagent blank may be used up to seven days for measurements using the same lots of
reagents. Store it in the dark at room temperature (1825 C). If a small amount of white floc
appears within a week, discard the reagent blank and prepare a new one.
Interferences
The substances in the Non-interfering substances table have been tested and found not to
interfere up to the indicated levels (in mg/L). Interfering substances that resulted in a concentration
change of 10% appear in the Interfering substances table.

Interference level
Barium
10.4 mg/L
Calcium
Chromium
1200 mg/L
(3+)
2 mg/L
Iron
8 mg/L
Lead
26.4 g/L
Magnesium
2000 mg/L
Organic Carbon
600 mg/L
Phosphorus
400 mg/L
Silica
600 mg/L
Silver
3.6 mg/L
Tin
6 mg/L

Interference level
Bromide
> 240 mg/L; positive interference
Chloride
Nitrogen, Total
Page 784
Nitrogen, Total
This test performed with standard nitrogen solutions prepared from the following compounds
obtained 95% recovery:
Ammonium chloride
Urea
Ammonium sulfate
Glycine
Ammonium acetate
Ammonium chloride or nicotinic-PTSA spikes in domestic influent, effluent and the ASTM standard
specification for substitute wastewater (D 5905-96) also resulted in 95% recovery.
The large amounts of nitrogen-free organic compounds in some samples may decrease digestion
efficiency by consuming some of the persulfate reagent. Samples known to contain high levels of
organics should be diluted and re-run to verify digestion efficiency.
analysis.
Preserve the sample by reducing the pH to 2 or less with concentrated (at least 2 mL/L)
Sulfuric Acid.
Store samples at 4 C (39 F) or less. Preserved samples may be stored up to 28 days.
Warm stored samples to room temperature and neutralize with 5 N Sodium Hydroxide before
analysis.
Accuracy check
This method generally yields 95100% recovery on organic nitrogen standards. For proof of
accuracy use Primary Standards for Kjeldahl Nitrogen.
1. Prepare one or more of the following three solutions. Each preparation is for an equivalent
120-mg/L N standard. Use the deionized water included in the kit or water that is free of all
organic and nitrogen-containing species.
Weigh 1.6208 g of Ammonium p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL

volumetric flask with deionized water. Add deionized water to the 1000-mL mark.
Weigh 2.1179 g of Glycine p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric

flask with deionized water. Add deionized water to the 1000-mL mark.
Weigh 2.5295 g of Nicotinic p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric

2. Analyze each of these solutions using the test procedure above. Calculate the percent
recovery for each using this formula. Refer to the Percent recovery table for more information.
measured concentration
% recovery = ---------------------------------------------------------------- 100
120
Nitrogen, Total
Page 785
Nitrogen, Total
Table 248 Percent recovery
Compound
Lowest Expected % Recovery
Ammonia-PTSA
95%
Glycine-PTSA
95%
Nicotinic-PTSA
95%
Analysts have found Ammonia-PTSA to be the most difficult to digest. Other compounds may yield
different percent recoveries.
ADDITIONS.
5. Follow the Persulfate digestion method test procedure for each of the spiked samples, starting
100-mg/L ammonia nitrogen standard solution
1. Substitute 0.5 mL of a 100-mg/L ammonia nitrogen standard solution in place of the sample.
Follow the Persulfate digestion method test procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST..
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
394
100 mg/L NH3N
98102 mg/L N
0.5 mg/L N
Nitrogen, Total
Page 786
Nitrogen, Total
Summary of method
An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfite is
added after the digestion to eliminate halogen oxide interferences. Nitrate then reacts with
chromotropic acid under strongly acidic conditions to form a yellow complex with an absorbance
maximum at 410 nm.

Required reagents
Description
Test N Tube Total Nitrogen Reagent Set
Unit
Catalog number
50 vials
2714100

Description
Quantity
Unit
Catalog number
each
LTV082.53.40001
DRB200 Reactor, 220 V, 15 x 16 mm
each
LTV082.52.40001
Funnel, micro
each
2584335
each
1970001
50/pkg
2185696
each
1970010
50/pkg
2199796

OR
Pipet,
TenSette,
0.1 to 1.0 mL
each
1864100
Finger cots
2/pkg
1464702
Unit
Catalog number
Ammonia Nitrogen Standard Sol., 1000-mg/L NH3N
1L
2354153
Ammonia Nitrogen Standard Sol., 100-mg/L NH3N
500 mL
2406549
Description
Analytical balance, 80 g capacity, 0.1 mg resolution
each
2936701
Cylinder, mixing with stopper, 25 mL
each
2088640
each
1457453

Pipet tips for 1970001
1000/pkg
2185628
Pipet tips for 1970010
250-pkg
2199725
Primary Standard Set, for Kjeldahl Nitrogen
set of 3
2277800
50 mL
245026
Sulfuric Acid, ACS
500 mL
97949
Wastewater Mixed Inorganic Standard for NH3-H, NO3-N, PO4, COD, SO4,TOC
500 mL
2833149
Water, deionized
500 mL
27249
Water, organic-free
500 mL
2641549
Weighing papers
500/pkg
1473800
each
2484600
each
2196800

Ammonia Nitrogen Standard Solution, 10-mL Voluette Ampules, 50 mg/L
16/pkg
1479110
16/pkg
2128410
Nitrogen, Total
Page 787
Nitrogen, Total
Description
Unit
Catalog number
Ammonia Nitrogen Standard Solution, 2-mL PourRite Ampules, 50 mg/L
20/pkg
1479120
Ammonia Nitrogen Standard Solution, 10-mg/L NH3N
500 mL
15349

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Total Inorganic, 10021
Nitrogen, Total Inorganic
DOC316.53.01090
Titanium Trichloride Reduction Method
Method 10021
0.2 to 25.0 mg/L N
Test N Tube Vials
Test preparation


Instrument
Sample cell
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

For safety, wear gloves while breaking ampules.
The ammonia salicylate reagent contains sodium nitroferricyanide. Cyanide solutions are regulated as hazardous wastes by
the Federal RCRA. Collect cyanide solutions for disposal as reactive (D001) waste. Be sure cyanide solutions are stored in a
caustic solution with pH >11 to prevent release of hydrogen cyanide gas. Refer to the current MSDS for safe handling and
disposal information.

Description
Quantity
Total Inorganic Nitrogen Pretreatment Reagent Set (TiCl3 Reduction Method)
Test N Tube AmVer Nitrogen-Ammonia Reagent Set
Water, deionized
Centrifuge
1 mL
1
Funnel, micro
Light Shield or adapter (see Instrument-specific information)
Pipet, TenSette, 1.010.0 mL with tips
Pipette, volumetric, Class A, 1.00-mL
Test Tube Rack

Page 789

Titanium Trichloride Reduction method, TNT
Stored Programs
346 N Inorganic TNT
Start
1. Select the test.

Instrument-specific
information).

one Total Inorganic
Nitrogen Reductant
ampule into the vial
containing the sample.
Pour the contents of
another Total Inorganic
Nitrogen Reductant
ampule into the vial
containing the blank.
A black precipitate will
form immediately.

Page 790
2. Pipet 1 mL of Total
Inorganic Nitrogen
Pretreatment Base
concentrate into each of
two Total Inorganic
Nitrogen Pretreatment
Diluent Vials.
3. Prepared Sample:
Pipet 1 mL of sample into
one vial.
6. Immediately cap the

vials. Shake gently for 30
seconds to mix the
reagents. Allow the vials to
stand for at least one
minute.
7. Insert the vials in a

centrifuge.

timer.
(If a centrifuge is not

available, wait at least
30 minutes for the solids to
settle at the bottom of the
vial. Proceed to step 9.)
A three-minute timer will

begin.
The precipitate should

remain black after
shaking. Excessive
shaking will cause the
precipitate to turn white
and cause low results.

shake for 30 seconds to
mix.
Blank Preparation:
Pipet 1 mL of deionized
water into the second vial.
Centrifuge the vials for

three minutes.

Titanium Trichloride Reduction method, TNT (continued)
9. Using a pipet, add

2 mL of centrifuged
sample to an AmVer
Tube for Low Range
Ammonia Nitrogen.

the contents of one
Ammonia Salicylate
Reagent Powder Pillow
(for 5-mL samples) to each
vial.

the contents of one
Ammonia Cyanurate
(for 5-mL samples) to each
vial.

if nitrogen is present.
Add 2 mL of centrifuged
blank to another AmVer
Tube for Low Range
Ammonia Nitrogen.
Pipet carefully to avoid
disturbing the sediment.
Zero

timer.
period will begin.
14.

0.0 mg/L N

holder.
READ the results in
mg/L N.

Page 791
Interferences
The substances in the Interfering substances table may interfere when present. The substances in
the Non-interfering substances table do not interfere below the levels listed.

Interference level
Calcium
Causes a positive interference at 1000 mg/L as CaCO3.
Manganese (IV)
Causes a negative interference at 3 mg/L.
Magnesium
Causes a positive interference at 1000 mg/L as CaCO3.
Sulfide
Sulfate

Interference level
Al3+
8 mg/L
Ba2+
40 mg/L
Cu2+
40 mg/L
Fe3+
8 mg/L
Zn2+
80 mg/L
40 mg/L
PO43P
8 mg/L
SiO2
80 mg/L
EDTA
80 mg/L
analysis.
Preserve samples by reducing the pH to 2 or less with concentrated (at least 2 mL)
Hydrochloric Acid*. Store at 4 C (39 F) or less.
Before analysis, warm stored samples to room temperature and neutralize with 5 N Sodium
Hydroxide*.

Page 792
Accuracy check
HR Nitrate Nitrogen PourRite Ampule Standard, 500-mg/L NO3N
Ampule breaker
TenSette Pipet 0.11.0 mL and tips
25 mL Mixing cylinders (3)
ADDITIONS.
6. Follow the Titanium Trichloride Reduction method, TNT test procedure for each of the spiked
instrument.
1. Use this solution in place of the sample. Follow the Titanium Trichloride Reduction method,
TNT test procedure.
Method performance
The total inorganic nitrogen test is designed to provide an estimate of the total nitrite, nitrate and
ammonia nitrogen load present in a water or wastewater sample. This test is most applicable to
the monitoring of samples taken from an industrial process stream or a wastewater treatment
stream where it is important to track the inorganic nitrogen load as it passes through the treatment
process. The test does exhibit different recoveries of each of the three nitrogen species, as
summarized below. The test is not recommended for use when quantifying only one of the three
species. In that case, specific procedures for each particular analyte would be more appropriate.

Page 793

Species Recovery
Nitrogen Form
Recovery
NH3N
112%
NO3N
100%
NO2
77%
Program
Standard

Distribution
Sensitivity
DConcentration
per 0.010 DAbs
346
20.0 mg/L NO3N
19.620.4 mg/L NO3N
0.2 mg/L NO3N
Summary of method
Titanium (III) ions reduce nitrate and nitrite to ammonia in a basic environment. After centrifugation
to remove solids, the ammonia is combined with chlorine to form monochloramine.
Monochloramine reacts with salicylate to form 5-aminosalicylate. The 5-aminosalicylate is oxidized
in the presence of a sodium nitroprusside catalyst to form a blue colored compound. The blue
color is masked by the yellow color from the excess reagent present to give a final green colored
solution. Test results are measured at 655 nm.

Page 794

Required reagents
Description
Quantity/Test
Unit
Catalog number
25 tests
2604945
25 tests
2604545
1 mL
100 mL
27242
Quantity
Unit
Catalog number
each
2676500
Centrifuge, 220 VAC, 6 x 15 mL
each
2676502
Funnel, micro
each
2584335
each
1970010
varies
50/pkg
2199796
each
1864100
1 pair
100/pkg
2550503
Unit
Catalog number
each
1457441
Total Inorganic Nitrogen Pretreatment Reagent Set (TiCl3 Reduction

Method)
Test N Tube AmVer Nitrogen-Ammonia Reagent Set
Water, deionized
Required apparatus
Description
Centrifuge, 115 VAC, 6 x 15 mL
OR

Test Tube Rack
Gloves, Nitrile, Large1
1
Description
Flask, volumetric Class A, 50-mL
Nitrate Nitrogen Standard Solution, 10-mg/L NO3N
Nitrate Nitrogen Standard Solution, 2-mL
PourRite
Ampule, 500 mg/L
500 mL
30749
20/pkg
1426020
each
1465100
each
1970001
50/pkg
2185696
1000/pkg
2185628
250-pkg
2199725
4 liters
27256
Water, deionized

Page 795

Description
Unit
Catalog number
Cylinder, 25 mL mixing, tall form
each
2088640
Hydrochloric Acid, concentrated
500 mL
13449
50 mL SCDB
245026
Sodium Thiosulfate, 0.1 N
100 mL
32332
each
1451535
Pipette, volumetric, Class A, 1.00-mL

each
2484600
each
2196800
Nitrate Nitrogen Standard Solution, 1-mg/L NH3N
500 mL
204649
500 mL
194749
500 mL
1279249
Nitrate Nitrogen Standard Solution, MDB, 15-mg/L NH3N
100 mL
2415132

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Total Kjeldahl, 8075
Nitrogen, Total Kjeldahl

Nessler Method1 (Digestion Required)
DOC316.53.01091
Method 8075
1 to 150 mg/L
Scope and Application: For water, wastewater and sludge; digestion is required.
1
Adapted from Hach, et. al., Journal of Association of Official Analytical Chemists, 70(5) 783-787 (1987); Hach, et. al., Journal of Agricultural
and Food Chemistry, 33(6) 1117-1123 (1985); Standard Methods for the Examination of Water and Wastewater
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

adjust.
If using a Pour-Thru system, periodically clean the cell by pouring a few sodium thiosulfate pentahydrate crystals into the cell
funnel or rinse with a solution of sodium thiosulfate. Flush it through the funnel and cell with enough deionized water to
dissolve. Rinse out the crystals.
Hold droppers and dropper bottles vertically, not at an angle, when dispensing reagent.
Nessler reagent contains mercuric iodide. Both the sample and blank will contain mercury (D009) at concentrations
regulated as a hazardous waste by the Federal RCRA. Do not pour these solutions down the drain. Refer to the current
MSDS for safe handling and disposal instructions.
Because of the methods sensitivity, it is recommended to use the Standards and Calibration Adjust feature..

Page 797

Description
Boiling Chips, silicon carbide
Quantity
23
Cots, finger
Digesdahl Digestion Apparatus
20 mL
Mineral Stabilizer
6 drops
Nessler Reagent
2 mL
6 drops
Potassium Hydroxide (KOH) Standard Solution, 1.0 N
varies
Potassium Hydroxide (KOH) Standard Solution, 8.0 N
varies
6 mL
TKN Indicator Solution
2 drops
Pipet, TenSette, 0.11.0 mL, plus tips
Safety Shield

Page 798

Nessler method
Stored Programs
399 Nitrogen, TKN
Start
1. Select the test.

for orientation.
5. Add one drop of TKN

Indicator to each cylinder.
2. Prepared Sample:
Digest the sample amount
as described in the
Digesdahl Digestion
Apparatus Instruction
Manual.
Digest an equal amount of
deionized water as the
blank.
4. Select the appropriate

analysis volume of the
digested sample given in
the Aqueous samples
(solutions or suspensions
in waterless than 1%
solids), Dry samples or
Oils and fats tables
depending on the sample.
Pipet the analysis volume
from the sample and the
blank into separate 25-mL
mixing graduated
cylinders.
6. If the aliquot is less

than 1 mL, proceed to
step 7.
7. Add 1.0 N KOH to

each cylinder, one drop at
a time, mixing after each
addition. Continue until the
first permanent blue color
appears.
8. Fill both cylinders to

the 20-mL mark with
deionized water.
If it is greater than 1 mL,

add drops of 8.0 N KOH to
each cylinder until the first
flash of blue color
appears. Stopper and
invert the cylinder after
each addition. Proceed to
the next step.

Page 799


Mineral Stabilizer to each

Polyvinyl Alcohol
Dispersing Agent to each
11. Fill both cylinders to

the 25-mL mark with
deionized water. Stopper
mix.
12. Pipet 1.00 mL of

Nessler Reagent to each
invert repeatedly.
The solution should not be
hazy. Any haze (turbidity)
will cause incorrect
results.
Zero

timer.
period will begin.

Page 800
14. When the timer

of the cylinders into
separate square sample
cells.


0 mg/L TKN

Read

the cell holder.

mg/L TKN.
19. Calculate sample TKN

as follows:
75 A
ppm TKN = ---------------BC
Where:
A = mg/L read from the

display
B = g (or mL of water)
sample taken for digest
C = mL analysis volume of
digested sample
Interferences
Table 253 Aqueous samples (solutions or suspensions in waterless than 1% solids)
Expected nitrogen concentration (mg/L)
Analysis volume (mL)
0.528
10.0
2112
5.0
11560
2.00
452250
1.00
42522500
0.50
Table 254 Dry samples

422200
10.0
1065600
5.00
35018,000
2.00
100056,000
1.00
4200220,000
0.50

Page 801

Table 255 Oils and fats
854500
10.0
21011,000
5.00
2100110,000
1.00
Collect samples in clean glass or plastic containers.
Adjust sample pH to 2 or less with Sulfuric Acid (about 2 mL per liter) and cool to 4 C (39 F).
Preserved samples can be stored up to 28 days.
Accuracy check
Kjeldahl Nitrogen standard method
This procedure checks digestion efficiency and indicates the amount of bound nitrogen that is
released during digestion. The methods and standards available to check digestion technique are
found in the Accuracy Check section following the procedure in the Digesdahl Digestion
Apparatus Instruction Manual. Using the digested Kjeldahl standard, perform the Nessler method
on a colorimeter. The TKN value should come within 3% of the value of the prepared
Kjeldahl standard.
Standard solution method (to check calibration accuracy only)
1.0-mg/L NH3N solution
TKN indicator
Dropper
Graduated cylinders (2)
Deionized water
Mineral Stabilizer
Polyvinyl Alcohol Dispersing agent
1. Add one drop of TKN Indicator to each 25-mL graduated mixing cylinder.
2. Fill one cylinder to the 20-mL mark with deionized water. Fill the other cylinder to the 20-mL
mark with a 1.0-mg/L NH3N solution.
3. Add 3 drops of Mineral Stabilizer to each cylinder. Invert several times to mix.
4. Add 3 drops of Polyvinyl Alcohol Dispersing agent to each cylinder. Invert several times to mix.
5. Perform the TKN procedure as described in step 11 to step 18. Accurate calibrations will show
2627 mg/L TKN.

Page 802
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
DConcentration
per 0.010 DAbs
399
76 mg/L NH3N
7082 mg/L NH3N
1 mg/L NH3N
Summary of method
The term Total Kjeldahl Nitrogen refers to the combination of ammonia and organic nitrogen.
However, only the organic nitrogen compounds appearing as organically bound nitrogen in the
trinegative state are determined in this test. Nitrogen in this form is converted into ammonium salts
by the action of sulfuric acid and hydrogen peroxide. The ammonia is then analyzed by a modified
Nessler method test. Test results are measured at 460 nm.

Required reagents
Description
Nitrogen Reagent Set, 0150 mg/L, Nessler Method, includes:
Quantity/Test
Unit
Catalog number
2495300
250 tests
20 mL
490 mL
2119649
Mineral Stabilizer
6 drops
50 mL SCDB
2376626
Nessler Reagent
2 mL
500 mL
2119449
6 drops
50 mL SCDB
2376526
varies
50 mL SCDB
2314426
varies
100 mL MDB
28232H
6 mL
500 mL
97949
2 drops
50 mL SCDB
2251926
Quantity
Unit
Catalog number
2055734
TKN Indicator Solution
Required apparatus
Description
23
500 g
Cots, finger
2/pkg
1464702
each
2636240
Digesdahl Digestion Apparatus, 115 VAC
each
2313020
Digesdahl Digestion Apparatus, 220 VAC
each
2313021
each
1970001
50/pkg
2185696
Safety Shield
each
5003000
OR

Page 803
Description
Nitrogen, Primary Standard Set for TKN
Unit
Catalog number
each
2277800
Nitrogen Standard Solution, 1-mg/L NH3N
500 mL
189149
Nitrogen Standard Solution, Voluette Ampule, 150-mg/L NH3N, 10-mL
16/pkg
2128410
Wastewater Influent Inorganics Standard for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833149
Description
Unit
Catalog number
Sodium Thiosulfate, Pentahydrate
454 g
46001
Pour-Thru Cell Kit (DR 2700, DR 2800)
each
5940400
Pour-Thru Cell Kit (DR 5000)
each
LZV479
each
2484600
each
2196800
each
1970010
50/pkg
2199796
250/pkg
2199725
1000/pkg
2185628
500 mL
15349
500 mL
2406549
1L
2354153
16/pkg
1479110
Balance, analytical, 80 g capacity
115 VAC
2936701
Weighing Paper, 76 x 76 mm
500/pkg
1473800
Ammonia Nitrogen Standard Solution, 2-mL PourRite Ampule, 50 mg/L
20/pkg
1479120

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen, Total, LR, 10071
Nitrogen, Total
DOC316.53.001086
Persulfate Digestion Method
Method 10071
LR (0.5 to 25.0 mg/L N)
Test N Tube Vials
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

Digestion is required for determining total nitrogen.
If the test overranges, repeat the digestion and measurement with diluted sample. The digestion must be repeated for
accurate results.
Use the deionized water provided in the reagent set or Organic-free Water to prepare the standards and perform the
procedure.

Description
Quantity
Test N Tube LR Total Nitrogen Reagent Set
DRB200 Reactor
Funnel, micro
Pipet, TenSette, 1.0 to 10.0 mL plus tips

Finger Cots
13
2
Nitrogen, Total
Page 805
Nitrogen, Total
Persulfate digestion method

Reactor and heat to
105 C.

Nitrogen Persulfate
each of two Total Nitrogen
Hydroxide Digestion
Reagent vials. Wipe off
any reagent that may get
on the lip or the tube
threads.
Note: One reagent blank is
sufficient for each set of
samples.
3. Prepared Sample:
Add 2 mL of sample to one
vial.
Blank Preparation: Add
2 mL of the deionized
water included in the kit to
a second vial.
4. Cap both vials. Shake

vigorously for at least
30 seconds to mix. The
persulfate reagent may not
dissolve completely after
shaking. This will not affect
accuracy.
Use only water that is free

of all nitrogen-containing
species as a substitute for
the provided deionized
water.
Stored Programs
350 N, Total LR TNT
Start

reactor and close the lid.
Heat for exactly 30
minutes.
Nitrogen, Total
Page 806
6. Using finger cots,

immediately remove the
hot vials from the reactor.
Cool the vials to room
temperature.
7. Select the test.


the digested vials and add
Nitrogen (TN) Reagent A
Nitrogen, Total

shake for 15 seconds.

timer.
period will begin.

timer.
period will begin.

remove the caps from two
TN Reagent C vials and
add 2 mL of digested,
treated sample to one vial.
Blank: Add 2 mL of
digested, treated reagent
blank to the second TN
Reagent C vial.

remove the caps from the
vials and add one TN
Reagent B Powder Pillow
to each vial.

shake for 15 seconds. The
reagent will not completely
dissolve. This will not
affect accuracy. The
solution will begin to turn
yellow.

Use slow, deliberate
inversions for complete
recovery.

timer.
The tubes will be warm to

the touch.
period will begin.
The yellow color will
intensify.
Nitrogen, Total
Page 807
Nitrogen, Total
Zero
17. Wipe the reagent

16-mm round cell holder.

0.0 mg/L N
Read
19. Wipe the reagent vial

and insert it into the 16mm round cell holder.

mg/L N.
Note: Multiple samples may

be read after zeroing on one
reagent blank.
Blanks for colorimetric measurement

The reagent blank may be used up to seven days for measurements using the same lots of
reagents. Store it in the dark at room temperature (1825 C). If a small amount of white floc
appears within a week, discard the reagent blank and prepare a new one.
Interferences
The Non-interfering substances table shows substances that have been tested and found not to
interfere up to the indicated levels (in mg/L). Interfering substances that resulted in a concentration
change of 10% appear in the Interfering substances table.

Interference level
Barium
2.6 mg/L
Calcium
300 mg/L
Chromium (3+)
0.5 mg/L
Iron
2 mg/L
Lead
6.6 g/L
Magnesium
500 mg/L
Organic Carbon
150 mg/L
Phosphorus
100 mg/L
Silica
150 mg/L
Silver
0.9 mg/L
Tin
1.5 mg/L

Interference level
Bromide
Chloride
Nitrogen, Total
Page 808
Nitrogen, Total
This test performed with standard nitrogen solutions prepared from the following compounds
obtained 95% recovery:
Ammonium chloride
Urea
Ammonium sulfate
Glycine
Ammonium acetate
Ammonium chloride or nicotinic-PTSA spikes in domestic influent, effluent and the ASTM standard
specification for substitute wastewater (D 5905-96) also resulted in 95% recovery.
The large amounts of nitrogen-free organic compounds in some samples may decrease digestion
efficiency by consuming some of the persulfate reagent. Samples known to contain high levels of
organics should be diluted and re-run to verify digestion efficiency.
analysis.
Preserve the sample by reducing the pH to 2 or less with concentrated (at least 2 mL/L)
Sulfuric Acid.
Store samples at 4 C (39 F) or less. Preserved samples may be stored up to 28 days.
Warm stored samples to room temperature and neutralize with 5 N Sodium Hydroxide before
analysis.
Accuracy check
This method generally yields 95100% recovery on organic nitrogen standards. For proof of
accuracy use Primary Standards for Kjeldahl Nitrogen.
1. Prepare one or more of the following three solutions. Each preparation is for an equivalent 25mg/L N standard. Use the deionized water included in the kit or water that is free of all organic
and nitrogen-containing species.
a. Weigh 0.3379 g of Ammonium p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL
volumetric flask with deionized water. Add deionized water to the 1000-mL mark.
b. Weigh 0.4416 g of Glycine p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric
c. Weigh 0.5274 g of Nicotinic p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric
2. Analyze each of these solutions using the test procedure above. Calculate the percent
recovery for each using this formula. Refer to the Percent recovery table for more information.
measured concentration
% recovery = ---------------------------------------------------------------- 100
25
Refer to the Percent recovery table.
Table 259 Percent recovery

Compound
Ammonia-PTSA
95%
Nitrogen, Total
Page 809
Nitrogen, Total
Table 259 Percent recovery (continued)
Compound
Glycine-PTSA
95%
Nicotinic-PTSA
95%
Analysts have found Ammonia-PTSA to be the most difficult to digest. Other compounds may yield
different percent recoveries.
Ampule breaker
ADDITIONS.
5. Follow the Persulfate digestion method test procedure for each of the spiked samples, starting
10-mg/L ammonia nitrogen standard solution
1. Substitute 2 mL of a 10-mg/L ammonia nitrogen standard solution in place of the sample.

Follow the Persulfate digestion method test procedure.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
350
10 mg/L NH3N
9.610.4 mg/L N
0.5 mg/L N
Nitrogen, Total
Page 810
Nitrogen, Total
Summary of method
An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfite is
added after the digestion to eliminate halogen oxide interferences. Nitrate then reacts with
chromotropic acid under strongly acidic conditions to form a yellow complex with an absorbance
maximum at 410 nm.

Required reagents
Description
Unit
Catalog number
50 vials
2672245
Quantity
Unit
Catalog number
each
LTV082.53.40001
DRB200 Reactor, 220 V, 15x16 mm
each
LTV082.52.40001
Funnel, micro
each
2584335
each
1970010
Test N Tube Total Nitrogen Reagent Set, LR

Description
DRB200 Reactor, 110 V, 15x16 mm
OR
50/pkg
2199796
13
each
1864100
2/pkg
1464702
Description
Unit
Catalog number
1L
2354153
500 mL
15349

Finger Cots

Primary Standard Set, for Kjeldahl Nitrogen
set of 3
2277800
Wastewater Mixed Inorganic Standard for NH3-H, NO3-N, PO4, COD, SO4,TOC
500 mL
2833149
Water, deionized
500 mL
27249
Water, organic-free
500 mL
2641549
Description
Unit
Catalog number
Balance, analytical, 80 g capacity, 115 VAC
each
2936701
Cylinder, mixing with stopper, 50 mL
each
2088641
each
1457453
each
1970001
50/pkg
2185696
1000/pkg
2185628
250-pkg
2199725
50 mL
245026
Nitrogen, Total
Page 811
Nitrogen, Total
Description
Unit

PourRite Ampule breaker, 2-mL
Catalog number
500 mL
97949
each
2484600
each
2196800
500 mL
189149
500 mL
2406549
Ammonia Nitrogen Standard Solution, 2-mL PourRite Ampule, 50 mg/L
20/pkg
1479120
16/pkg
1479110
16/pkg
2128410

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oil and Grease, LLE, 10056
Oil and Grease

USEPA1 Hexane Extractable Gravimetric Method
DOC316.53.01187
Method 10056
15 to 3000 mg/L HEM and SGT-HEM

1
Equivalent to USEPA Method 1664. Adapted from Standard Methods for the Examination of Water and Wastewater, Section 5520B.
Test preparation

Determine a blank value (350 mL) of distilled or deionized water) with each new lot of reagents. If the blank result is greater
than 5 mg, resolve the source of error or remove interferences before performing this procedure. Use the equivalent amount
of acid to determine the blank and all samples from each sampling source.
The sample must be at room temperature before analyzing.
Do not use plastic tubing to transfer the solvent between containers.
Do not pre-rinse the collecting vessel with sample.
Anhydrous sodium sulfate is used to remove traces of water from the hexane extraction layer. Dry the sodium sulfate at 200
to 250 C for 24 hours for best performance.
Spilled reagent will affect test accuracy and is hazardous to skin and other materials.
If determining both the HEM and the SGT-HEM, clean and dry two distillation flasks (one for each procedure) in advance.
HEM = n-hexane extractable materials
SGT-HEM = Silica Gel Treated n-hexane extractable materials
Oil and Grease

Page 813
Oil and Grease

Hexane Extractable Gravimetric Method
sample in a clean 500-mL
separatory funnel.
If the sample is not
collected in the separatory
funnel, set the empty
container and lid aside for
use in step 4.
2. Using a pipette and

pipette filler, add 4 mL of
1:1 Hydrochloric Acid
solution to the separatory
funnel. Mix well. The pH
must be 2 to hydrolyze
oils and grease and
prevent a sodium sulfate
interference.
Check sample pH after
acid addition by dipping a
glass rod into the sample
and allowing a few drops
to touch the pH paper. Do
not dip the pH paper into
the sample. Rinse the
glass rod with a small
portion of hexane back
into the separatory funnel
to remove any grease/oil
on the rod.
Oil and Grease

Page 814
3. Using an analytical
balance, weigh a
previously dried and
cleaned 125-mL distillation
flask containing 35
boiling chips to the nearest
0.1 mg. Record the weight
of the flask.
4. Add 20 mL of
n-hexane to the
separatory funnel.
If the sample was
collected in a separate
container or if repeating
this step, rinse the
collecting vessel/
volumetric flask which
contained the sample/
water layer with 20 mL of
n-hexane, then add the
20-mL n-hexane rinse to
the separatory funnel.
Oil and Grease

Hexane Extractable Gravimetric Method (continued)
5. Stopper the funnel.

Invert the funnel and
release the gases through
the stopcock. Then,
vigorously shake the
funnel for two minutes.
6. Let the funnel stand

undisturbed for at least 10
minutes to ensure
separation of the lower
water layer and the upper
solvent layer.
To release gases from the

separatory funnel, invert it
and shake it once very
hard (support the stopper
with your hand). Under a
hood, point the delivery
tube in a safe direction
and slowly open the
stopcock to release any
gas. Close the stopcock.
Repeat the venting
procedure until you no
longer hear the release of
gases.
The solvent layer may be

brown if a colored oil is
present.
If you repeat this step the
third time and the water
layer is cloudy, allow the
separatory funnel to stand
undisturbed for 20 minutes
for better separation of the
water and solvent layers.
7. Slowly drain the lower

water layer from the
separatory flask into the
original sample container
or 500-mL volumetric
flask. This should take
about 34 minutes. Save
the water layer for step 10.
To ensure that water is not
transferred in step 9, allow
several drops of solvent
layer to drain into the
water layer until the
solvent layer is visible on
top of the water.
If the water layer drains
too quickly, excess water
will be present in the
solvent layer. This causes
sodium sulfate and water
interference.
8. Set up the filtering

funnel. Put the glass
funnel in the neck of the
distillation flask. Place a
folded 12.5 cm filter paper
in the funnel. Add 10 g of
anhydrous sodium sulfate
to the filter paper. Rinse
the sodium sulfate with a
small amount of the
hexane. Discard the
hexane properly.
Use the same filter, funnel
and sodium sulfate when
repeating this step for the
second and third
extractions. Remove large,
hard sodium sulfate
chunks between
extractions to reduce
sodium sulfate
contamination.
Oil and Grease

Page 815
Oil and Grease

Repeat
Steps 410
9. Drip-drain the solvent

layer into the pre-weighed
boiling flask through a
funnel containing filter
paper and 10 g anhydrous
sodium sulfate. Gently stir
the sodium sulfate with a
glass stirring rod while the
solvent layer is draining.
Be careful not to rip the
filter paper.
Any spillage will cause
inaccurate results. To
reduce spillage, use a
glass rod to guide the
sample solution into the
filter.
Oil and Grease

Page 816
10. Return the water layer

to the separatory funnel.
Use the same glass funnel
for the second and third
extraction (referred to in
step 11).
To reduce spillage, a
second funnel can be
used when pouring the
water layer into the
separatory funnel.
11. Repeat step 4 through

step 10 two more times.
After the third extraction,
discard the water layer.
There may be small
amounts of acetone and/or
n-hexane in the water
layer.
Refer to your local waste
disposal procedure for
proper disposal
instructions.
12. Rinse the separatory

funnel with three separate
5-mL aliquots of fresh
n-hexane to remove any
oil film left on the funnel
walls. Drain each aliquot
through the funnel
containing the sodium
sulfate into the distillation
flask.
Oil and Grease

Go to
Step 15
13. Rinse the tip of the

glass funnel with 5 mL of
n-hexane while removing it
from the distillation flask.
Check for sodium sulfate
contamination.
Sodium sulfate
contamination will appear
as cubic crystals at the
bottom of the distillation
flask. If present, re-filter
the solvent layer through
filter paper without sodium
sulfate. You must re-clean,
dry and weigh the boiling
flask and boiling chips; or
have an extra flask ready
in case this is necessary.
14. If HEM is to be
determined, go to step 15.
If only the SGT-HEM is to
be determined and the
HEM is known, go to
step 21.
If only the SGT-HEM is to
be analyzed, the HEM is
needed in order to
determine the amount of
silica gel needed for the
SGT-HEM. For each group
of samples from a
discharge, determine the
HEM before the
SGT-HEM.
See
Figure 1
See
Figure 1
15. Using the distillation

assembly shown in
Figure 13, distill off the
n-hexane. Distillation is
complete when there are
no boiling bubbles or the
distillation flask appears
dry.
16. Disconnect the

condenser/connector
portion of the distillation
assembly at the pinch
clamp and remove the
distillation flask from the
heat source with an
anti-lint cloth or tongs.
Use a steam bath or a hot

plate to maintain a water
bath at the proper
temperature for the
distillation. Do not place
the flask directly on a hot
plate. This will cause low
results and is dangerous
because n-hexane is
volatile.
The distilled n-hexane

may be re-used in future
HEM extractions, but is not
recommended for
SGT-HEM due to the
potential increased water
content of the solvent.
Evaporation will be faster if

the long vertical arm of the
connector is wrapped with
insulation (paper towel,
cloth or asbestos
insulating tape). The
distillation should take less
than 30 minutes.
Oil and Grease

Page 817
Oil and Grease

17. Remove the remaining

solvent vapors from the
distillation flask by
attaching the vacuum
connector/gas inlet
adapter to the flask. Apply
a vacuum for 12 minutes
or until all n-hexane
solvent vapors have been
removed.
Crystals on the bottom of
the flask indicate that
sodium sulfate may have
been dissolved in the
extraction steps. Redissolve the extract in nhexane, filter into another
pre-weighed flask and
repeat steps 1416. This
is not necessarily true for
the standard extraction
since stearic acid is
crystalline below 69 C
(156 F). If sodium sulfate
is present in the standard,
big cubical crystals (not
the flattened stearic acid
crystals) will be visible.
Also, an unusually high
yield compared to the
expected value will result.
Oil and Grease

Page 818
18. Place the flask in a

desiccator for 30 minutes
(or longer if necessary)
until it cools to room
temperature.
If the silica gel indicator
has turned red, replace the
silica gel.
19. Using an analytical

balance, weigh the flask to
the nearest 0.1 mg.
Record this weight. Do not
touch the flask after
weighing; fingerprints will
add weight.
20. Calculate the test

results:
Always use a tong or a

lint-free wipe when
handling the flask.
Example:
Precise weighing is
necessary for accurate
results; multiple weight
measurements are
recommended. Re-wipe
the flask before each
measurement to ensure all
contaminants are
removed. Record each
weight; use the lowest
repeatable value for
calculations.
B = 92.4206 g
A B 1000 = mg/L HEM
A= Weight (mg) of residue

B= Weight (mg) of flask
with boiling chips (step 3).
A = 92.4659 g
Sample volume = 0.350 L
(350 mL)
92.4659 92.4206
------------------------------------------------- =
0.350
129.4 mg/L
If yield is less than

15 mg/L and additional
precision is needed, use a
1-liter sample.
Oil and Grease

21. If only calculating the

HEM, stop here.
If continuing with
SGT-HEM, re-dissolve
residue with approximately
85 mL of fresh n-hexane.
Heat slightly to ensure
re-dissolving of all HEM
materials.
22. For a 350-mL water

sample, dilution is
necessary if the HEM is
above 2850 mg/L (for a
1-liter sample, dilute if the
HEM is greater than
1000 mg/L).
To dilute to a 1000 mg/L
sample, pour the
re-dissolved HEM into a
100-mL volumetric flask.
Rinse the distillation flask
34 times with 23 mL of
n-hexane. Fill the
volumetric flask to volume
with n-hexane. Mix well.
Into a 100-mL beaker,
volumetrically pipet the
amount (Va) determined
by this equation:
23. Put a magnetic stir bar

and the correct amount of
silica gel (based on the
equation below) into the
flask with the solvent/
product from step 22.
24. Stir solution on a

magnetic stirrer for five
minutes (minimum).
3 mg/L HEM
-------------------------------------- =
100
silica gel (g 0.3)
10000
V a = ---------------Wh
where:
Va = Volume of aliquot to
be withdrawn (mL) to get
1000 mg of HEM.
Wh = Weight of HEM (mg)
(A B x 1000 in step 19
(mg)). Dilute to about
100 mL with n-hexane.
Oil and Grease

Page 819
Oil and Grease

Perform
Steps 1520.
25. Place a funnel on a

clean, dry distillation flask
with 35 boiling chips in it.
Place a 12.5-cm filter
paper in the funnel.
Pre-moisten the filter
paper with fresh n-hexane.
Filter the solution through
filter paper.
Rinse the beaker
containing remaining silica
gel 3 times with 5-mL
aliquots of fresh n-hexane
and pour the aliquots into
the distillation flask.
Oil and Grease

Page 820
26. Follow step 15

step 20. Weigh the
product remaining in the
bottom of the flask and
calculate the results using
the equation below:
AB
------------------------------------------ =
Sample Volume
mg/L SGT HEM
where:
A = Weight (mg) of residue
B = Weight (mg) of flask
with boiling chips.
Oil and Grease
Figure 13 Distillation assembly

1.
Pinch Clamp
8.
Water Bath
2.
J-Shaped Connector
9.
Hot Plate
3.
Clamp, 3-Prong
10. Receiving Flask
4.
Condenser
11. Water In
5.
Pinch Clamp
12. Clamp Holders
6.
Clamp, 3-Prong
13. Water Out
7.
Distillation Flask
14. Support Stand and Rod Assembly
Oil and Grease

Page 821
Oil and Grease
Interferences
Substances extracted from samples will vary from source to source, depending upon the diversity
of the site being sampled. Some samples may contain high amounts of detergents or particulates
that can interfere with the extraction procedure. For these samples, it may be necessary to use a
350-mL sample rather than 1-liter (which is an option). In this circumstance, the 350-mL sample is
EPA accepted for reporting. Wash all glassware in hot water with detergent, rinse with tap and
distilled water and rinse with n-hexane or acetone.
If an emulsion forms between the two phases (at step 6) and is greater than one-third the volume
of the solvent layer, filter the emulsion and solvent layer through a funnel with glass wool in it. If an
emulsion still exists, other possible solutions include: stirring the solvent and emulsion layer with a
stir bar, using solvent phase separation paper, centrifugation, using an ultrasonic bath with ice,
adding NaCl or other physical methods. (Solid phase or other extraction techniques would fall
under performance based modifications.)
A milky solvent/product layer in the distillation flask indicates water in the solvent layer. Let the
flask stand one hour to allow the water to settle. Re-filter the solvent layer through sodium sulfate
to remove remaining water.
Extremely low yields could mean a poor extraction (step 5 through step 8) and a high yield could
indicate a problem in the solvent drying (step 8). Follow step 5 through step 8 very carefully and
run your blank before you run samples in order to identify any possible interference due to these
steps. If your blank indicates a yield above 1 mg per test, you should identify the source of
contamination before continuing; likely sources are sodium sulfate contamination and improperly
rinsed glassware.
Collect samples in wide-mouth glass bottles or directly in the separatory funnel for immediate
analysis.
Collection of sample may be done directly into the separatory funnel. Measure 350 mL of
water with a graduated cylinder.
Pour this into the separatory funnel. Use a laboratory pen to mark the 350-mL level. Fill with
sample to this mark.
Do not pre-rinse the bottle or separatory funnel with the sample.
If analysis is delayed for more than a few hours, add 6 mL of 1:1 Hydrochloric Acid Solution for
each liter or quart of sample.
Check the pH to ensure it is 2 or less. Do not place the pH paper directly into the sample, but
dip a glass rod into the sample and touch the pH paper with a drop of sample.
Rinse the rod with n-hexane directly back into sample container after the pH has been
determined to make sure that no product has adhered to the glass rod.
If necessary, add more acid to bring pH below 2.
Store the sample between 04 C (3239 F). Preserved samples can be stored for 28 days.
Handling glassware
Before analysis, careful cleaning and drying of the glassware and boiling chips is necessary. Clean
the chips and distillation flask by washing with hot water and detergent, rinsing with distilled water
and then rinsing with acetone or n-hexane. Place the cleaned flask and boiling chips in a drying
oven at 105115 C (220240 F) for 2 hours. Cool to room temperature in a desiccator for at least
30 minutes. Store in the desiccator until needed.
To eliminate errors, always handle the flask with tongs or an anti-lint wipe. If the same flasks are
used repeatedly, record their weights after drying in the oven without boiling chips. The drying step
Oil and Grease

Page 822
Oil and Grease

may be skipped if the flasks weigh the same after the acetone or n-hexane rinse as it does after
drying. Boiling chips will vary in weight; their weight should be added to the flask weight.
Definition of HEM and SGT-HEM

Oil and grease is the conventional term used to define pollutants of this nature. The new term
n-Hexane Extractable Materials (HEM) indicates this method may be applied to materials other
than oils and greases.
Likewise, the term Total Petroleum Hydrocarbons (TPH) was traditionally used to classify aliphatic
hydrocarbon materials. The new term Silica Gel Treated n-Hexane Extractable Material
(SGT-HEM), indicates that this method may be applied to materials other than aliphatic petroleum
hydrocarbons that are not adsorbed by silica gel.
Detection limit
This method is not applicable to measurements of materials that volatilize at temperatures below
approximately 85 C (185 F). Petroleum fuels from gasoline through #2 fuel oil may be partially
lost in the solvent removal operation. Some crude oils and heavy fuel oils contain a significant
percentage of materials that are not soluble in n-hexane. Recoveries of these materials may
be low.
The method is capable of measuring HEM and SGT-HEM in the range of 15 to 3000 mg/L when
using a 350-mL sample and may be decreased to as low as 5 mg/L if a 1-liter sample is used.
When using the 1-liter sample volume, use the amount of reagents listed for the 1-liter size in EPA
monitoring, testing procedures and modifications.
1. Transfer 400 4 mg stearic acid and 400 4 mg hexadecane into a 100-mL volumetric flask.
2. Pour 75 mL of acetone into the flask. Cover with a small beaker and stir gently. Heat slightly
until all material is in solution. Over-heating with the lid on causes pressure build up.
3. Fill to volume with acetone. Cover. Allow to equilibrate to room temperature and continue to fill
to volume until solution is at stable volume.
4. Using a volumetric pipet, transfer 5 mL of the solution from step 3 into 350 mL of deionized
reagent water. This standard solution should be 114.3-mg/L HEM or 57.1-mg/L SGT-HEM. If
using a 1-liter water sample, 5 mL gives concentrations of 40 mg/L HEM and 20 mg/L
SGT-HEM. To verify the concentration, pipette 5 mL of the solution from step 3 in a
pre-weighed flask, place in hood and allow acetone to evaporate off. Weigh the flask. Verify
that the weight difference before and after solution addition is 40 (1) mg.
EPA monitoring, testing procedures and modifications

If the Oil and Grease tests are used for compliance reporting to the USEPA, make the following
changes to the procedure:
1. Use a 1-liter sample in a 2000-mL separatory funnel rather than a 350-mL sample in a 500-mL
separatory funnel (step 1).
2. Use 6 mL (instead of 4 mL) of 1:1 hydrochloric acid to bring the pH below 2 (step 2) and 30 mL
of n-hexane instead of 20 mL of n-hexane for the extraction (step 4).
Before testing real samples for oil and grease, you must be able to obtain a MDL (Minimum
Detection Limit) less than or equal to the EPA reported MDL and to report an IPR (Initial Precision
and Recovery). Before attempting the MDL and IPR, it is highly recommended to run
laboratory reagent water blanks to eliminate all interference.
Oil and Grease

Page 823
Oil and Grease

MDL: The USEPA-documented MDL is extremely difficult to reproduce if the slightest sodium
sulfate contamination occurs. EPA Method 1664 requires the MDL to be 1.4 mg/L for HEM and
1.6 mg/L for SGT-HEM. This is calculated by determining the standard deviation of seven
standard samples for HEM or seven standards for SGT-HEM and multiplying their respective
standard deviations by 3.143 (Students t test).
The recommended standard concentration for determining the MDL is about 5 mg/L. To prepare
the standard for HEM follow steps 13 in Standard preparation, but change step 1 to transfer
100 ( 4) mg stearic acid and 100 ( 4) mg hexadecane to a 250-mL volumetric flask. To prepare
the SGT-HEM standard, transfer 200 ( 4) mg of decahexane only into a 250-mL volumetric flask.
Transfer 5 mL of either standard into 1-liter of reagent water. Analysis of the standard should give
5 mg/L for either HEM or SGT-HEM.
IPR: Follow the procedure for HEM and SGT-HEM, using deionized water (void of any oil and
grease) as the blank. Perform the procedure four separate times using 5 mL of the standard
(40 mg/L 1:1 stearic acid/hexadecane) diluted into 1 liter of demineralized water. Report the
average percent recovery (X) and the standard deviation of the percent recovery(s) for both HEM
and SGT-HEM. X and s should fall within these parameters:
HEM: Precision(s) 10%; Recovery(X) 83101%
SGT-HEM: Precision(s) 13%; Recovery(X) 83116%
If not within these ranges, correct the problem and repeat IPR.
Once the MDL and IPR have been established, keep records for EPA verification or for future
reference.
Oil and grease reporting to EPA

To report the HEM and/or SGT-HEM, include the following data for each set of up to ten samples.
1. BLANK: Must be less than 5.0 mg/L for HEM and SGT-HEM.
Note: For compliance monitoring, it may be necessary to use a standard that either matches the
regulatory concentration limit or is 1 to 5 times higher than the concentration of the sample (B),
whichever is greater. The concentration of the spike (T) needs to be divided by 2 for SGT-HEM if
using the standard (40 mg/L 1:1 stearic acid/hexadecane).
2. OPR (Ongoing Precision and Recovery): Determine the amount of analyte in a one liter
sample containing 5 mL of the standard (40 mg/L 1:1 stearic acid/hexadecane). Continue on if
the HEM recovery is 70114% and the SGT-HEM recovery is 66114%. If recovery is lower,
an interference may be present or analysis technique may be faulty. Identify the cause and
repeat OPR until within range.
3. MS and MSD (matrix spike and matrix spike duplicate): Determine the background
concentration (B) of your sample by running HEM and SGT-HEM for the discharge water.
Spike two 1-liter discharge water samples with 5 mL of standard and measure the
concentration of the analyte after spiking (A).
Determine the Percent Recovery (P) as follows:
100 ( A B )
P HEM(40 mg/L) = ----------------------------------T
100 ( A B )
P SGT HEM = ----------------------------------T2
If HEM Recovery is 79114% and SGT-HEM Recovery is 66114%, then calculate the
Relative Percent Difference (RPD). If recovery is lower, an interference may be present.
Identify and correct the interference, then repeat MS and MSD.
Conc MS Conc MSD
RPD = ---------------------------------------------------- 200
Conc MS + Conc MSD
Oil and Grease

Page 824
Oil and Grease

If RPD for HEM 18 and RPD for SGT-HEM 24, then continue with step 4.
If recovery is lower, an interference may be present. Identify and correct the interference, then
repeat MS and MSD.
After every five MS/MSD tests, compute the average percent recovery (Pa) and standard
deviation of the percent recovery (sp). Record these numbers as Pa 2sp.
4. Calibrate your analytical balance at 2 mg and 1000 mg using class S weights to ensure
calibration within 10%.
5. Analyze up to 10 samples from the source used in the MS and MSD before starting back at
step 1. For every 10 samples you must determine a new blank, OPR, RPD, MS and MSD.
Summary
Each laboratory must first verify the MDL and IPR and ensure they are within proper parameters
before reporting oil and grease test results to the EPA. Once this is established for a laboratory, it
does not need to be done again.
For every 10 samples per discharge source, one must calibrate the balance, report one blank, one
OPR, one MS and one MSD. Logs must be kept on percent recovery and relative percent
differences for MS/MSD tests. For every five MS/MSD test, calculate and record the average
percent recovery and standard deviation.
Summary of method
Oil and Grease & Total Petroleum Hydrocarbons (TPH) include any material that may be
recovered as a substance that is soluble in the n-hexane extractant. These include substances
such as relatively non-volatile hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases
and related materials. When measuring oil and grease (HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then the n-hexane is evaporated. The residue left is
weighed to determine the concentration of oil and grease materials in mg/L.
When measuring Total Petroleum Hydrocarbons (SGT-HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then mixed with silica gel to absorb non-TPH
components. Then the n-hexane is evaporated. Like the HEM, the residue left is weighed to
determine the concentration of total petroleum hydrocarbons.
Note: Careful technique is required to obtain accurate and precise results.

Required reagents
Description
Quantity/Test
Unit
4 mL
500 mL
88449
100200 mL
500 mL
1447849
pH Paper, 014 pH units
varies
100/pkg
2601300
Silica Gel with indicator (for desiccator)
varies
454 g
1426901
Silica Gel, 100200 mesh
130 g
500 g
2665034
Sodium sulfate, anhydrous
10 g
113 g
709914
Hydrochloric Acid Standard Solution, 1:1 (6N)

Hexane, ACS grade
Catalog number
Oil and Grease

Page 825
Oil and Grease
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Adapter, vacuum connector/gas inlet, 28/15
each
1433900
Aspirator, vacuum
each
213100
Balance, Analytical, 115 VAC 60 Hz.
each
2936801
2055734
310
500 g
Clamp, 3-prong
Boiling Chips
each
42200
Clamp, holder
each
32600
Clamp, pinch type, No. 28, F/Glass Joints
each
1433800
Condenser, reflux, w/ground glass joints, 28/15
each
1433700
each
2088549
each
50841
Desiccator
each
2088800
Desiccator Plate
each
1428400
Filter Funnel, 65-mm, short stem
each
2664700
Filter Paper, 12.5-cm, folded, pore size 8 to 12 m
100/pkg
69257
each
50543
1434000
Flask, Erlenmeyer, 125-mL, w/ground glass joint 28/15
each
each
52049
Marker, Laboratory
each
2092000
Oven, drying, 120 VAC, 50 Hz
each
1428900
Pipette filler, safety bulb
each
1465100
Pipette, serological, 5-mL
each
53237
Ring Support, 4-inch
each
580-01
Rod, glass
3/pkg
177001
Steam bath, 8-inch, 5-ring
each
2347900
Hot Plate/Stirrer, 7.25 x 7.25", 120 VAC
each
2881600
Stir Bar, 22.2 x 7.9 mm
each
2095350
Support stand
each
56300
each
56900
Tube, connecting, J-shaped, w/ground glass joint, 28/15
each
1814300
Tubing, black rubber, 7.9 X 2.4 mm
3.6 m
56019
Unit
Catalog number
Description
Hexadecane, 99%, 400 mg
Stearic Acid, 400 mg
Oil and Grease

Page 826
100 mL
2664842
500 g
2664934
Oil and Grease

Description
Acetone, ACS
Unit
Catalog number
500 mL
1442949
Separatory funnel, 2-liters
each
52054
Beaker, 50 mL
each
50041H
Balance, Weight Set
each
2617601
Hotplate/Stirrer 7.25 x 7.25 in., 220240 VAC
each
2881602
Ring Support, 4.5 in.
each
2656300
Furnace, Muffle, 120 VAC
each
1429600
Pipet, Volumetric 5.0 mL

Flask, Volumetric
each
14515-37
100 mL
14574-452
each
508-42
Oil and Grease

Page 827

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oil and Grease, SPE, 10300
Oil and Grease

USEPA1 Solid Phase Extraction Method
5 to 1000 mg/L HEM and SGT-HEM
DOC316.53.01186
Method 10300
Hexane Extractable Gravimetry

1
Equivalent to USEPA Method 1664A, Solid Phase Extraction (SPE)
Test preparation

Follow the instructions for sample collection and acidification in Sample collection, preservation and storage.
Assemble the Xenosep SPE apparatus as shown in Figure 14. Be sure to put the waste collection tube in the large flask
before installing the funnel assembly. Put the pattern side of the SPE filter down in the SPE filter support (Figure 15).
Wash all glassware in hot water with detergent, rinse with tap and distilled water and rinse with acetone or n-hexane.
Rinse all glassware thoroughly with hexane to make sure the analyte is not retained on the apparatus. Inadequate rinsing will
result in low recovery.
Determine a blank value (1 liter of distilled or deionized water) with each new lot of reagents. If the blank result is greater
than 5 mg, resolve the source of error or remove interferences before performing this procedure.
If analyzing for SGT-HEM, rinse the inner cavity of the aluminum dish with a few milliliters of acetone and then with a few
milliliters of hexane to remove any potential artifacts.
Label the aluminum dish and heat in an oven for one hour at 105 C. Cool the dish in a dessicator for 30 minutes. Weigh the
dish on an analytical balance to the nearest 0.1 mg. Record this weight as "B" for HEM step 22 or SGT-HEM step 12.
Make sure that the aluminum dish is removed from the hot plate before the solution has completely evaporated. Heating the
solution too long will result in low recovery.
Use a vacuum pump that can generate a Free Air Flow of at least 1 CFM (3 CFM preferred). Insufficient SPE disk drying will
result in low recovery.

Description
Hydrochloric acid, 1:1
Quantity
6 mL
Hexane (n-hexane) in Teflon-FEP wash bottle
1 wash bottle
Methanol in PE wash bottle
1 wash bottle
Deionized water in PE wash bottle
1 wash bottle
SPE Starter Kit, EPA Method 1664A
1 kit
SPE Consumables Kit
1 kit
SPE Solvent Recovery Kit
1 kit
Aluminum weighing dish
Hot plate
Vacuum pump
Oil and Grease

Page 829
Oil and Grease

Description
Quantity
pH paper, 014 pH units
Lab stand
Clamp, swivel
Desiccator
For SGT-HEM:
Silica gel
1 bottle
125-mL Erlenmeyer flask
100-mL volumetric flask (for HEM results over 1000 mg/L)
Magnetic stir plate
Aluminum weighing dish
Additional sodium sulfate column
Figure 14 Solid phase extraction assembly

1.
Hot plate
7.
Eluter tube
2.
Aluminum dish
8.
Clamp, tubing
3.
Glass dome
9.
3-way valve
Oil and Grease

Page 830
Oil and Grease

4.
Solvent recovery assembly
10. 2-way valve
5.
Funnel assembly (see Figure 15)
11. To vacuum
6.
Sodium sulfate column
12. Waste collection tube with O-Ring
1.
Aluminum clamp
4.
SPE filterpattern side down
2.
Funnel
5.
Stainless steel support
3.
Coupler with O-Rings
6.
SPE starter holder
Figure 15 Funnel assembly
Solid phase extraction for HEM
1. Add approximately
10 mL of hexane to the
funnel. Wait 5 seconds.
Turn the vacuum on and
off to pull the solvent into
the waste collection tube.
Repeat with a second
10-mL portion of hexane.
2. Turn the vacuum on

for 1 minute to dry the
filter. Turn off the vacuum.
10 mL of methanol to the
funnel. Wait 5 seconds.
Turn the vacuum on and
off to pull the solvent into
the waste collection tube.
4. Remove the tube and

discard the solvent waste.
Refer to the MSDS and
local regulations for safe
disposal of the solvent.
Do not allow the filter to

become dry.
Make sure the valves are

set to pull vacuum from
the funnel holder and the
tubing clamp is open.
Oil and Grease

Page 831
Oil and Grease

Solid phase extraction for HEM (continued)
20 mL of deionized water
to the funnel. Wait 5
seconds. Turn the vacuum
on and off to pull the water
into the flask.
Do not allow the filter to
become dry.
If the filter becomes dry,
repeat steps 35.
9. Turn the valve to apply

vacuum to the eluter tube.
Close the tubing clamp on
the funnel holder tube.
6. Slowly pour the

acidified sample into the
funnel and turn on the
vacuum. Use deionized
water to rinse any debris
from the walls of the
funnel.
7. Leave the vacuum on

for 4 to 8 minutes to air dry
the filter. Turn off the
vacuum.
Do not leave the vacuum
on for more than
8 minutes.
See Sample collection,

preservation and storage
for sample acidification.
10. Add 10 mL of hexane

to the empty sample
bottle. Shake the bottle in
a horizontal, circular
motion for 10 seconds to
rinse the bottle.
11. Use a transfer pipet to

collect the hexane from
the shoulder of the sample
bottle. Slowly rinse the
walls of the funnel with the
hexane. Go around the
funnel at least 3 times.
Repeat steps 1011 two
more times.
Oil and Grease

Page 832
8. Put the funnel

assembly on the eluter
tube.
12. Turn the vacuum on

and off to pull the solvent
into the flat-sided flask.
Rinse the walls of the
funnel with approximately
10 mL of hexane. Wait 5
seconds. Turn the vacuum
on and off.
Oil and Grease

Solid phase extraction for HEM (continued)
13. Remove the flat-sided

flask from the eluter tube.
14. Pour the hexane into

a pre-weighed aluminum
dish.
15. Rinse the flat-sided

flask with approximately
5 mL of hexane. Add the
hexane to the dish.
16. Put the dish on the hot

plate and place the glass
cover over the dish.
17. Close the 3-way valve

and open the 2-way valve
to pull vacuum from the
solvent recovery
apparatus.
18. Turn on the vacuum.

Turn the hot plate on low
heat (3585 C). Heat for
approximately 2 minutes.
19. Examine the dish for

dry spots. As soon as
there is a dry spot remove
the dish from the hot plate.
Put the dish in a fume
hood until the rest of the
hexane has evaporated.
20. When the dish is dry,

put the dish in a desiccator
for 30 minutes.
21. After 30 minutes,

weigh the dish to the
nearest 0.1 mg. Repeat
steps 20 and 21 until the
weight loss is less than
0.5 mg from the previous
weight. Record the weight.

results:
Example:
AB
---------------------------------------- 1000
sample volume
B= 6.2318 g
A= weight of dish with

residue (g) (step 21)
B= weight of dish (g)
A= 6.2394 g
sample volume = 0.950 L
6.2394 g 6.2318 g
----------------------------------------------------- 1000 =
0.950 L
8.0 mg/L HEM
Oil and Grease

Page 833
Oil and Grease

Solid phase extraction for SGT-HEM (HEM < 1000 mg/L)
1. Add hexane to the

residue in the aluminum
dish from the HEM test
until the dish is
approximately half full
(approximately 60 mL).
2. Heat slightly to
dissolve all of the residue.
3. Pour the mixture into a

Rinse the pan and funnel
several times with hexane
and add to the flask. Add
hexane to approximately
the 100-mL mark.
4. Add 3 ( 0.3) g of
silica gel for every 100 mg
of HEM or fraction thereof:
3 mg/L HEM
-------------------------------------- =
100
silica gel (g)
Example: if the HEM value
was 735 mg, round 7.35 g up
to the next whole gram
increment (8 g or 800 mg)
and add 3 x 800/100= 24 g of
silica gel.
Do not add more than 30 g

silica gel.
5. Add a stir bar to the

flask and stir for 5 minutes.
Oil and Grease

Page 834
6. Put a new sodium

sulfate column into the
eluter tube. Put the funnel
on the eluter tube.
7. Install a clean
flat-sided flask.
8. Adjust the valves to

pull vacuum from the
eluter tube.
Oil and Grease

Solid phase extraction for SGT-HEM (HEM < 1000 mg/L) (continued)
Go to the HEM
procedure.
9. Pour the solution from

the Erlenmeyer flask into
the funnel. Turn the
vacuum on and off to pull
the solution into the flatsided flask.
10. Rinse the Erlenmeyer

flask with 5 mL hexane.
Add the hexane to the
funnel. Turn the vacuum
on and off.
11. Complete steps 1321

under Solid phase
extraction for HEM to
evaporate the solvent.

results:
AB
---------------------------------------- 1000
sample volume
A= weight of dish with

residue (g) (step 21)
Rinse the walls of the

funnel with approximately
5 mL of hexane. Turn the
vacuum on and off.
B= weight of dish (g)

Example:
A= 6.2360 g
B= 6.2320 g
sample volume = 0.950 L
6.2360 g 6.2320 g
----------------------------------------------------- 1000 =
0.950 L
4.2 mg/L SGT-HEM
Report result as 5 mg/L
SGT-HEM
Solid phase extraction for SGT-HEM (HEM > 1000 mg/L)
1. Add hexane to the

residue in the aluminum
dish from the HEM test
until the dish is
approximately half full
(approximately 60 mL).
Heat slightly to dissolve all
of the residue.
2. Pour the mixture into a

Rinse the pan and funnel
several times with hexane
and add to the flask.
3. Dilute to the mark with

hexane and mix well.
4. Calculate the volume

to remove for the silica gel
treatment:
100,000
V 1000 = ----------------------------HEM value
V1000 = volume that

contains 1000 mg HEM
Oil and Grease

Page 835
Oil and Grease

Solid phase extraction for SGT-HEM (HEM > 1000 mg/L) (continued)
Go to the SGT-HEM
(HEM < 1000 mg/L)
procedure.
5. Remove the amount

calculated in step 4 from
the volumetric flask and
add it to a 125-mL
Erlenmeyer flask. Add
hexane to approximately
the 100-mL mark.
6. Add 30 g of silica gel

to the flask.
If the volume added in
step 5 contained less than
1000 mg HEM, calculate
the amount of silica gel to
add based on the mg HEM
that was added to the
flask.
3 mg HEM in aliquot
---------------------------------------------------------- =
100
silica gel (g)
7. Complete steps 512

in the procedure for Solid
phase extraction for SGTHEM (HEM < 1000 mg/L).
8. Correct the test result

for the reduced volume
that was treated with the
silica gel:
100
W d -------------- = W c
V 1000
where:
Wd = result from step 12
V1000 = volume removed
for silica gel treatment
Wc = corrected SGT-HEM
result
Interferences
For samples that are known to contain extremely high levels of oil and grease, use a smaller
sample volume and correct for the volume difference to give the result as a 1-L sample.
High concentrations of particulates in the water sample can clog the SPE filter or retain high levels
of water, which can lower the extraction efficiency. Inorganic particulates are easier to filter than
organic particulates. The following techniques may help to filter samples containing high levels of
particulates:
Decantingallow the sample to settle and pour the top portion into the funnel first. Just before
dryness, add the rest of the sample. Remove any sediment from the bottle and add it to the
SPE filter. Use deionized water to rinse any sediment that remains in the bottle.
Prefilters or prefilter fibersplace the prefilter or prefilter fibers into the coupler before the
funnel is attached.
Drying agentsadd magnesium sulfate to the SPE filter or to the prefilter to remove water that
may be trapped in the particulates.
Filtration aidsadd materials such as sodium chloride, sand, diatomaceous earth or glass
beads to help speed the complete filtration of samples that contain organic particulates.
This method is not applicable to materials that volatilize at temperatures below approximately
85 C (185 F). Petroleum fuels from gasoline through #2 fuel oil may be partially lost in the solvent
removal operation. Some crude oils and heavy fuel oils contain a significant percentage of
materials that are not soluble in n-hexane. Recoveries of these materials may be low.
Oil and Grease

Page 836
Oil and Grease

Collect 1 L (9501050 mL) of sample in a wide-mouth glass bottle. Do not rinse the bottle with
sample before collection. The sample must be at room temperature before the test.
Sample volume
Complete the steps that follow to measure the sample volume.
6. Use a laboratory pen to mark on the sample bottle the liquid level of the sample.
7. When the test is complete, fill the bottle to this mark with tap water.
8. Pour the tap water into a 1-L graduated cylinder and write down the volume. Use this volume
for the sample volume in the final step of the test procedure for HEM or SGT-HEM.
Acidification
The sample must be acidified with 1:1 hydrochloric acid (HCl) solution to a pH of 2 or less before
the test is started. To find the volume of HCl to use, collect a separate aliquot, add HCl until the pH
is less than 2 and add this volume of acid to each sample bottle before collection. Do not dip pH
paper, a pH electrode, a glass rod or other materials into the sample because oil and grease in the
sample may adhere to these items.
If the test can not be started for more than a few hours after collection, cool and store the sample
at 04 C (3239 F). Acidified samples can be stored for 28 days.
Accuracy check
For USEPA reporting, each laboratory must first measure an acceptable MDL (Minimum Detection
Limit) and IPR (Initial Precision and Recovery) before samples can be measured. Before
measuring the MDL or IPR, run laboratory reagent water blanks to eliminate interferences.
Standard solution preparation
Required items:
Stearic acid, 98% minimum
Hexadecane, 98% minimum
Acetone for Organic Residue Analysis, residue less than 1 mg/L
10.0-mL Class A volumetric pipet
15.0-mL Class A volumetric pipet
1. Put 200 ( 2) mg stearic acid and 200 ( 2) mg hexadecane into a 100-mL volumetric flask.
2. Add 7585 mL of acetone and shake vigorously until all material has dissolved.
3. Allow to equilibrate to room temperature and fill to volume with acetone. Mix well. The
concentration of this stock solution is 4000 mg/L HEM (2000 mg/L SGT-HEM).
4. Use a volumetric pipet to dilute the stock solution for use in MDL, IPR or OPR measurements:
MDL standard solution:
a. Add 15 mL of the stock solution into a clean 100-mL volumetric flask. Dilute to the mark
with acetone and mix well.
b. Pipet 10 mL for HEM (or 20 mL for SGT-HEM) into a 1-L volumetric flask. Dilute to the
mark with deionized water at pH < 2 and mix well. The concentration of this standard
solution is 6 mg/L HEM (or 6 mg/L SGT-HEM).
Oil and Grease

Page 837
Oil and Grease

IPR or OPR standard solution:
Add 10 mL of the stock solution into 1 liter of deionized water and mix well. The concentration
of this solution is 40 mg/L HEM (20 mg/L SGT-HEM).
Note: To verify the concentration of the IPR/OPR standard, use a pipet to add 10 mL of the standard
solution into a pre-weighed flask or dish. Allow the acetone to evaporate in a fume hood. When dry,
weigh the flask or dish. The weight of the residue should be 40 (1) mg.
EPA requirements for MDL and IPR

MDL: measure seven replicates of a 6 mg/L standard solution, find the standard deviation and
multiply the standard deviation by 3.143 (Students t test). The acceptable limits are:
HEM: 1.4 mg/L
SGT-HEM: 1.6 mg/L
IPR: follow the procedure for HEM and SGT-HEM (if necessary) four separate times using a
40 mg/L HEM (20 mg/L SGT-HEM) standard solution. Report the average percent recovery (x)
and the standard deviation for both HEM and SGT-HEM. The acceptable limits are:
HEM: Precision(s) 11%; Recovery (x) 83101%
SGT-HEM: Precision(s) 28%; Recovery (x) 83116%
A low result can be an indication of loss during quantitative transfers or loss during heating. A high
result can be an indication of incomplete drying or contamination. Run a blank before running
samples. If the blank result is more than 5 mg, identify and remove the source of contamination.
After acceptable values are obtained for the MDL and IPR, keep records for USEPA verification.
Oil and grease reporting to USEPA

Include the following data with the HEM and/or SGT-HEM results for each set of up to 20 samples
per discharge source.
1. Blank value: must be less than 5.0 mg/L for HEM and SGT-HEM.
Note: It may be necessary to use a standard that matches the regulatory concentration limit, is 1 to 5
times higher than the concentration of the sample (B) or is at the concentration of the Ongoing
Precision and Recovery, whichever is highest. Divide the concentration of the spike (T) by 2 for
SGT-HEM if using the 40 mg/L HEM (20 mg/L SGT-HEM) standard.
2. OPR (Ongoing Precision and Recovery): add 10 mL of the 40 mg/L HEM (20 mg/L SGTHEM) standard to a 1-liter sample and run the test. The acceptable limits for recovery are:
HEM: 78114%
SGT-HEM: 64132%
3. MS and MSD (matrix spike and matrix spike duplicate): measure the HEM and SGT-HEM
concentration of the sample (B). Spike two 1-L samples with 10 mL of the 40 mg/L HEM
(20 mg/L SGT-HEM) standard and measure the concentration after spiking (A).
Calculate the Percent Recovery (P) as follows:
100 ( A B )
P HEM (40 mg/L) = ----------------------------------T
100 ( A B )
P SGT HEM = ----------------------------------T2
where:
A = concentration of the unspiked sample
B = concentration of the spiked sample
T = concentration of the spike solution
Oil and Grease
Page 838
Oil and Grease

If the recovery for HEM and SGT-HEM is within the acceptable limits for OPR (step 2), then
calculate the Relative Percent Difference (RPD).
Conc MS Conc MSD
RPD = ---------------------------------------------------- 200
Conc MS + Conc MSD
If the RPD for HEM is 18 and for SGT-HEM 34, then continue with step 4. If the recovery is
lower than the RPD, an interference may be present. Identify and correct the interference,
then repeat MS and MSD.
After every five MS/MSD tests, calculate the average percent recovery (Pa) and standard
deviation of the percent recovery (sp). Record these numbers as Pa 2sp. Update the
accuracy assessment on a regular basis (e.g., after five to ten new accuracy measurments).
4. Balance calibration: measure a 2 mg and a 1000 mg class S weight on the analytical
balance before and after each analytical batch. If the values are not within 10% of the actual
weight, recalibrate the balance.
Summary of method
Oil and Grease & Total Petroleum Hydrocarbons (TPH) include any material that may be
recovered as a substance that is soluble in the n-hexane extractant. These include substances
such as relatively non-volatile hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases
and related materials. When measuring oil and grease (HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then the n-hexane is evaporated. The residue left is
weighed to determine the concentration of oil and grease materials in mg/L.
When measuring Total Petroleum Hydrocarbons (SGT-HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then mixed with silica gel to absorb non-TPH
components. Then the n-hexane is evaporated. Like the HEM, the residue left is weighed to
determine the concentration of total petroleum hydrocarbons.
Definition of HEM and SGT-HEM
Oil and grease is the conventional term used to define pollutants of this nature. The newer term
n-Hexane Extractable Materials (HEM) indicates this method may be applied to materials other
than oils and greases.
Likewise, the term Total Petroleum Hydrocarbons (TPH) was traditionally used to classify aliphatic
hydrocarbon materials. The newer term Silica Gel Treated n-Hexane Extractable Material
(SGT-HEM), indicates that this method may be applied to materials other than aliphatic petroleum
hydrocarbons that are not adsorbed by silica gel.

Required reagents
Description
Quantity/Test
Unit
6 mL
500 mL
88449
100200 mL
1L
2510253
Methanol, ACS grade
10 mL
500 mL
1446449
Silica Gel, 100200 mesh (for SGT-HEM)
130 g
500 g
2665034
Hydrochloric Acid Standard Solution, 1:1 (6 N)

Hexane, for Organic Residue Analysis
Catalog number
Oil and Grease

Page 839
Oil and Grease

Required apparatus
Description
Quantity/Test
Unit
Catalog number
Balance, Analytical, 115 VAC 60 Hz.
each
2936801
Bottle, wash, 500-mL Teflon, FEP
each
2505100
Bottle, wash, 500-mL
3/pkg
2927204
Bottle, wide-mouth, 1-L
each
2951401
Clamp, swivel
each
2503400
Cylinder, graduated, 1-L
each
108153
Desiccator
each
2088800
1428400
Desiccator Plate
each
Flask, Erlenmeyer, 125-mL (for SGT-HEM)
each
50543
Hot Plate (Thermolyne), 120 VAC, 50 Hz
each
2344100
Marker, laboratory
each
2092000
Oven, drying, 120 VAC, 50 Hz
each
1428900
Pump, vacuum, 27 in. Hg, 1.3 CFM
each
2824800
SPE Consumables Kit
24/pkg
2943800
SPE Starter Kit, EPA Method 1664A
each
2943231
SPE Solvent Recovery Kit
each
2514300
Stirrer, magnetic, 120 VAC
each
2343600
each
2095350
Support stand
each
2504900
each
56900
Unit
Catalog number
Description
Hexadecane, 99%
Stearic acid
100 mL
2664842
500 g
2664934
Unit
Catalog number

Description
Acetone, for Organic Residue Analysis
Flask, volumetric, Class A, 1-L
pH Paper, 014 pH units
1L
2510153
each
1457442
each
1457453
100/pkg
2601300
each
1465100
each
53238
each
1451538
each
1451539
Silica Gel with indicator (for desiccator)
454 g
1426901

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Organic Carbon, Total HR, 10128
Organic Carbon, Total
DOC316.53.01095
Direct Method1
Method 10128
HR (100 to 700 mg/L C)

Scope and Application: For wastewater and industrial water
1
U.S. Patent 6,368,870
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

A reagent blank is required for each series of samples.
To test for TOC levels below 100 mg/L C, use Method 10173 or Method 10129.

Description
Quantity
Total Organic Carbon Direct Method High Range Test N Tube Reagent Set
DRB200 Reactor
pH Paper
Magnetic Stirrer
1
Stir Bar, magnetic
Test Tube Rack
Pipet,
TenSette,

Page 841

Description
Quantity
Water, organic-free
0.3 mL
Wipes, disposable
Direct method

reactor. Select the TOC
program.
2. Use a graduated
cylinder to add 10 mL of
sample to a 50-mL
Erlenmeyer flask that
contains a stir bar.
3. Add 0.4 mL of Buffer

Solution, pH 2.0. Use pH
paper to make sure the
sample pH is 2.
4. Place the flask on a

stir plate and stir at a
moderate speed for
10 minutes.
5. Label two High Range

Acid Digestion vials
sample and reagent blank.
6. Use a funnel to add

the contents of one TOC
Persulfate Powder Pillow
to each Acid Digestion vial
(colorless liquid).

to add 0.3 mL of
organic-free water to the
reagent blank vial and
0.3 mL of prepared
sample to the sample vial.
Swirl to mix.
8. Rinse two blue MR/HR

Indicator Ampules with
deionized water and wipe
them with a soft, lint-free
wipe. Do not touch the
ampules sides after
wiping. Pick them up by
the top.

Page 842

Direct method (continued)
Stored Programs
426 Organic Carbon HR
Start
9. Lower one unopened

ampule into each Acid
Digestion vial. When the
score mark on the ampule
is level with the top of the
Acid Digestion vial, break
the top off the ampule and
allow it to drop into the
Acid Digestion vial.
10. Cap the vial

assemblies tightly, insert
them in the reactor and
close the lid for 2 hours at
103105 C.
11. Carefully remove the

vial assemblies from the
reactor. Place them in a
test tube rack.
Allow the vials to cool for
one hour for accurate
results.

Insert a light shield or an
adapter if required (see
Instrument-specific
information).
The liquid in the reagent

blank vial should be dark
blue.
Do not invert or tilt the

vial after inserting the
ampule.
Zero

blank with a damp towel,
followed by a dry one, to
remove fingerprints or
other marks.
14.

0 mg/L C
16. Wipe the sample vial

assembly with a damp
towel, followed by a dry
one, to remove fingerprints
or other marks.
Read
17. Insert the sample vial

assembly in the 16-mm
round cell.

mg/L C.

Page 843
Interferences
The Interfering substances table lists substances that have been tested for interference and found
not to interfere up to the levels indicated.
If the sample contains greater than 1000 mg/L CaCO3 alkalinity, lower the sample pH to less than
7 before testing by adding Sulfuric Acid Solution.
Most sample turbidity is either dissolved during the digestion stage or settled during the cooling
period. Sample turbidities up to 50 NTU have been tested without interference.

Interference level
Aluminum
10 mg/L Al
Ammonia Nitrogen
1000 mg/L as N
ASTM Wastewater
No effect
Bromide
500 mg/L Br
Bromine
25 mg/L Br2
Calcium
2000 mg/L as CaCO3
Chloride
5000 mg/L Cl
Chlorine
10 mg/L Cl2
Chlorine Dioxide
6 mg/L ClO2
Copper
10 mg/L Cu
Cyanide
10 mg/L CN
Iodide
50 mg/L I
Iron (II)
10 mg/L Fe2+
Iron (III)
10 mg/L Fe3+
Magnesium
2000 mg/L as CaCO3
Manganese (VII)
1 mg/L Mn
Monochloramine
14 mg/L NH2Cl as Cl2
Nitrite
500 mg/L NO2
Ozone
2 mg/L O3
Phosphate
3390 mg/L PO43
Silica
100 mg/L SiO2
Sulfate
5000 mg/L SO42
Sulfide
20 mg/L S2
Sulfite
50 mg/L SO32
Zinc
5 mg/L Zn
Collect samples in clean glass bottles.
Rinse the sample bottle several times with the sample to be collected.
Fill the bottle completely full before capping.
Test samples as soon as possible.

Page 844
Acid preservation is not recommended.
Homogenize samples containing solids to assure representative samples.
Accuracy check
TOC Standard Ampule, 1000 mg/L C
Organic-free Reagent Water
1-L Class A volumetric flask
Acid Digestion Vials (3)
TOC Persulfate Powder Pillow (1)
50 mL Class A Volumentric flask
15 mL Volumetric Pipet and pipet filler
ADDITIONS.
4. Prepare a 300 mg/L C standard solution as follows:
a. Transfer 15.00 mL of the 1000-mg/L total organic carbon standard solution to a 50-mL
volumetric flask.
b. Dilute to the mark with Organic-free Reagent water. Insert the stopper and mix thoroughly.
Prepare this solution daily.
5. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of the
prepared 300-mg/L standard to three Acid Digestion Vials.
6. Add the contents of one TOC Persulfate Powder Pillow to each vial.
7. Add 0.3 mL of sample to each vial. Swirl to mix.
8. Continue the test starting at step 8 of the Direct method test procedure for each of the spiked
instrument.

Page 845
15 mL Volumetric pipet and pipet filler

a. Transfer 15.00 mL of the prepared 1000-mg/L organic carbon stock standard solution to a
2. Use this solution in place of the sample. Follow the Direct method test procedure.
Method performance
To test for TOC levels below 100 mg/L C, use Method 10173 or Method 10129.
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
426
350 mg/L C
337363 mg/L C
4 mg/L C
Summary of method
The total organic carbon (TOC) is determined by first sparging the sample under slightly acidic
conditions to remove the inorganic carbon. In the outside vial organic carbon in the sample is
digested by persulfate and acid to form carbon dioxide. During digestion, the carbon dioxide
diffuses into a pH indicator reagent in the inner ampule. The absorption of carbon dioxide into the
indicator forms carbonic acid. Carbonic acid changes the pH of the indicator solution which, in
turn, changes the color. The amount of color change is related to the original amount of carbon
present in the sample. Test results are measured at 598 and 430 nm.

Required reagents
Description
Total Organic Carbon Direct Method
High Range Test N Tube Reagent Set, includes:
Acid Digestion Solution Vials, High Range TOC1
Quantity/Test
Unit
Catalog number
50 vials
2760445
50/pkg
0.4 mL
25 mL
Funnel, micro
each
2584335
Indicator Ampules, MR/HR TOC1
10/pkg
TOC Persulfate Powder Pillows1
50/pkg
Buffer Solution, Sulfate1,2
pH Paper
Water, Organic-free
1
Not sold separately.
See alternate size below
5/pkg
39133
0.3 mL
500 mL
2641549
Quantity
Unit
Catalog number
each
50838
Required apparatus
Description

Page 846

Required apparatus
Description
Quantity
Unit
Catalog number
each
LTV082.53.40001
each
LTV082.52.40001
each
50541
Magnetic Stirrer
each
2881200
each
1970001
50/pkg
2185696
each
1970010
50/pkg
2199796
4531500

OR
Stir Bar, magnetic
each
13
each
1864100
280/pkg
2097000
Description
Unit
Catalog number
TOC Standard Solution Ampule (KHP Standard, 1000-mg/L C)
5/pkg
2791505
Test Tube Rack

Wipes, Disposable

Description
Unit
Catalog number
115 VAC
2936701
Buffer Solution, Sulfate, pH 2.0
500 mL
45249
Flask, volumetric class A, 50 mL
each
1457441
each
1457453
Pipet, Volumetric, 15 mL
each
1451539
Pipet Filler
each
1465100
1000/pkg
2185628
250/pkg
2199725
Potassium Acid Phthalate
500 g
31534
Sulfuric Acid Solution, 5.25 N
100 mL
244932
500/pkg
1473800

Page 847

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Organic Carbon, Total LR, 10129
DOC316.53.01093
Direct Method1
Method 10129
LR (0.3 to 20.0 mg/L C)

Scope and Application: For water, drinking water and wastewater
1
U.S. Patent 6,368,870
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

To test for higher ranges of TOC use Method 100173 or method 10128.

Description
Quantity
Total Organic Carbon Direct Method Low Range Test N Tube Reagent Set
DRB200 Reactor
pH Paper
Magnetic Stirrer
Pipet,
TenSette,
1

Page 849

Description
Quantity
Stir Bar, magnetic
Test Tube Rack
Water, organic-free
Wipes, disposable,
3.0 mL
1
Kimwipes
Direct method

program.
2. Use a graduated
sample to a 50-mL

sample pH is 2.

moderate speed for
10 minutes.
5. Label two Low Range


(colorless liquid).

to add 3.0 mL of
3.0 mL of prepared
Swirl to mix.
8. Rinse two blue Low

Range Indicator Ampules
wipe them with a soft, lintfree wipe.
Note: The organic free water

must contain less than 0.05
mg/L carbon.

Page 850
Do not touch the ampule

sides after wiping. Pick
them up by the top.

Stored Programs
427 Organic Carbon LR
Start

Acid Digestion vial, snap
10. Cap the vial

103105 C.

test tube rack.


results.
blue.

ampule.
Zero

other marks.
14. Insert the reagent

blank vial assembly in the

0.0 mg/L C

or other marks.
Read

round cell.

mg/L C.

Page 851
Interferences
The Interfering substances table lists substances that have been tested for interference and found
If the sample contains greater than 600 mg/L CaCO3 alkalinity, lower the sample pH to less than 7
before testing by adding Sulfuric Acid Solution.

Interference level
Aluminum
10 mg/L Al
Ammonia Nitrogen
1000 mg/L as N
ASTM Wastewater
No effect
Bromide
500 mg/L Br
Bromine
25 mg/L Br2
Calcium
2000 mg/L as CaCO3
Chloride
500 mg/L Cl
Chlorine
10 mg/L Cl2
Chlorine Dioxide
6 mg/L ClO2
Copper
10 mg/L Cu
Cyanide
10 mg/L CN
Iodide
50 mg/L I
Iron (II)
10 mg/L Fe2+
Iron (III)
10 mg/L Fe3+
Magnesium
2000 mg/L as CaCO3
Manganese (VII)
1 mg/L Mn
Monochloramine
Nitrite
500 mg/L NO2
Ozone
2 mg/L O3
Phosphate
3390 mg/L PO43
Silica
100 mg/L SiO2
Sulfate
5000 mg/L SO42
Sulfide
20 mg/L S2
Sulfite
50 mg/L SO32
Zinc
5 mg/L Zn

Page 852
Accuracy check
Organic-free water
ADDITIONS.
4. Prepare a 150-g/L C standard:
a. Transfer 15.00 mL of 1000-mg/L TOC standard solution to a 100-mL Class A volumetric
flask.
b. Dilute to volume with organic-free water. Mix.
prepared standard to three Acid Digestion Vials.
instrument.

Page 853
TOC Standard Ampule 1000 mg/L C
1. Prepare a 10.0 mg/L C standard solution as follows:

(1 liter) volumetric flask.
Reagent blanks
Water used for the reagent blank must contain less than 0.05 mg/L carbon. If the organic-free
water container is left open for extended periods, the water may absorb carbon dioxide (CO2) from
the atmosphere. To remove the dissolved CO2 from the organic-free water, it is necessary to acidsparge it (see steps 24 of the procedure).
Generally, water stored in plastic containers is not suitable for low-range TOC blanks. Water stored
in plastic may leach organic compounds from the container walls. The leached organic
compounds usually cannot be removed by acid sparging.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
427
10.0 mg/L C
9.110.9 mg/L C
0.2 mg/L C
Summary of method
conditions to remove the inorganic carbon. In the outside vial, organic carbon in the sample is

Page 854

Required reagents
Description
Reagent Set, Total Organic Carbon Direct Method
Low Range Test N Tube, includes:
Quantity/Test
Unit
Catalog number
50 vials
2760345
50/pkg
0.4 mL
25 mL
45233
2584335
Funnel, micro
each
Indicator Ampule, Low Range TOC1
10/pkg
50/pkg
pH Paper
Water, Organic-free
1
5/pkg
39133
3.0 mL
500 mL
2641549
Catalog number
Required apparatus
Description
Quantity
Unit
each
50838
each
LTV082.53.40001
each
LTV082.52.40001
each
50541
Magnetic Stirrer
each
2881200
each
1970001
2185696
OR
50/pkg
each
1970010
50/pkg
2199796
Stir Bar, magnetic
each
4531500
Test Tube Rack
each
1864100
Wipes, Disposable
280/pkg
2097000
Description
Unit
Catalog number
TOC Standard Solution Ampule (KHP Standard, 1000-mg/L C)
5/pkg
2791505

Description
Unit
Catalog number
115 VAC
2936701
Buffer Solution, Sulfate pH 2.0
500 mL
45249
each
1457453

Page 855

Description
Unit
Catalog number
each
1457442
Pipet filler
each
1465100
1000/pkg
2185628
250/pkg
2199725
each
1451539
each
1451538
500 g
31534
100 mL
244932
500/pkg
1473800

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Organic Carbon, Total MR, 10173
DOC316.53.01094
Direct Method1
Method 10173
MR (15 to 150 mg/L C)

Scope and Application: For water, drinking water and wastewater
1
U.S. Patent 6,368,870
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

To test for higher ranges of TOC use Method 10128. To test lower ranges of TOC use method 10129.

Description
Quantity
Total Organic Carbon Direct Method Mid Range Test N Tube Reagent Set
DRB200 Reactor
pH Paper
Magnetic Stirrer

Page 857

Description
Quantity
Stir Bar, magnetic
Test Tube Rack
Water, organic-free
3.0 mL
Wipes, disposable
Direct method

program.
2. Use a graduated
sample to a 50-mL

sample pH is 2.

moderate speed for
10 minutes.
5. Label two Mid Range


(colorless liquid).

to add 1.0 mL of
1.0 mL of prepared
Swirl to mix.
8. Rinse two blue MR/HR

Indicator Ampules with
deionized water and wipe
them with a soft, lint-free
wipe.

Page 858
Do not touch the ampule

sides after wiping. Pick
them up by the top.

Stored Programs
425 Organic Carbon MR
Start

Acid Digestion vial, snap
10. Cap the vial

103105 C.

test tube rack.
results.

Insert a light shield or an
adapter if required (see
Instrument-specific
information).

blue.

ampule.
Zero

other marks.
14.

The display will show
0 mg/L C.

or other marks.
Read

round cell.

mg/L C.

Page 859
Interferences
The Interfering substances table lists substance that have been tested for interference and found
If the sample contains greater than 1000 mg/L CaCO3 alkalinity, lower the sample pH to less than
7 before testing by adding Sulfuric Acid Solution.

Interference level
Aluminum
10 mg/L Al
Ammonia Nitrogen
1000 mg/L as N
ASTM Wastewater
No effect
Bromide
500 mg/L Br
Bromine
25 mg/L Br2
Calcium
2000 mg/L as CaCO3
Chloride
1500 mg/L Cl
Chlorine
10 mg/L Cl2
Chlorine Dioxide
6 mg/L ClO2
Copper
10 mg/L Cu
Cyanide
10 mg/L CN
Iodide
50 mg/L I
Iron (II)
10 mg/L Fe2+
Iron (III)
10 mg/L Fe3+
Magnesium
2000 mg/L as CaCO3
Manganese (VII)
1 mg/L Mn
Monochloramine
Nitrite
500 mg/L NO2
Ozone
2 mg/L O3
Phosphate
3390 mg/L PO43
Silica
100 mg/L SiO2
Sulfate
5000 mg/L SO42
Sulfide
20 mg/L S2
Sulfite
50 mg/L SO32
Zinc
5 mg/L Zn

Page 860
Accuracy check
1000-mg/L C TOC Standard Ampule
Organic-free water
ADDITIONS.
4. Prepare a 300-mg/L C standard:
a. Transfer 15.00 mL of 1000-mg/L TOC standard solution to a 50-mL Class A volumetric
flask.
b. Dilute to volume with organic-free water. Insert the stopper and mix thoroughly.
5. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of the 300mg/L standard to three Acid Digestion Vials.
instrument.

Page 861

TOC standard Ampule, 1000 mg/L C

volumetric flask.
b. Dilute to volume with Organic-free Reagent water. Insert the stopper and mix thoroughly.
Method performance
Program
Instrument
Standard
per 0.010 Abs
425
DR 5000
70 mg/L C
6872 mg/L C
1.2 mg/L C
Summary of method
conditions to remove the inorganic carbon. In the outside vial, organic carbon in the sample is

Required reagents
Description
Reagent Set, Total Organic Carbon Direct Method
Mid Range Test N Tube, includes:
Quantity/Test
Unit
Catalog number
50 vials
2815945
50/pkg
0.4 mL
25 mL
Funnel, micro
each
2584335
Indicator Ampules, MR/HR TOC1
10/pkg
50/pkg

Page 862

Required reagents
Description
pH Paper
Water, Organic-free
1
Quantity/Test
Unit
Catalog number
5/pkg
39133
1.0 mL
500 mL
2641549
Quantity
Unit
Catalog number
Required apparatus
Description
each
50838
each
LTV082.53.40001
each
LTV082.52.40001
each
50541
Magnetic Stirrer
each
2881200
each
1970001
50/pkg
2185696
each
1970010
50/pkg
2199796
Stir Bar, magnetic
each
4531500
Test Tube Rack
each
1864100
Wipes, Disposable
280/pkg
2097000
Description
Unit
Catalog number
TOC Standard Solution Ampule, 20 mL (KHP Standard, 1000-mg/L C)
5/pkg
2791505
Unit
Catalog number
OR

Description
100 mL-MDB
244932
1000/pkg
2185628
250/pkg
2199725
115 VAC
2936701
500/pkg
1473800
each
1457441
each
1457453

Buffer solution, sulfate, pH 2.0
500 g
31534
500 mL
45249
each
1451539
each
1451537
Pipet Filler
each
1465100

Page 863

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Organic constituents, UV absorbing, 10054
UV Absorbing (UV-254)
DOC316.53.01092
Direct Reading Method1
Method 10054
Scope and Application: To indicate the total concentration of UV-absorbing organic compounds in drinking water
and drinking water source waters.
1
Adapted from Standard Methods for the Examination of Water and Wastewater, Method 5910.
Test preparation


Instrument
Sample cell
Cell orientation
Adapter
DR 6000
2624410
Align clear windows facing right/left
DR 5000
2624410
Align clear windows facing the user
Multi-cell Adapter, 10-mm faces user

The sample pH should be between 4 and 10. If not, see Interferences in this procedure. Samples used for SUVA calculations
must not be pH adjusted
Any non-plastic filter assembly can be used for this test. Use a 0.45-m or glass fiber filter of nominal pore size (11.5 m)
without organic binder. A 0.45-m filter must be utilized if the results are to be used for SUVA calculations.
Handle the cell on the frosted sides only.
Use only Organic-Free Reagent Water to zero the instrument.
The Chromic Acid Cleaning Solution is regulated as a hazardous waste for chromium (D007) and corrosivity (D002) when
disposed per Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.

Description
Quantity
varies
Filter Assembly
Stand, buret
Organic Constituents UV Absorbing (UV-254)

Page 865

Stored Programs
410 Organics, UV - 254
Start
1. Select the test.


apparatus. Be sure to use
the white PTFE support
plate. Insert the filter with
the wrinkled surface
upward.
3. Mount the apparatus

into a support stand and
place a clean glass beaker
underneath.
4. Prewash the filter

assembly by pouring at
least 50 mL of OrganicFree Reagent Water
through the filter. Discard
the filtered water.
Pre-rinsing removes any
soluble impurities from the
filter.
Zero
5. Prepared Sample:
Pour 50 mL of sample
through the filter and
collect the filtered sample.
Rinse a clean 1-cm quartz
cell several times with
Organic-Free Reagent
Water. Fill the cell with
Organic-Free Reagent
Water. Wipe the cell walls
thoroughly.

Page 866
7. Align the clear

windows with the light
path. Insert the blank into
the cell holder.

0.000 cm1
1-cm cell
Lamp Warm Up will be
indicated if the UV lamp
has not been previously
on. This may take 23
minutes.

Organic Constituents (continued)
Read
9. Discard the contents

of the blank cell and rinse
the cell several times with
filtered sample.
10. After rinsing, fill the

cell with filtered sample.
Wipe the cell walls to
remove fingerprints.
11. Align the clear

windows with the light
path. Insert the cell
containing the prepared

absorbance per
centimeter (cm1).
For optimum results, the cm1 value should fall between 0.005 and 0.900. If the value is less than
0.005 absorbance using a 1-cm cell, use a 5-cm or 10-cm quartz cell.
To test with the 5-cm or 10-cm cell:
1. Press OPTIONS>MORE>CHEMICAL FORMS.
2. Press 5 CM or 10 CM. Press OK>RETURN.
3. The displayed results (in absorbance per centimeter) will be corrected for the 5-cm or 10-cm
cell pathlength selected. If cm1 results are greater than 0.900, accurately dilute the sample
with Organic-Free Reagent Water. Correct the test result by the appropriate dilution factor.
Interferences
Interference level
Sample pH outside 410
Add either 1 N Sodium Hydroxide or 1 N Sulfuric Acid to the sample to adjust sample
between pH 4-10.
UV-absorbing inorganics
(bromide, ferrous iron, nitrate,
nitrites)
Follow the UV scanning procedure below.
UV-absorbing Oxidants and

reductants (chloramines,
chlorates, chlorites, ozone,
thiosulfates)
Follow the UV scanning procedure below.
To determine the presence of interferences, a scan of the filtered sample versus Organic-Free
Reagent Water is recommended on a regular basis:
1. From the main menu, press WAVELENGTH SCAN>OPTIONS>.
2. Press 200>OK.
3. Press 400>OK.
4. Press 1 NM>OK.

Page 867

5. Insert the cell containing the Organic-Free Reagent Water (the blank) into the cell
compartment.
6. Press ZERO. The baseline scan from 200 to 400 nm will begin.
Note: Lamp Warm Up will be indicated if the UV lamp has not been previously on. This may take
23 minutes.
7. After the baseline scan is recorded, insert the cell containing the filtered sample into the cell
compartment. Press READ to scan the sample.
If the sample scan shows relatively sharp peaks, interferences may be present. Generally, natural
organic matter will show a relatively featureless curve in the UV region with increasing absorption
as the wavelength decreases. If the sharp peaks are indicated, an alternate wavelength should be
selected and reported.
Collect samples in cleaned glass containers. Do not use plastic containers.
Cell cleaning
New or dirty cells should be soaked with Chromic Acid Cleaning Solution to remove trace organic
contamination.
1. Allow to soak overnight or up to 12 hours.
2. After soaking, rinse with at least 10 volumes of Organic-Free Reagent Water.
Treatment of cells with chromic acid is required only occasionally if cells are rinsed with OrganicFree Reagent Water after use.
Method performance
Standard: There is no primary standard or calibration for the UV-254 method. Using a Potassium
Acid Phthalate solution equivalent to 30-mg/L as carbon, the following reproducibility data was
obtained using one instrument. Refer to Standard Methods for the standard preparation.
Program
Precision95% Confidence Limits of Distribution
410
0.431 - 0.433 cm-1
Summary of method
Filtered sample is measured at 254 nm against organic-free water as a indicator of organic
constituents in the sample water. Results are automatically reported in absorbance per centimeter
(cm1). The results can be used in calculating Specific Ultraviolet Absorbance (SUVA).
Estimated detection limit
Because this test is a non-specific measurement for organic constituents, there is no estimated
detection limit for program 410.

Page 868

Required reagents
Description
Quantity/Test
Unit
Catalog number
varies
500 mL
2641549
Organic-Free Reagent Water
Required apparatus
Description
Unit
Catalog number
Beaker, 100-mL
each
50042H
Buret Stand
each
32900
Clamp Holder
each
32600
Clamp, 3-Prong
each
42200
Filter Funnel Assembly, 7-cm
each
2164100
each
2164200
Filter Plate, PTFE, for 21641-00

Filter, glass fiber, 70-mm
100/pkg
253053
each
2624410
100 mL MDB
127032
Sample Cell, quartz, 1-cm (10 mm)

Sulfuric Acid, 1 N
Optional reagents
Description
Chromic Acid Cleaning Solution
1
Unit
Catalog number
500 mL
123349
100 mL
104532
MDB1
Larger sizes are available.
Optional apparatus
Description
Unit
Catalog number
Cell Holder for 10-cm (100 mm) sample cells (DR 5000 only)
each
LZY421
each
50841
Filter, membrane, 47-mm; 0.45-microns, hydrophilic, polyethersulfone SUVA
each
2894700
Filter Holder, glass for vacuum filtration (SUVA)
each
234000
Flask, filtering, glass, 1000-mL (SUVA)
each
54653
5 rolls/pkg
39133
Tubing, Latex rubber (SUVA)
12 ft
56019
500 g
31534
pair
4822800
Sample Cell, quartz, 1-cm (10 mm), matched pair

each
2624450
each
2624401
2270800
Aspirator (SUVA)
213100

Page 869

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen Demand, Biochemical, 8043
Oxygen Demand, Biochemical

Dilution Method1
DOC316.53.01200
Method 8043

1
Adapted from Standard Methods for the Examination of Water and Wastewater and from Klein, R.L.; Gibbs, C. Journal of Water Pollution
Control Federation, 1979, 51(9), 2257.
Test preparation

The BOD test is a 5-day test. Follow all steps carefully to make sure that the test does not have to be repeated.
The dilution water for this test must not have an oxygen demand or any toxins. When incubated for 5 days at 20 C, the
dissolved oxygen concentration in the dilution water must not change by more than 0.2 mg/L.
Carbonaceous BOD (CBOD) can be determined by the addition of nitrification inhibitor. A test for CBOD is recommended for
biologically treated effluents, samples seeded with biologically treated effluents and river water.
The TroubleshootingGraphical calculation method provides an alternate system for calculating results and is a convenient
tool for troubleshooting problems in BOD measurements. The graphical calculation method is not approved for regulatory
reporting.

Description
BOD bottles, 300-mL, glass, with glass stoppers and plastic caps
Dilution water containing nutrient buffer and seed (see Dilution water preparation)
Nitrification inhibitor (for CBOD only)
Quantity
6
varies
1 bottle
Pipet, serological
Incubator

Page 871

Dilution method
1. Prepare the dilution

water using a BOD
Nutrient Buffer Pillow. See
Dilution water preparation.

volumes. See Sample size
selection.
Note: If the minimum sample
volume is 3 mL or more,
determine the dissolved
oxygen in the undiluted
sample; this determination
can be omitted when
analysing sewage and settled
effluents known to have a
dissolved oxygen content
near 0 mg/L.
3. Stir the sample gently

with the pipet. Use the
pipet to add the minimum
sample volume to the first
BOD bottle.
4. Fill an additional BOD

bottle with dilution water
only. This will be the
dilution water blank.
Add the remaining four

sample volumes to four
more BOD bottles. Mark
the bottles and record the
contents of each bottle.
When analyzing
disinfected samples or
industrial effluents, refer to
Interferences.
5. If the test is for CBOD,

add two portions of
Nitrification Inhibitor
(approximately 0.16 g) to
each bottle.
The oxidation of nitrogen
compounds will be
prevented. Report results
as CBOD.
6. Fill each bottle to just

below the lip with dilution
water. Allow the dilution
water to flow down the
sides of the bottle to
prevent air bubbles from
becoming trapped in the
bottle.

Page 872
7. Stopper the bottles

carefully to prevent air
bubbles from becoming
trapped. Tightly twist the
stopper into place. Press
down on the stopper and
invert the bottles several
times to mix.
8. Measure the initial

dissolved oxygen
concentration in each
bottle. Use a probe and
meter or titration. If a
titration is used, two sets
of BOD bottles must be
prepared.
Be sure to measure the
DO of the dilution water
blank.

Dilution method

trapped. Add dilution water
to the lip of each BOD
bottle to make a water
seal.
10. Place a plastic cap

over the lip of each bottle.
Put the bottles in an
incubator at 20 (1) C.
Incubate for five days.
12. Calculate the BOD

value (see Calculation
MethodsStandard
Methods).
11. After five days,

measure the remaining
dissolved oxygen
bottle.
At least 1.0 mg/L DO
should be left in each BOD
bottle.
Dilution water preparation

The dilution water must be prepared very carefully to make sure that no source of oxygen demand
or toxins are added. The water that is used to prepare the dilution water must be of very high
quality. The water must not have any organic compounds or any toxic compounds such as
chlorine, copper and mercury.
Use the following guidelines to make sure the dilution water is of high quality.
Guidelines
Use distilled water from an alkaline permanganate distillation for the best results.
Do not use deionized water from ion exchange columns. The resins in the cartridges
(especially new cartridges) will occasionally release organic materials that have an oxygen
demand. In addition, bacteria can grow on the columns and contaminate the dilution water.
Store the distilled water in clean jugs in an incubator at 20 C. Fill containers till about full
and shake the jugs to saturate the water with air, or cap the jugs loosely and store for 24 hours
or more, to allow dissolution of oxygen.
A small aquarium pump or air compressor can be used to saturate the water with air. Make
sure that the air is filtered and that the filter does not grow bacteria. Clean the apparatus
before and after use.
Add the nutrients and seed (if necessary) to the distilled water immediately before the test.
The dissolved oxygen concentration in the dilution water must not change by more than
0.2 mg/L when incubated for 5 days at 20 C.
Procedure
1. Prepare and store the distilled water at 20 C (see Guidelines).
2. Select a BOD nutrient buffer pillow from the BOD nutrient buffer pillows table.
3. Tap the pillow on a hard surface then shake the pillow to mix the contents.

Page 873

4. Add the contents of the pillow to the distilled water in a jug with ample headspace above the
water. Cap the jug and shake vigorously for one minute to dissolve the nutrients and to
saturate the water with air.
5. If the sample is known to be low in bacteria, for example industrial waste or sewage that has
been disinfected, add 3 mL of bacterial seed to each liter of the dilution water. Use raw
sewage for the bacterial seed. Allow the sewage to stand undisturbed at 20 C for 24 to
36 hours before use. Pipet from the upper portion of the sewage. Make sure to measure the
BOD of the seed so that it can be subtracted from the BOD of the sample. A seed that has a
BOD of 200 mg/L (a typical range for domestic sewage) will typically deplete at least 0.6 mg/L
DO, when added at a rate of 3 mL/L of dilution water. If insufficient oxygen depletion occurs,
increase the quantity of the seed.
Table 268 BOD nutrient buffer pillows

Volume of dilution water to prepare
BOD nutrient buffer pillow catalog no.
300 mL (add pillow to each BOD bottle)
1416066
3 liters
1486166
4 liters
2436466
6 liters
1486266
19 liters
1486398
Note: To prepare dilution water by the conventional method, pipet 1 mL of each of the following solutions per
liter of distilled water at 20 C: Calcium Chloride Solution, Ferric Chloride Solution, Magnesium Sulfate
Solution, and Phosphate Buffer Solution. Cap the bottle and shake vigorously for one minute. The
Phosphate Buffer Solution should be refrigerated to decrease the rate of biological growth. Use care with
all solutions to avoid contamination.
Sample size selection

Make an estimation of the sample volumes that are necessary for the test. At least 2.0 mg/L of
dissolved oxygen (DO) should be consumed during the test and at least 1.0 mg/L DO should be
left in the BOD bottle.
Samples such as raw sewage will have a high BOD. Small sample volumes must be used
because large samples will consume all of the oxygen. Samples with a low BOD must use larger
sample volumes to make sure that enough oxygen is consumed to give accurate results.
The elevation of the laboratory changes the amount of oxygen that can dissolve in water (see
Oxygen saturation values at various altitudes (20 C)). At higher elevations, the amount of oxygen
that can dissolve in water decreases, so less oxygen is available to microorganisms.
Procedure
1. Refer to the Minimum sample volume table to select the minimum sample volume. For
example, if a sewage sample is estimated to contain 300 mg/L BOD, the minimum sample
volume is 2 mL. For sewage effluent with an estimated BOD of 40 mg/L, the minimum sample
volume is 15 mL.
2. Refer to the Maximum sample volume table to select the maximum sample volume. At 1000
feet, with an estimated BOD of 300 mg/L, the largest sample volume is 8 mL. For a BOD of 40
mg/L the maximum volume is 60 mL (also at 1000 feet).
3. Select two or more other sample volumes between the minimum and maximum volumes so
that there are four or five sample volumes total.

Page 874
Table 269 Minimum sample volume

Sample type
Estimated BOD (mg/L)
Minimum sample volume (mL)
Strong trade waste
600
Raw and settled sewage
300
200
150
120
100
Oxidized effluents
Polluted river waters
75
60
10
50
12
40
15
30
20
20
30
10
60
100
200
300
Table 270 Maximum sample volume1
BOD at sea level
BOD at 1000 ft
BOD at 5000 ft
Maximum sample volume (mL)
615
595
508
492
476
406
410
397
339
304
294
251
246
238
203
10
205
198
169
12
164
158
135
15
123
119
101
20
82
79
68
30
41
40
34
60
25
24
21
100
12
12
10
200
300
Samples with higher concentrations should be pre-diluted, per Standard Methods.

Page 875
Table 271 Oxygen saturation values at various altitudes (20 C)

Altitude (ft)
Average Pressure in mBar at this

altitude
Oxygen value (mg/L) in water

saturated with air
Sea level (0)
1013
9.09
1000
977
8.76
2000
942
8.44
3000
908
8.13
4000
875
7.82
5000
843
7.53
6000
812
7.24
Calculation MethodsStandard Methods

Use the Standard Methods calculation when the results must be reported to a regulatory agency.
When dilution water is not seeded:
D1 D2
BOD 5, mg/L = ------------------P
When dilution water is seeded:

( D 1 D 2 ) ( B 1 B 2 )f
BOD 5, mg/L = --------------------------------------------------------P
where:
BOD5 = BOD value from the 5-day test
D1 = DO of diluted sample immediately after preparation, in mg/L
D2 = DO of diluted sample after 5 day incubation at 20 C, in mg/L
P = Decimal volumetric fraction of sample used
B1 = DO of seed control before incubation, in mg/L
B2 = DO of seed control after incubation, in mg/L
f = ratio of seed in diluted sample to seed in seed control =
(% seed in diluted sample)/(% seed in seed control) OR
If seed material is added directly to sample or to seed control bottles:
f = (volume of seed in diluted sample)/(volume of seed in seed control)
Report results as CBOD5 if nitrification inhibitor was added.
Averaged results are acceptable if more than one sample dilution meets all of the following criteria:
The remaining DO is at least 1 mg/L
The final DO value is at least 2 mg/L lower than the initial DO value
There is no evidence of toxicity at higher sample concentrations
There are no obvious anomalies

Page 876
Interferences
Many chlorinated and industrial effluents require special handling to ensure reliable BOD results.
Usually, careful experimentation with the particular sample will indicate what modifications should
be made to the test procedure.
Toxins in the sample will adversely affect any microorganisms present and result in lower BODs.
To eliminate small amounts of residual chlorine, allow the sample to stand for one to two hours
at room temperature. For larger quantities, determine the amount of sodium thiosulfate to add to
the sample as follows:
c. Measure 100 mL of sample into a 250-mL Erlenmeyer flask. Using a 10-mL serological
pipet and a pipet filler, add 10 mL of 0.020 N Sulfuric Acid Standard Solution and 10 mL of
Potassium Iodide Solution, 100-g/L, to the flask.
d. Add three full droppers of Starch Indicator Solution and swirl to mix.
e. Fill a 25-mL buret with 0.025 N Sodium Thiosulfate Standard Solution and titrate the
sample from dark blue to colorless.
f.
Calculate the amount of 0.025 N Sodium Thiosulfate Standard Solution to add to the
sample:
mL titrant used x volume of remaining sample
mL 0.025 N sodium thiosulfate required = ------------------------------------------------------------------------------------------------------------------------100
g. Add the required amount of 0.025 N Sodium Thiosulfate Standard Solution to the sample.
Mix thoroughly. Wait 10 to 20 minutes before running the BOD test.
To eliminate the effect of phenols, heavy metals or cyanide, dilute the sample with high quality
distilled water. Alternately, the seed used in the dilution water may be acclimatized to tolerate such
materials. Acclimatize seed as follows:
a. Fill a one-gallon stainless steel or plastic container with domestic sewage and aerate for
24 hours. Allow the heavier material to settle.
b. After settling for one hour, siphon off three quarts of material and discard.
c. Fill the container with a mixture of 90% sewage and 10% wastes containing the toxic
material.
d. Aerate for 24 hours. Repeat steps b and c with increasing amounts of waste until the
container holds 100% toxic waste material.
Optimum pH for the BOD test is between 6.5 and 7.5. Adjust samples to pH 7.2 with Phosphate
Buffer Solution or 1 N Sulfuric Acid or Sodium Hydroxide Standard Solution if the pH is not in
this range.
Cold samples may be supersaturated with oxygen and will have low BOD results. Fill a one-quart
bottle about halfway with cold sample and shake vigorously for two minutes. Allow sample to reach
20 C. Then shake the bottle vigorously for two minutes.

Page 877
Accuracy check
ezGGA Method
BOD Standard Solution, Voluette Ampule, 300-mg/L, 10-mL (300-mg/L of glucose and 300mg/L of glutamic acid)
Seeded dilution water
4 BOD bottles
1.04.0 mL Class A volumetric pipets and pipet filler or 110 mL TenSette Pipet and Pipet tips
Dissolved oxygen measurement apparatus
DO measurement with the LBOD probe:

1. Add the necessary seed to a 300-mL BOD bottle.
2. Fill the BOD bottle with dilution water until the water level is approximately inch up the
ground glass portion of the neck. (See dimension x in illustration).
3. Put the 2-mL BOD standard ampule into the ampule breaker and rinse the assembly with
deionized water.
4. Hold the ampule and breaker over the rim of the BOD bottle.
5. Use the ampule breaker to open the ampule and allow it to fall into the BOD bottle. Leave
ampule in the BOD bottle during incubation period.
6. Follow the general procedure for the BOD test.
7. Calculate the BOD concentration of the standard solution. The 2 mL in the vial is equivalent to
6 mL as prepared by Standard Methods. Calculate the BOD concentration as though there
were 6 mL added to the bottle instead of 2 mL. The dilution factor for this standard is 50x.
DO measurement with the Clark Cell electrode:
1. Add the necessary seed to a 300-mL BOD bottle.
2. Use the ampule breaker to open the ampule.
3. Pour the contents of the ampule into the BOD bottle. Tap the ampule on the rim of the bottle to
dislodge the contents. Do not drop ampule into the bottle when using a Clark Cell.
4. Fill the ampule with buffered dilution water and add the water to the BOD bottle.
5. Repeat step 4.
6. Fill the BOD bottle with dilution water until the water level is approximately inch up the
ground glass portion of the neck.
7. Follow the general procedure for the BOD test.
8. Calculate the BOD concentration of the standard solution. The 2 mL in the vial is equivalent to
6 mL as prepared by Standard Methods. Calculate the BOD concentration as though there
were 6 mL added to the bottle instead of 2 mL. The dilution factor for this standard is 50x.
Note: The ampules include precisely 2 mL of 450 mg/L GGA. Pouring the entire solution into the bottle is
the same as adding 6 mL of 150 mg/L solution as per the Standard Methods.

Page 878
TroubleshootingGraphical calculation method

The Graphical Method helps troubleshoot problems in BOD measurements. This method cannot
be used when the results must be reported to a regulatory agency.
1. Plot the mg/L dissolved oxygen (DO) remaining in each diluted sample versus the mL sample
taken. Draw the best straight line through the plotted points. See Dissolved Oxygen per mL of
Sample.
Note: An erroneous point is visually evident at this time and can be disregarded. However, at least three
points should be on the line or very close to it. For unseeded dilution water, the line should cross the
mg/L DO Remaining scale near or below the oxygen saturation value for the altitude of the laboratory as
discussed in Dilution water preparation.
2. To calculate the BOD, use the following equation which is mathematically equivalent to the
BOD equation in Standard Methods.
mg/L BOD = (A x 300) B + C
where:
A = the slope
The slope of the line is equal to the mg/L DO consumed per mL of sample taken. Take any
point on the line and subtract the mg/L DO Remaining at that point from the mg/L DO where
the line crosses the DO scale (Y intercept, mg/L DO Remaining). Divide the difference by the
mL of sample at the point chosen.
300 = the volume of the BOD bottle
B = the Y intercept
This is the DO value where the line crosses the DO Remaining scale. (This should be very
close to the actual dilution water blank value.)
C = the sample DO
This is the DO of the undiluted sample.
Another way to write this equation is:
mg/L BOD = (Slope x 300) Y intercept + Sample DO
Note: If the best straight line is obtained by linear regression through use of a calculator, the sign (-) of
the slope must be changed (+) before multiplying by 300.
Example:
The mg/L DO remaining was determined for a series of four dilutions of domestic sewage after five
days of incubation. Results were as follows:
mL of sample taken
mg/L DO remaining
2.0
7.50
3.0
6.75
6.0
4.50
9.0
2.25
The DO values were plotted versus the mL of sample taken and a straight line drawn as in
Dissolved Oxygen per mL of Sample. If a set of BOD dilutions is run correctly with a homogeneous
sample, a graph of the mg/L DO remaining versus the sample volume would result in a straight
line. The value where the line intersects the y-axis is equal to the DO content of the dilution water
Page 879

after incubation, although this is not actually measured. In this case, it was equal to 9.0 mg/L and
the DO of the domestic sewage sample was assumed to be zero. If another type of sample is
used, the DO of an undiluted sample should be measured either by the Winkler titration or with a
luminescent or electrochemical probe.
The Calculation MethodsStandard Methods formula for calculating BOD also can be written as
follows (not approved for reporting purposes):
mg/L DO remaining w/smaller sample volume mg/L DO remaining w/larger sample volume
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 300 DO D + S = mg/L BOD
mL of larger sample volume mL of smaller sample volume
Using this information in the example:

mg/L DO remaining with smaller sample volume = 7.50
mg/L DO remaining with larger sample volume = 2.25
mL of larger sample volume = 9.0
mL of smaller sample volume = 2.0
300 = volume (mL) of BOD bottle
DOD = mg/L DO of dilution water = 9.0
S = mg/L DO of sample = assumed in this case to be zero
Therefore:
7.50
2.25--------------------------- 300 9 + 0 = mg/L BOD = 216 mg/L BOD
9.0 2.0
Using the equation below:

(slope x 300) Y-Intercept + sample DO = mg/L BOD
To determine slope, arbitrarily select point A in Figure 1. At this point the mg/L DO remaining is
equal to 3.0 mg/L. The mL of sample at this point is 8 mL.The difference between the yintercept of 9.0 mg/L and 3.0 mg/L equals 6 mg/L; 6 mg/L divided by 8 mL = 0.75 mg/L per mL.
slope = 0.75 mg/L per mL
Y intercept = 9.0 mg/L
sample DO = 0 (Because the sample is domestic sewage, this is assumed to be zero.)
Therefore:
(0.75 x 300) 9.0 + 0 = mg/L BOD = 216 mg/L BOD

Page 880
mg/L DO Remaining
y Intercept
mL of Sample
Figure 16 Dissolved Oxygen per mL of Sample

Page 881
Summary of method
Biochemical Oxygen Demand (BOD) is a measurement of the oxygen requirements of municipal
and industrial wastewaters and sewage. The test results are used to calculate the effect of waste
discharges on the oxygen resources of the receiving waters. The BOD test is of limited value in
measuring the actual oxygen demand because temperature change, biological population, water
movement, sunlight, oxygen concentration, and other environmental factors cannot be reproduced
accurately in the laboratory. The BOD test is of greatest value after patterns of oxygen uptake for a
specific effluent and receiving water have been established.
The BOD test is performed by incubating a sealed wastewater sample (or a prepared dilution) for
the standard five-day period and then determining the change in dissolved oxygen content. The
BOD value is then calculated from the results of the dissolved oxygen tests.

Required reagents
Description
BOD Nutrient Buffer Pillows, for 3 liters of dilution water
Quantity/Test
Unit
Catalog number
1 pillow
50/pkg
1486166
Catalog number
Required apparatus
Description
Quantity/Test
Unit
BOD Bottle, glass-stoppered, 300-mL, unlabelled
6/pkg
62106
BOD Bottle Cap
6/pkg
241906
each
62011
Clippers, large
each
96800
each
919002
each
53237
each
53238
Pipet Filler
each
1218900
Dissolved Oxygen measurement apparatus
Pipet, serological:
Description
BOD Standard Solution,
Voluette
Ampule, 300-mg/L, 10-mL
ezGGA BOD Standard Ampules, 450 mg/L, 2 mL

Page 882
Unit
Catalog number
16/pkg
1486510
20/pkg

Description
Unit
Catalog number
BOD Nutrient Buffer Pillows

for 300 mL of dilution water
50/pkg
1486166
for 4 liters of dilution water
50/pkg
2436466
50/pkg
1486266
25/pkg
1486398
Buffer Solution, APHA, for BOD, pH 7.2, phosphate type
500 mL
43149
Calcium Chloride Solution, APHA, for BOD
500 mL
42849
Ferric Chloride Solution, APHA, for BOD
1L
42953
500 L
43049
35 g
253335
Dispenser Cap, for Nitrification Inhibitor
each
45901
500 mL
1228949
Magnesium Sulfate Solution, APHA, for BOD

Sodium Hydroxide, pellets, ACS
Potassium Permanganate
Bottle, BOD, Serialized: 1-241
Bottle, BOD, Disposable
Stopper for Disposable BOD Bottle
Bottle Rack, BOD, 12 bottle
18734
104532
1L
35253
100 mL MDB
34932
1L
20353
1L
127053
454g
16801H
24/pkg
1486610
117/case
2943100
25/pkg
2943900
each
2094200
Brush, cylinder, 2-in. diameter
each
68700
Incubator, BOD, Compact Model 205, 110 Vac
each
2616200
Incubator, BOD, Compact Model 205, 220/240 Vac
each
2616202
Leash, rubber, for stopper & bottle
6/pkg
2091606
ATU (1-1-allyl-2-thiourea)
50 g
2845425
500 g
253334
50 capsules
2918700
BOD Seed Inoculum, Polyseed

1
500 g
100 mL MDB
Other numerical series are available

Page 883

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen Demand, Chemical with chloride removal 10067
Oxygen Demand, Chemical
DOC316.53.01101
Manganese III Reactor Digestion Method

(with optional chloride removal)1
Method 10067
30 to 1000 mg/L COD Mn

1
U.S. Patent 5,556,787
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

To determine if the sample contains chloride, use Quantab Titrator Strips for low range chloride.
If the sample COD is expected to exceed 1000 mg/L, dilute the sample as described in the Multiplication factors table.
Homogenize the sample for even distribution of solids and better accuracy and reliability.
Run one blank with each lot of reagent. Run all samples and blanks with the same lot of vials. The lot number appears on the
container label.
The stability of the reagent blank allows for reuse. Verify the reagent blank quality by measuring the absorbance of the blank
vs. a clean COD vial filled with deionized water. The absorbance range should be about 1.411.47.
If the sample boils during the digestion, the vial is not properly sealed. Test results will be invalid.
Spilled reagent will affect test accuracy and is hazardous. Do not run tests with spilled vials.
The maximum range of the VPD gauge is 40 inches of water; it will not indicate the full vacuum level obtained. Full vacuum is
2025 inches of mercury; this can be measured at the vacuum pump with a gauge calibrated for inches of mercury.

Page 885

Description
Quantity
Blender
DRB200 Reactor
Forceps, extra fine
Manganese III COD Reagent Vials, 201000 mg/L COD

Pipet,
TenSette
1
1 each
(0.1 to 1.0 mL and 1.0 to 10.0 mL)
2 of each
1 mL
Test Tube Rack
Vacuum pretreatment device
Vacuum Pump
Vial, glass, for sample and acid
Water, deionized
varies
Acidified sample preparation

Reactor and heat to
150 C or set to COD
program.
14. Homogenize 100 mL

of sample for 30 seconds
in a blender.
If suspended solids are
present, continue to mix
the sample while pipetting.

Page 886

water into an empty glass
mixing cell.

Pipet 9.0 mL of
homogenized sample into
another empty glass
mixing cell

Acidified sample preparation (continued)
17. Using an automatic

dispenser or TenSette
Pipet, add 1.0 mL of
concentrated sulfuric acid
to both the sample and the
blank.
18. Cap the cells tightly

and invert several times.
Proceed to Vacuum
pretreatment.
The solution will become

hot. Cool to room
temperature before
proceeding.
Acidified samples are
stable for several months
when refrigerated at 4 C.
Calculate the multiplication factor

All dilutions require that the ratio of sample to sulfuric acid remain at 9:1. For other dilutions that
are not listed in the Multiplication factors table, add the sample volume and the deionized water
and divide by the sample volume to obtain the multiplication factor.
Note: Mixing concentrated sulfuric acid and water is not additive. Adding 1.0 mL of concentrated sulfuric acid
to 9.0 mL of sample does not result in a final volume of 10.0 mL. This factor is built into the
calibration curve.
Table 273 Multiplication factors

Sample (mL)
Deionized Water (mL)
Range (mg/L COD)
Multiplication Factor
6.0
3.0
301500
1.5
3.0
6.0
603000
1.0
8.0
1809000
0.5
8.5
36018,000
18
For best results, use 0.5 mL or more of sample for diluting. If sample values exceed 18,000 mg/L
COD, use a separate sample dilution before performing the sample chloride removal procedure.
Example: Dilute the sample to a range of 904500 mg/L COD.
Sample Volume (2.0 mL) + Deionized water (7.0 mL) = Total Volume (9.0 mL)
Total Volume
9.0 mL
Multiplication Factor = ------------------------------------------ = ------------------ = 4.5
Sample Volume
2.0 mL
Standard test range is 50 to 1000 mg/L COD.

Example test range = 4.5(50) to 4.5(1000) = 225 to 4500 mg/L COD

Page 887

Vacuum pretreatment
1. Attach the Vacuum

Pretreatment Device
(VPD) to a vacuum pump
(not an aspirator-type
vacuum) that can create a
vacuum of 2025 inches of
mercury.
2. Label each Mn III

COD vial and remove the
cap. Insert the vials in the
numbered holes in the
VPD base.
3. Place the VPD top on

the base. Insert a fresh
Chloride Removal
Cartridge (CRC) directly
above each Mn III COD
Reagent Vial. Plug any
open holes in the VPD top
using the stoppers
provided.
4. Turn on the vacuum

pump and adjust the
vacuum regulator valve on
top of the VPD until the
internal gauge reads 20
inches of water.
5. Pipet 0.60 mL of
acidified sample (see
Acidified sample
preparation) into the CRC.
Pipet 0.60 mL of acidified
blank into another CRC. It
should take 3045
seconds to draw the liquid
through the CRC into each
vial.
6. Close the vacuum

regulator valve completely
to achieve full vacuum.
After one minute of full
vacuum, slide the VPD
back and forth several
times to dislodge any
drops clinging to the
cartridge.
7. Open the VPD

regulator valve to release
the vacuum. Turn the
pump off. Remove the
VPD top and set it beside
the base.
Proceed to Sample
preparation and
measurement.
If the sample does not flow

through the Chloride
Removal Cartridge (CRC),
increase the vacuum until
flow starts, then reduce
the vacuum down to 20
inches of water. Proceed
as usual.

Page 888
Dispose of the used

Chloride Removal
Cartridge. Do not reuse it.

Sample preparation and measurement
1. Use forceps to remove

the filter from the top of
each CRC.
If the sample does not
contain suspended solids,
it is not necessary to
transfer the filter to the
digestion vial.

DRB200 Reactor at
150 C. Close the
protective cover. Digest for
one hour.
To oxidize resistant
organics, samples can be
digested for up to four
hours. Digest the blank for
the same time period as
the samples.
2. Insert each filter in the

corresponding Mn III COD
Vial. (Use numbers on the
VPD as a guide.)
To avoid crosscontamination between
samples, clean forcep tips
between samples by
wiping with a clean towel
or rinsing with deionized
water.
6. Insert the vials in a

cooling rack for two
minutes.
If the solution develops a
colorless upper layer and
a purple lower layer, invert
the vial several times to
mix and proceed to the
next step.
3. Remove the Mn III

COD vial from the vacuum
chamber and replace the
original cap. Screw the
cap on tightly.
4. Invert the vials several

times to mix.
7. Cool the vials to room

temperature in a cool
water bath or with running
tap water for several
minutes.
8. Remove the vials from

the water and wipe with a
clean, dry paper towel.

Page 889

Sample preparation and measurement (continued)
Stored Programs
432 COD Mn III
Zero
Start
9. Select the test.

Insert an adapter or a light
Instrument-specific
information).

the 16-mm cell holder.

0 mg/L COD Mn
12. Make sure the filter

disc is not suspended in
the middle of the vial; it
can interfere with the
instrument reading.
The disc must be more
than 20 mm (0.8") or less
than 10 mm (0.4"), from
the bottom of the vial.
Move it by gently swirling
or by lightly tapping the
vial on the table top.
Read

the .

mg/L COD Mn.
Interferences
Inorganic materials may also be oxidized by trivalent manganese and constitute a positive
interference when present in significant amounts. Chloride is the most common interference and is
removed by sample pretreatment with the Chloride Removal Cartridge. If chloride is known to be
absent or present in insignificant levels, the pretreatment can be omitted. A simple way to
determine if chloride will affect test results is to run routine samples with and without the chloride
removal, then compare results. Other inorganic interferences (i.e., nitrite, ferrous iron, sulfide) are
not usually present in significant amounts. If necessary, these interferences can be corrected after
determining their concentrations with separate methods and adjusting the final COD test results
accordingly.
Ammonia nitrogen is known to interfere in the presence of chloride; it does not interfere if chloride
is absent.

Page 890
Use plastic bottles only if they are known to be free of organic contamination.
Test biologically active samples as soon as possible.
Samples treated with concentrated sulfuric acid to a pH of less than 2 (about 2 mL per liter)
and refrigerated at 4 C may be stored up to 28 days.
Accuracy check
COD standard solution, 800 mg/L
3. Use 0.60 mL of the 800-mg/L solution in place of the sample.

Method performance
Program
Instrument
Standard
per 0.010 Abs
432
DR 5000
600 mg/L COD
576624 mg/L COD
8 mg/L COD
Estimated detection limit (EDL)

The EDL for program 432 is 4 mg/L COD. The EDL is the calculated lowest average concentration
in a deionized water matrix that is different from zero with a 99% level of confidence.
Summary of method
Chemical Oxygen Demand (COD) is defined as ... a measure of the oxygen equivalent of the
organic matter content of a sample that is susceptible to oxidation by a strong chemical oxidant
(APHA Standard Methods, 19th ed., 1995). Trivalent manganese is a strong, non-carcinogenic
chemical oxidant that changes quantitatively from purple to colorless when it reacts with organic
matter. It typically oxidizes about 80% of the organic compounds. Studies have shown that the
reactions are highly reproducible and test results correlate closely to Biochemical Oxygen
Demand (BOD) values and hexavalent chromium COD tests. None of the oxygen demand tests
provide 100% oxidation of all organic compounds.
A calibration is provided which is based on the oxidation of Potassium Acid Phthalate (KHP). A
different response may be seen in analyzing various wastewaters. The KHP calibration is
adequate for most applications. The highest degree of accuracy is obtained when test results are
correlated to a standard reference method such as BOD or one of the chromium COD methods.
Special waste streams or classes will require a separate calibration to obtain a direct
mg/L COD reading or to generate a correction factor for the precalibrated KHP response. The

Page 891

sample digestion time can be extended up to four hours for samples that are difficult to oxidize.

Required reagents
Description
Quantity/Test
Unit
Catalog number
25/pkg
2623425
Chloride Removal Cartridge (CRC)
25/pkg
2661825
1 mL
2.5 L
97909
Water, deionized
varies
4L
27256
Quantity/Test
Unit
Catalog number
2616100
Required apparatus
Description
Blender, 120 VAC
each
varies
12/pkg
2401812
each
LTV082.53.40001
Cap, with inert Teflon liner, for mixing bottle

each
LTV082.52.40001
Forceps, extra fine point
each
2669600
each
1970010
Pipet Tips for TenSette Pipet 19700-10
50/pkg
2199796
each
1970001
50/pkg
2185696
Test Tube Rack
each
1864100
Vacuum Pretreatment Device (VPD)
each
4900000
Vacuum Pump, 1.2 CFM 115V
each
2824800
Vial, glass, for sample plus acid
each
2427700
Unit
Catalog number
Description
COD Standard Solution, 800-mg/L COD
200 mL
2672629
Oxygen Demand Standard for BOD, COD, TOC, 10-mL ampules
16/pkg
2833510
Potassium Acid Phthalate, ACS
500 g
31534
500 mL
2833149
Description
Unit
Catalog number
Dispenser, automatic, 1.05.0 mL
each
2563137
Wastewater Standard, Influent Inorganics, for NH3N, NO3N, PO4, COD, SO4, TOC
40 tests
2744940
Finger cots
2/pkg
1464702
Flask, volumetric class A, 1 L
each
1457453
Titrator Strips, Quantab, for low range chloride

Page 892

Description
Unit
Catalog number
100/pkg
2601300
each
2936701
500/pkg
1473800
Oven, Laboratory 120 VAC/60Hz
each
1428900
each
1428902
COD Standard Solution, 300-mg/L
200 mL
1218629
500 mL
1218649
200 mL
2253929
each
2270800
Weighing Paper, 76x76 mm
Standard Methods book, most recent edition

250/pkg
2199725
1000/pkg
2185628

Page 893

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen Demand, Chemical without chloride removal, 10067
DOC316.53.01102
(Manganese III Reactor Digestion Method)

Manganese III Reactor Digestion Method
(without chloride removal)1
Method 10067
30 to 1000 mg/L COD Mn

1
U.S. Patent 5,556,787
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

If the sample contains chloride, use the chloride removal method. To determine if the sample contains chloride, use
Quantab Titrator Strips for low range chloride.
If the sample COD value is not between 30 and 1000 mg/L, dilute the sample with deionized water to obtain this range.
Multiply the final result by the dilution factor.
Homogenize the sample for even distribution of solids and better accuracy and reliability.
Stability of the reagent blank allows for reuse. Verify the reagent blank quality by measuring the absorbance of the blank vs.
a clean COD vial filled with deionized water. The absorbance range should be about 1.41.5.
If the sample boils during the digestion, the vial is not properly sealed. Test results will be invalid.
Use finger cots to handle hot sample cells.
Spilled reagent will affect test accuracy and is hazardous. Do not run tests with spilled vials.
See the DRB200 User Manual for selecting pre-programmed temperature applications.

Page 895

Description
Quantity
Blender
DRB200 Reactor
Light Shield
Pipet,
TenSette
(0.1 to 1.0 mL)
Pipet tips for TenSette Pipet
Test Tube Rack
Water, deionized
varies
Sample preparation and measurement
Stored Programs
432 COD Mn III
Start

Insert an adapter or a light
Instrument-specific
information).

Reactor and heat to
150 C or use the COD
program.

Page 896

in a blender.
If suspended solids are
present, continue to mix
the sample while pipetting.
Pipet 0.5 mL of
one Mn III COD vial (the
prepared sample) and
0.5 mL of deionized water
into another Mn III COD
vial (the blank).

Sample preparation and measurement (continued)
18. Cap and invert several

times to mix.

DRB200 Reactor at
150 C. Close the
protective cover. Digest for
one hour.
Digest more resistant
organics and the blank for
up to four hours.
20. Remove the vials and

place them in a cooling
rack for two minutes.
If a vial develops a
colorless upper layer and
a purple lower layer, invert
the vial several times to
mix and proceed.
21. Cool the vials to room

temperature in a cool
water bath or with running
tap water. This takes
several minutes.
Zero
22. Invert the vials several

times to mix.

round cell holder.

0 mg/L COD Mn
25. Wipe the sample and

round cell holder.
COD Mn.
Interferences
Inorganic materials may also be oxidized by trivalent manganese and constitute a positive
interference when present in significant amounts. Chloride is the most common interference. If
chloride is known to be present in significant levels, the chloride needs to be removed with the
vacuum pretreatment device. A simple way to determine if chloride will affect test results is to run
routine samples with and without the chloride removal, then compare results. Other inorganic
interferences (i.e., nitrite, ferrous iron, sulfide) are not usually present in significant amounts. If
necessary, these interferences can be corrected after determining their concentrations with
separate methods and adjusting the final COD test results accordingly.
Ammonia nitrogen is known to interfere in the presence of chloride; it does not interfere if chloride
is absent.

Page 897
Samples treated with concentrated sulfuric acid to a pH of less than 2 (about 2 mL per liter)
and refrigerated at 4 C may be stored up to 28 days.
Accuracy check
COD Standard Solution, 800 mg/L
6. Use 0.50 mL of the 800-mg/L solution in place of the sample.

Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
432
600 mg/L COD
576624 mg/L COD
8 mg/L COD

Page 898

Estimated detection limit (EDL)
The EDL for program 432 is 4mg/L COD. The EDL is the calculated lowest average concentration
in a deionized water matrix that is different from zero with a 99% level of confidence.
Summary of method
Chemical Oxygen Demand (COD) is defined as ... a measure of the oxygen equivalent of the
organic matter content of a sample that is susceptible to oxidation by a strong chemical oxidant
(APHA Standard Methods, 19th ed., 1995). Trivalent manganese is a strong, non-carcinogenic
chemical oxidant that changes quantitatively from purple to colorless when it reacts with organic
matter. It typically oxidizes about 80% of the organic compounds. Studies have shown that the
reactions are highly reproducible and test results correlate closely to Biochemical Oxygen
Demand (BOD) values and hexavalent chromium COD tests. None of the oxygen demand tests
provide 100% oxidation of all organic compounds.
A calibration is provided which is based on the oxidation of Potassium Acid Phthalate (KHP). A
different response may be seen in analyzing various wastewaters. The KHP calibration is
adequate for most applications. The highest degree of accuracy is obtained when test results are
correlated to a standard reference method such as BOD or one of the chromium COD methods.
Special waste streams or classes will require a separate calibration to obtain a direct
mg/L COD reading or to generate a correction factor for the precalibrated KHP response. The
sample digestion time can be extended up to four hours for samples that are difficult to oxidize.

Required reagents
Description
Water, deionized
Quantity/Test
Unit
Catalog number
25/pkg
2623425
varies
4L
27256
Required apparatus
Description
Quantity
Unit
Catalog number
Blender, 2-speed, 120 VAC
each
2616100
each
2616102
each
LTV082.53.40001
each
LTV082.52.40001
each
1970001
50/pkg
2185696
Test Tube Rack
each
1864100
Pipet,
TenSette,
0.1 to 1.0 mL

Page 899

Description
Unit
Catalog number
COD Standard Solution, 800-mg/L COD
200 mL
2672629
Oxygen Demand Standard for BOD, COD, TOC, 10-mL ampules
16/pkg
2833510
500 g
31534
500 mL
2833149
Wastewater Standard, Influent Inorganics, for NH3N, NO3N, PO4, COD, SO4, TOC
Goggles, Safety vented
each
2550700
Gloves, Chemical Resistant, size 9-91
1 pair
2410104
Unit
Catalog number
40 tests
2744940

Description
Titrator Strips, Quantab, for low range chloride
Finger cots
2/pkg
1464702
Flask, volumetric class A, 1L
each
1457453
100/pkg
2601300
1000/pkg
2185628
each
2936701
500/pkg
1473800
each
1428900
each
1428902
200 mL
1218629
500 mL
1218649
200 mL
2253929
each
2270800
Standard Methods book, most recent edition

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, COD, MR, 8000
DOC316.53.01099
USEPA1 Reactor Digestion Method2
Method 8000
40.03
mg/L COD (ULR); 3 to 150 mg/L COD (LR);

0.7 to
20 to 1500 mg/L COD (HR); 200 to 15,000 mg/L COD (HR Plus)
Scope and Application: For water, wastewater; digestion is required
1
Ranges 3 to 150 mg/L COD and 20 to 1500 mg/L COD are USEPA approved for wastewater analyses (Standard Method 5220 D),
Federal Register, April 21, 1980, 45(78), 26811-26812.
Jirka, A.M.; Carter, M.J., Analytical Chemistry, 1975, 47(8), 1397
The ULR range is not available on the DR 2700 or the DR/2400.
Test preparation

information required to perform this test

Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800
LZV646

Some of the chemicals and apparatus used in this procedure may be hazardous to the health and safety of the user if
inappropriately handled or accidentally misused. Please read all warnings and associated MSDS sheets.
Run one blank with each set of samples. Run all tests (the samples and the blank) with the same lot of vials. The lot number
appears on the container label. See Blanks for colorimetric determination.
Spilled reagent will affect test accuracy and is hazardous to skin and other materials. Be prepared to wash spills with
running water.
Wear appropriate eye protection and clothing for adequate user protection. If contact occurs, flush the affected area with
running water. Review and follow reagent MSDS safety instructions carefully.
Store unused (light sensitive) vials in a closed box.
If high chloride samples are being tested, refer to the Alternate reagents section.

Page 901

Description
Quantity
Beaker, 250-mL
Blender
COD Digestion Reagent vials
varies
DRB200 Reactor
Magnetic stirrer and stir bar
Opaque shipping container for storage of unused, light-sensitive reagent vials

Pipet,
TenSette,
0.1 to 1.0 mL, with tips (for 20015,000 mg/L range)
varies
1
Test Tube Rack
Reactor digestion procedure
in a blender. For samples
containing large amounts
of solids, increase the
homogenization time.
If the sample does not
contain suspended solids,
omit steps 1 and 2.
2. For the 200

15,000 mg/L range or to
improve accuracy and
reproducibility of the other
ranges, pour the
a 250-mL beaker and
gently stir with a magnetic
stir plate.

Page 902

Reactor. Preheat to
150 C.
See the DRB200 User
Manual for selecting preprogrammed temperature
applications.

two COD Digestion
Reagent Vials. (Be sure to
use vials for the
appropriate range.)

Reactor digestion procedure (continued)
5. Prepared Sample:
Hold one vial at a
45-degree angle. Use a
clean volumetric pipet to
add 2.00 mL of sample to
the vial.
Hold a second vial at a
45-degree angle. Use a
clean volumetric pipet to
add 2.00 mL of deionized
water to the vial.
For the 20015,000 mg/L

vials: Use a TenSette
Pipet to add 0.20 mL of
sample to the vial.
For the 20015,000 mg/L

vials: Use a TenSette
Pipet to add 0.20 mL of
deionized water to the vial.
9. Heat the vials for two

hours.
10. Turn the reactor off.

Before removing the vials,
wait about 20 minutes for
the vials to cool to 120 C
or less.
7. Cap the vials tightly.

Rinse them with water and
wipe with a clean paper
towel.
8. Hold the vials by the

cap over a sink. Invert
gently several times to
mix. The sample vials
become very hot during
mixing.
Insert the vials in the
preheated DRB200
Reactor. Close the
protective lid.
11. Invert each vial

several times while still
warm.
12. Place the vials into a

rack and cool to room
temperature.
Proceed to Colorimetric
determination.

Page 903

Colorimetric determination
Stored Programs
431 COD ULR
430 COD LR
Zero
435 COD HR
Start
1. Select the test.

Instrument-specific
information).
2. Clean the outside of

the vials with a damp towel
followed by a dry one.

the 16-mm cell holder.

0 mg/L COD
or
0.0 mg/L COD

for orientation.
Read

into the 16-mm .

mg/L COD.
7. If using High Range

Plus COD Digestion
Reagent Vials, multiply the
result by 10.
For most accurate results
with samples near 1500 or
15,000 mg/L COD, repeat
the analysis with a diluted
sample.
Blanks for colorimetric determination

The blank may be used repeatedly for measurements using the same lot of vials. Store the blank
in the dark.
1. Monitor decomposition by measuring the absorbance at the appropriate wavelength. Refer to
the Range-specific test wavelengths table.
2. Zero the instrument in the absorbance mode. Use a vial containing 5 mL of deionized water
and measure the absorbance of the blank. Record the value.
3. Prepare a new blank when the absorbance has changed by about 0.01 absorbance units.

Page 904
Interferences
Chloride is the primary interference when determining COD concentration. Each COD vial
contains mercuric sulfate that will eliminate chloride interference up to the level specified in
Column 1 of the Interfering substances table. Dilute samples with higher chloride concentrations.
Dilute the sample enough to reduce the chloride concentration to the level given in Column 2.
Note: For best results, use the low range and ultra-low range test for samples with high chloride
concentrations (approaching maximum concentration) and low COD concentrations.
If sample dilution will cause the COD concentration to be too low for accurate determination, add
0.50 g of mercuric sulfate (HgSO4) to each COD vial before the sample is added.
The additional mercuric sulfate will raise the maximum chloride concentration allowable to the
level given in Column 3.

Column 1
(Maximum chloride
concentration )
Column 2
(Suggested chloride
concentration for
diluted samples)
Column 3
(Maximum chloride
concentration with
mercuric sulfate)
Ultra Low Range 1

(0.740.0 mg/L)
2000
1000
N/A
Low Range
(3150 mg/L)
2000
1000
8000
High Range
(201500 mg/L)
2000
1000
4000
20,000
10,000
40,000
Vial range
High Range Plus

(20015,000 mg/L)
1
Ultra Low Range is not available on the DR 2700.
Collect samples in glass bottles.
Samples treated with sulfuric acid* to a pH of less than 2 (about 2 mL per liter) and refrigerated
at 4 C can be stored up to 28 days.

Page 905
Accuracy check
1000 mg/L COD Standard Solution

OR
Potassium acid phthalate (KHP), dried overnight at 120 C
Deionized water, organic free
Class A volumetric flasks
Class A volumetric pipet
0.7 to 40.0 mg/L range

1. Prepare a 30-mg/L COD standard solution as follows:
e. Pipet 3.00 mL of the 1000 mg/L standard into a 100-mL volumetric flask.
f.
Dilute to volume with deionized water and mix well.
2. Use 2 mL of the 30 mg/L COD solution in place of the sample. Follow the Colorimetric
determination test procedure. The result should be 30 mg/L. Refer to the Standard adjust
instructions in this procedure to adjust the curve with the reading obtained from the standard.
3 to 150 mg/L range
1. Prepare a 100-mg/L COD standard solution as follows:
a. Pipet 10 mL of the 1000 mg/L standard into a 100-mL volumetric flask.
b. Dilute to volume with deionized water and mix well.
2. Use 2 mL of the 100-mg/L solution in place of the sample. Follow the Colorimetric
determination test procedure. The result should be 100 mg/L. Refer to the Standard adjust
instructions in this procedure to adjust the curve with the reading obtained from the standard
20 to 1500 mg/L range
Use 2 mL of 300 mg/L, 800 or 1000 mg/L COD standards for accuracy check.

Page 906

200 to 15,000 mg/L range
1. Prepare a 10,000-mg/L COD standard solution as follows:
a. Dissolve 8.500 g of dried KHP in 1000-mL of organic-free deionized water.
2. Use 0.2 mL of the 10,000 mg/L COD solution in place of the sample. Follow the Colorimetric
determination test procedure. The result should be 10,000 mg/L (after multiplying by 10).
Refer to the Standard adjust instructions in this procedure to adjust the curve with the reading
obtained from the standard.
Standard adjust
Alternate reagents
Mercury-free COD2 Reagents can provide a mercury-free testing option for non-reporting
purposes. For process control applications, COD2 Reagents will eliminate mercury waste and
save on disposal costs. These reagents are fully compatible with test procedures and calibration
curves programmed into the spectrophotometer. Determine chloride and ammonia for
accurate results.
Important Note: COD2 reagents are not approved for USEPA reporting purposes. Because
COD2 reagents do not contain mercury as a masking agent, they exhibit a positive
interference from chloride. Request a copy of the COD2 Reagent Vial Information Brochure,
Lit. No. 1356, for more information about specific applications.

Page 907
Method performance
Program
430
(Range,
3150 mg/L)
Standard
Precision
Distribution
Sensitivity
7783 mg/L COD
3 mg/L COD
28.831.2 mg/L COD
0.5 mg/L COD
785815 mg/L COD
23 mg/L COD
78508150 mg/L COD
230 mg/L COD
80 mg/L COD (Low Range)
431
(Range,
0.540.0 mg/L)
30 mg/L COD (Ultra Low

Range)
435
(Range,
201500 mg/L)
800 mg/L COD (High Range)
435
(Range,
20015,000 mg/L)
8000 mg/L COD (High Range

Plus)
Summary of method
The results in mg/L COD are defined as the milligrams of O2 consumed per liter of sample under
the conditions of this procedure. The sample is heated for two hours with sulfuric acid and a strong
oxidizing agent, potassium dichromate. Oxidizable organic compounds react, reducing the
dichromate ion (Cr2O72) to green chromic ion (Cr3+).
When the 0.740.0 or the 3150 mg/L colorimetric method is used, the amount of Cr6+ remaining
is determined. When the 201500 mg/L or 20015,000 mg/L colorimetric method is used, the
amount of Cr3+ produced is determined. The COD reagent also contains silver and mercury ions.
Silver is a catalyst, and mercury is used to complex chloride interferences.
Test results are measured at the wavelengths specified in the Range-specific test
wavelengths table.
Table 277 Range-specific test wavelengths

Range in mg/L COD
0.7 to 40.0
mg/L1
Wavelength
350 nm
3 to 150 mg/L
420 nm
20 to 1500
620 nm
2000 to 15,000 mg/L
620 nm
Not available on the DR 2700

Page 908

Required reagents
Description
Quantity/Test
Unit
Catalog number
Select the appropriate COD Digestion Reagent Vial:

Ultra Low Range, 0.7 to 40 mg/L COD
12 vials
25/pkg
2415825
Low Range, 3 to 150 mg/L COD
12 vials
25/pkg
2125825
High Range, 20 to 1500 mg/L COD
12 vials
25/pkg
2125925
High Range Plus, 200 to 15,000 mg/L COD
12 vials
25/pkg
2415925
varies
4L
27256
Quantity/Test
Unit
Catalog number
Water, deionized
Alternate reagents1
Description
Select the appropriate COD Digestion Reagent Vial:
COD2, Low Range, 0 to 150 mg/L COD
12 vials
25/pkg
2565025
COD2, High Range, 0 to 1500 mg/L COD
12 vials
25/pkg
2565125
COD2, High Range, 0 to 1500 mg/L COD
12 vials
150/pkg
2565115
COD2, High Range Plus, 0 to 15,000 mg/L COD
12 vials
25/pkg
2834325
COD Digestion Reagent Vials, 3 to 150 mg/L COD
12 vials
150/pkg
2125815
COD Digestion Reagent Vials, 200 to 1500 mg/L COD
12 vials
150/pkg
2125915
COD Digestion Reagent Vials, ULR 0.7-40.0 mg/L
1-2 vials
150/pkg
2415815
COD Digestion Reagent Vials, HR plus,200-25,000 mg/L
1-2 vials
150/pkg
2415915
These reagents are not approved for USEPA reporting purposes. Request a copy of the COD2 Reagent Vial Information Brochure,
Lit. No. 1356, for more information about specific applications.
Required apparatus
Description
Quantity
Unit
Catalog number
each
2616100
OR
each
2616102
each
LTV082.53.40001
each
LTV082.52.40001
each
1465100
each
1451536
OR

Page 909

Description
Beaker, 250-mL
Unit
Catalog number
each
50046H
200 mL
1218629
500mL
1218649
200 mL
2672629
200 mL
2253929
Oxygen Demand Standard (BOD, COD, TOC), 10-mL ampules
16/pkg
2833510
each
1970001
50/pkg
2185696
1000/pkg
2185628
500 g
31534
each
2095352
Stirrer, Electromagnetic, 120 VAC, with electrode stand
each
4530001
Stirrer, Electromagnetic, 230 VAC, with electrode stand
each
4530002
Test Tube Rack, 16 mm
each
1864100
70/pkg
2096900
Description
Unit
Catalog number
each
2936701
Flask, volumetric, 1000 mL Class A
each
1457453
1457442
Wipes, Disposable
Flask, volumetric, 100-mL Class A
each
Mercuric Sulfate
28 g
191520
Pipet, volumetric, 3-mL, Class A
each
1451503
each
1451538
Pipet, volumetric, 10-mL, Class A

Sulfuric Acid, conc
500 mL
97949
Wastewater Influent Standard for mixed parameters NH3N, NO3N, PO4, COD, SO4,
TOC
500mL
2833149
Wastewater Effluent Standard, for mixed parameters NH3N, NO3N, PO4, COD, SO4,
TOC
500mL
2833249
500/pkg
1473800
Finger cots
2/pkg
1464702
Gloves, chemical resistant 9-9 1
1 pair
2410104
Safety goggles, vented
each
2550700
EZ CODTM Recycling Service with 5-gal2 bucketmail back3
each
2895405
EZ CODTM Recycling Service with 5-gal bucketpick up3
each
2895405P
20 and 55 gal are available.
ez COD available to U.S. customers only

Page 910

Page 911

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, Dissolved, HR, 8166
Oxygen, Dissolved
DOC316.53.01096
HRDO Method
Method 8166
AccuVac Ampuls
HR (0.3 to 15.0 mg/L O2)

Test preparation


AccuVac Ampuls
Instrument
Sample cell
Adapter
DR 6000
2427606
DR 5000
2427606
DR 3900
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2122800
LZV584 (C)

Analyze samples on-site. Do not store for later analysis

Description
Quantity
High Range Dissolved Oxygen AccuVac Ampuls with reusable Ampul caps
Polypropylene Beaker, 50-mL
Oxygen, Dissolved
Page 913
Oxygen, Dissolved
HRDO Method
Stored Programs
445 Oxygen, Dis HR AV
Start
1. Select the test.

5. Hold the Ampul with

the tip pointing down and
immediately insert the
Ampul into the Ampul cap.
The cap prevents
contamination from
atmospheric oxygen.
mL of sample.
3. Fill a blue Ampul cap

with sample.

a High Range Dissolved
Oxygen AccuVac Ampul
fills completely.
6. Shake the Ampul for

30 seconds.

timer.
A small amount of
not affect results.
period will begin. This
enables the oxygen that
was degassed during
aspiration to redissolve
and react.
8. When the timer

expires, shake the Ampul
for 30 seconds.
Zero
9. Insert the blank in the

cell holder.

0.0 mg/L O2
Oxygen, Dissolved
Page 914
Allow any bubbles to

dissipate before
proceeding.
Read


mg/L O2.
Oxygen, Dissolved
Interferences
Interference level
Cr3+
Cu2+
Fe2+
Mg2+
Magnesium is commonly present in seawater and causes a negative interference. If the

sample contains more than 50% seawater, the oxygen concentration obtained by this
method will be 25% less than the true oxygen concentration. If the sample contains less than
50% seawater, the interference will be less than 5%.
Mn2+
Ni2+
NO2-

The main consideration in sampling with the High Range Dissolved Oxygen Ampul is to prevent
the sample from becoming contaminated with atmospheric oxygen between breaking open the
Ampul and reading the absorbance. This is accomplished by capping the Ampul with an Ampul
cap. If the Ampul is securely capped, the Ampul should be safe from contamination for several
hours. The absorbance will decrease by approximately 3% during the first hour and will not change
significantly afterwards.
Sampling and sample handling are important considerations in obtaining meaningful results. The
dissolved oxygen content of the water being tested may change with depth, turbulence,
temperature, sludge deposits, light, microbial action, mixing, travel time and other factors. A single
dissolved oxygen test rarely reflects the accurate overall condition of a body of water. Several
samples taken at different times, locations and depths are recommended for most reliable results.
Samples must be tested immediately upon collection, although only a small error results if the
absorbance reading is taken several hours later.
Accuracy check
The results of this procedure may be compared with the results of a titrimetric procedure or by
using a dissolved oxygen meter.*
Method performance
Program
Standard

Distribution
per 0.010 Abs
445
6.7 mg/L O2
6.27.3 mg/L O2
0.09 mg/L O2
Summary of method
The High Range Dissolved Oxygen AccuVac Ampul contains reagent vacuum-sealed in a 14-mL
Ampul. When the AccuVac Ampul is opened in a sample containing dissolved oxygen, it forms a
yellow color which turns purple. The purple color development is proportional to the concentration
of dissolved oxygen. Test results are measured at 535 nm.
* See Optional reagents, apparatus and meters.
Oxygen, Dissolved
Page 915
Oxygen, Dissolved

Required reagents
Description
Quantity/Test
Unit
Catalog number
25/pkg
2515025
Catalog number
High Range Dissolved Oxygen AccuVac Ampuls
Required apparatus
Description
Quantity
Unit
each
108041
each
2122800
6/pkg
2427606
2/pkg
2495402
Description
Unit
Catalog number
AccuVac
each
2405200
Optional reagents, apparatus and meters
snapper
AccuVac sampler
each
2405100
25/pkg
2677925
AccuVac stoppers
6/pkg
173106
HQ30d Meter with Standard LDO Dissolved Oxygen Probe1 (1 meter cable)
each
HQ30d53301000
HQ40d Meter with Standard LDO Probe1 (1 meter cable)
each
HQ40d53301000
Additional probes and cable lengths are available.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, Dissolved, LR, 8316
Oxygen, Dissolved
DOC316.53.01098
Indigo Carmine Method
Method 8316
AccuVac Ampuls
LR (6 to 800 g/L O2)

Scope and Application: For boiler feedwater
Test preparation


AccuVac Ampuls
Instrument
Sample cell
Adapter
DR 6000
2427606
DR 5000
2427606
DR 3900
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2122800
LZV584 (C)


Description
Quantity
Low Range Dissolved Oxygen AccuVac Ampuls
Oxygen, Dissolved
Page 917
Oxygen, Dissolved
Indigo Carmine method for AccuVac Ampuls
Stored Programs
446 Oxygen, Dis LR AV
Zero
Start
1. Select the test.

10 mL of sample.

the cell holder.

0 g/L O2
Read
5. Fill a Low Range

Dissolved Oxygen Ampul
fills completely.
6. Immediately insert the

Ampul into the cell holder.

g/L O2.
Use the initial reading. The

reading is stable for 30
seconds. After 30 seconds
the Ampul solution will
absorb oxygen from
the air.
Interferences
Excess amounts of thioglycolate, ascorbate, ascorbate + sulfite, ascorbate + cupric sulfate, nitrite,
sulfite, thiosulfate and hydroquinone will not reduce the oxidized form of the indicator and do not
cause significant interference.

Interference level
Hydrazine
100,000 fold excess will begin to reduce the oxidized form of the indicator solution.
Sodium hydrosulfite
Reduces the oxidized form of the indicator solution and will cause a significant interference.
Oxygen, Dissolved
Page 918
Oxygen, Dissolved

The main consideration in this procedure is to prevent contaminating the sample with
atmospheric oxygen.
For best results, sample from a stream of water that is hard plumbed to the sample source.
Use a funnel to maintain a continual flow of sample and yet collect enough sample to immerse
the Ampul.
Do not introduce air in place of the sample.
Rubber tubing, if used, will introduce unacceptable amounts of oxygen into the sample unless
the length of tubing is minimized and the flow rate is maximized.
Flush the sampling system with sample for at least 5 minutes.
Accuracy check
The reagent blank for this test can be checked by following these steps:
1. Fill a 50-mL beaker with sample and add one sodium hydrosulfite powder pillow.
2. Immerse the tip of a Low Range Dissolved Oxygen AccuVac Ampul in the sample into the tip.
Aspirate the sample into the Ampul.
3. Determine the dissolved oxygen concentration according to the preceding procedure. The
result should be 0 6 g/L.
Method performance
Sensitivity
Program
per 0.010 Abs
446
6 g/L O2
Summary of method
The Low Range Dissolved Oxygen AccuVac Ampul contains reagent vacuum-sealed in an Ampul.
When the AccuVac Ampul is broken open in a sample containing dissolved oxygen, the yellow
solution will turn blue. The blue color development is proportional to the concentration of dissolved
oxygen. Test results are measured at 610 nm.
Oxygen, Dissolved
Page 919
Oxygen, Dissolved

Required reagents
Description
Quantity/Test
Unit
Catalog number
25/pkg
2501025
Catalog number
Low Range Dissolved Oxygen AccuVac Ampuls
Required apparatus
Description
Quantity/Test
Unit
each
108041
each
2122800
6/pkg
2427606
2/pkg
2495402
Description
Hydrosulfite reagent powder pillows
Unit
Catalog number
100/pkg
2118869
Unit
Catalog number
each
2405200

Description
AccuVac
snapper
AccuVac sampler
each
2405100
25/pkg
2677925
AccuVac stoppers
6/pkg
173106

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, Dissolved, UHR, 8333
Oxygen, Dissolved
DOC316.53.01097
Ultra High Range Method
Method 8333
UHR (1.0 to 40.0 mg/L O2)
AccuVac Ampuls
Scope and Application: For aquaculture
Test preparation


AccuVac Ampuls
Instrument
Sample cell
Adapter
DR 6000
2427606
DR 5000
2427606
DR 3900
2427606
LZV846 (A)
DR 3800, DR 2700, DR 2800
2122800
LZV584 (C)


Description
Quantity
High Range Dissolved Oxygen AccuVacAmpuls, with reusable Ampul caps
Oxygen, Dissolved
Page 921
Oxygen, Dissolved
UHR Method for AccuVac Ampuls
Stored Programs
448 Oxygen, Dis UHR
Start
1. Select the test.

5. Hold the Ampul with

the tip pointing down and
immediately insert the
Ampul into the Ampul cap.
The cap prevents
contamination from
atmospheric oxygen.
mL of sample.
3. Fill a blue Ampul cap

with sample.

a High Range Dissolved
Oxygen AccuVac Ampul
fills completely.
6. Shake the Ampul for

30 seconds.

timer.
A small amount of
not affect results.
period will begin. This
enables the oxygen that
was degassed during
aspiration to redissolve
and react.
8. When the timer

expires, shake the Ampul
for 30 seconds.
Zero
9. Insert the blank in the

cell holder.

0.0 mg/L O2
Oxygen, Dissolved
Page 922
Allow any bubbles to

dissipate before
proceeding.
Read


mg/L O2.
Oxygen, Dissolved
Interferences
Interference level
Cr3+
Cu2+
Fe2+
Mg2+
Magnesium is commonly present in seawater and causes a negative interference. If the

sample contains more than 50% seawater, the oxygen concentration obtained by this
method will be 25% less than the true oxygen concentration. If the sample contains less than
50% seawater, the interference will be less than 5%.
Mn2+
Ni2+
NO2-

The main consideration in sampling with the High Range Dissolved Oxygen Ampul is to prevent
the sample from becoming contaminated with atmospheric oxygen between breaking open the
Ampul and reading the absorbance. This is accomplished by capping the Ampul with an Ampul
cap. If the Ampul is securely capped, the Ampul should be safe from contamination for several
hours. The absorbance will decrease by approximately 3% during the first hour and will not change
significantly afterwards.
dissolved oxygen content of the water being tested may change with depth, turbulence,
temperature, sludge deposits, light, microbial action, mixing, travel time and other factors. A single
dissolved oxygen test rarely reflects the accurate overall condition of a body of water. Several
samples taken at different times, locations and depths are recommended for most reliable results.
Samples must be tested immediately upon collection, although only a small error results if the
absorbance reading is taken several hours later.
Accuracy check
The results of this procedure may be compared with the results of a titrimetric procedure (request
Lit. Code 8042) or by using a dissolved oxygen meter.*
Method performance
Program
Standard
Precision
Distribution
Sensitivity
448
26.4 mg/L O2
23.629.2 mg/L O2
0.34 mg/L O2
0.45 mg/L O2
0.68 mg/L O2
* See Optional reagents, apparatus and meters.
Oxygen, Dissolved
Page 923
Oxygen, Dissolved
Summary of method
The High Range Dissolved Oxygen AccuVac Ampul contains reagent vacuum-sealed in an Ampul.
When the AccuVac Ampul is opened in a sample containing dissolved oxygen, it forms a yellow
color which turns purple. The purple color development is proportional to the concentration of
dissolved oxygen. Test results are measured at 680 nm.

Required reagents
Description
High Range Dissolved Oxygen
Ampul caps
AccuVac
Quantity/Test
Unit
Catalog number
25/pkg
2515025
Catalog number
Ampuls with 2 reusable

Description
Quantity
Unit
each
108041
each
2122800
6/pkg
2427606
2/pkg
2495402
Description
Unit
Catalog number
AccuVac snapper
each
2405200
AccuVac sampler
each
2405100
25/pkg
2677925
AccuVac stoppers
6/pkg
173106
HQ30d Meter with Standard LDO Dissolved Oxygen Probe (1 meter cable)1
each
HQ30d53301000
HQ40d Meter with Standard LDO Probe (1 meter cable)1
each
HQ40d53301000
Additional probes and cable lengths are available.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, Dissolved, DT, 8215 and 8332
Oxygen, Dissolved
DOC316.53.01179
Azide Modification of Winkler Method
1 to greater than 10 mg/L
Digital Titrator
Test preparation

Use the TitraStir apparatus for best results.

Description
Dissolved Oxygen Reagent Set
Quantity
1
varies
Bottle, with stopper, BOD, 300-mL
Clippers, for opening pillows
Digital Titrator
Dissolved Oxygen 1 Reagent Powder Pillows
Polypropylene Beaker, 50-mL, Low Form, with pour spout
Oxygen, Dissolved
Page 925
Oxygen, Dissolved
Method 8215, 300-mL BOD bottle

in a clean 300-mL BOD
bottle. Allow the sample to
overflow the bottle for 23
minutes to make sure that
a representative sample is
available.

one Manganous Sulfate
Powder Pillow and one
Alkaline Iodide-Azide
5. Remove the stopper

and add the contents of
one Sulfamic Acid Powder
Pillow. Replace the
stopper without trapping
air in the bottle. Invert the
prepared sample several
times to mix.
6. Select a sample
volume and Sodium
Thiosulfate Titration
Cartridge from the Volume
multipliers table that
corresponds to the
expected dissolved
oxygen (DO)
concentration.
The floc will dissolve and

leave a yellow color if
oxygen is present.
Oxygen, Dissolved
Page 926
3. Immediately and
without trapping air in the
bottle, insert the stopper.
Invert the bottle several
times to mix.
4. Again, invert the bottle

several times and wait
until the floc settles and
the top half of the solution
is clear again.
A flocculent precipitate will

form. It will be orangebrown if oxygen is present
or white if oxygen is
absent. The floc settles
slowly in salt water. The
settling normally takes
about five minutes. When
the floc settles, proceed to
step 4.
Wait until the floc settles

the second time to make
sure the reaction of the
sample and reagents is
complete.

cartridge.

Attach the cartridge to the

titrator body.
Oxygen, Dissolved
Method 8215, 300-mL BOD bottle (continued)
9. Use a graduated
cylinder to measure the
sample volume from the
Volume multipliers table.

titrating with sodium
thiosulfate to a pale yellow
color.
11. Add two 1-mL

droppers of Starch
Indicator Solution and
swirl to mix.

to a colorless end point.
Record the number of
digits required.

develop.
13. Calculate:
Digits Required x
Digit Multiplier =
mg/L Dissolved Oxygen
Table 284 Volume multipliers

Titration Cartridge (N Na2S2O3)
Range (mg/L DO)
Volume (mL)
100400
150
0.200
0.01
200800
75
0.200
0.02
6002400
25
2.000
0.10
Digit Multiplier
Oxygen, Dissolved
Page 927
Oxygen, Dissolved
Method 8332, 60-mL BOD bottle

in a clean 60-mL BOD
bottle. Allow the sample to
overflow the bottle for 23
minutes to make sure that
a representative sample is
obtained.

Powder Pillow and
Powder Pillow.

Powder Pillow. Replace
the stopper without
trapping air in the bottle
mix.
6. Accurately measure
20 mL of the prepared
sample and transfer it to a

oxygen is present.
Oxygen, Dissolved
Page 928
3. Immediately and
without trapping air in the
bottle, insert the stopper.
Invert the bottle several
times to mix.
4. Again invert the bottle

until the floc settles and
the top half of the solution
is clear again.

form. It will be orangebrown if oxygen is present
or white if oxygen is
absent. The floc settles
slowly in salt water. The
settling normally takes
about five minutes. When
the floc settles, proceed to
step 4.
Wait until the floc settles

the second time to make
sure the reaction of the
sample and reagents is
complete.
7. Insert a clean straightstem delivery tube to a

0.200 N Sodium
Thiosulfate Titration
Cartridge. Attach the
cartridge onto the titrator
body.

Oxygen, Dissolved
Method 8332, 60-mL BOD bottle (continued)
9. Titrate the prepared

solution with 0.2000 N
Sodium Thiosulfate until
the sample changes to a
pale yellow color. Record
the number of digits.
10. Add two 1-mL

droppers of Starch
Indicator Solution and
swirl to mix.

to a colorless end point.
digits required.
12. Calculate:
Digits Required x 0.1 =
mg/L Dissolved Oxygen

develop.
Interferences
Nitrite interference is eliminated by the azide in the reagents. Other reducing or oxidizing
substances may interfere. If these are present, use an alternate method, such as the High Range
Dissolved Oxygen Method (colorimetric, Method 8166) or a dissolved oxygen electrode (4500
OG).

Sampling and sample handling are important in obtaining meaningful results. The dissolved
oxygen content of the sample changes with depth, turbulence, temperature, sludge deposits, light,
microbial action, mixing, travel time and other factors. A single dissolved oxygen test rarely reflects
the over-all condition of a body of water. Several samples taken at different times, locations and
depths are recommended for most reliable results.
Collect samples in clean BOD Bottles.
If storage is necessary, run steps 1 through 4 of the procedure and store in the dark at
1020 C.
Seal the bottle with water by pouring a small volume of water into the flared lip area of a
stopper bottle.
Use a BOD Bottle Cap over the flared lip.
Samples preserved like this can be held 48 hours. Begin with step 5 when analyzing.
Accuracy check
Check the strength of the Sodium Thiosulfate Solution by using an Iodate-Iodide Standard
Solution, 10 mg/L as DO.
1. For the 300-mL procedure, begin at step 5, adding the Sulfamic Acid Powder Pillow to a
200-mL volume of Iodate-Iodide Standard Solution.
2. Use a 100-mL sample volume with the 0.200 N Sodium Thiosulfate Titration Cartridge. This
titration should take 500 digits. If more than 525 digits are required to reach the end point,
replace the Sodium Thiosulfate Cartridge.
Oxygen, Dissolved
Page 929
Oxygen, Dissolved
3. Use a 200-mL sample volume with the 2.00 N Sodium Thiosulfate Titration Cartridge. This
For the 60-mg/L procedure:
1. Begin the analysis at step 5, adding the Dissolved Oxygen 3 Powder Pillow to a 60 mL volume
of Iodate-Iodide Standard Solution.
2. Use a 20 mL sample volume with the 2.00 N Sodium Thiosulfate Titration Cartridge. This
Summary of method
Samples are treated with manganous sulfate and alkaline iodide-azide reagent to form an orangebrown precipitate. Upon acidification of the sample, this floc reacts with iodide to produce free
iodine as triiodide in proportion to the oxygen concentration. The iodine is titrated with sodium
thiosulfate to a colorless end point.

Required reagents for 300-mL BOD bottle
Description
Unit
Dissolved Oxygen Reagent Set (about 50 tests)
Catalog number
2272200
Includes:
(2) Alkaline Iodide-Azide Powder Pillows
50/pkg
107266
(2) Manganous Sulfate Powder Pillows
50/pkg
107166
each
2267501
100 mL MDB1
34932
50/pkg
2076266
each
1440101
Description
Unit
Catalog number
each
62100
each
96800
each
50846
Digital Titrator
each
1690001
each
50546
each
1720500
each
4157800

(2) Sulfamic Acid Powder Pillows
Sodium Thiosulfate Titration Cartridge. 2.00 N
1
MDB is Marked Dropper Bottle
Required apparatus for 300-mL BOD bottle
Oxygen, Dissolved
Page 930
Oxygen, Dissolved
Required reagents for 60-mL BOD bottle

Description
Unit
Catalog number
100/pkg
98199
100/pkg
98299
25/pkg
98768
each
2267501
Description
Unit
Catalog number
each
190902
each
96800
Digital Titrator
each
1690001
each
50543
Polypropylene Beaker, 50-mL, Low Form, with pour spout
each
108041
Required apparatus for 60-mL BOD bottle
each
1720500
each
4157800
Required standards
Iodate-Iodide Standard Solution, 10-mg/L as DO
Thermometer -10 225 C 405 mm
500 mL
40149
each
2635700
Cap, BOD Bottle Snap-over
6/pkg
241906
BOD Bottle, Serialized (#1-24)
24/pkg
2898700
Oxygen, Dissolved
Page 931

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, Dissolved, BT, 8229
Oxygen, Dissolved
USEPA1 Azide Modification of Winkler Method2
1 to more than 10 mg/L DO
DOC316.53.01161
Method 8229
Buret Titration

1
USEPA approved.
Adapted from Standard Methods for the Examination of Water and Wastewater, (Standard Method 4500 O C)
Test preparation

Dissolved oxygen can be lost from the sample during sample collection. Review the precautions in Sample collection,
preservation and storage before the test is started.
Standard APHA solutions for dissolved oxygen can be used in place of the powder pillow reagents by substituting 1 mL of
Manganous Sulfate Solution, 1 mL of Alkaline Iodide-Azide Reagent and 1 mL of Sulfuric Acid (concentrated) in place of the
powder pillows. These solutions must be dispensed below the surface of the liquid.

Description
BOD bottle, 300-mL
Quantity
1
Alkaline Iodide-Azide Reagent Powder Pillow
1 pillow
Manganous Sulfate Powder Pillow
1 pillow
Sulfamic Acid Powder Pillow
1 pillow
Sodium Thiosulfate Standard Solution (titrant), 0.025 N
1 bottle
1 bottle
Oxygen, Dissolved
Page 933
Oxygen, Dissolved
Buret titration

in a clean, 300-mL, glass
stoppered BOD bottle.
Overflow the bottle for two
or three minutes to remove
any trapped air bubbles to
make sure that a
representative sample is
available.

one Manganous Sulfate
Powder Pillow and one
Alkaline Iodide-Azide
3. Immediately insert the

stopper so that no air is
trapped in the bottle. Invert
several times to mix.
form. It will be
orange-brown if oxygen is
present or white if oxygen
is absent.
The floc will settle very
slowly in salt water. Wait
five more minutes before
proceeding to step 4.

Sodium Thiosulfate
Solution.

one Sulfamic Acid Powder
Pillow. Replace the
stopper without trapping
air in the bottle and invert
several times to mix. This
is the prepared sample.
oxygen is present.
Oxygen, Dissolved
Page 934

200-mL mark.
4. Again invert the bottle

until the floc has settled
and the top half of the
solution is clear again.
Waiting until floc has
settled twice makes sure
that the reaction is
complete. Results will not
be affected if the floc does
not completely settle.

the graduated cylinder into
a 250-mL Erlenmeyer
flask.
Oxygen, Dissolved

flask until it turns a very
pale yellow color.
10. Add two 1-mL

droppers of Starch
Indicator Solution. Swirl to
mix.

from dark blue to
colorless.
The solution will turn dark

blue.
The amount of titrant used

to reach the end point is
equal to the concentration
of dissolved oxygen in the
sample.
mL titrant used = mg/L DO
Interferences
Nitrite interference is eliminated by the azide in the reagents. Other reducing or oxidizing
substances may interfere. If these are present, use an alternate method, such as the High Range
Dissolved Oxygen Method, (Hach method 8166 - colorimetric) or a dissolved oxygen electrode
(Standard Method 4500 O G).
Pretreatment procedure for activated sludge samples
A sample pretreatment is necessary for activated sludge samples.
1. Add 10 mL of Copper Sulfate-Sulfamic Acid Inhibitor Solution to a clean 1000-mL graduated
cylinder.
2. Fill the cylinder with the sample using a tube that empties near the bottom of the cylinder and
allow the sample to overflow by about 200 mL.
3. Swirl the cylinder to mix the contents. Allow the suspended solids to settle.
4. Siphon the relatively clear top layer into a BOD bottle through a siphon tube extended to the
bottom of the bottle. Withdraw the siphon tube while the water is flowing. Make sure that no air
bubbles are trapped in the bottle. Continue with step 2step 11 of the test procedure.

dissolved oxygen content of the water being tested varies with depth, turbulence, temperature,
sludge deposits, light, microbial action, mixing, travel time and other factors. A single dissolved
oxygen test rarely reflects the overall condition of a body of water. Several samples taken at
different times, locations and depths are recommended for most reliable results.
Collect samples in a clean BOD bottle as described in step 1. If storage is necessary, complete
steps 14 of the test procedure and store in the dark at the temperature of the water source or
water seal at 1020 C. (Sealing with water is done by pouring a small amount of water into the
Oxygen, Dissolved
Page 935
Oxygen, Dissolved
flared lip area of a stoppered bottle. Snap a BOD bottle cap over the flared lip). Samples preserved
in this manner can be held four to eight hours. Start the test at step 6.
Accuracy check
The standard solution method can be used to confirm analytical technique and reagent
performance.
Complete the following test to make sure the concentration of the titrant is accurate.
Iodate-Iodide Standard Solution, 0.00125 N (equivalent to 10 mg/L as O2)
1. Add 200.0 mL of Iodate-Iodide Standard Solution, 0.00125 N, to an Erlenmeyer flask.

2. Add one Sulfamic Acid Powder Pillow and swirl to mix.
3. Follow steps 911 of the test procedure to titrate the standard to the end point. The titration
should use 10.0 mL of the titrant solution. If more than 10.5 mL is used, discard the titrant and
replace it with a fresh supply.
Summary of method
The Azide Modification of the Winkler Method is the standard test for dissolved oxygen. In the
analysis, manganous ion reacts with the dissolved oxygen present in the alkaline solution to form a
manganese (IV) oxide hydroxide flocculent. Azide is then added to suppress interference from any
nitrite, which would react with the iodide. The solution is then acidified and the manganese (IV) floc
is reduced by iodide to produce free iodine as I3 in proportion to the oxygen concentration. The
liberated iodine is then titrated to the starch-iodide end point.
Oxygen, Dissolved
Page 936
Oxygen, Dissolved

Required reagents
Description
Quantity/Test
Unit
Alkaline Iodide-Azide Reagent Powder Pillows
1 pillow
50/pkg
Catalog number
107266
Manganous Sulfate Powder Pillows
1 pillow
50/pkg
107166
2409353
Sodium Thiosulfate Standard Solution (titrant), 0.025 N
varies
1L
2 mL
100 mL MDB
34932
1 pillow
100/pkg
107399
Catalog number
Sulfamic Acid Powder Pillows
Required apparatus
Description
Quantity/Test
Unit
Bottle, glass-stoppered, BOD, 300-mL
each
62100
each
2636540
Buret Clamp, double
each
32800
each
96800
50846
each
each
50546
Support Stand
each
56300
Description
Unit
Catalog number
500 mL
40149
Unit
Catalog number
Alkaline Iodide-Azide Reagent Solution
500 mL
27749
Manganous Sulfate Solution
500 mL
27549
Iodate-Iodide Standard Solution, 0.00125 N

Description
APHA reagents:

Sulfuric Acid, ACS
1L
35253
500 mL
97949
each
2635700
each
50853
BOD bottle caps
6/pkg
241906
BOD bottles, serialized
24/pkg
2898700
Copper Sulfate-Sulfamic acid inhibitor
100 mL
35732
Copper Sulfate-Sulfamic acid inhibitor
500 mL
35749
Oxygen, Dissolved
Page 937

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen Scavengers, 8140
Oxygen Scavengers
DOC316.53.01105
Iron Reduction Method for Oxygen Scavengers

5 to 600 g/L carbohydrazide;
3 to 450 g/L DEHA;
9 to 1000 g/L hydroquinone;
13 to 1500 g/L iso-ascorbic acid [ISA];
15 to 1000 g/L methylethyl ketoxime [MEKO]
Method 8140
Powder Pillows
Scope and Application: For testing residual corrosion inhibitors (oxygen scavengers)
in boiler feed water or condensate
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

The sample temperature should be 25 3 C (77 5 F).
Soak glassware with 1:1 hydrochloric acid solution. Rinse several times with deionized water. These two steps will remove
iron deposits that can cause slightly high results.
To determine ferrous iron concentration, repeat the procedure, but do not add DEHA Reagent 2. Correct for the ferrous iron
concentration: OPTIONS>MORE>REAGENT BLANK>ON. The reading attributed to the ferrous iron concentration will appear.
Oxygen Scavengers
Page 939
Oxygen Scavengers

Description
Quantity
Bottle, glass mixing, with 25-mL mark
DEHA Reagent 1 Powder Pillow
DEHA Reagent 2 Solution
1 mL
Deionized Water
25 mL
Hydrochloric Acid, 1:1, 6.0 N
varies
Iron reduction method for Oxygen Scavengers

180 O Scav-Carbohy
181 O Scav-DEHA
182 O Scav-Hydro
183 O Scav-ISA
184 O Scav-MEKO
1. Select the test.

Oxygen Scavengers
Page 940

a mixing bottle with 25 mL
of sample.
When determining oxygen
scavengers that react
quickly with oxygen at
room temperature, cap the
bottle.
Fill a second mixing bottle
water.

one DEHA Reagent 1
Powder Pillow to each
mixing bottle. Swirl to mix.
Oxygen Scavengers
Iron reduction method for Oxygen Scavengers (continued)
5. Add 0.5 mL of DEHA

Reagent 2 Solution to
each bottle. Mix. Place
both sample cells in the
dark.
A purple color will develop
if an oxygen scavenger is
present.

timer.
period (or a two-minute
reaction period for
hydroquinone) will begin.
7. When the timer

expires, transfer the blank
and prepared samples into
the 10-mL sample cells.
8. Immediately after
transferring to the 10-mL
cell, wipe the blank and
Close the cover.
Keep the sample cells in

the dark during the
reaction period.
Zero
Read

For greater accuracy, read
the result immediately
after the timer expires.

prepared sample and
11. READ the results

in g/L.
Interferences
Substances which reduce ferric iron will interfere. Substances which complex iron strongly may
also interfere.

Interference level
Borate (as Na2B4O7)
Cobalt
Copper
Ferrous Iron
All levels.
Note: Determine and subtract (see Before starting the test:)
Hardness (as CaCO3)
Oxygen Scavengers
Page 941
Oxygen Scavengers
Interference level
Light
Light may interfere. Keep sample cells in the dark during color development.
Lignosulfonates
Manganese
Molybdenum
Nickel
Phosphate
Phosphonates
Sulfate
Temperature
Sample temperatures below 22 C or above 28 C (72 F or 82 F) may affect test accuracy.
Zinc
Collect samples in clean, dry, plastic or glass containers.
Avoid excessive agitation or exposure to sunlight when sampling.
Rinse the container several times with the sample prior to collection.
Allow the container to overflow and cap the container so that there is no headspace above the
sample.
Rinse the sample cell several times with the reacted sample, then carefully fill to the 10-mL
mark. Perform the analysis immediately.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
180
299 g/L
295303 g/L
4 g/L
181
226 g/L
223229 g/L
3 g/L
182
600 g/L
591609 g/L
8 g/L
183
886 g/L
873899 g/L
12 g/L
184
976 g/L
962990 g/L
14 g/L
Summary of method
Diethylhydroxylamine (DEHA) or other oxygen scavengers present in the sample react with ferric
iron in DEHA Reagent 2 Solution to produce ferrous ion in an amount equivalent to the DEHA
concentration. This solution then reacts with DEHA 1 Reagent, which forms a purple color with
ferrous iron proportional to the concentration of oxygen scavenger. Test results are measured at
562 nm. This method reacts with all oxygen scavengers and does not differentiate samples
containing more than one type of oxygen scavenger.
Oxygen Scavengers
Page 942
Oxygen Scavengers

Required reagents
Description
Oxygen Scavenger Reagent Set, includes:
(2) DEHA Reagent 1 Powder Pillows
(1) DEHA Reagent 2 Solution
Quantity/Test
Unit
Catalog number
2446600
100/pkg
2167969
1 mL
100 mL
2168042
Hydrochloric Acid, 1:1, 6.0 N
varies
500 mL
88449
Water, deionized
25 mL
4L
27256
Required apparatus
Description
Quantity
Unit
Catalog number
Bottle, glass mixing, with 25-mL mark
each
1704200
Dropper, 0.5 and 1.0-mL marks
20/pkg
2124720
2/pkg
2495402
Description
Unit
Catalog number
Thermometer, Non-Mercury, 10 to 225 C
each
2635700
Optional apparatus
Oxygen Scavengers
Page 943

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Ozone, 8311
Ozone
DOC316.53.01106
Indigo Method
Method 8311
LR (0.01 to 0.25 mg/L O3),

MR (0.01 to 0.75 mg/L O3),
HR (0.01 to 1.50 mg/L O3)
AccuVac Ampul
Test preparation


Instrument
Adapter
DR 6000
DR 5000
DR 3900
LZV846 (A)
DR 3800, DR 2800, DR 2700
LZV584 (C)

Analyze sample immediately. Do not preserve for later analysis.
Use tap water or deionized water for the blank (ozone-free water)
The sequence of measuring the blank and the sample is reversed in this procedure.

Description
Quantity
Select Ozone AccuVac Ampuls based on range:

00.25 mg/L
00.75 mg/L
01.50 mg/L
Beaker, Polypropylene, 50-mL, low form with pour spout

Water, ozone-free
1
varies
Ozone
Page 945
Ozone
Method Name for powder pillows
Stored Programs
454 Ozone LR AV
455 Ozone MR AV
456 Ozone HR AV
Start
1. Select the test.

ozone-free water in a
50-mL beaker.
3. Prepared Sample:
Gently collect at least
40 mL of sample in
another 50-mL beaker.
4. Fill one Indigo Ozone

with the sample and
another with the blank.
while the Ampul fills.
Zero
5. Quickly invert both

Ampuls several times to
mix.
Some of the blue color will
be bleached if ozone is
present.
6. Wipe the Ampuls with

a cloth to remove
fingerprints or other
marks.

0.00 mg/L O3

the cell holder.
Press READ. Results are in
mg/L O3.
Insert the sample into the

cell holder.

The most important consideration when collecting a sample is to prevent the escape of ozone from
the sample. The sample should be collected gently and analyzed immediately. Warming the
sample or disturbing the sample by stirring or shaking, will result in ozone loss. After collecting the
sample, do not transfer it from one container to another unless absolutely necessary.
Stability of Indigo reagent

Because indigo is light-sensitive, the AccuVac Ampuls should be kept in the dark at all times. The
indigo solution, however, decomposes slowly under room light after filling with sample. The blank
Ampul can be used for multiple measurements during the same day.
Ozone
Page 946
Ozone
Method performance
Program
Standard
Precision
Distribution
Sensitivity
454
0.15 mg/L
0.140.16 mg/L O3
0.01 mg/L O3
455
0.45 mg/L
0.430.47 mg/L O3
0.01 mg/L O3
456
1.00 mg/L
0.971.03 mg/L O3
0.01 mg/L O3
Summary of method
The reagent formulation adjusts the sample pH to 2.5 after the Ampule has filled. The indigo
reagent reacts immediately and quantitatively with ozone. The blue color of indigo is bleached in
proportion to the amount of ozone present in the sample. Other reagents in the formulation prevent
chlorine interference. No transfer of sample is needed in the procedure, therefore ozone loss due
to sampling is eliminated. Test results are measured at 600 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Select one or more Ozone AccuVac Ampules based on range:

00.25 mg/L
25/pkg
2516025
00.75 mg/L
25/pkg
2517025
01.5 mg/L
25/pkg
2518025
Ozone
Page 947
Ozone
Required apparatus
Description
Unit
Polypropylene Beaker, 50-mL, Low Form
Catalog number
each
108041
each
2122800
6/pkg
2427606
2/pkg
2495402
Unit
Catalog number
each
2405200

Description
Snapper,
AccuVac
Water, demineralized
4L
27256
SpecCheck Gel Secondary Standard Kit, Ozone, 0 0.75 mg/L set
each
2708000
Test tube stopper
6/pkg
173106

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
PCB in Soil, 10050
Polychlorinated Biphenyls (PCB)

in Soil
DOC356.53.01107
Immunoassay Method1
Method 10050
Scope and Application: For soil

1
Test preparation


Instrument
Adapter
DR 6000
DR 5000
A23618
DR 3900
LZV846 (A)
DR 3800, DR 2800, DR 2700
LZV583

This method analyzes for PCB that has been extracted from soil samples. Sample extracts, calibrators and reagents are
added to cuvettes coated with PCB-specific antibodies. The color that develops is then measured and compared with the
color measurements of the calibrators. The test requires about 20 minutes for complete analysis. As many as 10 cuvettes
can be run simultaneously
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes and other
rack and mixing as described in Use the 1-cm MicroCuvette rack. Cuvettes can be mixed individually, but test results may not
be as consistent.
There are two protocols in this procedure, one for levels of 1 ppm and 5 ppm and another for 10 ppm and 50 ppm. Each uses
a different quantity of calibrator and sample extract See PCB protocols for more information.
Store the reagents at 4 C when they are not in use. Allow the reagents to reach room temperature before using them in an
analysis. Actual testing may be done at temperatures ranging from 1 38 C.
The Soil Extractant contains methyl alcohol which is poisonous and flammable. Before using this and other reagents, read
the Material Safety Data Sheet (MSDS) for proper use of protective equipment and other safety information.
Polychlorinated Biphenyls (PCB) in Soil

Page 949
Description
Quantity
PCB Reagent Set
Water, deionized
varies
Caps, flip spout
Marker, laboratory
Wipes, disposable
Pipet, TenSette, 0.11.0 mL and pipet tips
Soil Extraction Kit and Soil Scoop
Analytical Balance
Cylinder, graduated
Scoop, 5 g
Soil extraction procedure
1. Weigh out 5 g of soil in

the plastic weighing boat.
2. Carefully pour the soil

into an extraction vial.

Page 950
3. Use the 5-gram scoop

to add one scoop of
sodium sulfate to the
extraction vial.
4. Use the graduated

cylinder to transfer 10 mL
of Soil Extractant into the
extraction vial.

5. Cap the extraction vial

tightly and shake
vigorously for one minute.
6. Allow to settle for at

least one minute. Carefully
open the extraction vial.
7. Using the disposable

bulb pipet, withdraw 1.0
1.5 mL from the liquid
layer at the top of the
extraction vial.
Transfer it into the filtration
barrel (the bottom part of
the filtering assembly into
which the plunger inserts).
Do not use more than
1.5 mL. The pipet is
marked in 0.25-mL
increments.
8. Insert the filtration

plunger into the filtration
barrel. Press firmly on the
plunger until the sample
extract is forced upward
into the center of the
plunger.
Use the resultant filtrate
for the immunoassay in
Immunoassay for soil
extracts.
It may be necessary to
place the filtration
assembly on a table and
press down on the
plunger.
Immunoassay for soil extracts
Single Wavelength
OK
1. Press
SINGLE WAVELENGTH

button.
OK.
for orientation.
tested.

the rack snugly.

diluent solution into each
cuvette. The same pipet
tip can be used repeatedly
for this step.
Have the necessary
apparatus at hand for
the next four steps; they
must be done without
delay.

Page 951

Immunoassay for soil extracts (continued)
5. Use a Wiretrol pipet

to transfer the appropriate
volume of calibrator or
sample extract into each
cuvette (see the PCB
protocols table).
Use a separate capillary
tube for each solution.
0.5 mL of PCB Enzyme
Conjugate into each
calibrator and sample
cuvette. The same pipette
tip can be used to add the
enzyme conjugate to each
cuvette.

cuvettes into an
appropriate waste
container.

container.
The sample PCBs and

calibrator PCBs remain
attached to the cuvette
walls.
Make sure that most of the

paper towel.

Page 952

Start the reaction period.
will begin. Immediately
begin mixing the cuvettes
for 30 seconds. See Use
the 1-cm MicroCuvette
rack.

30 seconds (Use the 1-cm
MicroCuvette rack.)

Immunoassay for soil extracts (continued)
Color Development

each cuvette.
each cuvette.

Start the reaction period.
in Use the 1-cm
MicroCuvette rack.
13. After 2.5 minutes, mix

same technique as
step 12.

was added in step 11.
seconds (see Use the 1cm MicroCuvette rack.)
the Stop Solution.
Measuring the Color
Zero

smudges and fingerprints.

cell holder see Table
Instrumentspecific information for cell
orientation.
cuvettes.

0.000 Abs

holder.
Record the results (ABS)
for each calibrator
and sample.
results.

Page 953
PCB protocols
There are two protocols in this procedure, one for levels of 1 ppm and 5 ppm and another for 10
ppm and 50 ppm. Each uses a different volume of calibrator and sample extract. Refer to the PCB
protocols table for range and volume information.
Table 289 PCB protocols

Range (as Arochlor 1248)
Volume of calibrator and sample extract used
1 ppm and 5 ppm
50 L
10 ppm and 50 ppm
10 L
To test across ranges, such as 1 and 50 ppm, test the lower concentration first. If the result is
positive then test at the higher level. If the result of the test at the lower concentration is negative,
the higher range test will be negative also and need not be performed.
The same filtered extract can be used for both protocols if it is tightly capped between assays. The
maximum time between assays cannot exceed one-half hour.
Using the Wiretrol* pipet

The Wiretrol Pipet can accurately measure small quantities of liquids. It consists of two parts: a
Teflon-tipped plunger and a calibrated capillary tube. The plunger can be reused; the capillary
tubes must be discarded after one use.
1. Wet the orange

Teflon tip of the Wiretrol
plunger in the sample and
carefully insert it into the
end of the capillary tube
with the colored band.
2. Push the tip to the

other end of the capillary
tube until it barely extends
beyond the end of the
capillary tube.
3. Submerge the
capillary tube below the
surface of the liquid to be
pipetted. Slowly and
smoothly draw the Wiretrol
plunger up until the bottom
of the plunger tips reaches
the appropriate volume
line.
Touch the end of the tube
to the side of the vessel to
release remaining drops
on the capillary tube tip.
* Wiretrol is a registered trademark of Drummond Scientific.

Page 954
4. To discharge the pipet,

place the tip of the
surface of the solution
and push the Wiretrol
plunger down in one
smooth motion. Change
capillary tubes for each
calibrator and sample.
Use the 1-cm MicroCuvette rack

The MicroCuvette rack (refer to The 1-cm MicroCuvette rack figure) has been designed
specifically to aid in achieving precise and accurate results when using the immunoassay
technique to analyze several samples at the same time.
Figure 1 The 1-cm MicroCuvette rack
Loading the rackThe cuvette rack is designed so that it may be inverted with the cuvettes in

Page 955

There is an inverse relationship between the concentration of PCB and the reading. In other
words, the higher the reading, the lower the concentration of PCB.
Table 290 Relative PCB Concentration

the sample PCB Concentration is...
Example
Readings:
1 ppb PCB Calibrator: 0.775 Abs
5 ppb PCB Calibrator: 0.430 Abs
Interpretation for a soil sample
concentration of PCB is greater than both 1 ppm and 5 ppm as Aroclor 1248.
Sample #2Sample reading is between the readings for the 1 ppm and 5 ppm PCB calibrators.
Therefore the sample concentration of PCB is between 1 ppm and 5 ppm as Aroclor 1248.
sample concentration of PCB is less than both 5 ppm and 1 ppm as Aroclor 1248.
Wear protective gloves and eyewear.
When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water

Page 956
Sensitivity
The PCB immunoassay cannot differentiate between the various Arochlors, but it detects their
presence in differing degrees.
Table 291 Various PCBs in soil

Concentration (ppm) to give a positive result at
Compound
1 ppm
5 ppm
10 ppm
50 ppm
1248
10
50
1016
20
67
1242
1.2
14
50
1254
1.4
4.6
11
28
1260
1.1
4.9
11
38
Table 292 Compounds not detectable at 1000 ppm

Biphenyl
2,4,6-trichlorophenyl
1,3-dichlorobenzene
2,4-dichlorophenyl
pentachlorophenol
1,4-dichlorobenzene
2,4,5-trichlorphenyl
1,2-dichlorobenzene
1,2,4-trichlorobenzene
Analyze the samples as soon as possible after collection.
If the samples must be stored, collect them in glass or Teflon containers that have been
washed with soap and water and rinsed with methanol. The container should be capped with a
Teflon-lined cap.
If a Teflon cap is not available, aluminum foil rinsed in methanol may be used as a substitute
cap liner.
Summary of method
and soil. Antibodies specific for PCB are attached to the walls of plastic cuvettes. They selectively
bind and remove PCB from complex sample matrices. A prepared sample and a reagent
containing enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme)
are added to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and PCB
compete for binding sites on the antibodies. Samples with higher levels of analyte will have more
antibody sites occupied by PCB and fewer antibody sites occupied by the enzyme-conjugate
molecules.
color. Therefore, there is an inverse relationship between color intensity and the amount of PCB in
the sample. The resulting color is then compared with a calibrator to determine whether the PCB
concentration in the sample is greater or less than the threshold levels. The PCB concentration is
inversely proportional to the color development: the lighter the color, the higher the PCB
The method reacts with all PCBs and cannot differentiate samples containing more than one type
of PCB.

Page 957

Required reagents
Description
Unit
Catalog Number
20 cuvettes
2773500
500 mL
27249
Description
Unit
Catalog Number
2581802
PCB Reagent Set1

Deionized Water
1
Required apparatus
Caps, flip spout
2/pkg
Marker, laboratory
each
2092000
each
1970001
1000/pkg
2185628
each
4879900

Wipes, disposable
280/box
2097000
each
2936701
each
108138
Soil Scoop, 5-g, 4.25-cc
each
2657205
Soil Extraction Refill Kit, includes:
each
2775200
Dropper, LDPE, 0.5 and 1.0-mL
20/pkg
2124720
Filter and Barrel Assembly
20/pkg
2567620
Sodium Sulfate, anhydrous
250 g
709929
Soil Extractant Solution
200 mL
2567729
2592920
Soil Sample Container
20/pkg
Weighing Boat, 8.9-cm square
20/pkg
2179020
Spatula, disposable
2/pkg
2569320
Unit
Catalog Number
100/pkg
2550502

Description
Gloves, Disposable, Nitrile,
Medium1

1
50/pkg
2185696
each
2550700
Other sizes are available.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phenols, 8047
Phenols
DOC316.53.01108
USEPA1 4-Aminoantipyrine Method2
Method 8047
0.002 to 0.200 mg/L

Scope and Application: For Water and Wastewater
1
USEPA accepted (distillation required); procedure is equivalent to USEPA method 420.1 for wastewater
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2612602
DR 5000
2612602
DR 3900
2612602
DR 3800, DR 2800, DR 2700
2612602

Analyze samples within four hours to avoid oxidation.
Spilled reagent affects test results and is hazardous to skin and other materials.
Use chloroform only with proper ventilation.
Phenol 2 Reagent Powder Pillows contain potassium ferricyanide. Both chloroform (D022) and cyanide (D001) solutions are
regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the drain. Chloroform solutions
and the cotton plug used in the delivery tube of the separatory funnel should be collected for disposal as a reactive waste. Be
sure that cyanide solutions are stored in a caustic solution with a pH >11 to prevent release of hydrogen cyanide gas. Refer
to a current MSDS for safe handling and disposal information.
Phenols
Page 959
Phenols

Description
Quantity
Chloroform, ACS
60 mL
Clippers
Cotton Balls
Hardness 1 Buffer Solution, pH 10.1
10 mL
Phenol 2 Reagent Powder Pillows
Phenol Reagent Powder Pillows
Support for Ring Stand, 5 x 8 inch base
Water, deionized
300 mL
4-Aminoantipyrine Method
Stored Programs
470 Phenol
Start
1. Select the test.

Phenols
Page 960
deionized water in a
500-mL graduated
cylinder.
Pour the measured
sample in a 500-mL
graduated cylinder.
Phenols
4-Aminoantipyrine Method (continued)
5. Prepared Sample:
Pour the measured
sample into another
6. Add 5 mL of Hardness
Buffer to each separatory
funnel. Stopper and shake
to mix.

one Phenol Reagent
separatory funnel. Stopper

one Phenol 2 Reagent
9. Add 30 mL of
chloroform to each
each funnel.
10. Invert each funnel and

temporarily vent. Shake
each funnel briefly and
vent. Then vigorously
shake each funnel for a
total of 30 seconds
(venting if necessary).
11. Remove the stoppers.

Allow both funnels to stand
until the chloroform settles
to the bottom of the funnel.
12. Insert a large,

pea-sized cotton plug into
the delivery tube of each
funnel.
The chloroform layer will

be yellow to amber if
phenol is present.
Filtering the chloroform

layer through the cotton
removes suspended water
or particles. The volume of
chloroform extract will be
about 25 mL.
Note: Make sure that steps

12 through 16 are performed
quickly because chloroform
will evaporate, causing high
readings.
Phenols
Page 961
Phenols
4-Aminoantipyrine Method (continued)
Zero
13. Drain the chloroform

layers into separate
sample cells (one for the
blank and one for each
sample).


0.000 mg/L Phenol

the cell holder
Phenol.
Stopper the cells.

The water phase contains
chloroform, which is
hazardous. Dispose
properly.
Interferences
Interference level
pH
The sample pH must be between 3 and 11.5 for best results.
Oxidizing or reducing agents
May interfere. Distill samples (see procedure below).

1.
2.
Distillation or the following pretreatment is necessary:

Fill a clean 500-mL graduated cylinder with 350 mL of sample. Pour the sample into a
clean 500-mL Erlenmeyer flask.
3.
4.
Filter 300 mL of the sample through a folded filter paper1. Use this solution in step 4.
Sulfides or suspended matter
See Distillation reagents and apparatus.

Most reliable results are obtained when samples are analyzed within four hours after collection.
Use the following storage instructions only if prompt analysis is not possible:
1. Collect 500 mL of sample in clean glass containers and add the contents of two Copper
Sulfate Powder Pillows.
2. Adjust the pH to 4 or less with 10% Phosphoric Acid Solution. Store at 4 C (39 F) or lower
and analyze within 24 hours.
Phenols
Page 962
Phenols
Accuracy check
Note: For greater accuracy, analyze standard solutions when new lots of reagent are first used.
Phenol, ACS
Deionized water
1000-mL Class A volumetric flasks
10 mL Class A volumetric pipet and safety bulb
1. Prepare a 1000-mg/L phenol stock solution as follows:

a. Weigh 1.000 g of Phenol ACS.
b. Transfer the phenol to a 1000-mL volumetric flask.
c. Dilute to the mark with freshly boiled and cooled deionized water and mix to dissolve.
2. Prepare a 10-mg/L working phenol solution as follows:
a. Pipet 10.0 mL of the 1000-mg/L stock solution to a 1000-mL volumetric flask.
b. Dilute to the mark with deionized water.
3. Prepare a 0.200-mg/L standard solution as follows:
a. Pipet 10.0 mL of the 10-mg/L working solution to a 500-mL volumetric flask.
b. Dilute to the mark with deionized water.
4. Use this solution in place of the sample. Follow the 4-Aminoantipyrine Method test procedure.
to Standard Adjust in the software: OPTIONS> MORE>STANDARD ADJUST.
Distillation
Sample distillation as described in the following steps will eliminate interferences. The sample pH
must be between 3 and 11.5 for the best results. See the Interfering substances table for
pretreatment guidelines.
1. Set up the Distillation Apparatus* by assembling the general purpose apparatus as shown in
the Distillation Apparatus Manual. Use the 500-mL Erlenmeyer flask to collect the distillate. It
may be necessary to use a laboratory jack to elevate the flask.
2. Place a stirring bar into the flask.
3. Measure 300 mL of water sample in a clean 500-mL graduated cylinder. Pour it into the
distillation flask.
* See Distillation reagents and apparatus.
Phenols
Page 963
Phenols
4. For proof of accuracy, use a 0.200-mg/L phenol standard (see Accuracy check) in addition to
the sample.
5. Using a serological pipet, add 1 mL of Methyl Orange Indicator to the distillation flask.
6. Turn on the stirrer power switch. Set the stir control to 5.
7. Add 10% Phosphoric Acid Solution drop-wise until the indicator changes from yellow
to orange.
8. Add the contents of one Copper Sulfate Powder Pillow and allow to dissolve (omit this step if
copper sulfate was used to preserve the sample). Cap the distillation flask.
9. Turn the water on and adjust it so a constant flow is maintained through the condenser. Set the
heat control to 10.
10. Collect 275 mL of distillate in the Erlenmeyer flask, then turn the heat off.
11. Fill a 25-mL graduated cylinder to the 25-mL mark with deionized water. Add the water to the
distillation flask.
12. Turn the still back on. Heat until another 25-mL of distillate is collected.
13. Using a clean graduated cylinder, re-measure the distillate to make sure that 300 mL has been
collected. The distillate is ready for analysis.
Method performance
Phenols
Page 964
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
470
0.100 mg/L phenol
0.0930.107 mg/L phenol
0.002 mg/L phenol
Phenols
Summary of method
The 4-aminoantipyrine method measures all ortho- and meta-substituted phenols. These phenols
react with 4-aminoantipyrine in the presence of potassium ferricyanide to form a colored antipyrine
dye. The dye is then extracted from the aqueous phase with chloroform and the color is measured
at 460 nm. The sensitivity of the method varies with the type of phenolic compound. Because
water samples may contain various types of phenolic compounds, the test results are expressed
as the equivalent concentration of phenol.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2243900
(2) Chloroform, ACS
60 mL
4L
1445817
(3) Hardness 1 Buffer Solution, pH 10.1
10 mL
500 mL
42449
100/pkg
183699
Phenols Reagent Set (100 Tests), includes:
(2) Phenol 2 Reagent Powder Pillows

(2) Phenol Reagent Powder Pillows
Water, deionized
100/pkg
87299
300 mL
4L
27256
Quantity
Unit
Catalog number
Required apparatus
Description
Clippers
each
96800
Cotton Balls
100/pkg
257201
50841
each
each
50849
each
52049
Pipet Bulb, safety
each
1465100
each
1451537
each
58001
Support for Ring Stand, 5 x 8 inch base
each
56300
Description
Unit
Catalog number
each
2936701
50/pkg
1481866
2274400
Copper Sulfate Powder Pillows

Distillation Heater and Support Apparatus, 115 VAC
each
Distillation Heater and Support Apparatus, 230 VAC
each
2274402
Distillation Apparatus Set, general purpose
each
2265300
100/pkg
189457
Filter Paper, 12.5 cm

each
50549
Funnel, 65 mm poly
each
108367
Methyl Orange Indicator Solution, 0.5-g/L

Phenol, ACS
100 mL MDB
14832
113 g
75814
Phenols
Page 965
Phenols
Distillation reagents and apparatus (continued)
Description
Phosphoric Acid Solution, 10%
Unit
Catalog number
100 mL MDB
1476932
100/pkg
241899
100/pkg
2601300
each
2635700
each
1970010
250/pkg
2199725
50/pkg
2199796
500/pkg
1473800
each
2550700
Gloves, Chemical Resistant, 99 inch1
1 pair
2410104
each
1457453
each
1457449

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphonates, 8007
Phosphonates
DOC316.53.01109
Persulfate UV Oxidation Method1
Method 8007
Multiple Ranges from 0.02 to 125.0 mg/L
Powder Pillows
Scope and Application: For boiler and cooling water, wastewater and seawater
1
Adapted from Blystone, P., Larson, P., A Rapid Method for Analysis of Phosphate Compounds, International Water Conference, Pittsburgh,
PA. (Oct 26-28, 1981)
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Clean glassware with 1:1 Hydrochloric Acid Solution, followed by a distilled water rinse. Do not clean glassware with
commercial detergent.
Wear UV safety goggles while the UV lamp is on.
Do not handle the UV lamp surface. Fingerprints will etch the glass. Wipe the lamp with a soft, clean tissue between samples
The digestion in step 7 is normally completed in less than 10 minutes. However, high-organic loaded samples or a weak
lamp can cause incomplete phosphate conversion. To check conversion efficiency, perform a longer digestion and make
sure the readings do not increase.

Description
Quantity
Bottle, square, with 25-mL mark
Cylinder, mixing, graduated, 50-mL
Goggles, UV safety
PhosVer
3 Phosphate Reagent Powder Pillows
Potassium Persulfate Powder Pillow for Phosphonate
Phosphonates
Page 967
Phosphonates
Description
Quantity
Safety bulb
Water, deionized
varies
UV Lamp with Power Supply
Persulfate UV Oxidation method for powder pillows
Stored Programs
501 Phosphonates
Start
1. Select the test.


one Potassium Persulfate
for Phosphonate Powder
Pillow to the bottle
containing 25 mL of
sample.
Swirl to dissolve the
powder.
Phosphonates
Page 968
2. Choose the
appropriate sample size
from the Expected ranges
with multipliers table. Pipet
the chosen volume into a
50-mL graduated cylinder.
If necessary, dilute the
sample to 50-mL with
deionized water and mix
well.
10-mL mark with diluted
sample from step 2.
4. Digested Sample:
Fill a sample mixing bottle
diluted sample from
step 2.
6. Insert the ultraviolet

(UV) lamp into the sample
bottle.
7. Turn on the UV lamp.
8. When the timer

expires, turn off the UV
lamp and remove it from
the sample.
WARNING
Wear UV safety goggles
while the lamp is on.
Start the instrument timer.

period will begin.
Phosphonates are
converted to
orthophosphate in this
step.
Phosphonates
Persulfate UV Oxidation method for powder pillows
9. Prepared Sample:
to the 10-mL mark with the
digested sample.

one PhosVer 3 Phosphate
the blank and prepared
sample. Immediately swirl
vigorously 2030 seconds
to mix. Some powder may
not dissolve.

timer.
period will begin.
If the sample is colder than
15 C, allow four minutes
for color development.
12. When the timer

Complete steps 1316
within three minutes after
the timer expires.
A blue color will develop if

phosphate is present. Both
sample and blank cells
may develop color. The
increase in sample color is
proportional to
the phosphonate
concentration.
Zero
Read

0.00 mg/L PO43

the cell holder.

mg/L PO43.
Read
16. Multiply the value in

step 15 by the appropriate
multiplier in the Expected
ranges with multipliers
table to obtain the actual
phosphonate
concentration.
Table 296 Expected ranges with multipliers

Expected range
(mg/L phosphonate)
Sample volume (mL)
Multiplier
0 2.5
50
0.1
05
25
0.2
Phosphonates
Page 969
Phosphonates
Table 296 Expected ranges with multipliers
Expected range
(mg/L phosphonate)
Sample volume (mL)
Multiplier
0 12.5
10
0.5
0 25
1.0
0 125
5.0
To express results in terms of active phosphonate, multiply the final value in step 16 by the
appropriate conversion factor list in the Conversion factors by phosphonate type table.
Table 297 Conversion factors by phosphonate type

Phosphonate type
Conversion factor
PBTC
2.84
NTP
1.050
HEDPA
1.085
EDTMPA
1.148
HMDTMPA
1.295
DETPMPA
1.207
HPA
1.49
active phosphonate (mg/L) = phosphonate concentration from step 16 x conversion factor
Phosphonates
Page 970
Phosphonates
Interferences
Interference levels decrease as the sample size increases. For example, copper does not interfere
at or below 100 mg/L for a 5.00 mL sample. If the sample volume is increased to 10 mL, copper
will begin to interfere above 50 mg/L.

Interference level (using 5 mL of sample)
Aluminum
100 mg/L
Arsenate
Benzotriazole
10 mg/L
Bicarbonate
1000 mg/L
Bromide
100 mg/L
Calcium
5000 mg/L
CDTA
100 mg/L
Chloride
5000 mg/L
Chromate
100 mg/L
Copper
100 mg/L
Cyanide
100 mg/L (Increase the UV digestion to 30 minutes.)
Diethanoldithiocarbamate
50 mg/L
EDTA
100 mg/L
Iron
200 mg/L
Nitrate
200 mg/L
NTA
250 mg/L
Orthophosphate
15 mg/L
Phosphites and
organophosphorus
compounds
Reacts quantitatively. Meta- and polyphosphates do not interfere.
Silica
500 mg/L
Silicate
100 mg/L
Sulfate
2000 mg/L
Sulfide
Sulfite
100 mg/L
Thiourea
10 mg/L

extreme sample pH
Collect samples in acid-cleaned (1:1 HCl) plastic or glass bottles that have been rinsed with
distilled water. Do not use a commercial detergent.
If prompt analysis is impossible, preserve the sample by adjusting to pH 2 or less with Sulfuric
Acid (about 2 mL per liter).
Store at 4 C (39 F). Preserved samples may be stored up to 24 hours.
Phosphonates
Page 971
Phosphonates
Accuracy check
Ideally, prepare a solution containing the exact phosphonate product to be tested. This will check
the UV conversion of phosphonate to orthophosphate. Alternatively, a phosphate standard can be
used to check the accuracy of the colorimetric part of the method.
Phosphate Standard Solution, 1 mg/L
Deionized water
Use 10 mL of a Phosphate Standard Solution, 1 mg/L solution in place of the sample
beginning at step 9 of the Persulfate UV Oxidation method for powder pillows test procedure.
Use deionized water for the blank. A multiplier value from the Expected ranges with multipliers
table is not needed. The expected result is 10.0 mg/L phosphate, due to a factor of 10
in the calibration.
Method performance
Program
Instrument
Standard
Precision95% Confidence Limits of

Distribution
501
DR 5000
2.00 mg/L PO43
1.972.03 mg/L PO43
Sensitivity
The sensitivity depends on the sample volume. Sensitivity is expressed as PO43 in this table. Use
the Conversion factors by phosphonate type table to express as a specific phosphonate.
Range (mg/L)
Volume (mL)
Change in concentration per 0.010 Abs
02.5
50
0.02 mg/L PO43
05
25
0.04 mg/L PO43
012.5
10
0.10 mg/L PO43
025
0.20 mg/L PO43
0125
1.00 mg/L PO43
Summary of method
This method is directly applicable to boiler and cooling tower samples. The procedure is based on
a UV-catalyzed oxidation of phosphonate to orthophosphate. The orthophosphate reacts with the
molybdate in the PhosVer 3 reagent to form a mixed phosphate/molybdate complex. This complex
is reduced by the ascorbic acid in the PhosVer 3, yielding a blue color that is proportional to the
phosphonate present in the original sample. The orthophosphate present in the original sample is
subtracted out by preparing the blank and using it to set zero concentration. Test results are
measured at 880 nm.
Phosphonates
Page 972
Phosphonates

Required reagents
Description
Quantity/Test
Unit
Catalog number
Phosphonate Reagent Set for 10-mL sample (100 tests), includes:
2429700
PhosVer 3 Phosphate Reagent Powder Pillows, 10-mL
100/pkg
2106069
Potassium Persulfate Powder Pillow for Phosphonate
100/pkg
2084769
varies
4L
27256
Water, deionized
Required apparatus
Description
Quantity
Unit
Catalog number
Bottle, square, with 25-mL mark
each
1704200
Polypropylene Beaker, 50-mL, low form
each
108041
Cylinder, mixing, graduated, 50-mL
each
189641
Goggles, UV safety
each
2113400
Pipet, serological, graduated, 10-mL
each
53238
Safety Bulb
each
1465100
UV Lamp with Power Supply, 115 VAC
each
2082800
OR
UV Lamp with Power Supply, 230 VAC
each
2082802
2/pkg
2495402
Description
Phosphate Standard Solution, 1-mg/L
Unit
Catalog number
500 mL
256949
Unit
Catalog number

Description
Hydrochloric Acid Solution, 1:1
500 mL
88449
Sulfuric Acid, ACS Grade
500 mL
97949
each
2635700
Thermometer, 10 to 225 C, 405 mm

pH paper, 014 pH
Ampule Breaker
Phosver 3 Phosphate Reagent Powder Pillows, 10 mL
UV Lamp, shortwave, pencil type
100/pkg
2601300
each
2196800
1000/pkg
2106028
each
2671000
Power Supply, 115V/60Hz
each
2670700
Power Supply, 220V/50Hz
each
2670702
Phosphonates
Page 973
Phosphonates
Optional standards
Description
Unit
Catalog number
946 mL
2059716
946 mL
1420416
100 mL
1424342
1436716
946 mL
Phosphate Standard Solution, 50 mg/L, 10 mL ampules
16/pkg
17110
100 mL
1436832
Phosphate Standard Solution, 500 mg/L, 10 mL ampules
10/pkg
1424210
Phosphate Standard Solution
100 mL
1424232

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Acid Hydrolyzable Digestion, 8180
Phosphorus, Acid Hydrolyzable

Digestion
USEPA1 Acid Digestion Method2
DOC316.53.01110
Method 8180
Scope and Application: For water, wastewater, and seawater

1
USEPA Accepted for wastewater analyses
Adapted from Standard Methods for the Examination of Water and Wastewater 4500-P B & E
Test preparation

Rinse all glassware with 1:1 hydrochloric acid. Rinse again with deionized water.
The results of the reactive phosphorus test after the digestion will include the orthophosphate and the acid-hydrolyzable
(condensed) phosphate. The condensed phosphate concentration is determined by subtracting the result of an
orthophosphate test from this result. Make sure that both results are in the same units, either mg/L PO43 or mg/L P before
subtracting. The result from this Acid Hydrolyzable test is subtracted from the result of a total phosphorus test to determine
organic phosphorus.

Description
Quantity
2 mL
2 mL
Water, deionized
varies
Hot Plate
Phosphorus, Acid Hydrolyzable Digestion

Page 975

Acid digestion
1. Use a graduated
of sample. Pour the
Erlenmeyer flask.
2. Use a 1-mL calibrated

dropper to add 2.0 mL of
Solution to the flask.

hot plate. Boil gently for 30
minutes. Do not boil dry.
Concentrate the sample to
less than 20 mL for best
recovery. After
concentration, maintain
the volume near 20 mL by
adding small amounts of
deionized water. Do not
exceed 20 mL.
480 P React. Mo
482 P React. Mo. AV
485 P React. Amino
490 P React. PV
492 P React. PV AV
535 P React. PV TNT
540 P React. HT TNT

Solution to the flask. Swirl
to mix.

Adjust the volume to 25
mL with deionized water
rinsings from the flask.
7. Proceed with a
reactive phosphorus test
of the expected acid
hydrolyzable phosphorus
concentration range.
Extend the color
development time to
10 minutes for the
PhosVer 3 method.

Page 976
4. Cool the sample to

room temperature.
Interferences
Interference level
Alkaline or highly buffered

samples
It may be necessary to add additional acid in step 2 to drop the pH of the solution below 1.
Turbidity
Use 50 mL of sample and double the reagent quantities. Use a portion of the digested
sample to zero the instrument in the reactive phosphorus procedure. This compensates for
any color or turbidity destroyed by this procedure.
Analyze the samples immediately for the most reliable results.
If prompt analysis is not possible, samples may be preserved up to 28 days. Filter immediately
and store at 4 C (39 F).
Summary of method
Phosphates present in condensed inorganic forms (meta-, pyro- or other polyphosphates) must be
converted to reactive orthophosphate before analysis. Pretreatment of the sample with acid and
heat hydrolyzes the condensed inorganic forms to orthophosphate.
This procedure must be followed by one of the reactive phosphorus (orthophosphate) analysis
methods for determining the phosphorus content of the sample. If the ascorbic acid (PhosVer 3)
method is used to measure the reactive phosphorus, this method is USEPA accepted for NPDES
reporting.

The following reagents and apparatus are required in addition to those required for the active
phosphorus test.
Required reagents
Description
Quantity/Test
Unit
Catalog number
2 mL
100 mL MDB
245032
2 mL
100 mL MDB
244932
Water, deionized
varies
4L
27256
Quantity
Unit
Catalog number
Required apparatus
Description
each
50840
each
50543
Hot Plate, 7 x 7 Digital, 120 VAC
each
2881500
Hot Plate Stirrer, 7 x 7 Digital, 220240 VAC
each
2881602

Page 977
Required apparatus (field applications)

Description
Unit
Heatab Cookit, with 1 box Heatabs

Heatab Replacements
Catalog number
each
220600
21/pkg
220700
Unit
Catalog number
245053

Description
Sodium, Hydroxide, 5.0 N
1000 mL
500 mL
97949
100/pkg
2601300
Filter Paper, folded 12.5 cm
100/pkg
69257
each
108367
each
2635700
Filter Funnel, Analytical, 65 mL

500 mL
88449
12/pkg
2087076

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Acid Hydrolyzable, 8180
DOC316.53.01111
PhosVer 3 with Acid Hydrolysis Method

0.06 to 3.50 mg/L
Method 8180
PO43
Test N Tube Vials
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

adjust.
Clean glassware with 1:1 Hydrochloric Acid Standard Solution. Rinse with deionized water. Do not use detergents that
contain phosphate to clean glassware.
Final samples will contain molybdenum. In addition, final samples will have a pH less than 2 and are considered corrosive
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.

Description
Total and Acid Hydrolyzable Phosphorus Reagent Set
Quantity
1
Deionized water
varies
DRB200 Reactor
Funnel, micro
Pipet, TenSette, 1 to 10 mL, plus tips

Test Tube Rack

Page 979

Acid Hydrolysis, TNT method
Stored Programs
536 P Total/AH PV TNT
Start

Reactor. Preheat to
150 C.
See the DRB200 User
Manual for selecting preprogrammed temperature
applications.

timer.
A 30-minute heating
period will begin.

Instrument-specific
information).
2. Select the test.

to add 5 mL of sample to a
Total and Acid
Hydrolyzable Test Vial.
Cap and mix.
4. Insert the vial into the

preheated DRB200
reactor. Close the
protective cover.

carefully remove the vial
from the reactor. Insert it in
a test tube rack and cool to
room temperature.
7. Using a TenSette
Pipet, add 2 mL of 1.00 N
sodium hydroxide to the
vial. Cap tightly and shake
to mix.

the vial with a towel to
other marks.

the contents of one
PhosVer 3 Powder Pillow
to the vial.
12. Immediately cap

tightly and shake to mix for
1015 seconds. The
powder will not completely
dissolve.
Zero

into the 16-mm round cell
holder.

0.00 mg/L PO43

Page 980

Acid Hydrolysis, TNT method (continued)
Read

timer.
period will begin.

the vial with a towel to
other marks.

holder.

mg/L PO43.
Read results within two to

eight minutes after adding
the PhosVer 3 reagent.
Interferences
Interference level
Aluminum
Arsenate
All levels
Chromium
Copper
Iron
Nickel
Silica
Silicate
Sulfide
Greater than 9 mg/L. Remove sulfide interference as follows:

1. Measure 25 mL of sample into a 50-mL beaker.
2. Swirling constantly, add Bromine Water drop-wise until a permanent
yellow color appears.
3. Swirling constantly, add Phenol Solution drop-wise just until the yellow color disappears.
Proceed with step 1.
Turbidity
Large amounts may cause inconsistent results in the test because the acid present in the
powder pillows may dissolve some of the suspended particles and because of variable
desorption of orthophosphate from the particles.
Zinc

extreme sample pH

Page 981

Collect samples in plastic or glass bottles that have been acid washed with 1:1 Hydrochloric Acid
Solution and rinsed with deionized water. Do not use commercial detergents containing phosphate
for cleaning glassware used in this test.
Analyze samples immediately for best results. If prompt analysis is not possible, preserve samples
by filtering immediately and storing the sample at 4 C (39 F) for up to 48 hours.
Accuracy check
Phosphate 2-mL Ampule Standard, 50-mg/L as PO43
Ampule breaker
ADDITIONS.
6. Follow the Acid Hydrolysis, TNT method test procedure for each of the spiked samples,
Phosphate Standard Solution. 3.0-mg/L
1. Use the Phosphate Standard Solution. 3.0-mg/L solution in place of the sample. Follow the
Acid Hydrolysis, TNT method test procedure.
to Standard Adjust in the softwarE: OPTIONS>MORE>STANDARD ADJUST. .

Page 982
Method performance
Program
Instrument
Standard
SensitivityDConcentration
per 0.010 DAbs
536
DR 5000
3.00 mg/L PO43
2.933.07 mg/L PO43
0.06 mg/L PO43
Summary of method
Phosphates present in condensed inorganic forms (meta-, pyro-, or other polyphosphates)
must be converted to reactive orthophosphate before analysis. Pretreating the sample with
acid and heat hydrolyzes the condensed inorganic forms to orthophosphate.
Orthophosphate reacts with molybdate in an acid medium to produce a mixed phosphate/
molybdate complex. Ascorbic acid then reduces the complex, giving an intense molybdenum
blue color. Test results are measured at 880 nm.

Page 983

Required reagents
Description
Quantity/Test
Total and Acid Hydrolyzable Phosphorus Reagent Set, includes:

PhosVer 3 Phosphate Reagent Powder Pillows
1 pillow
Potassium Persulfate Powder Pillows
Catalog number
2742745
50/pkg
2106046
1 pillow
50/pkg
2084766
varies
100 mL
2743042
2 mL
100 mL
104542
Total and Acid Hydrolyzable Test Vials1
1 vial
50/pkg
varies
100 mL
27242
Quantity
Unit
Catalog number
Water, deionized
1
Unit
50 tests
Required apparatus
Description
each
LTV082.53.40001
each
LTV082.52.40001
Funnel, micro
each
2584335
each
1451536
each
1451537
each
1465100
Pipet, TenSette, 1 to 10 mL
each
1970010
250/pkg
2199725
Test Tube Rack
each
1864100
Unit
Catalog number
500 mL
2833049
16/pkg
17110
Description
Drinking Water Standard, Mixed Parameter, Inorganic for F, NO3, PO4, SO4
Phosphate Standard Solution, 10-mL
Voluette
Ampule, 50-mg/L as PO4
Phosphate Standard Solution, 1-mg/L as PO43
500 mL
256949
946 mL
2059716
500 mL
2833249
Phosphate Standard Solution, 3 mg/L as PO4
Wastewater Standard, Effluent Inorganics, for NH3N, NO3N, PO4, COD, SO4, TOC

Page 984

Description
Unit
Catalog number
29 mL
221120
each
189640
Hydrochloric Acid Solution, 6.0 N, 1:1
500 mL
88449
29 mL
211220
100/pkg
2601300
100/pkg
69257
each
108367

each
2635700
12/pkg
2087076
50/pkg
2199796
each
50041H
Description
Unit
Catalog number
each
2196800
Beaker, 50 mL, Glass
Optional standards
Phosphate, Standard Solution, 3 mg/L
946 mL
2059716
946 mL
1420416
100 mL
1424342
946 mL
1436716
Phosphate, Standard Solution, 50 mg/L, 10 mL ampules
16/pkg
17110
100 mL
1436832
Phosphate, Standard Solution, 500 mg/L, 10 mL ampules
10/pkg
1424210
Phosphate, Standard Solution
100 mL
1424232

Page 985

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive, RSAV, 8114
(Orthophosphate)
DOC316.53.01115
Molybdovanadate Method1
0.3 to 45.0 mg/L
Method 8114
PO43

1
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

For best results, sample temperature should be 2025 C (6877 F).
After adding reagent, a yellow color will form if phosphate is present. The blank will be slightly yellow because of the reagent.
Description
Quantity
Liquid Reagent Test:

Molybdovanadate Reagent
1.0 mL
2
AccuVac Test:
Molybdovanadate Reagent AccuVac Ampuls
Beaker, 50-mL
Phosphorus, Reactive (Orthophosphate)

Page 987

Molybdovanadate reagent solution
Stored Programs
480 P React. Mo.
Start
1. Select the test.

10 mL of deionized water.

10 mL of sample.
4. Add 0.5 mL of
Molybdovanadate
Reagent to each sample
cell. Swirl to mix.
Zero

timer.
will begin.
6. When the timer

holder.
If the sample
concentration is greater
than 30 mg/L PO43, read
at exactly seven minutes
or make a 1:1 dilution of
the sample and repeat the
test

Page 988

0.0 mg/L PO43

the cell holder.
PO43.

Molybdovanadate for AccuVac Ampuls
Stored Programs
482 P React. Mo. AV
Start
1. Select the test.

for orientation.
2. Prepared Sample:
Collect 40 mL of sample in
one 50-mL beaker. Fill a
Molybdovanadate
with sample.
Collect 40 mL of deionized
water in another 50-mL
beaker.

timer.
Fill another Ampul with

tips immersed while the
Ampuls fill completely.
If the sample
the sample and repeat the
test.
Zero
5. When the timer

holder.

0.0 mg/L PO43

will begin.
Read

the cell holder.

mg/L PO43.
Interferences
The Interfering substances table shows interference levels and types of interference. The
Noninterfering substances at low concentrations (less than 1000 mg/L) table shows substances
that do not interfere in concentrations less than 1000 mg/L.

Interference level
Arsenate
Only interferes if sample is heated.
Iron, ferrous
Blue color caused by ferrous iron does not interfere if concentration is less than 100 mg/L.
Molybdate
Causes negative interference above 1000 mg/L.
Silica
Only interferes if sample is heated.

Page 989

Interference level
Causes a negative interference.
1. Measure 50 mL of sample into an Erlenmeyer flask.
Sulfide
2.
Add Bromine Water1 drop-wise with constant swirling until a permanent yellow color
develops.
3.
Add Phenol Solution1 drop-wise until the yellow color just disappears. Proceed with
step 3 (step 2 if using AccuVac procedure).
pH, extreme or
highly buffered samples
May exceed buffering capacity of reagents. May require pretreatment. pH should be about 7.
Fluoride, thorium, bismuth,

thiosulfate or thiocyanate
Cause negative interference.
Table 304 Noninterfering substances at low concentrations (less than 1000 mg/L)
Pyrophosphate
Tetraborate
Citrate
Lactate
Benzoate
Formate
Oxalate
Tartrate
Salicylate
Al3+
Fe3+
Mg2+
Ca2+
Ba2+
Sr2+
Li+
Na+
K+
NH4+
Cd2+
Mn2+
NO3
NO2
SO42
SO32
Pb2+
Hg+
Hg2+
Sn2+
Cu2+
Ni2+
Ag+
U4+
Zr4+
AsO3
CO3
IO3
ClO4
SiO44
Br
CN
Selenate
Collect samples in clean plastic or glass bottles that have been cleaned with 1:1 Hydrochloric
Acid Solution* and rinsed with deionized water.
Do not use a commercial detergent. The phosphate content will contaminate the sample.
Analyze samples as soon as possible for best results.
If samples cannot be analyzed promptly, store the sample for up to 48 hours at 4 C (39 F) or
below.
Warm stored samples to room temperature before analyzing.

Page 990
Accuracy check
Phosphate 2-mL Ampule Standard, 500-mg/L PO43
Ampule breaker
ADDITIONS.
6. Pour 10 mL of the spiked samples into three 10 mL sample cells or for AccuVacs, pour 10 mL
of the spiked samples into a beaker.
7. Follow the Molybdovanadate for AccuVac Ampuls test procedure for each of the spiked
instrument.
Phosphate standard solution, 10-mg/L
1. Use the Phosphate standard solution, 10-mg/L, in place of the sample. Follow the
Molybdovanadate for AccuVac Ampuls test procedure.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
480
30.0 mg/L PO43
29.630.4 mg/L PO43
0.3 mg/L PO43

Page 991
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
482
30.0 mg/L PO43
29.730.3 mg/L PO43
0.3 mg/L PO43
Summary of method
In the molybdovanadate method, orthophosphate reacts with molybdate in an acid medium to
produce a mixed phosphate/molybdate complex. In the presence of vanadium, yellow
molybdovanadophosphoric acid is formed. The intensity of the yellow color is proportional to the
phosphate concentration. Test results are measured at 430 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
1.0 mL
100 mL MDB
2076032
OR
Molybdovanadate Reagent AccuVac Ampuls
25/pkg
2525025
25 mL
4L
27256
Quantity
Unit
Catalog number
6/pkg
173106
Quantity
Unit
Catalog number
Beaker, 50-mL
each
50041H
AccuVac snapper
each
2405200
2122800
Water, deionized
Required apparatus (reagent liquid)

Description
Required apparatus
Description
Sample cell, 10 mL, round, 25 x 54 mm
each
6/pkg
2427606
2/pkg
2495402
Description
PourRite
Ampule, 500 mg/L as PO4
Wastewater Influent Standard, for mixed parameters NH3N, NO3N, PO4, COD, SO4,
TOC

Page 992
Unit
Catalog number
946 mL
1420416
16/pkg
1424210
500 mL
2833149
each
2196800

Description
Catalog number
29 mL
221120
each
189640
Hydrochloric Acid, 6.0 N 1:1
500 mL
88449
29 mL
211220
each
1970001
50/pkg
2185696
1000/pkg
2185628
100/pkg
2601300
each
2635700
12/pkg
2087076
each
4103600
Unit
Catalog number
946 mL
2059716
100 mL
1424342
946 mL
1436716

AccuVac ampule drainer
1
Unit
Optional standards
Description
Phosphate, Standard Solution, 50 mg/L, 10 mL Voluette Ampules
16/pkg
17110
100 mL
1436832
100 mL
1424232

Page 993

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive, Amino Acid, 8178
(Orthophosphate)
DOC316.53.01113
Amino Acid Method1

0.23 to 30.00 mg/L
Method 8178
PO43

1
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

adjust
The contents of one Amino Acid Reagent Powder Pillow may be substituted for 1 mL of amino acid reagent solution in
step 4.

Description
Amino Acid Reagent
Cylinder, 25-mL, graduated, mixing
Molybdate Reagent
Quantity
1 mL
1
1 mL
2

Page 995

Amino Acid Method
Stored Programs
485 P React. Amino
Start
1. Select the test.

2. Fill a 25-mL mixing

sample.
3. Add 1 mL of
Molybdate Reagent using
a 1-mL calibrated dropper.
4. Prepared Sample: Add

1 mL of Amino Acid
Reagent Solution. Stopper
mix.
A blue color will form if
phosphate is present.
Zero

timer.
period will begin. Continue
with step 6 while the timer
is running.
untreated sample.
7. When the timer

holder.
Read
9. Fill a second cell with

prepared sample.

the cell holder.

Page 996

mg/L PO43.

0.00 mg/L PO43
Interferences
Interference level
Calcium
Greater than 10,000 mg/L as CaCO3
Chloride
Greater than 150,000 mg/L Cl
Colored samples
Add 1 mL of 10 N Sulfuric Acid Standard Solution1 to another 25-mL sample. Use this
instead of untreated sample as the blank to zero the instrument. Use a pipet and pipet filler to
measure the sulfuric acid standard.
High salt levels (Na+)
May cause low results. To eliminate this interference, dilute the sample until two successive
dilutions yield about the same result.
Magnesium
Nitrite (NO2)
Bleaches the blue color. Remove nitrite interference by adding 0.05 g of sulfamic acid to the
sample. Swirl to mix. Continue with step 4.
Phosphates, high levels

(PO43)
As the concentration of phosphate increases, the color changes from blue to green, then
to yellow and finally to brown. The brown color may suggest a concentration as high as
100,000 mg/L PO43. If a color other than blue is formed, dilute the sample and retest.
Sulfide interferes. For samples with sulfide concentration less than 5 mg/L sulfide
interference may be removed by oxidation with Bromine Water as follows:
1. Measure 50 mL of sample into an Erlenmeyer flask.
Sulfide (S2)
2.
Add Bromine Water1 drop-wise with constant swirling until permanent

yellow color develops.
3.
Add Phenol Solution1 drop-wise until the yellow color just disappears. Use this solution
in steps 2 and 6.
Temperature
For best results, sample temperature should be 21 3 C (70 5 F).
Turbidity
May give inconsistent results for two reasons. Some suspended particles may dissolve
because of the acid used in the test. Also, desorption of orthophosphate from particles may
occur. For highly turbid samples, add 1 mL of 10 N Sulfuric Acid Standard Solution1 to
another 25-mL sample. Use this instead of untreated sample as the blank to zero the
instrument. Use a pipet and pipet filler to measure the sulfuric acid standard.

extreme sample pH
Acid Solution and rinsed with deionized water.
Do not use a commercial phosphate-based detergent for cleaning glassware. The phosphate
content will contaminate the sample.
Analyze samples immediately for best results.
If prompt analysis is not possible, preserve samples by filtering immediately and store at 4 C
(39 F) for up to 48 hours.
The sample should have a neutral pH (68) and be at room temperature before analysis.

Page 997
Accuracy check
Ampule breaker
ADDITIONS.
6. Follow the Amino Acid Method test procedure for each of the spiked samples, starting with the
10-mg/L Phosphate Standard Solution
1. Use the 10-mg/L Phosphate Standard Solution in place of the sample. Follow the Amino Acid
Method test procedure.
Method performance
Program
Instrument
Standard
per 0.010 Abs
485
DR 5000
10.00 mg/L PO43
9.8610.14 mg/L PO43
0.20 mg/LPO43
Summary of method
In a highly acidic solution, ammonium molybdate reacts with orthophosphate to form
molybdophosphoric acid. This complex is then reduced by the amino acid reagent to yield an
intensely colored molybdenum blue compound. Test results are measured at 530 nm.

Page 998

Required reagents
Description
Quantity/Test
Unit
Catalog number
100 tests
2244100
Amino Acid Reagent
1 mL
100 mL MDB
193432
Molybdate Reagent
1 mL
100 mL MDB
223632
Quantity/Test
Unit
Catalog number
each
189640
Unit
Catalog number
High Range Reactive Phosphorus Reagent Set, includes:
Required apparatus
Description
Cylinder, 25-mL, graduated, mixing
Description
946 mL
1420416
16/pkg
1424220
Wastewater Effluent Standard, for mixed parameters NH3N, NO3N, PO4, COD, SO4,
TOC
500 mL
2833249
Wastewater Influent Standard for mixed parameters NH3N, NO3N, PO4, COD, SO4,
TOC
500 mL
2833149
Water, deionized
4 liters
27256
each
2484600
PourRite Ampule breaker, 2 mL
PourRite
Ampule, 500-mg/L
PO43

Page 999

Description
Unit
Amino Acid Reagent Powder Pillow

100/pkg
80499
29 mL
221120
each
50543
Hydrochloric Acid Solution, 1:1, 6.0 N
500 mL
88449
Phenol Solution, 30g/L
29 mL
211220
454 g
234401
Sulfamic Acid
Sulfuric Acid Standard Solution, 10 N
1000 mL
93153
each
1970001
50/pkg
2185696
1000/pkg
2185628
100/pkg
2601300
100/pkg
69257
each
108367

Thermometer, Non-Mercury, -10 to 225C
each
2635700
12/pkg
2087076
Description
Unit
Catalog number
each
2196800

1
Catalog number
Optional Standards
946 mL
2059716
100 mL
1424342
1436716
946 mL
16/pkg
17110
100 mL
1436832
16/pkg
1424210
100 mL
1424232

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive HR, 8114
DOC316.53.01114
Molybdovanadate Rapid Liquid Method1

HR (0.3 to 45.0 mg/L PO4
Method 8114
3)
Pour-Thru Cell
Scope and Application: For treated and natural waters

1
Test preparation


Instrument
Pour-Thru Kit
Pour-Thru Cell Orientation
Adapter
DR 6000
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
DR 3800, DR 2800, DR 2700
LQV157.99.10002

compartment
5940400
1-inch (round) path aligned with arrow on

the adapter
LZV585 (B)

See the User Manual for Pour-Thru Module installation instructions.
Clean the Pour-Thru cell and all labware as specified in Analysis labware treatment.
Make sure the PourThru cell is completely seated in the sample cell compartment.
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal information.
Description
Quantity
2 mL
Water, deionized
25 mL
Dispenser, fixed volume, 1.0-mL, w/bottle
Flask, Erlenmeyer, 125-mL, PMP w/cap
Instrument-specific information
Page 1001
Description
Quantity
Molybdovanadate Rapid Liquid method
Stored Programs
489 P React. Mo. HR RL
Start
1. Select the test.
2.
5. Rinse another clean

plastic 125-mL Erlenmeyer
flask with deionized water.
6. Prepared Sample:
Measure 25 mL of sample
in the graduated cylinder.
Pour the water into the
flask.
3. Rinse a clean plastic

and a 25-mL graduated
cylinder with deionized
water.
Measure 25 mL of
deionized water in the
graduated cylinder. Pour
the water into the flask.
7. Add 1.0 mL of
Molybdovanadate
Reagent to each flask
using a Repipet Jr.
Dispenser. Swirl to mix.

timer.

in the sample if phosphate
is present. A small amount
of yellow may be present
in the blank due to the
reagent.
Page 1002

will begin.
If the sample
the sample and begin the
test again.
Molybdovanadate Rapid Liquid method (continued)
Zero
9. When the timer

expires, pour the blank
from the flask into the
Pour-Thru Cell.

0.0 mg/L PO43
Read

sample from the flask into
the Pour-Thru Cell.

mg/L PO43.
Flush the Pour-Thru Cell
water.
Interferences
See the Interfering substances table for a list of substances, interference levels and type of
interference. See the Noninterfering substances at low concentrations (less than 1000 mg/L) table
for a list of substances that do not interfere in concentrations less than 1000 mg/L.

Interference level
Arsenate
Negative interference. Positive interference if sample is heated.
Bismuth
Negative interference.
Fluoride
Iron, Ferrous
Blue color is caused by ferrous iron but this does not affect results if the ferrous iron
concentration is less than 100 mg/L.
Molybdate
Silica
Positive interference if sample is heated.

Negative interference. Sulfide interference may be removed by oxidation with Bromine Water
as follows:
1. Measure 25 mL of sample into a flask.
Sulfide
2.
Add Bromine Water1 drop-wise with constant swirling until permanent yellow color
develops.
3.
Add Phenol Solution1 drop-wise until the yellow color just disappears. Proceed with
step 7.
Thiocyanate
Thiosulfate
Thorium

extreme sample pH
Page 1003
Pyrophosphate
Tetraborate
Citrate
Lactate
Benzoate
Formate
Oxalate
Tartrate
Salicylate
Al3+
Selenate
Mg2+
Ca2+
Ba2+
Sr2+
Li+
Na+
K+
NH4+
Cd2+
Mn2+
NO3
NO2
SO42
SO32
Pb2+
Hg+
Hg2+
Sn2+
Cu2+
Ni2+
Ag+
Zr4+
AsO3
Br
CO32
ClO4
CN
IO3
Fe3+
SiO44
Do not use detergents that contain phosphate for cleaning labware.
If prompt analysis is not possible, preserve samples by filtering immediately and storing the
sample at 4 C (39 F) or below for up to 48 hours.
Analysis labware treatment

1. Clean containers by normal means (do not use detergents containing phosphorus), then rinse
2. Soak for several minutes in a 1:25 dilution of Molybdovanadate Reagent in deionized water.
3. Rinse well with deionized water. Dedicate these containers for HR PO43 analysis.
4. Fill the Pour-Thru Cell with this same mixture of Molybdovanadate reagent and deionized
water and let stand for several minutes.
5. Rinse the Pour-Thru Cell with 50 mL of deionized water.
Page 1004
How to clean the Pour-Thru Cell

The Pour-Thru Cell may accumulate a buildup of colored products, especially if the reacted
solutions are allowed to stand in the cell for long periods after measurement. To clean the PourThru Cell:
1. Remove the color by rinsing with a 1:5 dilution of ammonium hydroxide*
2. Follow with several deionized water rinses.
3. Invert a beaker over the glass funnel of the Pour-Thru Cell when not in use.
Accuracy check
Phosphate Voluette Ampule Standard Solution, 500-mg/L as PO43
Ampule breaker
ADDITIONS.
standard to three 25-mL mixing cylinders and fill to 25 mL with the fresh sample. Mix
thoroughly.
6. Follow the Molybdovanadate Rapid Liquid method test procedure for each of the spiked
instrument.
* Optional reagents and apparatus.
Page 1005
10.0-mg/L Phosphate Standard
1. Use the 10.0-mg/L Phosphate Standard solution in place of the sample. Follow the
Molybdovanadate Rapid Liquid method test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
489
10.0 mg/L PO43
9.910.1 mg/L PO43
0.34 mg/L PO43
Summary of method
In the molybdovanadate method, orthophosphate reacts with molybdate in an acid medium to
produce a phosphomolybdate complex. In the presence of vanadium, yellow
vanadomolybdophosphoric acid is formed. The intensity of the yellow color is proportional to the
phosphate concentration. Test results are measured at 430 nm.
Page 1006

Required reagents
Description
Quantity/Test
Unit
Catalog number
2 mL
500 mL
2076049
Water, deionized
25 mL
4L
27256
Catalog number
Required apparatus
Description
Quantity
Unit
each
108140
Dispenser, adjustable
each
2563137
each
2089843
Unit
Catalog number
946 mL
1420416
16/pkg
1424210
500 mL
2833149
Unit
Catalog number
Description
Phosphate Standard Solution, Voluette ampule, 10-mL, 500-mg/L as PO43

Wastewater Influent Standard for NH3N, NO3N, PO4, COD, SO4, TOC

Description
Ammonium Hydroxide, ACS
500 mL
10649
29 mL
221120
each
189640

500 mL
88449
29 mL
211220
each
1970001
Pipet, TenSette, Pipet, 0.1 - 1.0 mL

19700011
50/pkg
2185696
1000/pkg
2185628
100/pkg
69257
each
108367

1
each
2635700
12/pkg
2087076
Page 1007
Optional standards
Description
Unit
Catalog number
each
2196800
946 mL
2059716
100 mL
1424342
1436716
946 mL
16/pkg
17110
100 mL
1436832
100 mL
1424232

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive, RL, LR, 10055
DOC316.53.01117
Ascorbic Acid Rapid Liquid Method1

LR (19 to 3000 g/L
Method 10055
PO43)
Pour-Thru Cell
Scope and Application: For treated and natural waters

1
Test preparation


Instrument
Pour-Thru Kit
Adapter
DR 6000
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
DR 3800, DR 2800, DR 2700
LQV157.99.10002
5940400

compartment
1-inch (round) path aligned with arrow on the

adapter
LZV585 (B)

See the user manual for Pour-Thru Module installation instructions.
Clean the Pour-Thru cell and all labware as specified in Analysis labware treatment.
See Reagent preparation for preparing the Ascorbic Acid reagent.
Reaction time depends on sample temperature. For most accurate results, samples should be at room temperature
(about 20 C).
Obtain a reagent blank for each lot of reagent when the normal sample phosphate concentration is less than 750 g/L.
Follow the procedure using deionized water in place of the sample. Subtract the reagent blank value from the final results.
Page 1009

Description
Quantity
Ascorbic Acid Reagent Dilution Solution
1 mL
Ascorbic Acid Reagent Powder
varies
Cylinder, graduated, 25 mL, poly
Dispenser, bottle top
Molybdate Reagent Solution
2 mL
Pour-Thru Cell Assembly
Water, deionized
varies
Ascorbic acid method, rapid liquid
Stored Programs
488 P React. LR RL
Start
1. Select the test.


the 25-mL cylinder into
one of the flasks.
Page 1010
2. Rinse two clean

Erlenmeyer flasks three
times with the sample.
3. Rinse a clean 25-mL

three times with the
sample.

sample.
6. Measure a second
25-mL portion of sample
into the graduated cylinder
and pour the contents into
the second flask.
7. Add 1.0 mL of
Molybdate reagent to each
flask using a bottle-top
dispenser. Swirl to mix.
8. Prepared Sample:
Add 1.0 mL of prepared
Ascorbic Acid reagent to
one of the flasks with a
bottle-top dispenser. Swirl
to mix. The remaining flask
will be the blank.
Ascorbic acid method, rapid liquid (continued)
Zero

timer.
period will begin.
10. When the timer

of the flask that contains
the blank into the
Pour-Thru Cell.


sample into the Pour-Thru
Cell.
0 g/L PO43
Read

g/L PO43.

deionized water
Interferences
Interference level
Aluminum
200 mg/L
Arsenate
Interferes
Chromium
100 mg/L
Copper
10 mg/L
Hydrogen sulfide
Interferes
Iron
100 mg/L
Nickel
300 mg/L
Silica
50 mg/L
Silicate
10 mg/L
Turbidity
Samples with large amounts of turbidity may give inconsistent results because the acid
present in the reagents may dissolve some of the suspended particles and variable
desorption of orthophosphate from the particles may occur.
Zinc
80 mg/L
Page 1011
Interference level

extreme sample pH
Analysis labware treatment

All labware used in this test must be thoroughly cleaned to remove all traces of phosphate.
1. Clean containers with a non-phosphate detergent followed by a rinse with deionized water.
2. Fill and soak for 10 minutes with a 1:25 dilution of Molybdate Reagent in deionized water.
3. Rinse well with deionized water. Keep containers tightly closed when not in use.
4. Treat the Pour-Thru Cell with this same mixture of molybdate and water followed by thorough
rinsing with deionized water.
Dedicate these containers for low-level phosphate analysis. If these containers are rinsed and
capped after use, only occasional pre-treatment is necessary.
How to clean the Pour-Thru cell

solutions are allowed to stand in the cell for long periods after measurement. To remove color:
1. Rinse the Pour-Thru Cell with a 1:5 dilution of Ammonium Hydroxide.
2. Follow the Ammonium Hydroxide rinse with deionized water rinses.
3. Invert a beaker over the glass funnel of the cell when the Pour-Thru Cell is not in use.
Reagent preparation
The Ascorbic Acid reagent must be prepared before use.
1. Using a powder funnel, add the contents of one 48 g bottle of Ascorbic Acid Reagent Powder*
to one 450 mL bottle of Ascorbic Acid Reagent Dilution Solution.
2. Invert several times and swirl until the powder is completely dissolved.
3. Attach dispensers to the top of this bottle and the Molybdate Reagent bottle. This solution may
develop a yellow color with time but will still give accurate results for up to one month after
mixing if stored at 2025 C.
4. Record the date of preparation on the bottle and discard any remaining solution after one
month.
Do not add fresh reagent to previously mixed reagent. Use of this reagent after one month may
result in high reagent blanks and low values at high concentrations.
Page 1012
Do not use detergents that contain phosphate for cleaning labware.
If prompt analysis is not possible, preserve samples by filtering immediately and storing at 4
C (39 F) for up to 48 hours.
Accuracy check
Phosphate Standard Solution Ampule, 50-mg/L (50,000 g/L) as PO43 (for concentrations
greater than 1000 g/L)
Phosphate Standard Solution Ampule, 15-mg/L (15,000 g/L) as PO43 (for concentrations
less than 1000 g/L)
Ampule breaker
ADDITIONS.
4. Open the standard solution ampule needed for the expected concentration.
5. Measure three 25 mL portions of fresh samples into three flasks.
standard to the three 25-mL portions of fresh sample.
7. Follow the Ascorbic acid method, rapid liquid test procedure for each of the spiked samples,
Page 1013
1000-g/L (1.000-mg/L) phosphate standard solution
1. Use the 1000-g/L (1.000-mg/L) phosphate standard solution in place of the sample. Follow
the Ascorbic acid method, rapid liquid test procedure.
Method performance
Program
Instrument
Standard
per 0.010 Abs
488
DR 5000
1000 g/L PO43
9701030 g/L PO43
21 g/L PO43
Summary of method
Orthophosphate reacts with molybdate in an acid medium to produce a phosphomolybdate
complex. Ascorbic acid then reduces the complex, giving an intense molybdenum blue color.
Reactive phosphorus includes existing orthophosphate in the sample plus a small fraction of
condensed phosphate that may be hydrolyzed to orthophosphate during the test. Test results are
measured at 880 nm.
Page 1014

Required reagents
Description
Quantity/Test
Unit
Catalog number
Ascorbic Acid Reagent Dilution Solution
1 mL
450 mL
2599949
Ascorbic Acid Reagent Powder
varies
48 g
2651255
2 mL
500 mL
2599849
varies
4L
27256
Catalog number
Rapid Liquid Low Range Phosphorus Reagent Set, includes:
Molybdate Reagent Solution

Water, deionized
2678600
Required apparatus
Description
Quantity
Unit
each
108140
Adjustable Digital Dispenser
each
2563137
each
2089843
Powder funnel, 65 mm top ID
each
2264467
Description
Unit
Catalog number
Drinking Water Standard, Mixed Inorganics for NO3, PO4, SO4
500 mL
2833049
Phosphate Standard Solution, 1.00-mg/L as PO43
500 mL
256949
946 mL
2059716
Phosphate Standard Solution, 3-mg/L as
PO43
Phosphate Standard Solution, Voluette ampule, 10-mL, 50-mg/L PO43

PO43
Wastewater Effluent Standard for NH3N, NO3N, PO4, COD, SO4, TOC
16/pkg
17110
100 mL
1424342
500 mL
2833249
each
2196800
Unit
Catalog number

Description
Ammonium Hydroxide, ACS
Hydrochloric Acid Solution,6.0 N, 1:1
10649
each
189640
500 mL
88449
each
2635700
12/pkg
2087076
Filter Paper, fold 12.5 cm
100/pkg
69257
each
108367
each
1970001
50/pkg
2185696
1000/pkg
2185628
19700011

1
500 mL
Page 1015

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive, AVPP, 8048
(Orthophosphate)
DOC316.53.01119
USEPA1 PhosVer 3 (Ascorbic Acid) Method2
Method 8048
0.02 to 2.50 mg/L
PO43

1
USEPA Accepted for reporting for wastewater analyses. Procedure is equivalent to USEPA and Standard Method 4500-P-E for wastewater.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

adjust.

Description
Quantity
Powder Pillow Test:

PhosVer 3 Phosphate Reagent powder pillow
AccuVac Test:
PhosVer 3 Phosphate Reagent AccuVac Ampul
Beaker, 50-mL
Sample Cell, 10-mL round

Page 1017

Description
Quantity
Stopper for 18-mm Tube (supplied with PhosVer AccuVacs)
PhosVer 3 (Ascorbic Acid) method for powder pillows
Stored Programs
490 P React. PV
Start
1. Select the test.


10-mL of sample.
3. Prepared Sample:
PhosVer 3 phosphate
Powder Pillow to the cell.
Immediately stopper and
shake vigorously for 30
seconds.

timer.
period will begin. If the
sample was digested
using the Acid Persulfate
digestion, a ten-minute
reaction period is required.
Zero
6. When the timer

and insert it into the
cell holder.

Page 1018

0.00 mg/L PO43

the cell holder.
PO43.

PhosVer 3 (Ascorbic Acid) method for AccuVac Ampuls
Stored Programs
492 P React. PV AV
Start
1. Select the test.

10-mL of sample.

for orientation.

timer.
period will begin. If the
sample was digested
using the Acid Persulfate
digestion, a ten-minute
reaction period is required.
6. When the timer

holder.
3. Prepared Sample:
Fill a PhosVer 3
Phosphate AccuVac
4. Secure an Ampul cap

over the tip of the Ampul.
Shake the Ampul for
approximately
30 seconds.
Accuracy is unaffected by
undissolved powder.

the cell holder.
READ the results in
mg/L PO43.

0.00 mg/L PO43

Page 1019
Interferences
Interference level
Aluminum
Arsenate
Interferes at any level.
Chromium
Copper
Hydrogen Sulfide
Interferes at any level
Iron
Nickel
pH, excess buffering
reagents and require sample pretreatment. pH 210 is recommended.
Silica
Silicate
Turbidity or color
May cause inconsistent results because the acid in the powder pillow may dissolve some of
the suspended particles and because of variable desorption of orthophosphate from the
particles. For highly turbid or colored samples, add the contents of one Phosphate
Pretreatment1 Powder Pillow to 25 mL of sample. Mix well. Use this solution to zero the
instrument.
Zinc
Collect sample in plastic or glass bottles that have been cleaned with 1:1 Hydrochloric Acid
Solution* and rinsed with deionized water.
Do not use commercial detergents containing phosphate for cleaning glassware used in
phosphate analysis.
For best results, analyze samples immediately.
If prompt analysis is not possible, preserve samples by filtering immediately and storing at 4
C (39 F) for up to 48 hours.
Return the sample to room temperature before analysis.
Accuracy check
Ampule breaker
2. Select OPTIONS>MORE>STANDARD ADDITIONS from the instrument menu.

Page 1020

5. Prepare a 0.1-mL sample spike by adding 0.1 mL of standard to the unspiked sample. Press
the timer icon. After the timer expires, read the result.
6. Prepare a 0.2-mL sample spike by adding 0.1 mL of standard to the 0.1-mL sample spike.
Press the timer icon. After the timer expires, read the result.
7. Prepare a 0.3-mL sample spike by adding 0.1 mL of standard to the 0.2-mL sample spike.
Press the timer icon. After the timer expires, read the result. Each addition should reflect
1. Fill three mixing cylinders each with 50-mL of sample and spike with 0.2 mL, 0.4 mL and
0.6 mL of standard.
3. Analyze each standard addition sample as described in the PhosVer 3 (Ascorbic Acid) method
for AccuVac Ampuls.
recovery.
Phosphate standard solution, 50 mg/L
Deionized water
4 mL Class A volumetric pipet and pipet bulb
1. Prepare a 2.00 mg/L phosphate standard solution as follows:

a. Pipet 4.00 mL of Phosphate Standard, 50-mg/L, into a 100-mL volumetric flask.
b. Dilute to volume with demineralized water. Mix well. Prepare this solution daily.
Note: Alternately, use one of the mixed parameter standards listed in Recommended standards. These
contain 2.0 mg/L phosphate.
2. Use this solution in place of the sample. Follow the PhosVer 3 (Ascorbic Acid) method for

Page 1021
Method performance
Program
Standard
per 0.010 Abs
490
2.00 mg/L PO43
1.982.02 mg/L PO43
0.02 mg/L PO43
492
2.00 mg/L PO43
1.982.02 mg/L PO43
0.02 mg/L PO43
Summary of method
molybdate complex. Ascorbic acid then reduces the complex, giving an intense molybdenum blue
color. Test results are measured at 880 nm

Page 1022

Required reagents
Description
PhosVer 3 Phosphate Reagent Powder Pillows, 10-mL
Quantity/Test
Unit
Catalog number
100/pkg
2106069
25/pkg
2508025
Quantity
Unit
Catalog number
6/pkg
173106
OR
PhosVer 3 Phosphate Reagent AccuVac Ampuls

Description
Required apparatus
Description
Quantity
Unit
Catalog number
Beaker, 50-mL
each
50041H
6/pkg
173106
each
2122800
each
2122800
2/pkg
2495402
Unit
Catalog number
16/pkg
17110
Description
Voluette
Ampul, 50-mg/L as PO4
500 mL
17149
500 mL
256949
Standard, Drinking Water, Mixed Parameter, Inorganic: F, NO3, PO4, SO4
500 mL
2833049
Wastewater Effluent Standard, for mixed parameters: NH3N, NO3N, PO4, COD, SO4,
TOC
500 mL
2833249
4L
27256
Unit
Catalog number
Water, deionized

Description
Hydrochloric Acid Solution, 6.0N, 1:1
500 mL
88449
each
189641
Phosphate Treatment Powder Pillow
100/pkg
1450199
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1970010
Mixing Cylinder, 50 mL

Page 1023

Description
Unit
Catalog number
19700101
50/pkg
2199796
250/pkg
2199725
12/pkg
2087076
100/pkg
2601300
AccuVac snapper
AccuVac ampule blanks
each
2405200
25/pkg
2677925
each
1457442
each
1451504
AccuVac ampule drainer
each
4103600
Description
Unit
Catalog number
each
2196800
Optional standards
946 mL
1420416
100 mL
1424342
Phosphate; Standard Solution, 100 mg/L
100 mL
1436832
Phosphate; Standard Solution, 500 mg/L, 10 mL Voluette Ampules
16/pkg
1424210
Phosphate; Standard Solution, 500 mg/L
100 mL
1424232

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive Orthophosphate, TNT, 8048
(Orthophosphate)
DOC316.53.01118
USEPA1 PhosVer 3 Method
Method 8048
PO43
(0.06 to 5.00 mg/L

or 0.02 to 1.60 mg/L P)
Test N Tube Vials
Scope and Application: For water, wastewater and seawater;

1
USEPA accepted for reporting wastewater analysis. Procedure is equivalent to USEPA and Standard Method 4500-P E for wastewater.
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

DR 3900, DR 3800, DR 2800 and DR 2700 only: Install the light shield in Cell Compartment #2 before performing this test.
blank adjust.

Description
Quantity
PhosVer 3 Reagent Powder Pillow
Reactive Phosphorus Test N Tube Vial
Light Shield
Micro funnel
Pipet,
TenSette,
110 mL
Pipet tIps for TenSette Pipet

Test Tube Rack
1
1
varies

Page 1025

PhosVer 3 Method, TNT
Stored Programs
535 P React. PV TNT
Start
1. Select the test.


to add 5.0 mL of sample to
a Reactive Phosphorus
Test N Tube Dilution Vial.
Cap and mix.

the vial with a damp towel,
other marks.


the contents of one
PhosVer 3 Phosphate
Powder Pillow to the vial.
7. Immediately cap the

vial tightly and shake for at
least 20 seconds. The
powder will not dissolve
completely.

timer.
Zero

0.00 mg/L PO43
Read

the vial with a damp towel,
other marks.
10. When the timer

expires, insert the vial into
the 16 mm round cell.

Page 1026
READ the
period will begin. Read
samples between two and
eight minutes after adding
the PhosVer 3 reagent.
Interferences
Interference level
Aluminum
Arsenate
All levels
Chromium
Copper
Iron
Nickel
Silica
Silicate
Sulfide
Greater than 6 mg/L. Remove sulfide interference as follows:

2. Swirling constantly, add Bromine Water drop-wise until a permanent yellow color
appears.
3. Swirling constantly, add Phenol Solution drop-wise just until the yellow color disappears.
Proceed with step 1 of the PhosVer 3 Method, TNT test.
Turbidity
Large amounts may cause inconsistent results in the test because the acid present in the
powder pillows may dissolve some of the suspended particles and because of variable
desorption of orthophosphate from the particles.
Zinc

extreme sample pH
Collect samples in plastic or glass bottles that have been acid cleaned with 1:1 Hydrochloric
Do not use commercial detergents containing phosphate for cleaning glassware used in this
test.
Analyze samples immediately after collection for best results.
If prompt analysis is impossible, preserve samples up to 48 hours by filtering immediately and

storing at 4 C.
Accuracy check
Ampule breaker
2. Select standard additions from the instrument menu: Options>More>Standard Additions.

Page 1027

6. Follow the PhosVer 3 Method, TNT test procedure for each of the spiked samples, starting
3.0-mg/L phosphate standard solution
1. Use the 3.0-mg/L phosphate standard solution in place of the sample. Follow the PhosVer 3
Method, TNT test procedure.
to Standard Adjust in the software: Options>More>Standard Adjust.
Method performance
Program
Instrument
Standard
per 0.010 Abs
535
DR 5000
3.00 mg/L PO43
2.943.06 mg/L PO43
0.06 mg/L PO43
Summary of method
color. Test results are measured at 880 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
2742545
50/pkg
2106046
Reactive Phosphorus Test N Tube Dilution Vials
50/pkg
NA1
Reactive Phosphorus Test N Tube Reagent Set (50 tests), includes:
Not available separately

Page 1028
Required apparatus
Description
Quantity
Funnel, micro
each
2584335
each
1970010
50/pkg
2199796
13
each
1864100

Test Tube Rack
Unit
Catalog number

Page 1029
Description
Phosphate Standard Solution,
PouRite
Ampule, 50-mg/L as
PO43,
2-mL
Unit
Catalog number
20/pkg
17120H
500 mL
17149
500 mL
256949
946 mL
2059716
500 mL
2833049
500 mL
2833249
each
2484600
Unit
Catalog number
Bromine Water 30 g/L
29 mL
221120
500 mL
88449
Phenol Solution 30 g/L
29 mL
211220

Standard, Drinking Water, Mixed Parameter, Inorganic for
F,
NO3, PO4, SO4
Wastewater Effluent Standard, for mixed parameters: NH3N, NO3N, PO4, COD, SO4,
TOC
PouRite Ampule breaker, 2 mL

Description
each
1970001
50/pkg
2185696
1000/pkg
2185628
100/pkg
69257
each
108367

Beaker, 50-mL
1
each
2635700
12/pkg
2087076
each
50041H
Unit
Catalog number
each
2196800
Optional standards
Description
946 mL
1420416
100 mL
1424342
16/pkg
17110
100 mL
1436832
16/pkg
1424210
100 mL
1424232

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Reactive Orthophosphate, HR, TNT, 8114
(Orthophosphate)
DOC316.53.01116
Molybdovanadate Method1
HR (1.0 to 100.0 mg/L PO4
Method 8114
3)
Test N Tube Vials

1
Test preparation


Instrument
DR 6000
Light shield
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

DR 3900, DR 3800, DR 2800 and DR 2700 only: Install the light shield in Cell Compartment #2 before performing this test.
Reagent blanks for each lot of reagents may be used more than once. At room temperature, the reagent blank is stable for
up to three weeks.
The seven-minute reaction time in step 5 is for samples at 23 C. For samples at 13 C, wait 15 minutes. For samples at
33 C, wait two minutes.
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal information.

Description
High Range Reactive Phosphorus Test N Tube Vials
Water, deionized
Quantity
1
5 mL
Light Shield
Test Tube Rack

Page 1031

Molybdovanadate method, TNT
Stored Programs
540 P React. HR TNT
Start
1. Select the test.

Use a TenSette Pipet to
water to a Reactive High
Range Phosphorus Test N
Tube Vial.
3. Prepared Sample:
add 5.0 mL of sample to a
Reactive High Range
Phosphorus Test N Tube
Vial.
Zero
5. Wipe the reagent


0.0 mg/L PO43

Page 1032

timer.
A seven-minute reaction
period will begin.
Read the sample within
two minutes after the timer
expires.
Read

sample vial and insert it
into the 16-mm round cell
holder.

mg/L PO43.
Interferences
The Interfering substances table lists interference types and levels. The Noninterfering substances
at low concentrations (less than 1000 mg/L) table shows substances that do not interfere in
concentrations less than 1000-mg/L.

Interference level
Arsenate
Only interferes if the sample is heated.1
Iron, ferrous
Blue color caused by ferrous iron does not interfere if iron concentration is less than
100 mg/L.
Molybdate
Silica
Only interferes if the sample is heated.1

Causes a negative interference. Remove interference as follows:
Sulfide
2.
Add Bromine Water2 drop-wise with constant swirling until a permanent yellow
color develops.
3.
Add Phenol Solution2 drop-wise until the yellow color just disappears.
Proceed with step 3.
Extreme pH or highly buffered

samples
May exceed buffering capacity of the reagents. Samples may require pretreatment. Sample
pH should be about 7.

Cause a negative interference.
Temperature, Cold (less than

20 C)
Causes a negative interference.
Temperature, Hot (greater

than 25 C)
Causes a positive interference.
Gentle warming of the sample to room temperature will not cause this substance to interfere.
Pyrophosphate
Tetraborate
Citrate
Lactate
Formate
Oxalate
Tartrate
Salicylate
Al3+
Selenate
Mg2+
Ca2+
Ba2+
Sr2+
Li+
Na+
K+
NH4+
Cd2+
Mn2+
NO3
NO2
SO42
SO32
Pb2+
Hg+
Hg2+
Sn2+
Cu2+
Ni2+
Ag+
Zr4+
AsO3
Br
CO32
ClO4
CN
Fe3+
SiO44
IO3
Benzoate

Page 1033
Collect samples in plastic or glass bottles that have been cleaned with 1:1 Hydrochloric Acid
Solution* and rinsed with deionized water.
Do not use commercial detergents containing phosphate for cleaning glassware used in
this test.
For best results, analyze the samples immediately after collection.
If prompt analysis is impossible, preserve the samples for up to 48 hours by filtering

immediately and storing at 4 C.
Bring the stored sample to room temperature before analysis.
Accuracy check
10-mL Voluette Ampule of Phosphate Standard Solution, 500-mg/L PO43
1:1 Hydrochloric acid solution
Deionized water
Ampule breaker
1. Clean the glassware with 1:1 hydrochloric acid solution. Rinse again with deionized water. Do
not use detergents containing phosphate to clean the glassware.
3. Select standard additions from the instrument menu:OPTIONS>MORE>STANDARD
ADDITIONS.
7. Follow the Molybdovanadate method, TNT test procedure using 5 mL of each of the spiked
instrument.

Page 1034
50-mg/L PO43 standard
1. Use the 50-mg/L PO43 standard solution in place of the sample. Follow the Molybdovanadate
method, TNT test procedure.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
540
50.0 mg/L PO43
49.150.9 mg/L PO43
0.7 mg/L PO43
Summary of method
molybdate complex. In the presence of vanadium, yellow molybdovanadophosphoric acid forms.
The intensity of the yellow color is proportional to the phosphate concentration. Test results are
measured at 420 nm.

Page 1035

Required reagents
Description
Quantity/Test
Unit
Catalog number
50 vials
2767345
High Range Reactive Phosphorus Test N Tube Reagent Set,

includes:
(1) Reactive High Range Phosphorus Test N Tube Vials1
(2) Water, deionized
1
50/pkg
5 mL
100 mL
27242
Catalog number
Not available separately.
Required apparatus
Description
Quantity
Unit
each
1970010
50/pkg
2199796
Test Tube Rack
each
1864100
Unit
Catalog number
Description
Phosphate Standard Solution, 50-mg/L, as PO4
500 mL
17149
Phosphate Standard Solution, Voluette ampule, 500-mg/L as PO43, 10-mL
16/pkg
1424210
Wastewater Influent Standard for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833149
each
2196800
Unit
Catalog number

Description
Ammonium Hydroxide
500 mL
10649
29 mL
221120
each
189640

Hydrochloric Acid Solution 6.0 N, 1:1
500 mL
88449
29 mL
211220
Dropper, LDPE, 0.51.0 mL
20/pkg
2124720
250/pkg
2199725
100/pkg
2601300
each
2635700
12/pkg
2087076
100/pkg
69257
each
108367
Beaker, 50-mL
each
50041H -
Finger cots
2/pkg
1464702

Page 1036
Optional standards
Description
Unit
Catalog number
946 mL
2059716
946 mL
1420416
100 mL
1424342
1436716
946 mL
16/pkg
17110
100 mL
1436832
Phosphate, Standard Solution, 500 mg/L; 10 mL Voluette Ampules
16/pkg
1424210
100 mL
1424232

Page 1037

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Total, 8190
Phosphorus, Total
DOC316.53.01121
USEPA1
PhosVer 3 with Acid Persulfate Digestion Method
Method 8190
0.06 to 3.50 mg/L PO43 or
0.02 to 1.10 mg/L P
Test N Tube Vials

1
USEPA Accepted for reporting wastewater analyses (Standard Methods 4500 P-E).
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

adjust.
The test range for total phosphate is limited to 0.06 to 3.5 mg/L PO43. Values greater than 3.5 mg/L may be used to
estimate dilution ratios, but should NOT be used for reporting purposes. If the value is greater than 3.5 mg/L, dilute the
sample and repeat the digestion and the colorimetric test.
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.
Phosphorus, Total
Page 1039
Phosphorus, Total

Description
Quantity
Total Phosphorus Test N Tube Reagent Set
Deionized water
varies
DRB200 Reactor
Funnel, micro

Pipet,
TenSette,
1
1
1 to 10 mL, plus tips
Test Tube Rack
PhosVer 3, acid persulfate digestion
Stored Programs
536 P Total/AH PV TNT
Start

Reactor. Preheat to
150 C.
5. Cap tightly and shake

to dissolve.

to add 5.0 mL of sample to
a Total Phosphorus Test
Vial.

the contents of one
Potassium Persulfate
Powder Pillow for
Phosphonate to the vial.

DRB200. Close the
protective cover.

timer to 30 minutes and
start.
8. When the timer

expires, carefully remove
the hot vial from the
reactor. Insert it in a test
tube rack and cool to room
temperature.
2. Select the test.
A 30-minute heating
period will begin.
Phosphorus, Total
Page 1040
Phosphorus, Total
PhosVer 3, acid persulfate digestion (continued)
Zero

to add 2 mL of 1.54 N
Sodium Hydroxide
vial. Cap and mix.

the vial with a damp cloth
followed by a dry one.

16 mm cell holder.

the contents of one
PhosVer 3 Powder Pillow
to the vial.
14. Immediately cap

tightly and shake to mix for
2030 seconds.

timer.
The powder will not

dissolve completely.

0.00 mg/L PO43
period will begin.
Read the sample within
28 minutes after the timer
expires.

wipe the outside of the vial
with a wet towel, then a
dry one. Insert the
prepared sample vial into
the 16 mm cell.
PO43.
Interferences
Interference level
Aluminum
Arsenate
Interferes at any level
Chromium
Copper
Iron
Nickel
pH, excess buffering
Silica
Phosphorus, Total
Page 1041
Phosphorus, Total
Interference level
Silicate
Sulfide
Turbidity or color
May cause inconsistent results because the acid in the powder pillow may dissolve some of
the suspended particles and because of variable desorption of orthophosphate from
the particles.
Zinc
Collect samples in plastic or glass bottles that have been acid washed with 1:1 Hydrochloric
Do not use commercial detergents containing phosphate for cleaning glassware used in this
test.
If prompt analysis is not possible, samples may be preserved up to 28 days by adjusting the
pH to 2 or less with concentrated Sulfuric Acid* (about 2 mL per liter) and storing at 4 C.
Warm stored samples to room temperature and neutralize with 5.0 N Sodium Hydroxide*
before analysis.
Accuracy check
Ampule breaker
Mixing cylinders, (3)
1. Clean glassware with 1:1 Hydrochloric Acid Standard Solution. Rinse again with deionized
water. Do not use phosphate detergents to clean glassware.
ADDITIONS.
7. Use a 5-mL aliquot of the spiked sample in place of the sample. Follow the PhosVer 3, acid
persulfate digestion test procedure for each of the spiked samples, starting with the 0.1 mL
sample spike. Measure each of the spiked samples in the instrument.
Phosphorus, Total
Page 1042
Phosphorus, Total
Phosphate standard solution, 3.0-mg/L
1. Use the 3.0 mg/L phosphate standard solution in place of the sample. Follow the PhosVer 3,
acid persulfate digestion test procedure.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
536
3.00 mg/L PO43
2.933.07 mg/L PO43
0.06 mg/L PO43
Summary of method
Phosphates present in organic and condensed inorganic forms (meta-, pyro- or other
polyphosphates) must be converted to reactive orthophosphate before analysis. Pretreatment of
the sample with acid and heat provides the conditions for hydrolysis of the condensed inorganic
forms. Organic phosphates are converted to orthophosphates by heating with acid and persulfate.

'
Required reagents
Description
Total Phosphorus Test N Tube Reagent Set, 50 tests, includes:
Unit
Catalog number
2742645
50/pkg
2106046
50/pkg
2084766
2 mL
100 mL
2743042
50/pkg
varies
100 mL
27242
Quantity
Unit
Catalog number
each
LTV082.53.40001

Total and Acid Hydrolyzable Test Vials1
Water, deionized
1
Quantity/Test
Not sold separately
Required apparatus
Description
Phosphorus, Total
Page 1043
Phosphorus, Total
Required apparatus
Description
Quantity
Unit
Catalog number
each
LTV082.52.40001
Funnel, micro
each
2584335
Light shield, DR 3900
each
LZV849
Light shield, DR 3800, DR 2800, DR 2700
each
LZV646
Pipet, TenSette, 1.0 to 10 mL
each
1970010
250/pkg
2199725
Test Tube Rack
each
1864100
Unit
Catalog number
Description
Drinking Water Standard, Mixed Parameter, Inorganic for F-, NO3, PO4, SO4
500 mL
2833049
Phosphate Standard Solution, 10-mL Voluette Ampule, 50-mg/L as PO43
16/pkg
17110
500 mL
256949
946 mL
2059716
Wastewater Standard, Effluent Inorganics, for NH3N, NO3N, PO4, COD, SO4, TOC
500 mL
2833249
each
2196800
Description
Unit
Catalog number
each
189640
each
1451536
500 mL
88449
1000 mL
245053
500 mL
97949
each
1970001
Pipet,
TenSette
Pipet, 0.11.0 mL
50/pkg
2185696
1000/pkg
2185628
12/pkg
2087076
100/pkg
2601300
Deionized Water
4L
27256
each
2635700
Finger cots
2/pkg
1464702
Unit
Catalog number
1420416
Optional standards
Description
946 mL
100 mL
1424342
100 mL
1436832
16/pkg
1424210
Phosphorus, Total
Page 1044
Phosphorus, Total
Optional standards
Description
Unit
Catalog number
100 mL
1424232
Phosphorus, Total
Page 1045

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Total Digestion, 8190
Phosphorus, Total, Digestion

USEPA1 Acid Persulfate Digestion Method2
DOC316.53.01112
Method 8190

1
USEPA Accepted for wastewater analyses when used with the ascorbic acid (PhosVer 3) method.
Adapted from Standard Methods for the Examination of Water and Wastewater 4500-P B & E
Test preparation

Rinse all glassware with 1:1 hydrochloric acid. Rinse again with deionized water.

Description
Quantity
1
2 mL
2 mL
Water, deionized
varies
Hot Plate

Page 1047

Acid Persulfate Digestion
17. Use a graduated

of sample. Pour the
Erlenmeyer flask.

one Potassium Persulfate
Powder Pillow.
Swirl to mix.

Solution to the flask.

hot plate. Boil gently for 30
minutes. Do not boil dry.
Concentrate the sample to
less than 20 mL for best
recovery. After
concentration, maintain
the volume near 20 mL by
adding small amounts of
deionized water. Do not
exceed 20 mL.
480 P React. Mo
482 P React. Mo. AV
485 P React. Amino
490 P React. PV
492 P React. PV AV
535 P React. PV TNT
540 P React. HT TNT
21. Cool the sample to

room temperature.

Solution to the flask. Swirl
to mix.

Adjust the volume to 25
mL with deionized water
rinsings from the flask.
24. Proceed with a

reactive phosphorus test
of the expected total
phosphorus concentration
range.
Extend the color
development time to
10 minutes for the
PhosVer 3 (ascorbic acid)
method.

Page 1048
Interferences
Interference level
Alkaline or highly buffered

samples
It may be necessary to add additional acid in step 19 to drop the pH of the solution below 1.
Turbidity
Use 50 mL of sample and double the reagent quantities. Use a portion of the reacted sample
to zero the instrument in the reactive phosphorus procedure. This compensates for any color
or turbidity destroyed by this procedure.
If prompt analysis is not possible, samples may be preserved up to 28 days.
To preserve samples. adjust the pH to 2 or less with Concentrated Sulfuric Acid* (about 2 mL
per liter). Store at 4 C.
Warm the sample to room temperature and neutralize with 5.0 N Sodium Hydroxide before
analysis.
Summary of method
forms. Organic phosphates are converted to orthophosphate by heating with acid and persulfate.
Organically bound phosphates are thus determined indirectly by subtracting the result of an acid
hydrolyzable phosphorus test from the total phosphorus result.
This procedure must be followed by one of the reactive phosphorus (orthophosphate) analysis
methods for determining the phosphorus content of the sample. If the ascorbic acid (PhosVer 3)
method is used to measure the reactive phosphorus, this method is USEPA accepted for NPDES
reporting.
The following reagents and apparatus are required in addition to those required for the active
phosphorus test.
* See Optional reagents.

Page 1049

Required reagents
Description
Quantity/Test
Unit
Catalog number
245199
100/pkg
2 mL
100 mL MDB
245032
2 mL
100 mL MDB
244932
Water, deionized
varies
4L
27256
Quantity
Unit
Catalog number
Required apparatus
Description
each
50840
each
50543
Hot Plate, 7 x 7 digital, 115 VAC
each
2881500
Hot Plate, Stirrer, 7 x 7 digital, 230 VAC
each
2881602
Optional reagents
Description
Unit
Catalog number
Sodium, Hydroxide, 5.0 N
1000 mL
245053
500 mL
97949
100/pkg
2601300
each
2635700
Sampling bottle with cap, low density polyethylene, 250 mL
Thermometer, Non-Mercury, 10 to 225C
12/pkg
2087076
500 mL
88449

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Phosphorus, Total, TNT, 10127
Phosphorus, Total
DOC316.53.01123
Molybdovanadate Method with Acid

Persulfate Digestion1
Method 10127
HR (1.0 to 100.0 mg/L PO43)
Test N Tube Vials

1
Adapted from Standard Methods for the Examination of Water and Wastewater., (4500 B-C)
Test preparation


Instrument
Light shield
DR 6000
DR 5000
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

Reagent blanks can be used more than once, but should not be used more than one day.
The final samples will contain molybdenum. In addition, the final samples will have a pH less than 2 and are considered
corrosive (D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.

Description
Quantity
Total High Range Phosphorus Test N Tube Reagent Set
DRB200 Reactor, 15 x 16 mm

Pipet,
TenSette,
1 to 10 mL, plus tips
1
1
Test Tube Rack
Funnel, Micro
Phosphorus, Total
Page 1051
Phosphorus, Total
Molybdovanadate method, acid persulfate digestion
Stored Programs
542 P Total HR TNT
Start

Reactor. Heat to 150 C.

the contents of one
Potassium Persulfate
Cap tightly and shake to
dissolve.
Phosphorus, Total
Page 1052
2.
Select the test.

DRB 200 Reactor.
Close the protective cover.
water to a Total
Phosphorus Test N Tube
Vial.
4. Prepared Sample:
add 5.0 mL of sample to a
Total Phosphorus Test N
Tube Vial.

timer to 30 minutes and
start the timer.

carefully remove the hot
vials from the reactor.
Insert them in a test tube
rack and allow to cool to
room temperature
(1825 C).
A 30-minute heating
period will begin.
Phosphorus, Total
Molybdovanadate method, acid persulfate digestion (continued)

to add 2.0 mL of 1.54 N
sodium hydroxide to each
vial.
10. Use a polyethylene

Molybdovanadate
Reagent to each vial.

timer.
will begin. Read the
sample between seven
and nine minutes after
adding the
Molybdovanadate
Reagent.
Zero
13. When the timer

into the 16-mm cell holder.

0.0 mg/L PO43
12. Wipe the vials with a

damp towel, followed by a
dry one, to remove
fingerprints or other
marks.
Read

sample into the 16-mm cell
holder.

mg/L PO43.
Interferences
Sample turbidity may cause inconsistent results in the test because the acid present in the
reagents may dissolve some of the suspended particles and because of variable desorption of
orthophosphate from the particles.
The Interfering substances table shows interference levels and types. The Noninterfering
substances, (at concentrations less than 1000 mg/L) table shows substances that do not interfere
in concentrations less than 1000 mg/L.

Interference level
Arsenate
Causes positive interference if the sample is warm when the molybdovanadate reagent is
added (after the digestion).1 Cool the sample after digestion before adding reagent.
Iron, ferrous
Blue color caused by ferrous iron does not interfere if iron concentration is less than
100 mg/L.
Phosphorus, Total
Page 1053
Phosphorus, Total
Interference level
Molybdate
Silica
Causes positive interference if the sample is warm when the molybdovanadate reagent is
added (after the digestion). Cool the sample after digestion before adding reagent.
Extreme pH or highly buffered

samples
May exceed buffering capacity of the reagents. Samples may require pretreatment. Sample
pH should be about 7.

Temperature, Cold (less than

18 C)
Temperature, Hot (greater

than 25 C)
Causes a positive interference.

Post-digestion samples should be brought to room temperature (1825 C) before the
addition of the Molybdovanadate Reagent or sodium hydroxide.
Gentle warming of the sample to reach room temperature will not cause this substance to interfere.
Table 324 Noninterfering substances, (at concentrations less than 1000 mg/L)
Pyrophosphate
Tetraborate
Selenate
Benzoate
Citrate
Oxalate
Lactate
Tartrate
Formate
Salicylate
Al3+
Fe3+
Mg2+
Ca2+
Ba2+
Sr2+
Li+
Na+
K+
NH4+
Cd2+
Mn2+
NO3
NO2
SO42
SO32
Pb2+
Hg+
Hg2+
Sn2+
Cu2+
Ni2+
Ag+
U4+
Zr4+
AsO3
ClO4
CN
Br
CO3
IO3
SiO44
Collect samples in plastic or glass bottles that have been acid washed with 1:1 Hydrochloric
Do not use commercial detergents containing phosphate for cleaning the glassware used in
this test.
Analyze samples immediately after collection for best results.
If prompt analysis is impossible, preserve samples up to 28 days by adjusting the pH to 2 or

less with concentrated Sulfuric Acid* (about 2 mL per liter) and storing at 4 C.
Warm stored samples to room temperature and neutralize with 5.0 N Sodium Hydroxide*
before analysis.
Phosphorus, Total
Page 1054
Phosphorus, Total
Accuracy check
10-mL Voluette Ampule of Phosphate Standard Solution, 500 mg/L as PO43
Ampule breaker
1. Clean glassware with 1:1 Hydrochloric Acid Standard Solution. Rinse again with deionized
water. Do not use detergents containing phosphate to clean glassware.
ADDITIONS
standard to three 10-mL portions of fresh sample in 25 mL mixing cylinders.
7. Follow the Molybdovanadate method, acid persulfate digestion test procedure for each of the
spiked samples, starting with the 0.1 mL sample spike. Measure each of the spiked samples in
the instrument.
Phosphorus, Total
Page 1055
Phosphorus, Total
Phosphate standard solution, 50-mg/L
1. Use the 50-mg/L phosphate standard solution in place of the sample. Follow the
Molybdovanadate method, acid persulfate digestion test procedure.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
542
50 mg/L PO43
49.450.6 mg/L PO43
0.7 mg/L PO43
Summary of method
forms. Organic phosphates are converted to orthophosphates by heating with acid and persulfate.
molybdate complex. In the presence of vanadium, yellow molybdovanadophosphoric acid forms.
The intensity of the yellow color is proportional to the phosphate concentration. Test results are
measured at 420 nm.
Phosphorus, Total
Page 1056
Phosphorus, Total

Required reagents
Description
Quantity/Test
Unit
Catalog number
50 vials
2767245
0.5 mL
25 mL
50/pkg
2084766
2 mL
100 mL
2743042
50/pkg
5 mL
100 mL
27242
Quantity
Unit
Catalog number
Total High Range Phosphorus Test N Tube Reagent Set, includes:

(1) Molybdovanadate Reagent1
(1) Potassium Persulfate powder Pillows
(1) Sodium Hydroxide Solution, 1.54 N
(1) Total Phosphorus Test Vials1
(2) Water, deionized
1
Not available separately.
Required apparatus
Description
each
LTV082.53.40001
each
LTV082.52.40001
Dropper, 0.5 and 1.0 mL marks, plastic
20/pkg
2124720
Light shield, DR 3900
each
LZV849
Light shield, DR 3800, DR 2800, DR 2700
each
LZV646
each
1970010
250/pkg
2199725
Test Tube Rack
each
1864100
Funnel, micro
each
2584335
Unit
Catalog number
Description
Phosphate Standard Solution, Voluette ampule, 500-mg/L as PO43, 10-mL
Phosphate Standard Solution, 50-mg/L as
PO43
Wastewater Standard, Influent Inorganics for NH3N, NO3N, PO4, COD, SO4, TOC
16/pkg
1424210
500 mL
17149
500 mL
2833149
each
2196800
Unit
Catalog number

Description
500 mL
88449
1000 mL
245053
500 mL
97949
100 mL
2076032
50/pkg
2199796
Pipet,
TenSette,
Pipet, 0.11.0 mL
each
1970001
50/pkg
2185696
Phosphorus, Total
Page 1057
Phosphorus, Total
Description
Unit
19700011

Deionized Water
Catalog number
1000/pkg
2185628
100/pkg
2601300
4L
27256
each
2635700
Finger cots
2/pkg
1464702
12/pkg
2087076
each
189640
Funnel, micro
each
2584335
Optional standards
Description
Unit
Catalog number
946 mL
1436716
100 mL
1436832
16/pkg
17110
100 mL
1424232

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Potassium, 8049
Potassium
DOC316.53.01127
Tetraphenylborate Method
Method 8049
0.1 to 7.0 mg/L
Powder Pillows
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Program # 905 has a calibration curve for potassium; however, due to potential variation between lots of Potassium 3
Reagent, it is recommended that a new calibration for each lot of reagent is performed to obtain best accuracy. Prepare and
store the calibration as directed under Calibration.
Filter highly colored or turbid samples before analysis.
Refer to a current MSDS for pollution prevention and waste management information.
After the test, clean the cells with soap and a brush.

Description
Quantity
Potassium Reagent 1 Powder Pillow
Clippers
Pipet, TenSette, 110 mL, plus tips

Water, deionized
varies
2
varies
Potassium
Page 1059
Potassium
Tetraphenylborate method for powder pillows
Stored Programs
905 Potassium
Start
1. Select the test.

2. Fill a graduated mixing

sample.

one Potassium 1 Reagent
Pillow. Add the contents of
Pillow. Stopper and invert

Pillow after the solution
clears. Stopper and shake
the solution for 30
seconds.
A white turbidity will form if
potassium is present.

timer.
period will begin.
6. Prepared Sample:
Pour at least 10-mL of the
solution from the cylinder
into a sample cell.
Zero

0.0 mg/L K
Potassium
Page 1060
the second sample cell
with 10 mL of unreacted
sample.
Read
10. Within seven minutes

holder.

mg/L K.

Potassium
Interferences
The substances in the Interfering substances table have been tested and will not interfere at or
below the levels stated. If these substances are present at higher levels, conduct interference
studies at the higher levels to determine if the substance interferes.

Interference level
Ammonium Nitrogen
15 mg/L as N
Calcium
7000 mg/L as CaCO3
Chloride
15,000 mg/L
Magnesium
6000 mg/L as CaCO3
Adjust the pH to 2 or less with Nitric Acid (about 2 mL per liter)*.
Preserved samples may be stored at least six months at room temperature.
Before analysis, adjust the pH to 45 with 5.0 N Sodium Hydroxide*.
Do not measure pH in the sample container with a pH electrode, as this will introduce
potassium from the filling solution. Use pH Paper* or pour off sample and test pH in a separate
beaker.
Accuracy check
Important Note: This procedure is applicable only to Stored Program 905 and not to User
Programs.
Potassium Voluette Ampule Standard, 250-mg/L K
Ampule breaker
ADDITIONS.
Potassium
Page 1061
Potassium
6. Follow the Tetraphenylborate method for powder pillows test procedure for each of the spiked
instrument.
Calibration
An approximate calibration curve is preprogrammed within Program 905. For improved accuracy,
a new calibration should be performed with each new lot of reagents. Prepare calibration
standards containing 1, 2, 3, 4, 5, 6, 7 and 8 mg/L potassium as follows:
1. Into eight different 100-mL Class A volumetric flasks, pipet 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and
8.0 mL of the 100-mg/L Potassium Standard Solution using class A glassware or TenSette
Pipet.
2. Dilute to the mark with deionized water. Mix thoroughly.
3. Use deionized water for the 0-mg/L potassium standard.
User-entered Program
Refer to the user manual for instrument-specific information on user-entered programs.
1. Access the User Program feature.
2. For the initial potassium calibration, assign a new program number.
3. Enter a name for the potassium test.
4. Set up the following parameters
Program Type: Single wavelength
Concentration Resolution: 0.1
Units: mg/L
Chemical Form: K
Wavelength l (nm): 650
Calibration: Read Standards
5. Set up the following parameters:
Upper Limit: On, 8.0
Timer 1: Timer 3:00
Lower Limit: On, -0.2
Press Calibration: C=a + bA >Edit>OK
6. Enter the concentrations for the calibration, starting with 0.0, in the left column.
7. When all standard concentrations have been entered, navigate to the 0.0 line.
8. Insert the cell containing the blank (deionized water) and zero the instrument.
9. Perform the potassium test on each standard and insert the first prepared standard into the
cell holder. Navigate to the line corresponding to this standard concentration. Read the result.
Repeat for each standard concentration.
10. If the graph result is acceptable, exit the program. It may be possible to obtain a better fit to the
data by reading another curve. The curve which results in the highest r2 value is generally the
best fit. After selection of the best curve, exit the program.
11. Save the calibration.
Potassium
Page 1062
Potassium
Method performance
Program
Standard
per 0.010 Abs
905
5.0 mg/L K
4.75.3 mg/L K
0.1 mg/L K
Summary of method
Potassium in the sample reacts with sodium tetraphenylborate to form potassium
tetraphenylborate, an insoluble white solid. The amount of turbidity produced is proportional to the
potassium concentration. Test results are measured at 650.

Required reagents
Description
Potassium Reagent Set:
Quantity/Test
Unit
Catalog number
2459100
25/pkg
1432198
25/pkg
1432298
100/pkg
1432399
varies
4L
27256
Catalog number
Water, deionized
Required apparatus
Description
Quantity
Unit
Clippers
each
96800
each
189640
each
1457442
each
1970010
varies
50/pkg
2199796
2/pkg
2495402
Unit
Catalog number
Pipet,
TenSette,
110 mL

Description
Potassium Standard Solution, 10-mL
Voluette
Potassium Standard Solution, 100-mg/L

Ampule, 250 mg/L
16/pkg
1479010
500 mL
2351749
each
2196800

Description
Unit
Catalog number
Nitric Acid, 1:1
500 mL
254049
50 mL
245026
100/pkg
2601300
Potassium
Page 1063
Potassium
Description
Unit
2199725
each
1970001
50/pkg
2185696
1000/pkg
2185628
12/pkg
2087076
Pipet
19700101

Brush, test tube
Liqui-Nox detergent
1
Catalog number
250/pkg
Pipet Tips, for
TenSette
each
69000
946 mL
2088153

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Quaternary Ammonium Compounds, 8337
Quaternary Ammonium
Compounds
DOC316.53.01128
Direct Binary Complex Method
Method 8337
0.2 to 5.0 mg/L as CTAB
Powder Pillows
Scope and Application: For cooling tower water and pool/spa water
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Description
Quantity
QAC Reagent 1 Powder Pillows
2 pillows
QAC Reagent 2 Powder Pillows
2 pillows
Bottle, square, with 25 mL mark
Sample Cells, 1-inch, 10 mL
Quaternary Ammonium Compounds

Page 1065

Direct Binary Complex Method
Stored Programs
401 QAC
Start
1. Select the test.

5. Swirl the bottles to

Do not shake! Shaking
creates air bubbles that
interfere with test results.
Fill one 25-mL mixing
bottle with 25 mL of
deionized water.
3. Prepared Sample:
Fill another mixing bottle

one QAC Reagent 1
bottle.

one QAC Reagent 2
bottle.
7. Swirl the bottles to

dissolve the reagent. Do
not shake.

timer.
A purple color will form if

a quaternary ammonium
compound is present
period will begin.
Zero
the solutions from the
bottles into the sample
cells.
10. When the timer


Page 1066

0.0 mg/L CTAB

CTAB (cetyltrimethylammonium
bromide).
Interferences
Interference studies were conducted by preparing a CTAB standard solution of approximately
3 mg/L as well as a solution of the potential interference. The constituent was said to interfere
when the resulting concentration changed by 10%. The Interfering substances table shows
interfering substances and levels. The Noninterfering substances table shows substance that do
not interfere up to the tested concentrations.
After several samples have been analyzed, the sample cells may exhibit a build-up of a pink or
purple color. A rinse with 1.0 N Sodium Hydroxide Solution followed by an Alconox detergent
wash and deionized water rinse will eliminate the build-up when it occurs.

Interference level
Calcium (as CaCO3)
Positive interference above 1350 mg/L
Chlorine, HOCl and OCl
Cyanuric acid
Negative interference above 70 mg/L
Igepal nonionic surfactant
Iodine, I3
Iron, Fe3+
Liquimine 14P,
filming amine
Magnesium, Mg2+
Niaproof anionic surfactant
Polyacrylic acid
Sodium lauryl sulfate
Sodium polyphosphate
Tribenzylamine
Triton X-100 nonionic

surfactant
Urea

extreme sample pH
May exceed the buffering capacity of the reagents and require sample pretreatment. Adjust
the sample pH between 3 and 5 by using a pH meter or pH paper and adding dropwise an
appropriate amount of acid or base such as 1.0 N Sulfuric Acid Standard Solution or 1.0 N
Sodium Hydroxide Standard Solution. If significant volumes of acid or base are used, a
volume correction should be made.
Table 329 Noninterfering substances

Non-interfering Substance
Highest Concentration Tested (mg/L)
Silica, SiO2
400
Potassium alum, AlKS2O8
500
Sodium thiosulfate, Na2S2O3
30

Page 1067
Collect samples in glass bottles that have been rinsed several times with sample before final
sample filling.
Do not use plastic containers; plastic adsorbs QACs.
Acidify the sample to a pH of less than 2.
Store at 4 2 C.
Accuracy check
QAC Standard Solution, 100-mg/L CTAB
Mixing bottles (3)
ADDITIONS.
6. Follow the Direct Binary Complex Method test procedure for each of the spiked samples,
QAC Standard, 100-mg/L as CTAB
Deionized water
Class A 5 mL volumetric pipet and pipet bulb
1. Prepare a 5.0 mg/L CTAB standard solution as follows:

a. Pipet 5.0 mL of QAC Standard, 100-mg/L as CTAB, into a 100-mL volumetric flask.
2. Use this solution in place of the sample. Follow the Direct Binary Complex Method test
procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST

Page 1068

Method performance
Program
Instrument
Standard
per 0.010 Abs
401
DR 5000
3.0 mg/L CTAB
2.73.3 mg/L CTAB
0.04 mg/L CTAB
Summary of method
The test method makes use of a colorimetric chemistry in which a quaternary ammonium
compound reacts with an indicator to produce a color change from pale pink to vivid purple. The
test is conducted in a stabilized, acid-buffered solution containing a masking agent to eliminate
potential interferences. This test is applicable to the monitoring of QACs in swimming pools and
cooling towers. Test results are measured at 575 nm.

Page 1069

Required reagents
Description
Quantity/Test
Unit
Catalog number
2459200
(4) QAC Reagent 1 Powder Pillows
2 pillows
50/pkg
2401066
(8) QAC Reagent 2 Powder Pillows
2 pillows
25/pkg
2401268
Quaternary Ammonium Compounds Reagent Set (100 tests), includes:

Description
Quantity
Unit
Catalog number
Bottle, square, with 25 mL mark
each
1704200
each
96800
2/pkg
2495402
Description
Unit
Catalog number
QAC Standard Solution, 100-mg/L as CTAB
100 mL
2415342
Water, deionized
4 liters
27256
Unit
Catalog number
Alconox detergent
1.8 kg
2088000
100 mL
104532
1000 mL
104553

Description
100 mL
127032
each
2635700
each
1970001
50/pkg
2185696
19700011
1000/pkg
2185628
each
1457442
each
1451537

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Salinity, DT, 10073
Salinity
Mercuric Nitrate Method
0100
ppt1
Salinity
DOC316.53.001180
Method 10073
Digital Titrator
Scope and Application: For seawater and brackish water

1
parts per thousand (ppt)
Test preparation

ppt salinity x 569 = mg/L chloride (Cl).
ppt salinity x 940 = mg/L sodium chloride (NaCl)
Use the TitraStir apparatus for best results
Both the sample and the blank will contain mercury (D009) at a concentration regulated as a hazardous waste by the Federal
RCRA. Do not pour these solutions down the drain. Refer to the MSDS for more information on proper disposal of these
materials.

Description
Diphenylcarbazone Reagent Powder Pillows
Deionized Water
Digital Titrator
Mercuric Nitrate Titration Cartridge, 2.570 N
Quantity
1
varies
1
varies
Syringe, 3-cc, Luer lock tip
Vial with 2-, 5-, 10-, 15-, 20- and 25-mL marks
Salinity
Page 1071
Salinity
Iodate-Iodide Method

tube into the Mercuric
Nitrate Titration Cartridge.
titrator body.

3. Use the 3-mL syringe

to collect a 2.0-mL water
sample. Add to the vial,
provided.
4. Fill the vial to the

10-mL mark with
deionized water.
Note: 2 mL = 2 cc

one Diphenylcarbazone
the vial and mix.
6. Titrate the sample with

mercuric nitrate until the
color changes from yellow
to light pink.
A small amount of
not affect results.

digits.
7. Calculate sample
salinity in parts per
thousand:
Digits Required x 0.1 =
ppt Salinity
Summary of method
The mercuric nitrate method of chloride analysis has become popular due to the sharp yellow to
pinkish-purple end point of diphenylcarbazone. A single, stable powder has been developed,
combining the color indicator with an appropriate buffer to establish the correct pH.
Salinity
Page 1072
Salinity

Required reagents
Description
Unit
Catalog number
Diphenylcarbazone Reagent Powder Pillows
100/pkg
96799
Mercuric Nitrate Titration Cartridge, 2.570 N
each
2393701
Water, Deionized
Chloride Standard, 12,500 mg/L as Cl-, (22 ppt Salinity) 10 mL AMP
Sodium Chloride standard, 10,246 mg/L as NaCl, 100 mL AMP
4L
27256
16/pkg
1425010
10.9 ppt salinity
2307442
Required apparatus
Description
Unit
Catalog number
each
1720500
each
4157800
Digital Titrator2
each
1690001
4321300
Syringe, 3-cc, Luer lock tip
each
Vial with 2-, 5-, 10-, 15-, 20- and 25-mL marks
each
219300
Voluette breaker
each
2196800
Salinity
Page 1073

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Selenium, 8194
Selenium
DOC316.53.01129
Diaminobenzidine Method1
Method 8194
0.01 to 1.00 mg/L

1
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2612602
DR 5000
2612602
DR 3900
2612602
DR 3800, DR 2800, DR 2700
2612602

Distillation is required for determining total selenium. See Distillation at the end of the procedure. Use the distillate as the
sample in step 2.
Acetone1 is a suitable solvent for removing toluene from glassware after results are measured.
Toluene (F005) solutions are regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the
drain. Water saturated with toluene, toluene solutions and the cotton plug used in the delivery tube of the separatory funnel
should be collected for disposal with laboratory solvent wastes. Refer to the current MSDS for safe disposal and handling
information.
If there are visible water bubbles on the bottom of the cell, decant the top portion into a clean, dry cell prior to reading the
sample.
1
Selenium
Page 1075
Selenium

Description
Quantity
Buffer Solution, sulfate type, pH 2.0
10 mL
Cotton Ball
Cylinder, Graduated: 50- and 100-mL
1 of each
Diaminobenzidine, tetrahydrochloride
0.1 g
Dropper, 0.5 and 1.0 mL marks, one glass and one plastic
1 of each
Funnel, separatory, 250 mL
Hot Plate, 7-inch
Pipet, volumetric, 5-mL, plus safety bulb filler
4 mL
Ring support (3-inch) and stand
Spoons, measuring, 0.2 and 0.05 g

TitraVer
1 of each
0.4 g
Hardness Reagent
Toluene, ACS
60 mL
Water, Deionized
100 mL
Diaminobenzidine method
Stored Programs
640 Selenium
Start
1. Select the test.

Label the flask blank.
Measure 100 mL of
Erlenmeyer flask. Label
the flask sample.
Selenium
Page 1076
3. Add a 0.2-g spoonful

of TitraVer Hardness
Reagent to each flask.
Swirl to mix.
4. Add a 0.05-g spoonful

of diaminobenzidene
tetrahydrochloride to each
Selenium
Diaminobenzidine method (continued)
5. If you have not

distilled the sample, add
5.0 mL of Buffer Solution,
sulfate type, pH 2.0 to
each flask. Swirl to mix.
6. Heat each flask on a

hot plate. Bring the
contents to a gentle boil.
If the sample has been

distilled, adjust the pH of
the distillate to pH 2.7
0.2 using 5 N Sodium
Hydroxide Standard
Solution. Adjust the blank
to pH 2.7 0.2 using 5.25
N Sulfuric Acid Standard
Solution.
9. Transfer the contents

of each flask to separate
250-mL separatory
funnels. Label the funnels
blank and sample.
Place each flask in a
support ring on a stand.

timer.
period will begin. Continue
to boil the contents gently
during this time period.
if selenium is present.
10. Add 2.0 mL of 12 N

Potassium Hydroxide
Standard Solution to each
funnel using a calibrated
1.0-mL plastic dropper.
Stopper. Shake each
funnel to mix.
11. Add 30-mL of toluene

to each funnel. Stopper.
Swirl and invert each
funnel, then open the
stopcock to vent the
funnel. Close the
stopcock. Repeat twice
with each funnel.
8. When the timer

expires, remove both
flasks. Cool to room
temperature using a water
bath.
Do not boil more than one
minute after the timer
expires.

timer.
period will begin. During
this time, vigorously shake
the funnel that contains
the blank.
Use toluene only with

adequate ventilation.
Selenium
Page 1077
Selenium
Diaminobenzidine method (continued)

timer.

timer.
this time, vigorously shake
the funnel that contains
the sample.
A four-minute reaction
period will begin.
15. When the timer

expires, remove the
stopper and drain the
lower water layer from
each funnel and discard.
Complete steps 1619
within five minutes after
the timer expires. The
developed color is stable,
but should be measured
as soon as possible.
Zero


0.00 mg/L Se
16. Insert a cotton plug

into the delivery tube of
each separatory funnel.
Slowly drain the toluene
into respective sample
cells labeled blank and
sample. Stopper the
sample cells.
Filtering the toluene
through dry, absorbent
cotton will remove water or
suspended particles.
Read

the cell holder.

mg/L Se.
Interferences
There are no positive inorganic interferences with this method. Any other interferences can be
removed by distilling the sample.

Interference level
Ferric iron
Up to 2.5 mg/L. Distill sample to eliminate interference.
Manganese
Will not interfere.
Strong oxidizing agents (i.e.,

iodine, bromine or chlorine)
Can react with the indicator to give low results. Distill sample to eliminate interference.
Selenium
Page 1078
Selenium
Collect samples in clean glass or plastic containers.
Adjust the pH to 2 or less with Nitric Acid* (about 1.5 mL per liter).
Preserved samples can be stored for up to six months at room temperature.
Distillation
CAUTION
Always perform this procedure under a fume hood.
This distillation involves the use of a strong acid and oxidizer at high temperatures. To avoid
personal injury, observe all laboratory safety precautions when operating the distilling apparatus.
2. Add 1 mL of Methyl Orange Indicator Solution. Stir with a glass rod.
3. Use a dropper to add 0.1 N Hydrochloric Acid Standard Solution dropwise until the solution
becomes pink. Then add an additional 2 mL.
4. Use a pipet to add 5.0 mL Calcium Chloride Solution. Mix well.
5. Use a dropper to add 1-g/L Potassium Permanganate Standard Solution drop-wise until the
solution is purple.
6. Place the beaker on a hot plate. Evaporate the solution to approximately 250 mL. Periodically
add 1-g/L Potassium Permanganate Solution to keep the solution purple.
7. Any precipitate formed at this step is manganese dioxide and may be ignored.
8. Cool the solution. While cooling, set up the distillation apparatus for the general purpose
distillation as shown in the distillation manual.
9. Pour the treated sample solution into the distillation flask. Add a stirring bar to the flask.
10. Pipet 5.0 mL of 0.1 N Sodium Hydroxide Standard Solution into the flask. Turn the stirrer
power switch to ON. Set the stir control to 5.
11. Turn on the water and adjust so a constant flow is maintained through the condenser. Set the
heat control to 10.
12. When only a few milliliters are left in the distillation flask, turn the power switch off. The
distillate in the Erlenmeyer flask may be discarded.
CAUTION
Perform step 13 under a fume hood.
13. When the flask has cooled, add 50 mL of 19.2 N Sulfuric Acid Standard Solution to the flask.
Add 10 grams of Potassium Bromide to the flask.
14. Fill a 250-mL beaker to the 75-mL mark with deionized water. Place it under the drip tube.
Elevate the beaker with a laboratory jack so the tube extends below the level of the water.
Purchase Potassium Bromide from a local chemical supplier.
Selenium
Page 1079
Selenium
15. Add 1.0 mL of 30% hydrogen peroxide solution to the flask. Turn the stir control to 5 and the
heat control to 10. Cap the distillation flask.
16. Heat the distillation flask until the yellow color is gone from the complete distillation apparatus,
including the J-tube and condenser. Remove the beaker from under the drip tube.
17. Turn off the heater switch. When the J-tube and condenser have cooled, rinse them with
deionized water. Add the washings to the 250-mL beaker. Total volume in the beaker should
be approximately 100 mL.
18. Add the Phenol Solution drop-wise to the distilled sample to discharge the bromine color (a
white precipitate of tribromophenol will form).
19. Allow the precipitate to settle. Using a dropper, collect about 5 mL of the clear, colorless
distillate and transfer to a test tube.
20. Test the solution for completeness of precipitation by adding 2 drops of Phenol Solution. If the
solution becomes cloudy or white precipitate forms, residual bromine is still present (proceed
to next step). If no cloudiness occurs, the sample is ready for analysis.
21. Transfer the 5-mL aliquot back to the beaker and continue to add Phenol Solution until no
turbidity is formed in subsequent 5-mL aliquots.
22. Transfer the entire sample into a 500-mL volumetric flask. Rinse the beaker with deionized
water and add to the flask.
23. Dilute to volume with deionized water, stopper and mix well. The distillate is now ready
for analysis.
Accuracy check
Selenium Standard Solution, 1000 mg/L
10 mL volumetric pipet and pipet bulb
Deionized water
ADDITIONS.
4. Prepare a 100-mg/L selenium standard solution:
a. Pipet 10 mL of 1000-mg/L selenium standard solution into a 100-mL volumetric flask.
b. Dilute to volume with demineralized water.
prepared standard to three 100-mL portions of fresh sample.
Selenium
Page 1080
Selenium
6. Follow the Diaminobenzidine method test procedure for each of the spiked samples, starting
1000 mg/L Selenium Standard Solution
Deionized water
Volumetric pipet
TenSette Pipet
1. Prepare a 0.5 mg/L Selenium standard solution:

a. Prepare a 100-mg/L selenium standard solution: Pipet 10 mL of 1000-mg/L selenium
standard solution into a 100-mL volumetric flask. Dilute to volume with demineralized
water.
b. Pipet 1.00 mL of the 100-mg/L solution into a 200-mL volumetric flask. Dilute to volume
c. Transfer 100 mL of the standard into a 500-mL Erlenmeyer flask
2. Use this solution in place of the sample. Follow the Diaminobenzidine method test procedure.
Method performance
Program
Standard
Precision95%
Distribution
Sensitivity
Concentration
per 0.010 Abs
640
0.50 mg/L Se
0.470.53 mg/L Se
0.01 mg/L Se
Summary of method
An EDTA masking agent is added to the sample to remove interferences such as iron prior to the
test. The addition of a sulfate buffer adjusts the sample to the optimum pH of 1 to 2. Under these
conditions, diaminobenzidine reacts with all selenium present as selenite (Se4+) to give a yellowcolored piazselenol complex which is extracted and the color intensity measured colorimetrically.
Selenium present as Se2+ and Se6+ is not detected unless the sample is distilled. Test results are
measured at 420 nm.
Selenium
Page 1081
Selenium

Required reagents
Description
Quantity/Test
Unit
(1) Buffer Solution, sulfate type, pH 2.0
10 mL
500 mL
45249
(1) Diaminobenzidine, tetrahydrochloride
0.1 g
5g
706222
4 mL
100 mL
23032
(1) TitraVer Hardness Reagent, ACS
0.4 g
100 g
20426
(1) Toluene, ACS
60 mL
4L
1447017
100 mL
4L
27256
Water, deionized
1
Catalog number
2244200
Selenium Reagent Set (100 tests1), includes:
This test requires a reagent blank. The number of tests shown refers to any combination of samples and blanks.
Required apparatus
Description
Quantity
Unit
Catalog number
257201
100/pkg
each
50841
each
50842
Dropper, 0.5 & 1.0 mL marks, glass
5/pkg
1419705
Dropper, 0.5 & 1.0 mL marks, plastic
20/pkg
2124720
50549
each
each
52046
Hot Plate, 7x7-inch, 120 VAC
each
2881500
Hot plate stirrer 7x7-inch, 220-240 VAC
each
2881602
each
1451537
each
1465100
Ring, support, (3-inch) 83-mm
each
58000
Sample Cells, 1-inch square, 25 mL with stopper, matched pair
2/pkg
2612602
49200
each
each
63800
Support, ring stand, (5 x 8 inch) 127 x 203 mm
each
56300
Unit
Catalog number

Description
Calcium Chloride Solution
1000 mL
42853
Hydrochloric Acid Standard Solution, 0.1 N
1000 mL
1481253
473 mL
14411
Methyl Orange Indicator Solution, (0.50-g/L)
500 mL
14849
29 mL
211220
Potassium Permanganate Solution, 1-g/L
100 mL
1416442
Potassium Bromide, ACS grade1

Selenium
Page 1082
1000 mL
19153
Selenium
Description
Unit
500 mL
203849
Distillation Apparatus Set, general purpose
each
2265300
Distillation Apparatus Heater, 115 VAC
each
2274400
Distillation Apparatus Heater, 230 VAC
each
2274402
Beaker, 1000 mL
each
50053
Glass stirring rod
3/pkg
177001
Stir bar, magnetic
each
2095351
Beaker, 250 mL
each
50046H
10/pkg
56524
Test tubes, 24 mL, 16 mm diameter

1
Catalog number
Purchase Potassium Bromide from a local chemical supplier
Description
Selenium Standard Solution, 1000-mg/L
Unit
Catalog number
100 mL
2240742

Description
Unit
Catalog number
Acetone, ACS
500 mL
1442949
Nitric Acid, ACS
500 mL
15249
100 mL
245032
100 mL
244932
each
1970001
50/pkg
2185696

1000/pkg
2185628
12/pkg
2087076
100/pkg
2601300
each
1457442
19700011
each
1457445
each
1451538
each
1451535
Selenium
Page 1083
Selenium

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Silica, HR, 8185
Silica
DOC316.53.01133
Silicomolybdate Method
Method 8185
HR (1 to 100 mg/L)
Powder Pillows
Scope and Application: For water and seawater
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Sample temperature should be 1525 C (5977 F)
This method is very sensitive to small differences in the measurement wavelength. For best results, use the Standard Adjust
feature (as described in Accuracy check, Standard solution method) to optimize results on each instrument.

Description
Quantity
High Range Silica Reagent Set
Silica
Page 1085
Silica
Silicomolybdate method for powder pillows
Stored Programs
656 Silica HR
Start
1. Select the test.


timer.
period will begin.

10-mL of sample.
6. When the timer

of one Citric Acid Powder
Pillow to the sample cell.
Swirl to mix.
Any yellow color due to
phosphorus is removed in
this step.
3. Prepared Sample:
Molybdate Reagent
Powder Pillow for High
Range Silica to the sample
cell. Swirl until completely
dissolved.

Pillow for High Range
Silica. Swirl to mix.

timer.
with 10 mL of the original
sample.
period will begin.
Perform steps 311 within
three minutes after the
timer expires.
Zero

insert the blank into the
cell holder.
Silica
Page 1086

0 mg/L SiO2

if silica or phosphorus is
present.
Read

sample and insert the
cell holder.

mg/L SiO2.
Silica
Interferences
Occasionally a sample contains silica which reacts very slowly with molybdate. The nature of
these molybdate-unreactive forms is not known. A pretreatment with Sodium Bicarbonate*, then
Sulfuric Acid* will make these forms reactive to molybdate. The pretreatment is given in Standard
Methods for the Examination of Water and Wastewater under Silica-Digestion with Sodium
Bicarbonate. A longer reaction time with the sample and the molybdate and acid reagents (before
adding citric acid) may help instead of the bicarbonate treatment.

Interference level
Color
Eliminated by zeroing the instrument with the original sample.
Iron
High levels of Fe2+ and Fe3+ interfere.

Does not interfere below 50 mg/L PO43. At 60 mg/L PO43, a negative 2% interference
Phosphate
occurs. At 75 mg/L PO43, the interference is negative 11%.
Sulfides (S2)
All levels interfere.
Turbidity
Collect samples in clean plastic bottles.
If prompt analysis is not possible, store samples at 4 C (39 F) for up to 28 days.
Accuracy check
Silica Standard Solution, 1000 mg/L
chemical form.
ADDITIONS.
6. Follow the Silicomolybdate method for powder pillows test procedure for each of the spiked
instrument.
Silica
Page 1087
Silica
Silica Standard Solution, 50-mg/L
1. Use the Silica Standard Solution, 50-mg/L in place of the sample. Use deionized water as the
blank. Follow the Silicomolybdate method for powder pillows test procedure.
Method performance
Program
Standard
per 0.010 Abs
656
50 mg/L SiO2
4852 mg/L SiO2
1.0 mg/L SiO2
Summary of method
Silica and phosphate in the sample react with molybdate ion under acidic conditions to form yellow
silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. Silica is then determined by measuring the remaining yellow

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
2107469
High Range Silica Reagent Set for 10-mL samples (100 tests), includes:
Acid Reagent Powder Pillows for High Range Silica
2429600
Citric Acid Powder Pillows
100/pkg
2106269
Molybdate Reagent Powder Pillows for High Range Silica
100/pkg
2107369
10 mL
4L
27256
Unit
Catalog number
200 mL
111729
500 mL
19449
Water, deionized
Description
Silica
Page 1088
Silica

Description
Unit
Catalog number
Sodium Bicarbonate
454 grams
77601
Sulfuric Acid 1.00 N
100 mL
127032
12/pkg
2087076
each
2635700
Silica
Page 1089

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Silica, LR, 8186
Silica
DOC316.53.01132
Heteropoly Blue Method1
Method 8186
LR (0.010 to 1.600 mg/L as SiO2)
Powder Pillows
Scope and Application: For boiler and ultrapure water

1
Test preparation


Powder pillows
Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

The four-minute reaction time in step 4 is for samples at 20 C; for samples at 10 C, wait eight minutes; for samples at
The one-minute reaction time in step 6 is for samples at 20 C; for samples at 10 C, wait two minutes; for samples at 30 C,
wait 30 seconds.
If testing for very low levels of silica, use Method 8282.
For best results, use matched cells.

Description
Quantity
Amino Acid F Reagent Powder Pillows (for 10-mL sample)
1 pillow
2 pillows
Molybdate 3 Reagent Solution

Sample Cells see Instrument-specific information
1 mL
2
Silica
Page 1091
Silica
Heteropoly Blue
Stored Programs
651 Silica LR
Start
1. Select the test.
2. Insert an adapter if
Fill two sample cells
(Instrumentspecific information) with
10 mL of sample.
5. When the timer

of one Citric Acid Reagent
6. Start the timer. A oneminute reaction period

will begin.
The destruction of
possible phosphate
interference occurs during
this period.
3. Add 14 drops of
Molybdate 3 Reagent to
each sample cell. Swirl to
mix.
4. Start the timer. A fourminute reaction period will

begin.
7. Prepared Sample:
Amino Acid F Reagent
Powder Pillow to one of
the sample cells. Swirl to
mix.
8. Start the timer.
Blank Preparation: The

sample without the Amino
Acid F Reagent is the
blank.
Silica
Page 1092
period will begin.
silica is present.
Silica
Heteropoly Blue (continued)
Read
Zero


0.000 mg/L SiO2.

the cell holder with the fill
line facing the user.

mg/L (SiO2)
Interferences
Interference level
Color
Iron
Large amounts interfere.
Phosphate
Does not interfere at levels less than 50 mg/L PO4. At 60 mg/L PO4, an
interference of 2% occurs. At 75 mg/L PO4 the interference is 11%.
Slow reacting forms of silica
Occasionally a sample contains silica which reacts very slowly with

molybdate. The nature of these molybdate-unreactive forms is not known. A
pretreatment with Sodium Bicarbonate, then Sulfuric Acid will make these
forms reactive to molybdate. The pretreatment is given in Standard Methods
for the Examination of Water and Wastewater under Silica-Digestion with
Sodium Bicarbonate. A longer reaction time with the sample and the
molybdate and acid reagents (before adding citric acid) may help instead of
the bicarbonate pretreatment.
Sulfides
Interfere at all levels.
Turbidity

Collect samples in clean plastic bottles. Analyze samples as soon as possible after collection. If
prompt analysis is not possible, store samples for up to 7 days by cooling to 4 C (39 F) or below.
Silica
Page 1093
Silica
Accuracy check
Silica standard solution, 25 mg/L
ADDITIONS.
default values for standard concentration, sample volume, and spike volumes. After the values
5. Follow the test procedure for each of the spiked samples starting with the 0.1 mL sample
1. Use a 1.00-mg/L SiO2 Standard Solution in place of the sample. Perform the silica procedure.
Solution, navigate to Standard Adjust in the software OPTIONS>MORE>STANDARD
ADJUST.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
651
1.000 mg/L SiO2
0.9901.010 mg/L SiO2
0.012 mg/L SiO2
Summary of method
Silica and phosphate in the sample react with molybdate ion under acidic conditions to form yellow
silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. An Amino Acid is then added to reduce the yellow
silicomolybdic acid to an intense blue color, which is proportional to the silica concentration. Test
Silica
Page 1094
Silica

Required reagents
Description
Low Range Silica Reagent Set (100 tests), includes:
Amino Acid F Reagent Powder Pillows (for 10-mL sample)
Quantity/Test
Unit
Catalog Number
2459300
100/pkg
2254069
100/pkg
2106269
1 mL
50 mL
199526
Recommended Standard
Description
Unit
Deionized Water
4L
Catalog Number
27256
Silica Standard Solution, 1-mg/L SiO2
500 mL
110649
Silica Standard Solution, 25-mg/L as SiO2
236 mL
2122531
Unit
Catalog Number
Optional Reagents and Apparatus

Description
Sodium Bicarbonate
454 g
77601
1000 mL
127053
each
1970001
50/pkg
2185696

Silica
Page 1095

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Silica, Rapid Liquid, 8282
Silica
DOC316.53.01131
Heteropoly Blue Rapid Liquid Method1
Method 8282
ULR(3 to 1000 g/L as SiO2)
Pour-Thru Cell
Scope and Application: For testing trace levels of soluble silica in pure and ultrapure water
1
Test preparation


Instrument
Pour-Thru Cell
Adapter
DR 6000
LQV175.99.20002
Arrow faces right
DR 5000
LZV479
DR 3900
DR 3800, DR 2800, DR 2700
LQV157.99.10002
5940400

compartment
1-inch (round) path aligned with arrow on the
adapter
LZV585 (B)

Clean the Pour-Thru cell and all labware as specified in Labware
See Reagent preparation for instructions on preparing the Amino Acid F Reagent.
The one-minute reaction time in step 12 is for samples at 20 C; for samples at 10 C, wait two minutes; for samples at
30 C, wait 30 seconds.
Silica
Page 1097
Silica

Description
Quantity
Amino Acid F Reagent Powder
varies
Amino Acid Reagent Dilution Solvent
varies
Citric Acid F Reagent
1 mL
Molybdate 3 Reagent
1 mL
Flask, Erlenmeyer, 250-mL, PMP, w/cap
Pour-Thru Cell Module (see Instrument-specific information)
Heteropoly Blue Rapid Liquid method
Stored Programs
645 Silica ULR
Reagent Blank
ON
Start
1. Select the test.

2. Activate the reagent

blank option to account for
the Molybdate 3 reagent
blank. The value is printed
on the bottle label.
3. Use the numeric

keypad on the instrument
to manually adjust the
4. Fill two clean 250-mL

Erlenmeyer flasks to
Repeat
Steps 57
5. Fill a clean 50-mL

with sample from one of
the flasks; then discard the
contents of the cylinder.
Repeat three times.
Silica
Page 1098

sample from the same
flask. Discard any
flask.

into the original flask.
8. Repeat steps 5
through 7 for the second
flask containing sample.
Silica
Heteropoly Blue Rapid Liquid method (continued)
9. Add 1.0 mL of

timer.
period will begin.
11. When the timer

expires, add 1.0 mL of
Citric Acid F Reagent to

timer.
15. Add 1.0 mL of Amino

Acid F Reagent to the
remaining flask. Swirl to
mix.
16. Wait at least 15

seconds, then pour the
contents of the second
flask into the Pour-Thru
Cell.
period will begin. The
destruction of possible
phosphate interference
occurs during this period.
Zero
13. When the timer

of one flask into the
Pour-Thru Cell.


0 g/L SiO2
A faint blue color will

develop if silica is present.
Read

mg/L SiO2.

deionized water
Silica
Page 1099
Silica
Interferences
Interference level
Color
Eliminated by zeroing the instrument with the blank (follow procedure).
Iron
Interferes at high levels.
pH (extreme)
Adjust pH to less than 7.
Phosphate (PO43)
Interferes at levels greater than 50 mg/L PO43.
Sulfides
Turbidity
Use only plastic containers with tight-fitting closures. Do not use glass containers; they will
contaminate the sample with silica.
Soak sampling containers with a solution made of one part Molybdate 3 Reagent to 50 parts of
high quality deionized water of low silica concentration. Fill the containers completely and let
them stand for several hours. Rinse thoroughly with low-level silica water, drain and close.
Repeat this cleaning periodically.
Allow the sample stream to flow for 12 minutes before collection. Do not adjust the flow
during the sampling period as this may introduce particulates.
Rinse the container well with sample before collecting the portion for analysis.
Analyze samples as soon as possible.
Reagent preparation
1. Dissolve the contents of one bottle of Amino Acid F Reagent Powder in one bottle of Amino
Acid Reagent Dilution Solvent.
2. Install a bottle-top dispenser on this bottle, as well as on the Molybdate 3 Reagent and Citric
Acid Reagent bottles.
3. Prepare smaller volumes of Amino Acid F Reagent by dissolving Amino Acid F Reagent
Powder in Amino Acid F Reagent Solvent at a ratio of 11 grams per 100 mL of reagent solvent.
This prepared solution has limited stability; test routinely with the 1-mg/L (1000 g/L) Silica
Standard Solution to confirm performance.
Reduced sensitivity at high concentrations (1000 g/L) indicates reagent instability. If the
concentration is less than 950 g/L, use fresh Amino Acid F Reagent Solution.
Labware
All containers used in this test must be cleansed thoroughly to remove any traces of silica. Use
plastic containers for all analysis and storage because glass can contaminate the sample with
silica. Small bottles or flasks with screw-type closures work well.
1. Clean containers by normal means (do not use phosphate detergents), then rinse with high
quality deionized water of low-level silica concentration.
2. Soak for 10 minutes with a 1:50 dilution of Molybdate 3 Reagent in low-level silica water.
3. Rinse repeatedly with either low-level silica water or the sample before use. Keep containers
tightly closed when not in use.
Silica
Page 1100
Silica
4. Fill the Pour-Thru Cell with this same mixture of Molybdate 3 and water and let stand for
several minutes before use.
5. Rinse with low-level silica water.

solutions are allowed to stand in the cell for long periods after measurement.
1. Remove the color by rinsing with a 1:5 dilution of ammonium hydroxide and deionized water.
2. Rinse several times with deionized water.
3. Cover the Pour-Thru Cell when it is not in use.
Accuracy check
1-mg/L (1000-g/L) Silica standard
TenSette Pipet and Pipet Tips.
250-mL plastic Erlenmeyer flasks (3)
chemical form.
ADDITIONS.
4. Prepare three samples. Fill three plastic Erlenmeyer flasks with 50 mL of prepared sample.
1-mg/L standard to each flask and mix thoroughly.
6. Follow the Heteropoly Blue Rapid Liquid method test procedure for each of the spiked
instrument.
Silica
Page 1101
Silica
500-g/L SiO2 Standard Solution
1. Use a 500-g/L SiO2 Standard Solution in place of the sample. Follow the Heteropoly Blue
Rapid Liquid method test procedure.
to Standard Adjust in the software:OPTIONS>MORE>STANDARD ADJUST.
Method performance
Program
Standard
per 0.010 Abs
645
500 g/L silica
496504 g/L silica
13 g/L silica
Summary of method
A number of modifications are necessary to adapt the Low Range Silica method for analyzing
trace levels in the Ultra Low Range method. It is absolutely necessary to use the one-inch PourThru Cell and liquid reagents. The Pour-Thru Cell increases the reproducibility of the optics and
reduces the instability of the readings that result from moveable sample cells. Liquid reagents
produce more reproducible readings and lower blank values by eliminating slight turbidity that may
remain when using powdered reagents. Use of liquid reagents in continuous monitors for silica
provides a means of confirming the analyzer performance.
Silica and phosphate in the sample react with molybdate ions under acidic conditions to form
yellow silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. Amino Acid F Reagent is then added to reduce the yellow
Silica
Page 1102
Silica

Required reagents
Description
Quantity/Test
Unit
Amino Acid F Reagent Powder
varies
55 g
2651155
Amino Acid Reagent Dilution Solvent
varies
475 mL
2353011
Rapid Liquid ULR Silica Reagent Set, includes:
Catalog number
2678500
1 mL
500 mL
2254249
Molybdate 3 Reagent
1 mL
500 mL
199549
Catalog number
Required apparatus
Description
Quantity
Unit
each
108141
each
2111302
each
2089846
Funnel, powder
each
2264467
Unit
Catalog number
Description
500 mL
110649
Silica Standard Solution, 500-g/L SiO2
3.78 L
2100817
4L
27256
Unit
Catalog number
Water, deionized

Description
Molybdate 3 Reagent
500 mL
10649
2.9 L
199503
199517
Molybdate 3 Reagent
3.78 L
Molybdate 3 Reagent
100 mL
199532
Molybdate 3 Reagent
1L
199553
each
1970001
50/pkg
2185696
12/pkg
2087079
each
2635700
Silica
Page 1103

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Silica, 8282
Silica
DOC316.53.01130
Heteropoly Blue Method1
Method 8282
ULR(3 to 1000 g/L as SiO2)
Pour-Thru Cell
Scope and Application: For testing trace levels of soluble silica in pure and ultrapure water
1
Test preparation


Instrument
DR 6000
Pour-Thru Kit
LQV175.99.20002
DR 5000
DR 3900
DR 3800, DR 2800, DR 2700
Cell Orientation
LZV479
Adapter
Arrow faces right
LQV157.99.10002

compartment
5940400
1-inch (round) path aligned with arrow on

the adapter
LZV585 (B)

Clean the Pour-Thru cell and all labware as specified in Labware.
See Reagent preparation for instructions on preparing the Amino Acid F Reagent.
The one-minute reaction time in step 12 is for samples at 20 C; for samples at 10 C, wait two minutes; for samples at
30 C, wait 30 seconds.

Description
Quantity
Amino Acid F Reagent Solution
1 mL
1 mL
Molybdate 3 Reagent
1 mL
Flask, Erlenmeyer, 250-mL, PMP, with cap
Pipet, TenSette, 0.1 to 1.0 mL with tips
Silica
Page 1105
Silica
Description
Quantity
Heteropoly Blue method
Stored Programs
645 Silica ULR
Reagent Blank
ON
Start
1. Select the test.

2. Activate the reagent

blank option to account for
the Molybdate 3 reagent
blank. The value is printed
on the reagent blank.
3. Use the numeric

keypad on the instrument
to manually adjust the
4. Fill two clean 250-mL

Erlenmeyer flasks to
Repeat
Steps 57

with sample from one of
the flasks; then discard the
contents of the cylinder.
Repeat three times.
Silica
Page 1106

sample from the same
flask. Discard any
flask.

into the original flask.
8. Repeat steps 5
through 7 for the second
flask containing sample.
Silica
Heteropoly Blue method (continued)

to add 1.0 mL of

timer.
period will begin.
11. When the timer

expires, add 1.0 mL of
Citric Acid F Reagent to

timer.
15. Add 1.0 mL of Amino

Acid F Reagent to the
remaining flask. Swirl to
mix.
16. Wait at least 15

seconds, then pour the
contents of the second
flask into the Pour-Thru
Cell.
period will begin. The
destruction of possible
phosphate interference
occurs during this period.
Zero
13. When the timer

of one flask into the
Pour-Thru Cell.


0 g/L SiO2
A faint blue color will

develop if silica is present.
Read

mg/L SiO2.

deionized water
Silica
Page 1107
Silica
Interferences
Interference level
Color
Iron
Interferes at high levels.
pH (extreme)
Adjust pH to less than 7.
Phosphate (PO43)
Interferes at levels greater than 50 mg/L PO43.
Sulfides
Turbidity
Silica
Page 1108
Silica
Use only plastic containers with tight-fitting closures. Do not use glass containers; they will
contaminate the sample with silica.
Soak sampling containers with a solution made of one part Molybdate 3 Reagent to 50 parts of
high quality deionized water of low silica concentration. Fill the containers completely and let
stand for several hours. Rinse thoroughly with low-level silica water, drain and close. Repeat
this cleaning periodically.
Allow the sample stream to flow for 12 minutes before collection. Do not adjust the flow
during the sampling period as this may introduce particulates.
Rinse the container well with sample before collecting the portion for analysis.
Analyze samples as soon as possible.
Reagent preparation
Amino Acid F Reagent Solution is available in either 100-mL bottles or a package of 20 unit-dose
ampules. The bottled reagent is stable for up to one year if the bottle is kept closed when not in
use. The ampuled reagent is sealed under argon and is more stable with a shelf life greater than 1
year. Reduced sensitivity at high concentrations (1000 g/L) indicates reagent instability. Check
the bottled reagent on a routine basis by performing an analysis on a 1-mg/L (1000 g/L) Silica
Standard Solution. If the concentration is less than 950 g/L, use a fresh bottle of Amino Acid F
Reagent Solution.
Prepare larger or smaller volumes of Amino Acid F Reagent by dissolving Amino Acid F Reagent
Powder in Amino Acid F Reagent Solvent at a ratio of 11 grams per 100 mL of reagent solvent.
These reagents are available as the Amino Acid F Reagent Package. This prepared solution has
limited stability; test routinely with the 1-mg/L Silica Standard Solution.
Labware
All containers used in this test must be cleansed thoroughly to remove any traces of silica. Use
plastic containers for all analysis and storage because glass can contaminate the sample with
silica. Small bottles or flasks with screw-type closures work well.
1. Clean containers by normal means (do not use phosphate detergents), then rinse with high
quality deionized water of low-level silica concentration.
2. Soak for 10 minutes with a 1:50 dilution of Molybdate 3 Reagent in low-level silica water.
3. Rinse repeatedly with either low-level silica water or the sample before use. Keep containers
tightly closed when not in use.
4. Fill the Pour-Thru Cell with this same mixture of Molybdate 3 and water and let stand for
several minutes before use.
5. Rinse with low-level silica water.

solutions are allowed to stand in the cell for long periods after measurement.
1. Remove the color by rinsing with a 1:5 dilution of ammonium hydroxide and deionized water.
2. Rinse several times with deionized water.
3. Cover the Pour-Thru Cell when it is not in use.
Silica
Page 1109
Silica
Accuracy check
1-mg/L (1000-g/L) Silica standard
TenSette Pipet and Pipet tips.
250-mL plastic Erlenmeyer flasks (3)
chemical form.
ADDITION.
4. Prepare three samples. Fill three plastic Erlenmeyer flasks with 50 mL of prepared sample.
1-mg/L standard to each flask and mix thoroughly.
6. Follow the Heteropoly Blue method test procedure for each of the spiked samples, starting
500-g/L SiO2 Standard Solution
1. Use a 500-g/L SiO2 Standard Solution in place of the sample. Follow the Heteropoly Blue
method test procedure.
Method performance
Program
Instrument
Standard
per 0.010 Abs
645
DR 5000
500 g/L silica
496504 g/L silica
13 g/L silica
Summary of method
A number of modifications are necessary to adapt the Low Range Silica method for analyzing
trace levels in the Ultra Low Range method. It is absolutely necessary to use the one-inch PourThru Cell and liquid reagents. The Pour-Thru Cell increases the reproducibility of the optics and
reduces the instability of the readings that result from moveable sample cells. Liquid reagents
produce more reproducible readings and lower blank values by eliminating slight turbidity that may
Silica
Page 1110
Silica
remain when using powdered reagents. Use of liquid reagents in continuous monitors for silica
provides a means of confirming the analyzer performance.
Silica and phosphate in the sample react with molybdate ions under acidic conditions to form
yellow silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. Amino Acid F Reagent is then added to reduce the yellow

Required reagents
Description
ULR Silica Reagent Set (using Amino Acid F solution, 100 tests)
Quantity/Test
Unit
Catalog number
2553500
2581400
1.0 mL
100 mL
2386442
Includes: (2) 199532, (2) 2254232, (1) 2386442

ULR Silica Reagent Set (using Amino Acid F ampules, 40 tests)
Includes: (1) 199532, (1) 2254232, (2) 2386420
Amino Acid F Reagent Solution
OR
Amino Acid F Reagent Solution, 1.2-mL Ampules
Citric Acid Reagent Solution
20/pkg
2386420
2 mL
500 mL
2254249
2.0 mL
500 mL
199549
Quantity
Unit
Catalog number
Required apparatus
Description
each
108141
each
2089846
each
1970001
50/pkg
2185696
Description
Unit
Catalog number
500 mL
110649
Silica Standard Solution, 500-g/L SiO2
3.78 L
2100817
4L
27256
Water, deionized
Silica
Page 1111

Description
Unit

Molybdate 3 Reagent
Catalog number
500 mL
10649
2.9 L
199503
199517
Molybdate 3 Reagent
3.78 L
Molybdate 3 Reagent
100 mL
199532
Molybdate 3 Reagent
1L
199553
PourRite ampule breaker
each
2484600
Amino Acid F reagent package
each
2254117
12/pkg
2087079
each
2635700


HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Silver, 8120
Silver
DOC316.53.01134
Colorimetric Method
Method 8120
(0.02 to 0.70 mg/L)
Powder Pillows
Test preparation


Instrumetn
Sample cell
Cell orientation
DR 6000
2495402

DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800 DR 2700
2495402

Digestion is required for samples with interferences. See Digestion.
For best results, measure a reagent blank value for each new lot of reagent (follow the procedure using deionized water in
place of the sample). Subtract the reagent blank value from the final results or enter the value as a reagent blank adjust for
automatic subtraction.
The graduated cylinder must be completely dry before beginning the test. If the Silver 1 Powder becomes moist, it
will not dissolve completely, which will inhibit color development.
The sample pH for this test must be between 9 and 10. Do not use a pH meter to adjust the sample pH as it will contaminate
the sample. See Digestion for the procedure to adjust pH.
The Pour-Thru Cell cannot be used with this procedure.

Description
Quantity
Silver 1 Reagent Powder Pillow
Sodium Thiosulfate Powder Pillow
Clippers
Silver
Page 1113
Silver
Colorimetric Method
Stored Programs
660 Silver
Start
1. Select the test.


one Silver 1 Powder Pillow
to a dry 50-mL graduated
mixing cylinder.
If the Silver 1 Powder
becomes wet at this
point, the powder will
not dissolve completely,
which will inhibit color
development.

a sample cell to at least
the 10 mL mark with the
mixture.
Discard all but 25 mL of
the sample from the
mixing cylinder.

one Silver 2 Reagent
cylinder. Swirl to
completely wet the
powder.
If clumps of dry powder
are present when the
sample is poured in, the
powder will not dissolve
completely, which will
inhibit color development.

one Sodium Thiosulfate
the sample in the mixing
invert to mix.
Be sure to prepare a blank
for each sample.
Silver
Page 1114
4. Use a 50-mL
graduated cylinder to add
50 mL of sample to the
50-mL graduated mixing
invert repeatedly for one
minute.
Be sure to first adjust the
pH of samples if they were
preserved (see Sample
collection, preservation
and storage).

timer.
will start.
Silver
Colorimetric Method (continued)
Zero
9. Fill a second sample

cell to at least the 10 mL
mark with the mixture.
10. When the timer

expires, insert the blank in
the cell holder.

0.00 mg/L Ag
Read

sample in the cell holder.
READ the results in
mg/L Ag.
Immediately rinse the

cells.
Interferences
Standard solutions of approximately 0.4 mg/L Ag with different concentrations of a potential
interfering ion were prepared. The concentration of silver was measured. Interfering substances
shows the ions that caused a change in the silver concentration of more than ten percent (10%).

Interference level
Aluminum
Ammonia
Cadmium
Calcium
Chloride
Chromium6+
Copper
Iron
Lead
Manganese
Magnesium
Mercury
Nickel
Zinc
Silver
Page 1115
Silver
Collect samples in acid-cleaned glass or plastic bottles.
Use pH paper to adjust the pH to 2 or less with concentrated Nitric Acid (about 2 mL/liter).
Store preserved samples at room temperature for up to 6 months.
If the sample contains particulates or for a dissolved metal analysis, filter through a 0.45 m
filter at collection. After filtration, adjust the pH to 2 or less for storage.
Before analysis, adjust the pH to 910 with 5.0 N sodium hydroxide. (See steps 1112 of
Digestion.)
Do not use a pH meter because the pH electrode will contaminate the sample.
Accuracy check
Silver Standard Solution, 1000 mg/L Ag
5.0 mL Class A volumetric pipet and pipet bulb
TenSette Pipet, 0.11.0 mL and tips
5. Select OPTIONS>MORE>STANDARD ADDITIONS from the instrument menu. Accept the
are accepted, the unspiked sample reading will appear in the top row. See the user manual for
more information.
6. Prepare a 50.0 mg/L silver standard solution as follows. Add 5.00 mL of 1000 mg/L Silver
Standard Solution to a 100-mL volumetric Class A flask. Dilute to volume with deionized water.
This is a 50.0 mg/L standard solution.
7. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of the 50.0
mg/L standard to three 50-mL portions of fresh sample. Mix thoroughly.
8. Follow the Colorimetric Method test procedure for each of the spiked samples, starting with
the 0.1 mL spiked sample. Measure each of the spiked samples in the instrument.
Silver
Page 1116
Silver
1. Prepare a 0.5 mg/L silver standard solution as follows: Pipet 0.5 mL of silver standard,
1000 mg/L as Ag, into a 1000-mL (1 liter) volumetric flask. Dilute to the mark with deionized
water. Mix well. Prepare this solution daily.
2. Use the 0.5-mg/L silver standard solution in place of the sample. Follow the Colorimetric
Method test procedure.
navigate to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST.
4. Turn on the Standard Adjust option and accept the displayed concentration. If an alternate
Digestion
If the sample contains organic matter, thiosulfate or cyanide, digest the sample before analysis.
Possible sources for these compounds are wastewater, silver electroplating baths and silver strike
solutions. Use the Digesdahl Digestion Apparatus.
DANGER
Chemical hazard. Poisonous hydrogen cyanide gas may be generated during the digestion.
Operate the Digesdahl in a closed fume hood.
CAUTION
Chemical hazard. Always wear safety glasses and use a safety shield or operate the
Digesdahl in a closed fume hood. Follow the safety precautions in the Digesdahl Digestion
Apparatus Manual.
1. Add an appropriate size sample to the 100-mL digestion flask for use with the Digesdahl. Add
several boiling chips to prevent bumping.
Note: Appropriate sample size is determined experimentally. The final sample concentration (after dilution to
100 mL) should be 00.6 mg/L. Larger dilutions may be necessary for electroplating baths and silver
strike solutions. Do not exceed the maximum sample volume of 25 mL. Several 25-mL aliquots may be
digested in succession to concentrate a very dilute sample.
2. Turn on the water aspirator and make sure there is suction in the fractionating head.
3. Carefully add 3 mL of concentrated sulfuric acid to the sample in the volumetric flask.
Immediately place the head on the digestion flask. Never use less than 3 mL of acid.
4. Place the digestion flask on the heater. Turn the temperature dial to 440 C (825 F).
5. When the sulfuric acid reflux line becomes visible, wait 35 minutes.
6. Make sure there is acid in the flask before adding hydrogen peroxide!
7. Place the capillary funnel on the fractionating head. Fill the funnel to the 10-mL line with
50% hydrogen peroxide.
Note: If the sample completely evaporates, turn the Digesdahl off and cool completely. Carefully add water to
the flask before handling. Start the digestion over with a fresh sample.
Silver
Page 1117
Silver
8. Use the capillary funnel to add 5 mL of hydrogen peroxide. Check the solution in the flask for
digestion completion. If digestion is not complete, continue adding hydrogen peroxide in 5 mL
to 10 mL portions. Several portions may be necessary.
Note: The digestion is complete when the digestate is colorless or the color of the digestate does not change
after hydrogen peroxide is added. A completely digested sample will not cause foam to form.
9. After digestion is complete and all the hydrogen peroxide has boiled away, reduce the volume
of the digestate to near dryness. Do not allow the sample to become completely dry! Remove
the flask from the heater. Cool to room temperature.
10. Slowly add approximately 25 mL of deionized water to the cooled flask. Swirl to mix.
11. Add 2 drops of 1 g/L Phenolphthalein Indicator Solution. Add 2 drops of 1 g/L Thymolphthalein
Indicator Solution.
12. Use sodium hydroxide to adjust the pH of the solution to 910. The solution will be pink in this
pH range.
Note: A purple color indicates a pH greater than 10. If this occurs, add a drop of sulfuric acid and 2 drops of
each indicator and repeat the pH adjustment. Initially, use 50% sodium hydroxide, then 1 N sodium
hydroxide as the end point is approached.
13. Filter turbid digestates. Quantitatively transfer the filtrate (or unfiltered sample) to a clean
100-mL volumetric flask. Dilute to the mark with deionized water and mix. Follow the
Colorimetric Method for silver.
Method performance
Standard
Precision
Distribution
Sensitivity
0.50 mg/L Ag
0.490.51 mg/L Ag
0.005 mg/L Ag
Summary of method
Silver ions in basic solution react with cadion 2B to form a green to brown to red-purple complex.
The sodium thiosulfate acts as a decolorizing agent for the blank. The Silver 1 and Silver 2
reagents contain the buffer, indicator and masking agents. Organic extractions are not necessary
and this method does not have as many interferences as the traditional dithizone method. Test

Required reagents
Description
Quantity/Test
Unit
Catalog number
50/pkg
2293566
Silver 2 Reagent Solution Pillow
50/pkg
2293666
Sodium Thiosulfate Powder Pillow
50/pkg
2293766
Silver Reagent Set (50 tests), includes:
Silver
Page 1118
2296600
Silver
Required apparatus
Description
Quantity
Unit
Catalog number
Clippers
each
96800
each
2117941
each
189641
2/pkg
2495402
Description
Water, deionized
Unit
Catalog number
100 mL
1461342
4L
27256
Unit
Catalog number
490 mL
2119649
Digestion reagents and apparatus

Description
Phenolphthalein Indicator Solution, 1 g/L
Sodium Hydroxide Solution, 50%
Thymolphthalein Indicator Solution, 1 g/L
Water, deionized
15 mL SCDB
189736
500 mL
218049
100 mL MDB
104532
2.5 L
97909
15 mL SCDB
2185336
4L
27256
500 g
2055734
Digesdahl Digestion Apparatus, 115 VAC, 50/60 Hz
each
2313020
Digesdahl Digestion Apparatus, 230 VAC, 50/60 Hz
each
2313021
Safety Shield, for Digesdahl
each
5003000
Silver
Page 1119
Silver
Description
Unit
Catalog number
each
189641
Nitric Acid, concentrated ACS
500 mL
15249
100 mL
245032
pH paper, 014
100/pkg
2601300
Membrane filter paper, 0.45 m
100/pkg
2618800
each
1457442
each
1451537
each
1970001
50/pkg
2185696
1000/pkg
2185628
each
1457453
Volumetric pipet, Class A, 0.5 mL
each
1451534
Safety bulb
each
1465100
Finger cots
2/pkg
1464702

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Solids, Settleable Matter, 8165
Solids, Settleable Matter

Direct Measurement1
DOC316.53.001202
Method 8165
Scope and Application: For sewage and wastewater.

1
Adapted from Standard Methods for the Examination of Water and Wastewater, Section 2540E.
Test preparation

Description
Quantity
Imhoff Cone
Imhoff Cone Brush
Imhoff Cone Support
Direct Measurement
1. Fill an Imhoff cone to

the 1-liter mark with a
thoroughly mixed sample.
2. Wait 45 minutes for

the undisturbed sample to
settle.
3. Spin the cone forward

and backward several
times to dislodge materials
on the inclined side of the
cone.
4. Wait 15 minutes for

the sample to settle.
5. Read the graduated

scale on the Imhoff cone
at the top of the solids
layer as mL/L settleable
matter.

Page 1121
Refrigerate at 4 C up to the time of analysis to minimize microbiological decomposition of

solids.
Analyze within 24 hours. Bring to room temperature before analysis.
Summary of method
The amount of settleable matter in sewage treatment plant influent and effluent gives an empirical
estimate of the type and extent of treatment required and the general quality of the water being
discharged.

Required apparatus
Description
Quantity/Test
Unit
Catalog number
Imhoff Cone
each
206700
Imhoff Cone Brush
each
68800
Imhoff Cone Support
each
57200
Quantity/Test
Unit
Catalog number
2000 mL
each
50054
pair
each
2410104
Optional apparatus
Description
Beaker, Glass, low form
Gloves, Chemical resistant, size 99 1/2
Pitcher, Graduated
Sampler, Dipper
2000 mL
each
2612854
39 ft handle
each
2929501

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Solids, Nonfilterable; Total and Volatile, 8158 and 8164
Solids, Nonfilterable Suspended

Solids; Total and Volatile
USEPA1 Gravimetric Method2
DOC316.53.001204
Methods 8158 and 8164

1
USEPA accepted.
Adapted from Standard Methods for the Examination of Water and Wastewater Section 2450
Test preparation

The Total Nonfilterable Solids are the same as the Total Suspended Solids (TSS)

Description
Quantity
Filter flask
Filter holder
Filter, 47-mm
Tongs
Tweezers
Watch glass
Desiccator with desiccant
Muffle Furnace
Drying Oven
Deionized Water
varies
Solids, Nonfilterable Suspended Solids; Total and Volatile

Page 1123

Gravimetric MethodTotal Nonfilterable Residue, Method 8158
1. Use tweezers to place

a 47-mm glass fibre filter
disc in the filter holder.
Always use tweezers to
handle filter discs.
Moisture from fingers can
add moisture to the disc
and cause a weighing
error.
5. If volatile nonfilterable
solids are also being
measured, use tongs to
place the watch glass with
the disc into a muffle
furnace and ignite at
550 C for 15 minutes. If
not, omit this step.
Partially preheat the muffle
furnace before inserting
the watch glass. Placing
the watch glass in a
550 C furnace could
cause it to shatter. Bring
the temperature up to
550 C 15 minutes after
placing the filter and watch
glass in the furnace.
2. Place the filter holder

assembly in the filtering
flask and add 100 mL of
deionized water. Apply
vacuum to the flask until
all the water is drawn
through the filter.
3. Slowly release the

vacuum from the filtering
system and remove the
disc from the filter holder
and transfer to a watch
glass.
4. Place the disc in a

preheated drying oven at
103 C for one hour.
6. Use metal tongs to

remove the disc and watch
glass from the oven or
furnace and place in a
desiccator. Cover
immediately. Allow the
watch glass to cool slightly
before sealing the
desiccator as pressure
from the heated air inside
the desiccator can force
the cover off.
7. Remove the watch

glass and disc from the
desiccator as a unit and
place beside the analytical
balance.
8. Again, place the disc

in the filter holder/flask
assembly. Wet the disc
with deionized water to
ensure adhesion to the
holder.
Use plastic tweezers to

remove the disc from the
watch glass and weigh to
the nearest 0.1 mg
(0.0001 g). Record this
value as B.
Allow the filter and glass to

cool to room temperature.

Page 1124

Gravimetric MethodTotal Nonfilterable Residue, Method 8158 (continued)
9. Filter 100 mL (or

more, if solids content is
low) of well-mixed,
representative water
sample by applying
vacuum to the flask.
Follow with three separate
10-mL washings of
deionized water.
For greatest accuracy,
filter as much sample as
possible. However, using a
sample that contains more
than 15 mg of solids will
clog the filter prematurely.
Adjust the exact volume of
the water sample to
achieve the optimum
condition. Several
completed tests will show
whether any adjustment is
necessary.
10. Remove any residue

that remains on the sides
or bottom lip of the filter
holder. A rubber
policeman on the end of a
stirring rod is very helpful
to scrape the residue
loose. Small amounts of
deionized water will help
wash the residue down
onto the filter disc.
Slowly release the vacuum
from the filtering system
and gently remove the
filter disc from the holder.
Place the disc on a watch
glass. Inspect the filtrate
(filtered water in flask) to
make sure that the solids
are properly trapped on
the disc.
11. Place the watch glass

and filter in a drying oven
at 103 C for one hour.

desiccator. Cover
before sealing the
the cover off.

Page 1125

Gravimetric MethodTotal Nonfilterable Residue, Method 8158 (continued)
13. Remove the watch

balance.
the nearest 0.1 mg
(0.0001 g). Record this mg
value as A.
14. Return the disc to the

watch glass if the mg/L
Volatile Nonfilterable
Residue is to be
determined. If not, discard
the disc.
If Volatile Nonfilterable
Residue is to be
determined, do not lose
any of the suspended
matter on the disc.
15. Calculate Total Non-filterable Residue (TNR):

AB
----------------------------------------------------------------- = mg/L TNR
Sample Volume in Liters
Where:
A = Weight (mg) of disc with residue
B = Weight (mg) of disc
Example:
A = 95.5 mg
B = 81.5 mg
Volume of sample = 0.1 L
95.5 mg 81.5 mg
------------------------------------------------- = 140 mg/L TNR
0.1 L

Page 1126

Gravimetric MethodVolatile Nonfilterable Solids, Method 8164
1. Place the watch glass

and filter disc from the
Total Nonfilterable
Residue procedure
(step 14) in the muffle
furnace and ignite at
550 C for 15 minutes.
Partially preheat the muffle
furnace before inserting
the watch glass. However,
placing the watch glass in
a 550 C furnace could
cause it to shatter. Bring
the temperature up to
550 C 15 minutes after
placing the filter and watch
glass in the furnace.

desiccator. Cover
before sealing the
the cover off.
3. Remove the watch

balance.
the nearest 0.1 mg
value as C.

4. Calculate Volatile
Non-filterable Residue
(VNR):
AC
----------------------------------------------------------------- =
Sample Volume in Liters
mg/L VNR
where:
A = Weight (mg) of disc
with residue
C = Weight (mg) of disc
and residue after ignition
Example:
A = 95.5 mg
C = 91.2 mg
Volume of sample = 0.1 L
95.5 mg 91.2 mg
-------------------------------------------------- =
0.1 L
43 mg/L VNR
Samples should be analyzed as soon as possible after collection but can be stored up to
seven days by cooling to 4 C (39 F).

Page 1127

Required apparatus
Description
Unit
Aspirator, vacuum
each
Catalog number
213100
Balance, Analytical, 115 VAC, 60 Hz
each
2936701
each
62011
each
50842
Desiccant, indicating Drierite
each
2088701
each
1428500
Desiccator Plate, ceramic
each
1428400
100/pkg
253000
Filter Holder, magnetic
Filter disc, glass fiber, 47-mm
each
1352900
each
54653
Furnace, muffle 240 VAC, 50/60 Hz
each
1429624
Furnace, muffle, 120 VAC, 50/60 Hz
each
1429600
Oven, laboratory, 240 VAC, 50 Hz
each
1428902
each
1428900
Stopper, rubber, one-hole, No. 8
6/pkg
211908
Tongs
each
56900
3.6 m
56019
Tweezers, plastic
each
1428200
Watch Glass, 100-mm
each
57870
4L
27256
Unit
Catalog number
Water, deionized

Description
Ammonium Hydroxide, approx.. 58% ACS
500 mL
10649
Bottle, w/cap, wide mouth 500 mL poly
12/pkg
2087079
Brush
each
68700
Pump, vacuum, hand-operated
each
1428300
Pump, vacuum, 1.2 CFM, 220 VAC, w/Europeon plug
each
Pump, vacuum, 1.2 CFM, 115 VAC, 60 Hz
each
2824800
Rubber policeman for 3/16" rod
each
1430900
Stirring rod, glass
3/pkg
177001

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Solids, Total Filterable, 8163
Solids, Total Filterable

(Total Dissolved Solids)
DOC316.53.001246
Method 8163

1
USEPA accepted.
Adapted from Standard Methods for the Examination of Water and Wastewater, Part 2540C
Test preparation

Total Filterable Solids = Total Dissolved Solids (TDS)
Samples with high bicarbonate concentrations may require increased drying at 180 C to make sure that the conversion of
bicarbonate to carbonate is complete.
Limit sample size to no more than 200 mg residue for best results.
When measuring volatile dissolved solids, heat the evaporating dish to 550 C for 1 hour before beginning the test.
Sample residue from this procedure can be used directly in Method 8277, Solids, Total Volatile and Fixed.

Description
Quantity
Evaporating dish
Filter flask
Filter holder
Filter, 47-mm
Hot Plate
Steam Bath, 8" diameter
Analytical Balance
Desiccator
Tongs
Tweezers
Drying Oven
Deionized Water
varies
Solids, Total Filterable (Total Dissolved Solids)

Page 1129

USEPA Gravimetric MethodTotal Filterable Solids, Method 8163
5. Heat a clean
evaporating dish in a
drying oven at 180 C for
one hour.
Using metal tongs, remove
the evaporating dish from
the furnace and place in a
desiccator. Cover
dish to cool slightly before
sealing the desiccator as
pressure from the heated
air inside the desiccator
can force the cover off.

holder/flask assembly
using a clean filter flask.
Before using the flask,
remove all residue from
the flask by thoroughly
cleaning with a dilute
solution of ammonium
hydroxide and rinsing with
deionized water.
7. Place a 47-mm filter

With vacuum applied to
the flask, wash the filter
with three separate 20-mL
volumes of deionized
water. Remove all traces
of water by continuing
vacuum for two to three
minutes after the water
has passed through the
filter. Disconnect the
vacuum and discard these
washings from the flask.
8. Reconnect vacuum to
the filter holder/flask
assembly. Use a clean
100-mL graduated cylinder
to filter 100 mL (or more if
solids content is low) of a
well-mixed representative
water sample.
10. Use tongs to transfer

the desiccator to the
balance. Weigh to the
nearest 0.1 mg (0.0001 g)
and record this weight
as B.
11. Place the steam bath

on the hot plate, add water
and transfer the
evaporating dish from the
balance to the steam bath.
12. Pour the 100-mL

filtrate sample from the
filter flask into the
evaporating dish.
Evaporate to dryness (this
may take up to four hours).

the water sample to
achieve the optimum
condition. Several
necessary.
Apply vacuum
9. Apply vacuum for two

to three minutes after the
sample has passed
through the filter.
Disconnect the vacuum.

Page 1130
Periodically check the

reservoir of the water bath.
Add more water if needed.

USEPA Gravimetric MethodTotal Filterable Solids, Method 8163 (continued)
Repeat steps
9 and 10

the evaporating dish
residue to a drying oven
and dry at 180 C for one
hour. Transfer to a
desiccator and cool.
14. Weigh the evaporating

dish to the nearest 0.1 mg
(0.0001 g) on an analytical
balance and record this
weight as A.
Allow the dish to cool

slightly before sealing the
desiccator; pressure from
the heated air inside the
desiccator can force the
cover off.
15. Repeat steps 13 and

14 until a constant weight
is obtained or until the
weight change is less than
4% of the previous weight
or 0.5 mg, whichever is
less.
16. Calculate the Total

Filterable Residue (TFR).
Refer to Total filterable
residue calculation.
The solid residue can be

used directly in method
8277 to determine Volatile
Dissolved Solids.
Total filterable residue calculation

To calculate TFR:
AB
-------------------------------------------------------------- = mg/L TFR
Sample volume in liters
Where:
A = Weight (mg) of residue and dish after drying
B = Weight (mg) of dish Sample Volume = 0.1 L
Example:
A = 20187.3 mg
B = 20140.1 mg
20187.3 20140.1
------------------------------------------------- = 472 mg/L TFR
0.1
Samples should be analyzed as soon as possible after collection but can be stored up to

Page 1131
Summary of method
A well-mixed sample is filtered through a standard glass fiber filter. The filtrate is evaporated to
dryness in a weighed dish and dried to a constant weight at 180 C. The increase in the weight of
the dish after drying represents the total filterable solids (total dissolved solids).

Required apparatus
Description
Unit
Aspirator, vacuum
each
Catalog number
213100
each
2936701
each
62011
each
50842
each
2088701
each
1428500
each
1428400
Evaporating dish, porcelain, w/lip, 120-mL, 90-mm

Filter disc, glass fiber, 47-mm
100/pkg
253000
Filter Holder, magnetic
each
1352900
each
54653
Hot plate/stirrer, 7 x 7 inch, 115 VAC
each
2881600
each
2881602
each
1428902
each
1428902
each
Steam bath, 8-inch diameter
1428900
2347900
6/pkg
211908
Tongs
each
56900
3.6 m
56019
Tweezers, plastic
each
1428200
Water, deionized
4L
27256
Unit
Catalog number

Description
Ammonium Hydroxide, 58% ACS
500 mL
10649
Bottle, w/cap, wide mouth 500 mL poly
12/pkg
2087079
Brush
each
68700
Pump, vacuum, hand-operated
each
1428300
Pump, vacuum, 1.2 CFM, 220 VAC, w/Europeon plug
each
Pump, vacuum, 1.2 CFM, 115 VAC, 60 Hz
each
2824800
Rubber policeman for 3/16" rod
each
1430900
Stirring rod, glass
3/pkg
177001

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Solids, Volatile Dissolved and Fixed Dissolved, 8277
Solids, Volatile Dissolved and

Fixed Dissolved
DOC316.53.001206
Gravimetric Method1
Method 8277
Scope and Application: For wastewater.

1
Test preparation

The residue from Method 8163Solids, Total Filterable can be used in this method. Start at Step 12..

Description
Quantity
Filter flask
Filter holder assembly with stopper
Filter, 47-mm, glass fiber
Tongs
Evaporating dish
Analytical balance
Muffle furnace
Vacuum sourceand tubing
Deionized water
Steam bath
Hot plate
See Sample collection, preservation and storage for reorder information.
Solids, Volatile Dissolved and Fixed Dissolved

Page 1133

Gravimetric Method
1. Heat an evaporating
dish at 550 C for one
hour. Cool and store the
dish in a desiccator until
needed. Allow the dish to
cool slightly before sealing
the desiccator, as
pressure from the heated
air can force the cover off.

holder/flask assembly
using a clean filter flask.
All residue should be
removed from the flask by
cleansing thoroughly with
a dilute solution of
ammonium hydroxide and
rinsing with deionized
water.
3. Place a 47-mm filter

With vacuum applied to
the flask, wash the filter
with three separate 20-mL
volumes of deionized
water. Remove all traces
of water by continuing
vacuum for two to three
minutes after the water
has passed through the
filter. Disconnect the
vacuum and discard these
washings from the flask.
4. Reconnect vacuum to
the filter holder/flask
assembly. Using a clean
100-mL graduated
cylinder, filter 100 mL (or
more if solids content is
low) of a well-mixed
representative water
sample.

the desiccator to the
and record this weight as
A.
7. Place the steam bath

on the hot plate, add water
and transfer the
evaporating dish from the
balance to the steam bath.
8. Pour the 100-mL

filtrate sample from the
filter flask into the
evaporating dish.
Evaporate to dryness (this
may take up to four hours).

the water sample to
achieve the optimum
condition. Several
necessary.
Apply Vacuum
5. Apply vacuum for two

to three minutes after the
sample has passed
through the filter.
Disconnect the vacuum.

Page 1134
Periodically check the

reservoir of the water bath.
Add more water if needed.

Gravimetric Method (continued)
9. Place the dish in a

pre-heated oven at 103105 C and dry to a
constant weight. This will
take 3060 minutes.
10. Take the dish out of

the oven and allow it to
cool to room temperature
in a desiccator.
Allow the dish to cool
slightly in the desiccator
before sealing the
desiccator, as pressure
from the heated air can
force the cover off.

the evaporating dish to the
nearest 0.1 mg (0.0001 g).
Record this as B.
12. Transfer the dish into

a preheated muffle
furnace at 550 C for
30 minutes.
15. The loss of weight is

total volatile solids.
Weighed residue is total
fixed solids.
Calculate:
Repeat
Steps 910

the furnace with tongs and
in a desiccator. Weigh the
(0.0001 g) using an
analytical balance. Record
this mg value as D.
14. Repeat the ignition

(steps 12. and 13.) until
two successive weighings
do not differ by more than
4% or 0.5 mg, whichever
is greater.
mg/L Volatile Diss. Solids =

( B D ) 1000
--------------------------------------C
mg/L Fixed Diss. Solids =
( A D ) 1000
--------------------------------------C
Where:
B = Weight (mg) of solids
+ dish before ignition
D = Weight (mg) of solids
+ dish after ignition
C = Volume in mL of
filtrate transferred to the
aluminum dish
A = Weight (mg) of dish

Page 1135
Samples should be analyzed as soon as possible after collection, but can be stored up to
Summary of method
A well-mixed sample is filtered through a glass fiber filter. An aliquot of the filtrate is evaporated in
a weighed dish and dried to constant weight in a 103105 C oven. The dish and sample residue
are ignited at 550 C for 30 minutes. The loss of sample mass upon ignition represents the volatile
dissolved solids. The remaining residue after ignition represents the fixed dissolved solids.

Required apparatus
Description
Unit
Catalog number
Balance, Analytical, SA80, 115 VAC, 60 Hz
each
2936701
each
50842
each
2088701
each
1428500
each
1428400
Evaporating dish, porcelain, w/lip, 120-mL, 90-mm
each
52561
Furnace, muffle 120 VAC 50/60 Hz
each
1429600
Furnace, muffle 240 VAC 50/60 Hz
each
1429624
Steam bath, 8 diameter
each
2347900
Tongs
each
56900
Aspirator, vacuum
each
213100
Filter disc, 47 mm, glass fiber
100/pkg
253000
Filter Holder, 47 mm magnetic
each
1352900
Flask, filtering 1000 mL
each
54653
each
2881600
Hot plate/stirrer, 7 x 7 inch, 220-240 VAC
each
2881602
each
1428900
each
1428902
Stopper, rubber, one-hole No. 8
6/pkg
211908
4L
27256
Water, deionized

Page 1136

Description
Unit
Catalog number
Dish, aluminum (63 x 17.5 mm)
100/pkg
2164000
500 mL
1473649
Blender,115 VAC, 1.2 L
each
2616100
Blender, 240 VAC, 1.2 L
each
2616102
12/pkg
2087079
Bottles, w/cap, wide mouth, 500 mL poly

Beaker, glass, 250 mL
each
50046H
Pump, Vacuum, 1.2 cfm, 115 VAC
each
2824800
Pump, Vacuum, 1.2 cfm, 220 VAC, European plug
each
2824802
Pump, Vacuum, Hand-operated
each
1428300
Stir Bar, 22 x 8 mm
each
2095350
Stirrer, magnetic, 4 x 4, 115 VAC
each
2881200
each
2881202

Page 1137

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Solids, Total Volatile and Fixed, 8276
Solids, Total Volatile and Fixed
DOC316.53.001205
Gravimetric Method1
Method 8276
Scope and Application: For potable, surface and saline water and domestic and industrial wastewater.
1
Test preparation

When measuring volatile solids, ignite the aluminum dishes for one hour at 550 C prior to use.
Larger sample sizes may be used. For larger samples, use an evaporating dish and a steam bath to evaporate the liquid.
Samples from Method 8271 may be used directly in this procedure. Use the resulting sample and start this method at step 7.

Description
Quantity
Weighing dish, aluminum
Drying oven
Desiccator and desiccant
Analytical balance
Muffle furnace

Page 1139

Gravimetric Method
1. Heat an aluminum
dish to 550 C for one
hour. Store and cool the
dish in a desiccator until
needed.
1. Weigh an aluminum
value as C.
If a sample from Method

8271 is used, omit steps
26. Proceed to step 7.
2. Mix the sample and

add 50 mL to the
aluminum dish. Use a
blender or a beaker with
stir bar and stir plate to mix
the samples.
3. Place the sample in a

preheated oven and
evaporate at 103105 C
for approximately six
hours.
Highly mineralized water
may prolong the drying
time.
Note: A steam bath and
evaporating dish may be
used in place of the drying
oven for larger samples.
After evaporation on the
steam bath, dry the dish to
constant weight in a
103105 C drying oven.

in a desiccator.
5. Weigh the dish to the

using an analytical
balance. Record this mg
value as A.

Page 1140
6. Transfer the aluminum

dish into a pre-heated
muffle furnace at 550 C
for 30 minutes.
After 30 minutes, take the
dish out of the furnace with
tongs and cool to room
temperature in a
desiccator.

nearest 0.1 mg using an
analytical balance.
Repeat ignition until two
successive sample
weighings (A B) do not
differ by more than 4% or
0.5 mg, whichever is less.
Record this mg value as B.

8. The loss of weight is

total volatile solids.
Weighed residue is total
fixed solids.
Calculate:
mg/L Volatile Solids
( A B ) 1000
= --------------------------------------------------------------sample in volume in mL
mg/L Fixed Solids
( B C ) 1000
= -------------------------------------------------------sample volume in mL
where:
A = Weight (mg) of solids
+ dish before ignition at
550 C
B = Weight (mg) of solids
+ dish after ignition at
550 C
C = Weight (mg) of empty
dish
Summary of method
A well-mixed sample is evaporated in a weighed dish and dried to a constant weight in a
103105 C oven. The dish and sample are ignited at 550 C for 30 minutes. The loss of sample
mass upon ignition represents the volatile solids. The remaining sample after ignition represents
the fixed solids.

Page 1141

Required apparatus
Description
Unit
Catalog number
each
2936701
each
50841
each
2088701
each
1428500
each
1428400
100/pkg
2164000
Furnace, muffle, 120 Vac, 50/60 Hz
each
1429600
Furnace, muffle, 240 Vac, 50/60 Hz
each
1429624
Oven, laboratory, 240 Vac, 50 Hz
each
1428902
4L
27256
Water, deionized
each
1428900
Tongs
each
56900
Description
Unit
Catalog number
Blender, 1.2 liter, 120 VAC
each
2616100
each
2616102
Stirrer, Magnetic, 4 x 4, 115 VAC
each
2881200
each
2881202
Beaker, Glass, 250 mL
each
50046H
Stir Bar, 22 x 8 mm
each
2095350
Steam Bath, 8 inch diameter
each
2347900
Evaporating Dish, porcelain, 120 mL
each
52561
12/pkg
2087079

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Solids, Total, 8271
Solids, Total
DOC316.53.001203
Method 8271
Scope and Application: For potable, surface and saline water and for domestic and industrial wastewater.
1
USEPA Accepted.
Adapted from Standard Methods for the Examination of Water and Wastewater, Section 2540B
Test preparation

Test results may be used directly in continuation with Method 8276Solids, Total Volatile and Fixed
Dry the aluminum dishes at 103-105C for one hour prior to use. Store dried dishes in a desiccator.
Larger samples can be used. For larger samples, use a steam bath and evaporating dishes in place of the aluminum dishes.

Description
Quantity
Weighing dish, aluminum
Drying oven
Analytical balance
Tongs
See Sample collection and storage for reorder information.
Solids, Total
Page 1143
Solids, Total
Gravimetric Method
1. Heat an aluminum
dish at 103105 C for one
hour. Store and cool the
dish in a dessicator.
2. Weigh the aluminum

(0.0001 g). Record the mg
value as B.
3. Mix the sample and

add 50 mL to the
aluminum dish. Use a
blender or a beaker with
stirbar and stir plate to mix
samples.
4. Place the sample in a

preheated oven and
evaporate at 103105 C
for approximately six
hours.
Highly mineralized water
may prolong the drying
time.
Note: A steam bath and
evaporating dish may be
used in place of the drying
oven for larger samples.
After evaporating on the
steam bath, dry the dish to
constant weight in a 103105C drying oven.
Repeat
Steps 46

in a desiccator.
Solids, Total
Page 1144

using an analytical
balance. Record this mg
value as A.
7. Repeat step 3step 5

until results do not differ by
more than 0.4 mg.
Successive weighings that
are identical for some
wastewater samples are
unlikely due to slow
organic volatilization.
Solids, Total
8. Calculate:
( A B ) 1000
mg/L Total Solids = ---------------------------------------------------------Sample Volume in mL
Where:
A = Weight (mg)1 of
sample + dish
B = Weight (mg) of dish
1
Note: Continue with Method

8276 if Volatile and Fixed
Solids results are required.
Weight in mg = grams x 1000
Summary of method
A well-mixed sample is evaporated in a weighed dish and dried to constant weight in an oven at
102105 C. The increase in weight over that of the empty dish represents the total solids.

Required apparatus
Description
Unit
Catalog number
each
2936701
Cylinder, 50-mL
each
50841
each
2088701
each
1428500
each
1428400
Water, deionized
4L
27256
100/pkg
2164000
1428900
each
Tongs
each
56900
each
1428902
Solids, Total
Page 1145
Solids, Total

Description
Unit
Catalog number
each
2616100
each
2616102
Stirrer, Magnetic, 4 x 4, 115 VAC
each
2881200
each
2881202
Beaker, Glass, 250 mL
each
50046H
Stir Bar, 22 x 8 mm
each
2095350
Steam Bath, 8 inch diameter
each
2347900
Evaporating Dish, porcelain, 120 mL
each
52561
12/pkg
2087079

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Suspended Solids, 8006
Suspended Solids
DOC316.53.01139
Photometric Method1
Method 8006
(5 to 750 mg/L)
1
Adapted from Sewage and Industrial Wastes, 31, 1159 (1959).
Test preparation

TheInstrument-specific information table displays information that may vary from instrument to
instrument. Select your spectrophotometer from the instrument column on the left. Read across to
find the corresponding sample cells and adapters required to perform this test on
your spectrophotometer.

Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402


Description
Quantity
Beaker, 600-mL, polypropylene
Blender
Cylinder, 500-mL polypropylene, graduated
Sample Cells (see the Instrument-specific information table)
Suspended Solids
Page 1147
Suspended Solids
Photometric Method
Stored Programs
630 Suspended Solids
Start
1. Select the test.

required (see the
Instrument-specific
information table).
2. Blend 500 mL of
sample in a blender at
high speed for exactly two
minutes.
3. Pour the blended

beaker.
4. Prepared Sample:
Stir the sample and
immediately pour 10 mL of
the blended sample into a
sample cell.

for orientation.
Zero
with 10 mL of tap water or
deionized water.


0 mg/L TSS
8. Swirl the prepared

sample to remove any gas
bubbles and uniformly
suspend any residue.
Read

cell holder.

mg/L TSS.
Interferences
Samples that absorb strongly at 810 nm, such as blue dyes, may give false, high-bias readings. A
user-entered calibration is advised for these samples.
Suspended Solids
Page 1148
Suspended Solids

Collect samples in clean plastic or glass bottles. Analyze samples as soon as possible after
collection. The sample may be stored for seven days by cooling to 4 C (39 F).
Accuracy check
Calibration for this test is based on parallel samples using the gravimetric technique on sewage
samples from a municipal sewage plant. For most samples, this calibration will provide satisfactory
results. When higher accuracy is required, run parallel spectrophotometric and gravimetric
determinations with portions of the same sample. Make the new calibration on the particular
sample using a gravimetric technique as a basis.
Summary of method
This method of determining suspended solids is a simple, direct measurement which does not
require the filtration or ignition/weighing steps that gravimetric procedures do. The USEPA
specifies the gravimetric method for solids determinations, while this method is often used for
checking in-plant processes. Test results as mg/L total suspended solids (TSS) are measured at
810 nm.

Required apparatus
Description
Quantity
Unit
each
Catalog number
108052
Blender, 1.2-L, 120 VAC
each
2616100
Blender, 1.2 L, 240 VAC
each
2616102
Cylinder, 500-mL graduated, polypropylene
each
108149
Suspended Solids
Page 1149

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Sulfate, 8051
Sulfate
DOC316.53.01135
USEPA1 SulfaVer 4 Method2
Method 8051
(2 to 70 mg/L)
1
USEPA accepted for reporting wastewater analyses. Procedure is equivalent to USEPA method 375.4 for wastewater.
Test preparation


Powder pillows
AccuVac Ampuls
Instrument
Sample cell
Cell orientation
Sample cell
Adapter
DR 6000
2495402
2427606
DR 5000
2495402
2427606
DR 3900
2495402
2427606
LZV846 (A)
DR 3800, DR 2800, DR 2700
2495402
2122800
LZV584 (C)

Adjust the standard curve for each new lot of reagent (Standard solution method).
For best results, calibrate the instrument with each new lot of reagent (see Calibration).
Filter highly colored or turbid samples using filter paper and a funnel. Use this sample in step 2 and 5.
SulfaVer 4 contains barium chloride. The final solution will contain barium chloride (D005) at a concentration regulated as a
hazardous waste by the Federal RCRA. Refer to a current MSDS for safe handling and disposal instructions.
Use a blank AccuVac Ampule in place of the sample cell in Step 5 .if necessary.
Sulfate
Page 1151
Sulfate

Quantity
Description
Powder Pillow Test:
SulfaVer 4 Reagent Powder Pillows
AccuVac Test:
SulfaVer 4 Reagent AccuVac Ampuls
Beaker, 50-mL
Stopper
SulfaVer 4 powder pillow procedure
Stored Programs
680 Sulfate
Start
1. Select the test.

for orientation.

of sample.

one SulfaVer 4 Reagent
sample cell. Swirl
vigorously to dissolve the
powder.
White turbidity will form if
sulfate is present.
Sulfate
Page 1152

timer.
will begin. Do not disturb
the cell during this time.
Note: Accuracy is not
affected by undissolved
powder.
Sulfate
SulfaVer 4 powder pillow procedure (continued)
6. When the timer

and insert it in the cell
holder (fill lines face right.)
Zero
Read

wipe the cell and insert the
prepared sample in the
cell holder.

0 mg/L SO42
READ the results in

mg/L SO42.
Clean sample cells with

soap and a brush.
SulfaVer 4 AccuVac Ampuls procedure
Stored Programs
685 Sulfate AV
Start
1. Select the test.

for orientation.
2. Prepared sample:
3. Cap or stopper the

Ampul and quickly invert
Fill a SulfaVer 4 Reagent

AccuVac Ampul with
completely.
White turbidity will form if

sulfate is present.

timer.
Note: Accuracy is not
affected by undissolved
powder.
Sulfate
Page 1153
Sulfate
SulfaVer 4 AccuVac Ampuls procedure (continued)
Fill a clean sample cell
6. When the timer

holder.

it in the cell holder.
READ the results in

mg/L SO42.

0 mg/L SO42
Interferences
Interference level
Calcium
Chloride
Greater than 40,000 mg/L as Cl
Magnesium
Silica
Greater than 500 mg/L as SiO2

Collect samples in clean plastic or glass bottles. Samples may be stored up to 7 days by cooling to
4 C (39 F) or lower. Warm to room temperature before analysis.
Accuracy check
Sulfate Ampule Standard Solution, 2500 mg/L sulfate
Ampule breaker
TenSette Pipet and pipet tips
Mixing cylinder, 25 mL or 50 mL
Beaker, 50 mL
2. Select standard additions from the instrument menu:
OPTIONS>MORE>STANDARD ADDITIONS.
Sulfate
Page 1154
Sulfate
5. Fill three mixing cylinders with 25 mL of sample. Use the TenSette Pipet to add 0.1 mL, 0.2 mL
and 0.3 mL of standard, respectively, to each mixing cylinder and mix thoroughly. Transfer 10
mL of each sample spike to a clean sample cell.
Note: For AccuVac Ampuls, fill three mixing cylinders with 50 mL of sample and spike with 0.2 mL,
0.4 mL and 0.6 mL of standard. Transfer 40 mL from each of the three mixing cylinders to three
50-mL beakers.
6. Follow the SulfaVer 4 powder pillow procedure for each of the spiked samples, starting with
Note: For AccuVac Ampuls, follow the SulfaVer 4 AccuVac Ampuls procedure for each of the spiked
samples, starting with the 0.2 mL sample spike. Measure each of the spiked samples in
the instrument.
Sulfate standard solution, 1000 mg/L
Tensette pipet, 110 mL and pipet tips
1. Prepare a 70 mg/L sulfate standard solution as follows: use a pipet to add 7.0 mL of sulfate
standard solution, 1000 mg/L as SO42, to a 100-mL volumetric flask. Dilute to the mark with
deionized water. Mix well. Prepare this solution daily.
2. Follow the SulfaVer 4 powder pillow procedure or SulfaVer 4 AccuVac Ampuls procedure
and use the 70-mg/L SO42 standard solution in place of the sample.
3. To adjust the calibration curve using the reading obtained with the standard solution, set
standard adjust to on (OPTIONS>(MORE)>STANDARD ADJUST) and accept the concentration.
Calibration
A calibration is recommended for the SulfaVer 4 method for the best accuracy. Complete the
following steps to enter a new calibration curve in the instrument. Perform this procedure for each
new lot of reagent.
Required items:
Sulfate standard solution, 1000 mg/L
Seven 100 mL Class A volumetric flasks
110 mL TenSette pipet and tips
1. Prepare seven calibration standards (10, 20, 30, 40, 50, 60 and 70 mg/L SO42) as follows.
Use the Tensette pipet to add 1, 2, 3, 4, 5, 6 and 7 mL of the 1000-mg/L sulfate standard
solution to seven different 100-mL Class A volumetric flasks.
2. Dilute each flask to the mark with deionized water. Mix thoroughly.
3. Use each standard solution in place of the sample and follow the SulfaVer 4 powder pillow
procedure or SulfaVer 4 AccuVac Ampuls procedure.
Sulfate
Page 1155
Sulfate
4. Refer to the user manual (user programs section) to enter the calibration in the instrument as
a user program.
Method performance
Precision
Distribution
Sensitivity
40 mg/L SO42
3050 mg/L SO42
0.4 mg/L SO42
0.7 mg/L SO42
Program
Standard
680
685
40 mg/L SO4
3248 mg/L SO4
Summary of method
Sulfate ions in the sample react with barium in the SulfaVer 4 and form a precipitate of barium
sulfate. The amount of turbidity formed is proportional to the sulfate concentration. Test results are
measured at 450 nm.
Sulfate
Page 1156
Sulfate

Required reagents
Description
SulfaVer 4 Reagent Powder Pillows
Quantity/Test
Unit
Catalog number
100/pkg
2106769
25/pkg
2509025
Quantity
Unit
Catalog number
2405200
OR
SulfaVer 4 Sulfate Reagent AccuVac Ampuls
Required apparatus
Description
AccuVac snapper
each
Beaker, 50-mL
each
50041H
each
2122800
each
2427606
Sample cell, 10 mL, square, matched pair
2/pkg
24954025
Stopper
6/pkg
173106
Unit
Catalog number
2175749
Description
Sulfate Standard Solution, 1000-mg/L
500 mL
Sulfate Standard Solution, 2500-mg/L, 10-mL Ampules
16/pkg
1425210
Mixed Parameter Standard for sulfate, fluoride, nitrate and phosphate
500 mL
2833049
Description
Unit
Catalog number
each
189640
each
189641
Blank AccuVac Ampules
25/pkg
2677825
Ampule Breaker, for 10 mL Ampules
each
2196800
Tensette Pipet 0.11.0 mL
each
1970001
Tips for Tensette Pipet 110

Tensette Pipet 110
Tips for tensette Pipet 110
50/pkg
2185696
each
1970010
50/pkg
2199796
each
1457442
Sulfate
Page 1157

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Sulfide, 8131
Sulfide
DOC316.53.01136
USEPA1 Methylene Blue Method2
Method 8131
(5 to 800 g/L)
Scope and Application: For testing total sulfides, H2S, HS, and certain metal sulfides in groundwater,
wastewater, brines and seawater.
1
USEPA approved for reporting wastewater analysis. Procedure is equivalent to Standard Method 4500-S2 D.
Test preparation


Instrument
Sample volume
Sample cell
Cell orientation
DR 6000
10 mL
2495402
DR 5000
10 mL
2495402
DR 3900
10 mL
2495402
DR 3800, DR 2800, DR 2700
10 mL
2495402

Avoid excessive agitation of samples to minimize sulfide loss.
Some sulfide loss may occur if dilution is necessary.
Sulfide 2 reagent contains potassium dichromate. The final solution will contain hexavalent chromium (D007) at a
concentration that is regulated as a hazardous waste by Federal RCRA. Refer to the current MSDS for safe handling and
disposal instructions.

Description
Quantity
Sulfide 1 Reagent
12 mL
Sulfide 2 Reagent
12 mL
Water, deionized
1025 mL
Stoppers
Sulfide
Page 1159
Sulfide
Methylene Blue Method
Stored Programs
690 Sulfide
Start
1. Select the test.

required (see Instrumentspecific information). Refer
orientation.
5. Use the dropper to

add 0.5 mL Sulfide 2
Reagent to each cell.
Measure 10 mL of
deionized water in a
sample cell.
3. Prepared Sample:
Use a pipet to add 10 mL
of sample to a second
sample cell. Do not mix
the sample more than
necessary to prevent
sulfide loss.
4. Use the dropper to

add 0.5 mL Sulfide 1
Reagent to each cell.
6. Cap or stopper the cell

and immediately invert to
mix.

timer.
8. When the timer

holder.
The solution will turn pink

initially and then turn blue
if sulfide is present.
Zero

0.00 g/L S2
Sulfide
Page 1160

will begin.
Read

cell holder.

g/L S2.
Swirl to mix.
Sulfide
Soluble sulfides
Complete the following steps to measure soluble sulfides.
1. Centrifuge a sample in completely filled, capped tubes.
2. Use the supernatant in place of the sample and follow the Methylene Blue Method procedure.
To estimate insoluble sulfides, subtract the soluble sulfide concentration from the total sulfide
concentration.
Interferences
Interference level
Strong reducing substances such as

sulfite, thiosulfate and hydrosulfite.
Interfere by reducing the blue color or preventing its development.
Sulfide, high levels
High concentrations of sulfide may inhibit full color development and require sample
dilution. Some sulfide loss may occur when the sample is diluted.
Turbidity
For turbid samples, prepare a sulfide-free blank as follows. Use this blank in place of
the deionized water blank in the Methylene Blue Method test procedure.
1. Measure 25 mL of sample into a 50-mL Erlenmeyer flask.
2. Add bromine water by drops with constant swirling until a permanent yellow color
just appears.
3. Add phenol solution by drops until the yellow color just disappears. Use this
solution to replace the deionized water in step 2 of the procedure.
This pretreatment procedure removes sulfide from the sample, but the turbidity and
any color will remain. The interference from turbidity or color will be corrected when
the instrument is set to zero with this solution (step 9).

Collect samples in clean plastic or glass bottles. Fill completely and cap tightly. Prevent excessive
shaking or prolonged exposure to air. Analyze samples immediately.
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
690
DR 5000
520 g/L S2
504536 g/L S2
5g/L S2
Summary of method
Hydrogen sulfide and acid-soluble metal sulfides react with N,N-dimethyl-p-phenylenediamine
sulfate to form methylene blue. The intensity of the blue color is proportional to the sulfide
concentration. High sulfide levels in oil field waters may be determined after proper dilution. Test
Sulfide
Page 1161
Sulfide

Required reagents
Description
Quantity/Test
Unit
Catalog number
2244500
Sulfide 1 Reagent
1 mL
100 mL MDB
181632
Sulfide 2 Reagent
1 mL
100 mL MDB
181732
10 mL
4 liters
27256
Catalog number
Sulfide Reagent Set, includes:
Water, deionized
Required apparatus
Description
Quantity
Unit
each
53238
each
1465100
Stopper, for 18-mm Tube
6/pkg
173106
Unit
Catalog number

Description
29 mL
221120
29 mL
211220
Stopper, for 18-mm Tube
25/pkg
173125
each
50541

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Sulfite, BT, 8071
Sulfite
DOC316.53.01162
Iodate-Iodide Buret Titration Method1

0 to 500 mg/L as SO3
2 (or
Method 8071
0 to > 500 mg/L)
Buret Titration
Scope and Application: For boiler water.

1
Test preparation

Sulfite is easily destroyed by atmospheric oxygen. Violent shaking or swirling will cause low results.
Analyze samples immediately. Cool hot samples to 50 C (122 F) or lower before analysis.

Description
Quantity
Dissolved Oxygen Reagent Powder Pillow
1 pillow
1 bottle
1 bottle
Graduated cylinder (see Range-specific information)
Buret titration
See
Table 1
1. Select a sample
2. Fill the buret to the

zero mark with 0.0125 N
Potassium Iodide-Iodate
Standard Solution.
3. Use a graduated
measure the sample
volume. Add the sample to
the Erlenmeyer flask.

Powder Pillow and mix
gently.
If the sample volume is

less than 50 mL, gently
dilute to approximately
50 mL with deionized
water. Do not agitate.
Sulfite
Page 1163
Sulfite

Swirl gently to mix.

the Range-specific
information to calculate
the concentration:

flask until the color
changes to a permanent
blue color (end point).

= mg/L sulfite (SO32)
Write down the volume of

titrant that was used.
was titrated and 5 mL of
titrant was used to reach
the end point. The
concentration is 5 x 10 =
50 mg/L as SO32

Range (mg/L as SO32)
Sample volume (mL)
Multiplier
0100
50
10
40200
25
20
100500
10
50
Over 500
100
Interferences

Interference level
Nitrite
Nitrite will react with sulfite to cause a negative error in the titration.
Metals
Some metals, especially copper, catalyze the oxidation of sulfite to sulfate. Immediate fixing
of sample with the contents of one Sulfamic Acid Powder Pillow per liter of sample will help
minimize the interference.
Organic compounds
Oxidizable organic compounds could cause high results.
Oxidizable compounds
Will cause high results.
Sulfide
Will cause high results.
Sulfite
Page 1164
Sulfite
Avoid excessive agitation or prolonged exposure to air. Complete the test procedure as soon
as possible after collection for best accuracy. Samples cannot be preserved or stored.
Cool hot samples to 50 C (122 F) or lower.
Accuracy check
The standard additions method can be used to find if the sample has an interference. The
standard solution method can be used to confirm analytical technique and reagent performance.
Sulfite Voluette Ampule Standard, 5,000-mg/L SO32
Ampule breaker
Pipet filler

8. Each 0.1 mL of standard that was added will use 1.0 mL of titrant to reach the endpoint. If
Complete the following test to make sure the concentration is accurate.
0.025 N Sodium Thiosulfate Standard Solution
10-mL volumetric pipet, Class A
1. Use a pipet to add 10.0-mL of 0.025 N Sodium Thiosulfate Standard Solution to a 250-mL
volumetric flask. Dilute to the mark with deionized water. The diluted standard is equivalent to
40 mg/L sulfite.
2. Use a graduated cylinder to measure 50 mL of the diluted standard. Add the standard to the
Erlenmeyer flask.
3. Titrate the standard to the end point with the titrant solution. The result should be close to
40 mg/L sulfite.
Sulfite
Page 1165
Sulfite
Summary of method
The water sample is acidified, treated with a starch indicator and titrated with a potassium iodideiodate standard solution. The acid releases free iodine, which is reduced to colorless iodide by the
sulfite in the sample. When all of the sulfite is gone, excess iodine will react with the starch to form
a blue color.

Required reagents
Description
Quantity/Test
Unit
1 pillow
100/pkg
98799
varies
1L
1400153
1 mL
100 mL MDB
34932
100 tests
2459800
Reagent set, Sulfite Buret Titration
Catalog number
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Buret, Class A, Teflon Stopcock plug, Certified 10-mL
each
2636538
Buret Clamp, double
each
32800
each
96800
50837

each
each
50838
each
50840
each
50841
each
50546
Support Stand
each
56300
each
1451537
each
1451538
Pipet filler, Safety Bulb
each
1465100
Description
Unit
Catalog number
1L
2409353
16/pkg
1426710
Description
Unit
Catalog number
each
1970001
50/pkg
2185696
each
2196800
Sulfite Standard Solution,
Voluette
Ampule, 5,000-mg/L SO3 10-mL
Pipet tips
Ampule breaker

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Sulfite, DT, 8216
Sulfite
DOC316.53.001181
4 to greater than 400 mg/L as SO3
Method 8216
2
Digital Titrator
Scope and Application: For boiler water
Test preparation

Analyze samples immediately. Cool hot samples to 50 C (122 F) or lower before analysis.
Sulfite is easily destroyed by atmospheric oxygen. Violent shaking or swirling will cause low results. Avoid unnecessary
agitation throughout the procedure.
The Dissolved Oxygen 3 Reagent Powder Pillow can be replaced with 0.5 mL of 19.2 N Sulfuric Acid Standard Solution.
mg/L Sulfite (SO32) x 1.01 = Bisulfite, Hydrogen Sulfite (HSO3)
mg/L Sulfite (SO32) x 1.30 = Sodium Bisulfite, Sodium Hydrogen Sulfite (NaHSO3)
mg/L Sulfite (SO32) x 2.37 = Sodium Metabisulfite, Sodium Pyrosulfite (Na2S2O5)
mg/L Sulfite (SO32) x 1.58 = Sodium Sulfite (Na2SO3)

Description
Quantity
Sulfite Reagent Set
Clippers
Deionized Water
varies
Digital Titrator
Graduated Cylinder, 10-, 25- or 50-mL, based on sample concentration
varies
Sulfite
Page 1167
Sulfite
8. Select a sample
volume from the Volume
multipliers table that
corresponds to the
expected sulfite (SO32)
concentration.

tube into the Iodate-Iodide
Titration Cartridge
(KIO3KI).


titrator body.

volume into a clean,
Dilute to the 50-mL mark

to measure the sample
volume from the Volume
multipliers table.

and swirl gently to mix.
13. Add one dropperful of

and swirl to mix.

titrating with the
iodate-iodide to a
permanent blue end point.
digits required.
15. Calculate:
Digits Required x
Digit Multiplier =
mg/L Sulfite (SO32)

Range (mg/L as
SO32)
Volume (mL)
Cartridge Strength
Digit Multiplier
Up to 160
50
0.3998 N
0.4
100400
20
0.3998 N
1.0
Over 400
0.3998 N
4.0
200800
10
0.3998 N
2.0
Sulfite
Page 1168
Sulfite
Accuracy check
This accuracy check should be performed when interferences are suspected or to verify analytical
technique.
1. Snap the top off a Sulfite Voluette Ampule Standard, 5,000-mg/L SO32.
2. Use a TenSette Pipet to add 0.1 mL of standard to the sample titrated. Resume titration back
to the same end point. Record the number of digits required.
3. Repeat, using additions of 0.2 and 0.3 mL, titrating to the end point after each.
4. Each 0.1-mL addition of standard should require 25 additional digits of titrant. If these uniform
increases do not occur, determine the cause.
A standard solution equivalent to 40-mg/L sulfite can be prepared by diluting 10.0 mL of 0.025 N
Sodium Thiosulfate Titrant to 250 mL in a volumetric flask. Titrate a 50-mL sample, using the
above procedure.
Interferences
Sulfide, organic matter and other oxidizable substances will cause positive error in the titration.
Nitrite will react with sulfite to cause low results.
Some metals, especially copper, catalyze the oxidation of sulfite to sulfate.
Addition of one Dissolved Oxygen 3 Powder Pillow per liter of sample immediately upon
sampling will help eliminate the effects of nitrite and copper.
Summary of method
Sulfite ion is titrated with potassium iodate-iodide standard solution under acidic conditions to a
blue starch end point. The volume of titrant used is proportional to the sulfite concentration.

Required reagents
Description
Unit
Sulfite Reagent Set (about 100 tests), includes:

Iodate-Iodide Titration Cartridge, 0.3998 N
Starch indicator Solution
Water, deionized
Catalog number
2272300
100/pkg
98799
each
1496101
100 mL MDB
34932
4L
27256
Sulfite
Page 1169
Sulfite
Required apparatus
Description
Unit
Clippers for opening pillows
each
Catalog number
96800
Digital Titrator
each
1690001
50838
each
each
50840
each
50841
each
50543
each
1720500
each
4157800
Thermometer -10225 C, 405 mm
each
2635700
Volumetric Pipet, 10 mL
each
1451538
each
1457446
Safety bulb
each
1465100
Tensette Pipet
each
1970001
Pipet tips
50/pkg
2185696
Pipet tips
1000/pkg
2185628
Unit
Catalog number
Required standards
Description
Sulfite Standard Solution, Voluette Ampule, 5,000-mg/L SO3 10-mL
Ampule Breaker
Sulfite Standard Solution, 15 mg/L

1000 mL
2409353
16/pkg
2267410
100 mL MDB
203832
each
2196800
500 mL
2408449
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Sulfite, Europe only
Sulfite
DOC316.53.01137
Colorimetric Method1
(0.10 to 5.00 mg/L)
Scope and Application: For boiler water and foodstuffs.
1
Reagent sets for this method are only available in Europe.
Test preparation

information required to perform this test

Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Samples must be analyzed immediately.
The temperature of samples and reagents must be between 1525 C (5977 F).
Adjust the pH of the sample to a value between 310.

Description
Quantity
Sulfite Reagent A
5 drops
Sulfite Reagent B
2 drops
Water, deionized
varies
Pipet, 10-mL serological
Sulfite
Page 1171
Sulfite
Colorimetric Method
Stored Programs
692 Sulfite HPT 430
Start
1. Select the test.

required (see the
Instrument-specific
information table). Refer to
the user manual for
orientation.
5. Swirl to mix.
Fill a clean sample cell
3. Prepared Sample:
Pipet 10 mL of sample into
a second clean sample
cell.
4. Add 5 drops of Sulfite

Reagent A (HPT 430 A) to
6. Add 2 drops of Sulfite

Reagent B (HPT 430 B) to
the prepared sample. Swirl
to mix.

timer.

insert it in the cell holder.
Zero

0.00 mg/L SO32
Sulfite
Page 1172

Read
10. When the timer

expires, wipe the prepared
cell holder.

mg/L SO32.
Sulfite
Interferences
Interference level
Sulfide
Greater than 5 mg/L

Collect samples in clean plastic or glass bottles. Analyze immediately.
Method performance
Program
Instrument
Standard
Precision
Distribution
Sensitivity
692
DR 5000
3.00 mg/L SO32
2.513.49 mg/L SO32
0.04 mg/L SO32
Summary of method
The reagents react with sulfite to form a yellow complex. The color is measured at 435 nm.

Required reagents
Description
Quantity/Test
Unit
Catalog number
100/pkg
HPT430
Sulfite Reagent A1
5 drops
28 mL
Sulfite Reagent B1
2 drops
8.7 mL
Quantity/Test
Unit
Catalog number
Pipet, 10-mL serological
each
53238
each
1465100
Sulfite Colorimetric Reagent Set, includes:
Not available separately. Reagent sets for this method are only available in Europe.
Required apparatus
Description
Sulfite
Page 1173

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Surfactants, Anionic (Detergents), 8028
Surfactants, Anionic (Detergents)
DOC316.53.01138
Crystal Violet Method1
Method 8028
(0.002 to 0.275 mg/L as LAS)

1
Adapted from Analytical Chemistry, 38, 791 (1966).
Test preparation

The Instrument-specific information table displays information that may vary from instrument to
instrument. Select the spectrophotometer from the instrument column on the left. Read across to
the spectrophotometer.

Instrument
Sample cell
Cell orientation
DR 6000
2612602
DR 5000
2612602
DR 3900
2612602
DR 3800, DR 2800, DR 2700
2612602

Use benzene only in a well-ventilated area.
Benzene (D018) solutions are regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the
drain. Collect water that is saturated with benzene and benzene solutions for disposal with laboratory solvent wastes. Refer
To prevent water droplets from forming in the sample cells, use only dry sample cells and discard the first few mL of benzene.
Additionally, it can help to transfer the liquid from the funnel to a sample cell, let it sit for a few seconds and decant to a
second cell for reading.
Excessive shaking may cause an emulsion to form, which makes the phases separate more slowly. If this occurs, remove
most of the water layer, then gently mix the contents of the funnel with a clean Teflon-coated rod or other inert tool.
Spilled reagent will affect test accuracy and is hazardous to the skin and other materials.
Acetone may be used to clean benzene from glassware.
In bright light conditions (e.g. direct sunlight) be sure to cover the cell compartment during measurements.

Page 1175

Description
Quantity
Benzene, ACS
55 mL
Buffer Solution, sulfate-type
10 mL
Detergent Reagent Powder Pillows
1 pillow
Support Ring, 4-inch
Support, Ring Stand, 5 x 8 inch base
Crystal Violet Method
Stored Programs
710 Surfactants
Start
1. Select the test.

required ( Instrumentspecific information table).

300 mL mark with sample.

for orientation.

Page 1176

clean 500-mL separatory
funnel.
4. Add 10 mL of Sulfate
Buffer Solution. Stopper
the funnel. Shake the
funnel for five seconds.

Crystal Violet Method (continued)

one Detergents Reagent
funnel.
6. Stopper the funnel

and shake until the powder
dissolves completely. The
powder will dissolve
slowly.
7. Add 30 mL of benzene
to the funnel. Stopper the
funnel and shake gently
for one minute.
8. Place the separatory

funnel in a support stand.

timer.

remove the stopper and
drain the bottom water
layer. Discard this layer.

Drain the top benzene
layer into a clean 25-mL
sample cell. Stopper the
cell.

Fill another sample cell to
the 10-mL mark with pure
benzene. Stopper the cell.
period will begin.
Do not filter the benzene

layer before color
measurement. Filtration
removes the blue color.
Zero

blank cell into the cell
holder.

0.000 mg/L LAS
Read

sample cell into the cell
holder.

mg/L LAS.

Page 1177
Interferences
Interference level
Chloride
High amounts of chloride, such as those levels found in

brines and seawater, will cause low results.
Perchlorate ions
Periodate ions

Collect samples in clean plastic or glass bottles. Analyze samples as soon as possible. Samples
may be stored at least 24 hours by cooling to 4 C (39 F). Warm to room temperature
before testing.
Accuracy check
Detergent Voluette Ampule Standard, 60-mg/L LAS
Ampule breaker
TenSette Pipet, 0.11.0 mL and Tips
ADDITIONS.
5. Prepare three sample spikes as follows. Measure 300 mL of sample into each of the three
beakers. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively,
to each sample and mix thoroughly.
6. Follow the test procedure for each of the spiked samples, starting with the 0.1 mL sample

Page 1178

Detergent Voluette Ampule Standard, 60-mg/L LAS
1. Prepare a 0.180 mg/L LAS standard solution as follows: Pipet 3.0 mL of Detergent Standard,
60-mg/L as LAS, into a 1000-mL (1 liter) volumetric flask. Dilute to the mark with deionized
water. Mix well. Prepare this solution daily.
2. Follow the Crystal Violet Method test procedure.
3. To adjust the calibration curve with the reading obtained with the standard solution, navigate to
Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST.
4. Turn on the Standard Adjust option and accept the displayed concentration. If an alternate
Method performance
Program
Standard
Precision
Distribution
Sensitivity
710
0.180 mg/L LAS
0.1720.188 mg/L LAS
0.002 mg/L LAS
Summary of method
Detergents, ABS (alkyl benzene sulfonate) or LAS (linear alkylate sulfonate) are determined by
association with crystal violet dye and extraction of the ion-pair complex into benzene. Test results

Required reagents
Description
Quantity/Test
Unit
Catalog number
2446800
Benzene, ACS
40 mL
4 liters
1444017
Buffer Solution, sulfate-type
10 mL
500 mL
45249
Detergent Reagent Powder Pillows
1 pillow
25/pkg
100868
Detergents Reagent Set, includes:
Required apparatus
Description
Quantity
Unit
Catalog number
each
96800
each
50840
each
50841
each
50849
each
52049
Support Ring, 4-inch
each
58001
Support, Ring Stand, 5 x 8 inch base
each
56300

Page 1179
Description
Detergent Standard Solution, 10-mL
Voluette
Ampule, 60-mg/L LAS
Unit
Catalog number
16/pkg
1427110
Unit
Catalog number
500 mL
1442949

Description
Acetone, ACS
Beaker, 600 mL
each
50052
each
1457453
Pipet filler bulb
each
1465100
each
1970001
Pipet tips for TenSette Pipet 19700-01


50/pkg
2185696
each
1451503
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Tannin and Lignin, 8193
Tannin and Lignin
DOC316.53.01140
Tyrosine Method1
Method 8193
(0.1 to 9.0 mg/L)

Scope and Application: For water, wastewater and boiler water.
1
Adapted from Kloster, M.B., Journal American Water Works Association, Vol. 66, No. 1, p. 44 (1974).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Filter turbid samples and report results as mg/L soluble tannic acid.
For best accuracy, use a pipet to add the TanniVer 3 solution.
The Pour-Thru Cell can be used as an alternative to the sample cells, when using a full 25 mL sample..
Results will be given in mg/L tannins (as tannic acid).

Description
Quantity
Tannin and Lignin Reagent Set:

Sodium Carbonate Solution
10 mL
TanniVer 3 Tannin-Lignin Reagent
1 mL
Pipet Filler
Pipet, volumetric Class A, 5.0-mL
Pipet, volumetric Class A, 0.5-mL
Water, deionized
25 mL
Tannin and Lignin

Page 1181
Tannin and Lignin

Tyrosine Method
Stored Programs
720 Tannin & Lignin
Start
1. Select the test.


invert to mix.
Fill a 25-mL graduated
mixing cylinder to the
25-mL mark with
deionized water.
3. Prepared Sample:
Fill a second 25-mL
sample.
4. Pipet 0.5 mL of
TanniVer 3 Tannin-Lignin
Reagent into each
cylinder.
6. Pipet 5.0 mL of
Sodium Carbonate
Solution into each cylinder.
Insert the stopper and
invert to mix.
7. Pour 10 mL of each
solution into two sample
cells.

timer.
period will begin.

tannins and/or lignins are
present.
Zero
9. When the timer

Tannin and Lignin

Page 1182

0.0 mg/L Tannins
(as Tannic Acid).
Read
11. Insert the sample into

the cell holder.

mg/L Tannins
(as Tannic Acid).
Tannin and Lignin
Interferences
Interference level
Ferrous iron
Causes a positive interference. (2 mg/L of ferrous iron produces a color equivalent to

about 1 mg/L of tannic acid.) To eliminate interference of ferrous iron up to 20 mg/L,
add one 0.2 g scoop of Sodium Pyrophosphate1 to the sample before testing.
Sulfite
To eliminate sulfite interference, add 1 mL of formaldehyde1 to the sample before

testing the sample.

Accuracy check
Tannic acid, analytical grade
15-mL Class A volumetric pipet and pipet bulb
Deionized water
1. Prepare a tannic acid stock solution as follows: dissolve 0.200 grams of tannic acid in
deionized water and dilute to 1000 mL. Prepare this solution monthly.
2. Prepare a 6.0-mg/L tannic acid standard solution by diluting 15.00 mL of the stock solution to
500 mL with deionized water. Prepare this standard daily.
3. Follow the tannin and lignin procedure as described above.
4. To adjust the calibration curve using the reading obtained with the 6.0-mg/L Standard Solution,
navigate to Standard Adjust in the software:OPTIONS>MORE>STANDARD ADJUST.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
720
6.0 mg/L tannic acid
5.86.2 mg/L tannin
0.1 mg/L tannin
Summary of method
This test measures all hydroxylated aromatic compounds, including tannin, lignin, phenol and
cresol. This method produces a blue color proportional to the amount of these compounds present
Tannin and Lignin

Page 1183
Tannin and Lignin

in the sample. The results are reported as total tannin and lignin and expressed as mg/L tannic
acid. Test results are measured at 700 nm.

Required reagents
Description
Quantity/Test
Unit
(2) Sodium Carbonate Solution
10 mL
500 mL
67549
(1) TanniVer 3 Tannin-Lignin Reagent
1 mL
100 mL
256032
25 mL
4L
27256
Tannin and Lignin Reagent Set (up to 100 tests), includes:
Catalog number
2244600
Water, deionized
Required apparatus
Description
Quantity
Unit
Catalog number
Cylinder, mixing, with stopper, 25-mL
each
2088640
each
1465100
each
1451537
each
1451534
Description
Unit
Catalog number
Tannic Acid, Analytical Grade
113 g
79114
Description
Unit
Catalog number
each
1457453
each
1457449
Formaldehyde
100 mL
205932
each
1451539
Sodium Pyrophosphate
50 g
1429525
each
1970001
1000/pkg
2185628

Weighing papers
Spatula, micro

each
2936701
500/pkg
1473800
each
1225600
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Toxicity, 10017
Toxicity
DOC316.53.01141
ToxTrak Method 1, 2
Method 10017
(0 to 100% Inhibition)
Scope and Application: For drinking water, wastewater and natural waters.
1
Liu, D., Bull. Environ. Contm. Toxicol. 26, 145-149 (1981)
Environmental Technology Verification ETV Program evaluated, November, 2003
Test preparation


Instrument
DR 6000, DR 5000
Light shield
DR 3900
LZV849
DR 3800, DR 2800, DR 2700
LZV646

Do not leave the cells in the instrument during incubation. All samples and control cells should be allowed to react under
similar conditions of temperature and light.
If testing chlorinated samples, add two drops of sodium thiosulfate to each blank and sample to remove the chlorine.
If testing drinking water, take the control sample from a reservoir of tap water known to be free of toxins, if possible.

Description
Bacterial Count Broth Tube
Quantity
1 tube
Pipet, transfer, sterile
Reaction tubes, with cap
Sodium Thiosulfate
ToxTrak Reagent Powder Pillows
ToxTrak Accelerator Solution
Water, deionized
varies
2
4 drops
varies
Clippers
Incubator
Pipet, volumetric, Class A, 5.00 mL and pipet filler
Toxicity
Page 1185
Toxicity
Inoculum development using indigenous biomass
1. Using one of the

dropper pipets provided,
add 1.0 mL of source
culture (indigenous
biomass) to a Total
Bacteria Count Broth
Tube.
Note: Commercial sources of
freeze-dried bacteria may
also be used.
2. Incubate the tube

contents at 35 C (95 F)
until the broth is visibly
turbid (approximately 12
hours).
Note: The culture can be kept
for several days in the
incubator or at room
temperature. Use before
72 hours for best results.
Reaction tube colorimetric test
Single Wavelength
Zero
6
OK
1. Press
Single Wavelength.
button.
OK.
required ( Instrumentspecific information).
Toxicity
Page 1186
Fill an empty reaction tube


0.000 Abs
Toxicity
Reaction tube colorimetric test (continued)
5. Label a reaction tube

control. Open one
ToxTrak Reagent Powder
Pillow and add the
contents to the empty
tube.
6. For each sample or

dilution, label a reaction
tube with the sample
number.
9. Add two drops of

Accelerator Solution to
each vial. Cap and shake
to mix.
10. Add 0.5 mL of

inoculum (previously
prepared) to each tube.
Open one ToxTrak

and add the contents to
each empty sample tube.
7. Add 5.0 mL of
deionized water to the
control tube.
8. Add 5.0 mL of sample

(or dilution) to each
sample tube.
Use deionized water that

is free of toxicity or
another water source that
represents baseline
toxicity.
To find the approximate

threshold level of toxicity
for a sample, see No
observed effect
concentration (NOEC).
11. Insert the control in

the cell holder. Record the
absorbance.
12. Repeat step 11 for all

samples and dilutions. Be
sure to record each
absorbance.
Shaking fully oxygenates

the samples and assures
that the oxygen
concentration is not a
factor in determining the
respiration rate.
Toxicity
Page 1187
Toxicity
Reaction tube colorimetric test (continued)
13. Allow the solutions in

the tubes to react until the
absorbance of the control
has decreased by
0.60 ( 0.10) Abs. This
takes 4575 minutes.
Invert occasionally.
14. After the absorbance

of the control has
decreased by 0.60 ( 0.10)
Abs. Remove the control
and insert the blank into
the cell holder.
Zero
Read
16. Insert the control into

the cell holder.

0.000 Abs

absorbance reading.
Record this value.
The reaction time varies

according to temperature,
age of the culture, bacteria
concentrations, etc.
17. Insert each sample or

dilution into the cell holder
and READ the results.
18. Calculate the % Inhibition:
Record each absorbance

value.
where:
A sample
% I = 1 ---------------------- 100
A control
A = Initial absorbance value Final absorbance value

Example:
Absorbance of control: initial = 1.500 abs, final = 0.900 abs; Acontrol = 0.600
Absorbance of sample: initial = 1.700 abs, final = 1.300 abs; Asample = 0.400
0.400
% I = 1 --------------- 100 = 33% I
0.600
Interpreting results
The results as percent inhibition (% I) are a relative measurement. They do not represent a true
quantitative measurement of toxic concentration. The percent inhibition does not necessarily
increase in direct proportion to the concentration of toxins.
Results below 10% are not reliable, but can be used to make an estimate of toxicity when the
results are consistent. If a sample shows less than 10% inhibition, repeat the test several times.
Toxicity
Page 1188
Toxicity
Look at the series of data points to find the likelihood of toxicity
(refer to the Interpreting results that are less than 10% inhibition table).
Some toxins will increase respiration and give a negative percent inhibition on this and all other
respiration-based toxicity tests. After repeated testing, samples that always give a percent
inhibition that is more negative than 10% should be considered toxic.
Table 357 Interpreting results that are less than 10% inhibition
Data points: percent inhibition
Conclusion
7%, 9%, 5%, 8%, 5%
May be slightly toxic
7%, 4%, 5%, 5%, 1%
Most likely not toxic
7%, 9%, 5%, 8%, 5%
May be slightly toxic
No observed effect concentration (NOEC)

To determine the minimum inhibition concentration of a toxin:
1. Dilute 1 mL of sample to 10 mL with deionized water.
2. Run the test and find the percent inhibition for the dilution.
3. Dilute 1 mL of the sample dilution from step 1 to 10 mL with deionized water.
4. Run the test and find the percent inhibition for the dilution.
5. Continue to make serial 1:10 dilutions of the sample (1:10, 1:100, 1:1000, etc.) until a level is
reached that gives 0% inhibition in the final calculation.
When 0% inhibition is found, the dilution represents the approximate threshold level of toxicity
for a sample. This is the No Observed Effect Concentration (NOEC).
Lowest observable effect concentration (LOEC)
Due to the many variables involved in the test, the limit of detection is approximately
10% inhibition. This correlates to the Lowest Observable Effect Concentration (LOEC).
Disposal of test cultures

Use one of the following methods to dispose of active bacterial cultures:
Autoclave used test containers at 121 C (250 F) for 15 minutes at 15 pounds of pressure.
Once the containers are sterile, pour the contents down the drain with running water. The
reaction tubes may be washed and re-used.
Sterilize test containers by using a 1:10 dilution of commercial laundry bleach. Pour the test
container contents and test containers into the bleach solution. Allow 1015 minutes of contact
time with the bleach solution. Then pour the liquid down the drain and wash the reaction tubes
for reuse.
Summary of method
This method is based on the reduction of resazurin, a redox-active dye, by bacterial respiration.
When it is reduced, resazurin changes color from blue to pink. Toxic substances can inhibit the
rate of resazurin reduction. A chemical accelerant has been added to shorten the reaction time.
The absorbance of the color change is measured at 603 nm.
Toxicity
Page 1189
Toxicity

Required reagents
Description
Quantity/Test
ToxTrak Reagent Set, includes:

Media Set, Total Bacteria Count Tubes
Pipet, transfer, sterile
ToxTrak Reagent Powder Pillows
ToxTrak Accelerator Solution
Tubes, 16 x 100 mm (sold as 6/pkg)
Tube caps for 22758-00 (sold as 6/pkg)
Water, deionized
Unit
Catalog number
25/set
2597200
15/pkg
2277700
2232512
15/pkg
varies
100 mL
2409232
50/pkg
2560766
4 drops
15 mL SCDB
2560836
30 tubes
2275806
30 caps
2241106
varies
500 mL
27249
Unit
Catalog number
Required apparatus
Description
Clippers
each
93600
20/pkg
2124720
1453700
Forceps, flat square tip
each
Incubator, Dri-Bath, 12 well, 120 VAC
each
2281400
each
1451537
each
1465100
Description
Unit
Catalog number
each
1970001

Rack, test tube
50/pkg
2185696
each
1970010
50/pkg
2199796
each
1864100
2185628
1000/pkg
250/pkg
2199725
BOD seed (polyseed)
50/pkg
2918700
each
2092000
Laboratory pen, permanent marker

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
TPH, 10050
TPH (Total Petroleum Hydrocarbons)

DOC316.53.01142
Immunoassay1
Method 10050
Scope and Application: For soil and water

1
Test preparation


Instrument
Adapter
DR 6000
DR 5000
A23618
DR 3900
LZV846 (A)
DR 3800, DR 2800, DR 2700
LZV583

The TPH test can be used for both soil and water testing. When testing soil, start with the Soil Extraction Procedure. When
testing water samples only, start with the Immunoassay Procedure for Soil Extracts and Water Samples. The test requires
about 20 to 30 minutes for complete analysis. As many as 10 cuvettes can be run simultaneously.
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes and other
Store the reagents at 4 C when they are not in use. Allow the reagents to reach room temperature before using them in an
analysis. Actual testing may be done at temperatures ranging from 1 38 C.
The Soil Extractant contains methyl alcohol which is poisonous and flammable. Before using this and other reagents, read
the Material Safety Data Sheet (MSDS) for proper use of protective equipment and other safety information.

Page 1191

Description
Quantity
TPH Reagent Set
Caps, flip spout
Marker, laboratory
Wipes, disposable
Pipet,
TenSette,
1
1
0.11.0 mL
1. Weigh out 10 g of soil

in the plastic weighing
boat.
2. Carefully pour the soil

into an extraction vial.

Page 1192
3. Use the 5-gram scoop

to add one scoop of
sodium sulfate to the
extraction vial.
4. Use the graduated

cylinder to transfer 10 mL
of Soil Extractant into the
extraction vial.

Soil extraction procedure (continued)
5. Cap the extraction vial

tightly and shake
vigorously for one minute.
6. Allow to settle for at

least one minute. Carefully
open the extraction vial.
7. Using the disposable

bulb pipet, withdraw 1.0
1.5 mL from the liquid
layer at the top of the
extraction vial.
Transfer it into the filtration
barrel (the bottom part of
the filtering assembly).
Do not use more than
1.5 mL. The bulb is
marked in 0.25-mL
increments.
8. Insert the filtration

plunger into the filtration
barrel. Place the filtration
assembly on a table and
press firmly on the plunger
until the sample extract is
forced upward into the
center of the plunger.
Use the resultant filtrate as
the sample in the
Immunoassay procedure
for soil extracts and water
samples.
Immunoassay procedure for soil extracts and water samples
Single Wavelength
OK
1. Press
Single Wavelength.
button.
OK.
tested.

the rack snugly.
4. Pipet 0.5 mL of Diluent

Solution into each
Calibrator cuvette.
The same pipette tip can
be used repeatedly for this
step.

Page 1193

Immunoassay procedure for soil extracts and water samples (continued)
5. If testing soil: Pipet

0.5 mL of Diluent Solution
into each sample cuvette.
If testing water: Pipet
0.5 mL of each water
sample into each sample
cuvette. Use a new pipette
tip for each sample.

timer for 10 minutes.
will begin. Immediately mix
the contents of the
cuvettes for 30 seconds
using the technique
described in Using the 1cm MicroCuvette Rack.
6. Have the necessary

apparatus at hand for the
next four steps as they
must be done without
delay.
Use a Wiretrol pipet to
transfer 50 L of each
calibrator to be used into
the calibrator cuvettes.
7. If testing soil: Use a

Wiretrol pipet to transfer
50 L of the filtered extract
from step 8 of the Soil
extraction procedure into
cuvette. Use a separate
capillary tube for each
solution.
0.5 mL of TPH Enzyme
Conjugate into each
calibrator and sample
cuvette.
The same pipette tip can
be used repeatedly for this
step.
Mix the contents of the

cuvettes after each
addition. Use a separate
capillary tube for each
solution.

cuvettes after the addition
of each sample.

same technique.

cuvettes into an
appropriate waste
container.

container.
The simple TPH and

calibrator TPH will remain
attached to the cuvette
walls.
Make sure that most of the

cuvettes. Turn the
tap them lightly on a paper
towel.

Page 1194
If testing water: Use a

Wiretrol pipet to transfer
50 L of methanol into
each sample cuvette.

Immunoassay procedure for soil extracts and water samples (continued)
Color Development

each Cuvette.
each cuvette.

timer for 10 minutes.
will begin. Immediately mix
the contents of the
using the technique
described in Using the 1cm MicroCuvette Rack.

same mixing technique
that was used in step 14.

in the same order that was
used in step 13. The same
pipette tip can be used
using the same mixing
technique as in step 14.
yellow.
Zero

smudges and fingerprints.

cell holder.
Refer to Instrumentspecific information for cell
orientation.

0.000 Abs

holder.
absorbance reading.
Record the results for
each calibrator and
sample.

reporting results to find the
relative concentration.

Page 1195
Using the Wiretrol* Pipet

The Wiretrol Pipet can accurately measure small quantities of liquids. It consists of two parts: a
Teflon-tipped plunger and a calibrated capillary tube. The plunger can be reused. The capillary
tubes must be discarded after one use.
1. Wet the orange

Teflon tip of the Wiretrol
plunger in the sample and
carefully insert it into the
end of the capillary tube
with the colored band.
2. Push the tip to the

other end of the capillary
tube until it barely extends
beyond the end of the
capillary tube.
3. Submerge the
surface of the liquid to be
pipetted. Slowly and
smoothly draw the Wiretrol
plunger up until the bottom
of the plunger tips reaches
the appropriate volume
line.
Touch the end of the tube
to the side of the vessel to
release remaining drops
on the capillary tube tip.
* Wiretrol is a registered trademark of Drummond Scientific.

Page 1196
4. To discharge the pipet,

place the tip of the
surface of the solution
and push the Wiretrol
plunger down in one
smooth motion. Change
capillary tubes for each
calibrator and sample.

The MicroCuvette rack (Figure 1) has been designed to aid in achieving precise and accurate
results when using the immunoassay technique to analyze several samples at the same time.
Loading the RackThe cuvette rack is designed so that it may be inverted with the cuvettes in

Page 1197

There is an inverse relationship between the concentration of TPH and the absorbance reading. In
other words, the higher the absorbance reading, the lower the concentration of TPH:
If sample reading < calibrator reading, TPH concentration in sample > calibrator reading
If sample reading > calibrator reading, TPH concentration in sample < calibrator reading
Example:
Readings
TPH Calibrator #1: 0.480 Abs
TPH Calibrator #2: 0.360 Abs
Interpretation for a Soil Sample
Sample #1The sample reading (0.200 Abs) is less than the readings for both calibrators.
The concentration of TPH in the sample is greater than 50 ppm diesel fuel.
Sample #2The sample reading (0.400 Abs) is between the readings for the TPH calibrators.
The concentration of TPH in the sample is between 20 ppm and 50 ppm diesel fuel.
Sample #3The sample reading (0.550 Abs) is greater than the readings for both calibrators.
The concentration of TPH in the sample is less than 20 ppm diesel fuel.
Interpretation for a Water Sample
Sample #1The sample reading (0.200 Abs) is less than the readings for both calibrators.
The concentration of TPH in the sample is greater than 5 ppm diesel fuel.
Sample #2The sample reading (0.400 Abs) is between the readings for the TPH calibrators.
The concentration of TPH in the sample is between 2 ppm and 5 ppm diesel fuel.
Sample #3The sample reading (0.550 Abs) is greater than the readings for both calibrators.
The concentration of TPH in the sample is less than 2 ppm diesel fuel.

1. Wear protective gloves and eyewear.
2. When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
3. Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
4. If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water

Page 1198
Sensitivity
The antibodies used in the TPH Test Kit react with a variety of compounds found in petroleum
fuels; however, each TPH calibrator has been formulated to represent a specific concentration of
diesel fuel. To use the calibrators for other TPH compounds, see TPH compounds in soil for soil or
TPH compounds in water for water to select the proper TPH calibrator for the compound, sample
and range you want to test.
Example:
To use the TPH calibrators for gasoline, find Gasoline in the first column of theTPH compounds
in soil table or theTPH compounds in water table. Read across the column to find the ppm
represented by each calibrator. For gasoline, calibrator #1 = 15 ppm, calibrator #2 = 35 ppm, etc.
Table 359 TPH compounds in soil

Compound
TPH calibrator #1
(ppm)
TPH calibrator #2
(ppm)
TPH calibrator #3
(ppm)
TPH calibrator #4
(ppm)
Diesel fuel
20
50
100
200
Gasoline
15
35
70
140
Kerosene
35
75
140
250
Benzene
20
45
85
160
Toluene
15
30
50
90
Ethylbenzene
15
35
75
m-Xylene
20
35
70
o-Xylene
10
20
40
80
p-Xylene
16
BTEX
15
25
45
Table 360 TPH compounds in water

Compound
TPH calibrator #1
(ppm)
TPH calibrator #2
(ppm)
TPH calibrator #3
(ppm)
TPH calibrator #4
(ppm)
Diesel fuel
10
20
Gasoline
1.5
3.5
14
Kerosene
3.5
7.5
14
25
Benzene
4.5
8.5
16
Toluene
1.5
Ethylbenzene
0.5
1.5
3.5
7.5
m-Xylene
0.9
3.5
o-Xylene
p-Xylene
0.3
0.5
0.9
16
BTEX
0.5
1.5
2.5
4.5

Page 1199
Diluting water samples

To test for TPH in water at concentrations that are higher than those shown in theTPH compounds
in water table, dilute the sample with deionized water. Add a volume of sample from the Dilution
multipliers table to a graduated cylinder and dilute to 50 mL with deionized water. Run the test.
Multiply the calibrator levels shown in theTPH compounds in water table by the dilution multiplier in
theDilution multipliers table.
Example:
If a 0.5 mL water sample is diluted to 50 mL and tested, the calibrator levels shown in theTPH
compounds in water table for diesel fuel would represent 200, 500, 1000 and 2000 ppm
respectively.
Table 361 Dilution multipliers

mL Sample
Dilution multiplier
0.5
100
1.0
50
2.0
25
5.0
10
10.0
25.0
Interferences
Interference level
Chlorine in water samples
Interferes above 2 ppm. Remove with 1 drop per 100 mL sodium thiosulfate (0.1 N).
Analyze the samples as soon as possible after collection.
To store the samples, collect them in glass or Teflon containers that have been washed with
soap and water and rinsed with methanol. The container should be capped with a Teflon-lined
cap. If a Teflon cap is not available, aluminum foil rinsed in methanol may be used as a
substitute cap liner.
When collecting water samples, fill the container completely (no head space) and cover the
container with a tightly-sealed lid immediately after collection.
SoilStore the samples at 4 C (40 F) for no longer than 14 days.
WaterChill the sample in an ice bath or refrigerator to limit the loss of volatile compounds.
Store samples no longer than 24 hours.

Page 1200
Summary of method
This method provides semi-quantitative screening for TPH based on thresholds as diesel fuel in
the following concentrations:
Soil20, 50, 100, 200 ppm as diesel fuel
Water2, 5, 10, 20 ppm as diesel fuel
and soil. Antibodies specific for TPH are attached to the walls of plastic cuvettes. They selectively
bind and remove TPH from complex sample matrices. A prepared sample and a reagent
containing enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme)
are added to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and TPH
compete for binding sites on the antibodies. Samples with higher levels of analyte will have more
antibody sites occupied by TPH and fewer antibody sites occupied by the enzyme-conjugate
molecules.
color. Therefore, there is an inverse relationship between color intensity and the amount of TPH in
the sample. The resulting color is then compared with a calibrator to determine whether the TPH
concentration in the sample is greater or less than the threshold levels. The TPH concentration is
inversely proportional to the color development: the lighter the color, the higher the TPH

Required reagents
Description
Unit
Catalog Number
20 cuvettes
2774300
500 mL
27248
Description
Unit
Catalog Number
Caps, flip spout
2/pkg
2581802
Marker, laboratory
each
2092000
TPH Reagent
Set1
Deionized water
1
Required apparatus

each
1970001
1000/pkg
2185628
each
4879900
Wipes, disposable
box
2097000

Page 1201
Soil extraction reagents and apparatus

Description
Unit
Catalog Number
Balance, AccuLab Pocket Pro 250 B
each
2796900
Gloves, disposable, Nitrile, medium1
each
2550502
Pipet tips for 1970001 Ten Sette Pipet
50/pkg
2185696
Soil Scoop, 5-g, 4.25-cc
20/pkg
2657205
each
2775200
Dropper, LDPE, 0.5 and 1.0-mL
20/pkg
2124720
Filter and Barrel Assembly
20/pkg
2567620
Soil Extractant Solution
200 mL
2567729
Soil Sample Container
20/pkg
2592920
Weighing Boat, 8.9-cm square
20/pkg
2179020
Spatula, disposable
2/pkg
2569320
Sodium Sulfate, anhydrous
250 g
709929
Soil Extraction Refill Kit, includes:

Description
Unit
Catalog number
each
2936701
Weighing papers
Safety goggles, vented
500/pkg
1473800
each
2550700
each
108138
Pipet, Wiretrol, 1050 L
20/pkg
2852200
Pipet, Wiretrol, 501000 L
250/pkg
2568905
100 mL MDB
32332
Sodium Thiosulfate standard solution, 0.1 N

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Trihalomethanes, 10132
Trihalomethanes
DOC316.53.01143
THM Plus Method
Method 10132
(10 to 600 ppb as Chloroform)
Water Bath Method
Scope and Application: For screening THMs in drinking water samples and Formation Potential tests.
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2427606 and 2495402
Sample cell faces right
DR 5000
2427606 and 2495402
Sample cell faces user
DR 3900
2427606 and 2495402
Sample cell faces user
DR 3800, DR 2800, DR 2700
2427606 and 2495402
Sample cell faces right

Analyze the samples immediately after collection or refrigerate the samples until the analysis is complete.
If the samples were refrigerated after collection, do not warm the samples to room temperature prior to analyzing. This will
minimize volatilization of the disinfection by-products (DBPs). If refrigerated samples are analyzed, heat the samples for an
additional two minutes (total of seven minutes) in step 12 of the procedure.
If analyzing more than four samples, use 450 mL of water in the water bath.
THM Plus Reagent 2 must be at room temperature before use.
A bottle-top dispenser may be used in place of the TenSette Pipet.
Trihalomethane compounds are extremely volatile. Immediately cap sample cells after filling with sample.
Reagent blank is stable for 12 hours and need not be prepared for each test.
Do not mark below the 10 mL fill line.
Trihalomethanes
Page 1203
Trihalomethanes
Description
Quantity
THM Plus Reagent Set
varies
Beaker, 600-mL
Cell Holder Assembly, TTHM
Evaporating Dish, 125 mm x 65 mm
Hot Plate, 7 x 7 inch
Pipet,
TenSette,
0.11.0 mL and tips
Pipet,
TenSette,
110 mL and tips
Sample Cells, 10-mL (see the Instrument-specific information table)
Wipers, disposable
varies
Ice1
varies

1
Depending on the temperature of the tap water, ice may be needed for the cooling baths used in steps 14 and 17.
THM Plus Method

Important Note: Perform steps 49 rapidly to avoid loss of THMs from the sample. When testing more than one
sample, complete steps 49 for one sample before going on to another. If dispensing sample with a pipette, the
pipette must dispense quickly without causing aeration or back pressure.
Stored Programs
725 THM Plus
Start
1. Select the test.

required (see
the Instrument-specific
information table).
for orientation.
Trihalomethanes
Page 1204
2. Prepare a hot water

bath by adding 500 mL of
water to an evaporating
dish. Put the dish on a hot
plate and turn the heater
on high.
3. Prepare a cooling bath

by adding 500 mL of cold
(1825 C) tap water to a
second evaporating dish.
Maintain the temperature
in this range.

one round sample cell to
the 10-mL mark with
sample. Cap and label as
sample.
Trihalomethanes
THM Plus Method (continued)
Fill the second sample cell
with deionized water. Cap
and label as blank.
9. Cap tightly and mix by

shaking.
Thorough mixing makes
sure that all of the THM
goes into the liquid and
does not accumulate in the
air above the sample.

THM Plus Reagent 1 to
each cell.
7. Cap tightly and mix

gently by swirling each cell
three times.
Vigorous shaking can
cause loss of THMs into
the sample cell
headspace.
10. Place the sample cells

in the cell holder
assembly.
11. Place the assembly in

the hot water bath when
the water is boiling rapidly.
Do not allow the water to
rise above the white
diamond near the top of
the sample cells.

to add 3 mL of THM Plus
Reagent 2 to each cell.
Avoid excess agitation of
the sample when
dispensing the reagent.
The reagent is viscous and
a small amount may
remain on the tip after
dispensing. This will not
affect the results.

timer.
period will begin.
Heat for 7 minutes if
refrigerated samples are
being analyzed.
Trihalomethanes
Page 1205
Trihalomethanes
13. When the timer

expires, remove the
assembly and sample
cells from the hot water
bath. Place in the cooling
bath.
Use ice to cool the tap
water if necessary.

timer.
A three-minute cooling
period will begin.
remove the cells from the
cooling bath.
The temperature of the
sample should be
1525 C.
Trihalomethanes
Page 1206

timer.
A three-minute cooling
period will begin.
remove the cells from the
cooling bath.

to add 1 mL of THM Plus
Reagent 3 to each cell.
The sample and blank will
become warm.
Use ice to cool the tap

water if necessary.
Invert each cell a few

times to make sure that a
uniform temperature of the
sample is maintained.

one THM Plus Reagent 4
sample cell and one to the
blank.
16. Replace the cooling

water with fresh, cold tap
water. Place the assembly
that contains the sample
and blank cells into the
cooling bath.
19. Cap each cell tightly

and mix by shaking until all
the powder dissolves.
The powder dissolves
slowly. Intermittent
shaking during the first five
minutes of the color
development period will
help dissolve the reagent
powder.

timer.
A 15-minute development
time will begin.
The color is stable for at
least 30 minutes after the
15-minute development
time.
Trihalomethanes
Zero

pour the prepared sample
and prepared blank into
two square sample cells.
22. When the timer

holder.

0 ppb CHCl3
Allow the solution to settle

in the square cells for 30
seconds to enable any
turbidity that may be
present to settle.
Read

the cell holder.
READ the results in ppb
chloroform (CHCl3).
Interferences
Table 364 Interfering substances1
Interference level
Chlorine
10 mg/L
Copper
1000 mg/L
Hardness, Ca
1000 mg/L as CaCO3

May have some turbidity until Reagent 3 is added
Hardness, Mg
4000 mg/L as CaCO3

May have some turbidity until Reagent 3 is added
Iron
10 mg/L
Lead
2 mg/L
Mercury
10 mg/L
Monochloramine
20 mg/L
Nickel
10 mg/L
Sodium Bisulfite
100 mg/L
EDTA
Interferes negatively at all levels
The substances in the Interfering substances table have been tested and found to cause no interference up to the indicated levels.
Table 365 Additional disinfection by-products (DBPs) that are included in results
Compound
Effect
1,1,1-trichloro-2-propanone
Interferes positively
1,1,1-tricholoacetonitrile
Chloral hydrate
Dibromochloroacetic acid
Trihalomethanes
Page 1207
Trihalomethanes
Table 365 Additional disinfection by-products (DBPs) that are included in results
Compound
Effect
Dichlorobromoacetic acid
Tribromoacetic acid
Trichloroacetic acid
Collect samples in 40-mL glass bottles sealed with Teflon-lined septa caps.
Fill the bottles slowly to overflowing so that no air is included with the sample.
Seal the bottles tightly and invert to check that no air has been trapped.
Because trihalomethane compounds (THMs) are extremely volatile, immediate analysis yields
the greatest accuracy. If the samples cannot be analyzed immediately, cool samples to 4 C.
This will slow the formation of any additional THM compounds in chlorinated samples.
Store the samples at 4 C in an atmosphere free of organic vapors. Samples should not be
held more than 14 days. 0.1 N Sodium Thiosulfate can be used to dechlorinate samples for
longer storage.
Add 1 drop of 0.1 N Sodium Thiosulfate to dechlorinate a finished or distribution system

sample collected in a 125 mL bottle.
Trihalomethanes
Page 1208
Trihalomethanes
Accuracy check
THM Standard Ampule, 10 mg/L as chloroform
Ampule breaker
Wiretrol Pipet
Note: Make sure that the chloroform is not lost to volatilization when attempting to add the chloroform to
the solution. Make sure that the chloroform ampule is kept cold (can use a small ice-bath).

1. Open a THM Standard Ampule, 10 ppm as chloroform.
2. Use a Wiretrol Pipet to transfer 0.100 mL (100 L) of the chloroform standard into a fresh
10 mL portion of sample.
3. Immerse the end of the pipet tip under the water and slowly dispense the chloroform.
4. Cap the sample cell immediately and swirl three times to mix.
Note: The accuracy check methods require careful attention to technique, for it is very easy to lose the
chloroform to volatilization when attempting to add it to the solution. Make sure the chloroform ampule is
kept cold (may wish to use a small ice-bath)
5. Immediately start steps 624 of the procedure to analyze the spiked sample.
6. The value of the spiked sample should increase 100 +/- 20 ppb over the value obtained on the
original unspiked sample.
7. Calculate the % Recovery:
ppb THMs Spiked Sample ppb THMs Unspiked Sample
% Recovery = -------------------------------------------------------------------------------------------------------------------------------------------------------- 100
100 ppb THM Added

1. Prepare a 99 ppb chloroform standard by pipetting 10.0 mL of organic-free water into a sample
cell. Open a THM Standard Ampule, 10 ppm as chloroform. Use a Wiretrol Pipet to transfer
0.100 mL (100 L) of the chloroform standard into the organic-free water. When adding the
standard into the sample, discharge the pipet slowly at or near the bottom of the sample cell
with a slight swirling motion.
Note: If the aliquot of the standard is discharged too quickly, the solution will form a single bubble which
will rise to the top of the solution and volatilize, without being absorbed in the solution.
2. Cap the sample cell immediately and swirl three times to mix.
3. Immediately start steps 624 of the procedure. Do not make up the standard in advance. Use
the standard immediately upon preparation.
4. To adjust the calibration curve using the reading obtained with the 99 ppb Standard Solution,
navigate to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST
Trihalomethanes
Page 1209
Trihalomethanes
Method performance
Program
Standard
Precision
Distribution
Sensitivity
725
66 ppb CHCl3
5379 ppb CHCl3
19 ppb CHCl3
Summary of method
The THM Plus method reacts with the trihalogenated disinfection by-products formed as the result
of the disinfection of drinking water with chlorine in the presence of naturally occurring organic
materials. These disinfection by-products (DBPs) may be produced in the treatment plant or the
distribution system as long as the water is in contact with free chlorine residual. The formation of
the DBPs is influenced by chlorine contact time, chlorine dose and residual, temperature, pH,
precursor concentration, and bromide concentration.
The predominant DBPs formed by the chlorination of drinking water are the trihalomethanes or
THMs. The four trihalogenated compounds that form are chloroform, bromoform,
dichlorobromomethane, and dibromochloromethane. These four compounds comprise the Total
Trihalomethanes (TTHMs) group which is regulated under the Safe Drinking Water Act. The
combined concentration of the TTHMs, is regulated in drinking water samples. Other DBPs that
may be present and react under the conditions of the THM Plus method are listed in Interferences.
In the THM Plus method, THM compounds present in the sample react with
N, N,-diethylnicotinamide under heated alkaline conditions to form a dialdehyde intermediate. The
sample is then cooled and acidified to pH 2.5. The dialdehyde intermediate formed is then reacted
with 7-amino-1,3 napthalene disulfonic acid to form a colored Schiff base. The color formed is
directly proportional to the total amount of THM compounds present in the sample. Test results are
measured at 515 nm and reported as ppb chloroform.

Required reagents
Description
Quantity/Test
Unit
THM Plus Reagent 1
6 drops
15 mL
Catalog number
2753929
THM Plus Reagent 2
6 mL
330 mL
2754048
2790800
Reagent Set (50 tests1), includes:
THM Plus Reagent 3
2 mL
110 mL
2754142
THM Plus Reagent 4
2 pillows
100 pillows
2756699
Fifty tests equals 25 samples and 25 individual blanks. Additional tests can be obtained when multiple samples are run using a single blank.
Required apparatus
Description
Quantity
Unit
Catalog number
Beaker, 600-mL
each
50052
Cell Holder Assembly, TTHM
each
4788000
each
2764700
Hot Plate, 7 x 7 in., 115 VAC, digital
each
2881600
Hot Plate, 7 x 7 in., 230 VAC, digital
each
2881602
each
1970001
Trihalomethanes
Page 1210
Trihalomethanes
Required apparatus
Description
Pipet, TenSette, 110 mL
Quantity
Unit
Catalog number
varies
50/pkg
2185696
each
1970010
varies
50/pkg
2558996
Wipers, disposable
varies
280/pkg
2097000
Description
Unit
Catalog number
Chloroform, 10-ppm ampule
7/pkg
2756707
Water, Reagent, Organic-free
500 mL
2641549
Description
Unit
Catalog number
Pipet, filler, safety bulb
each
1465100
Pipet, volumetric, class A, 10 mL
each
1451538
Pipettes, Wiretrol, 50100 L
250/pkg
2568905
5/pkg
2794005
100 mL
323-32
Vials, glass, 40-mL, with Septa cap

Sodium thiosulfate standard solution, 0.1 N
Trihalomethanes
Page 1211

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Trihalomethane Formation Potential
Trihalomethane Formation
Potential (THMFP)
DOC316.53.01147
Method 102241
THM Plus
Scope and Application: For determining the potential of potable source waters to form trihalomethanes and other
disinfection by-products when under the influence of direct chlorination. For evaluating water treatment processes,
water sources or for predicting THM concentrations in a distribution system. For running Simulated Distribution
System Trihalomethanes (SDS-THM) studies.
1
Adapted from Standard Methods for the Examination of Water and Wastewater, Section 5710
Test preparation

Develop a formation potential test plan. See Trihalomethane formation potential test plan on page 1216.
Precondition sample containers, test bottles and glassware to be chlorine demand free. See Treating analysis labware on
page 1217.
Bring sample to the temperature prescribed in the formation potential plan before preparing the samples.

Description
Quantity
DPD Free Chlorine Reagent
Varies
Chlorine Dosing Solution Ampules
Varies
THM Plus Reagent
Varies
Sample Bottles/Caps
Bottle Labels
pH Meter
Thermometer
Pipet, TenSette, 0.1 to 1.0 mL and tips
Sample Cells, 10 mL, with caps
Stirrer/Hotplate
Stir Bar Magnet
Cell Holder Assembly and Evaporating Dish (water bath)
Spectrophotometer
Trihalomethane Formation Potential (THMFP)

Page 1213

THM Plus
1. Complete a
Trihalomethane formation
potential test plan
(page 1216).
Measure and record the
temperature and pH of the
sample water to be tested.
If the test plan temperature
is different from the
collected sample,
condition the sample to the
test plan temperature
before continuing the test.
2. Prepare six chlorine

demand-free bottles.
Rinse each bottle with
sample and fill each
118-mL bottle (bottles
contain 125 mL when filled
to overflowing) with
approximately 100 mL of
Label the bottles 1
through 6.
3. Do not handle the

stir bar with fingers.
Use tweezers or tongs to
insert a stir bar magnet
into each bottle. Set Bottle
#1 on a stir plate and stir
gently. A small vortex
should be visible on the
surface of the liquid.
4. Open a Chlorine
Dosing Solution Ampule.
Using a TenSette Pipet,
add 0.2 mL of the chlorine
solution to Bottle #1 while
stirring. Immerse the end
of the pipet tip under the
water to dispense the
chlorine.
Mixing while adding the
chlorine is imperative to
avoid highly localized
areas of chlorine
concentration.
If the study is run with

fixed pH, add the pH buffer
to the sample before filling
the bottles.
Repeat steps 45
5. Turn off the stirrer and

fill the bottle until it is
Cap in a manner to avoid
trapping any air bubbles
and invert to mix. Put the
sample bottle in the dark
or wrap with foil.
6. Calculate the actual

amount of chlorine added
(Equation 1 on page
1215).
Each 0.2 mL of Dosing

Solution added will add
approximately 2.0 mg/L
Cl2 to the sample.

Page 1214
7. Repeat Steps 45 for

bottles 2 through 6.
Increase the amount of
chlorine added in
increments of 0.2 mL
(Table 366).
The amounts added may
be increased or decreased
based on the expected
chlorine demand of the
sample water and chlorine
contact time.
8. Incubate or refrigerate
the bottles at the
temperature and contact
time specified in the
test plan.

THM Plus
Method 8021
or
Method 10069
9. After the prescribed

chlorine contact time is
completed, analyze the
samples for residual Free
Chlorine using DPD Free
Chlorine Reagent (Method
8021 or Method 10069).
Follow the chlorine
procedure supplied with
the spectrophotometer
being used.
Method 10132

bottle or bottles having the
prescribed chlorine
residual from the test plan
and analyze for THMs
using the
THM Plus method
(Method 10132)1, 2.
Follow the procedure
supplied with the
spectrophotometer being
used. Results are g/L
(ppb) chloroform.
Recheck the pH of the sample remaining in the analyzed bottle to check for pH shifts that may occur in low alkalinity waters.
The selected bottle or bottles to be analyzed for THMs should be analyzed as soon as possible, especially bottles that have had sample
removed when testing for chlorine residual. If this is not possible, add 1 drop of 0.1 N sodium thiosulfate and store the samples at 4 C for up to
14 days.
Chlorine addition calculation

Use Equation 1 to calculate the concentration of the chlorine added in step 6.
Equation 1
0.2 mL volume of standard added ampule certificate value mg/L Cl 2
mg/L Cl 2 = ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------125 mL
Example:
0.2 mL certificate value of 1250 mg/L Cl
mg/L Cl 2 = ----------------------------------------------------------------------------------------------------------------2125 mL
mg/L Chlorine = 2.0

Page 1215
Incremental reagent addition

Table 366 defines the incremental addition of chlorine dosing solution.
Table 366 Incremental addition of a 1250 mg/L Cl2 dosing solution

Bottle #
Cl2 dosing solution added (mL)
Increases sample concentration (in mg/L Cl2) by
0.2
2.0
0.4
4.0
0.6
6.0
0.8
8.0
1.0
10.0
1.2
12.0
Trihalomethane formation potential test plan

Formation potential tests are run to determine the potential of a water to form THMs or other
disinfection by-products under a standard or a user-specified set of reaction conditions. The goals
of the test plan should be well defined and the reaction conditions clearly documented to support
the goals of the plan.
Formation potential plans are often structured to have a fixed sample pH, sample temperature,
chlorine dose, chlorine contact time and a desired chlorine residual after the contact time.
Formation plans with fixed pH, temperature, chlorine concentration and chlorine contact times are
designed to compare the variability of various waters while holding the variables responsible for
THM formation constant. This allows the evaluation of different source waters and allows the
results to be duplicated at different utilities or by different analysts under similar reaction
conditions. Some standard or fixed reaction condition plans available in the published literature or
in Standard Methods are listed here as examples.
Reaction conditions such as the standard 7-day reaction (incubation) period at pH 7.0 0.2 with
phosphate buffer at 25 2 C with a remaining chlorine residual between 3.0 and 5.0 mg/L are not
intended to simulate water treatment processes. These conditions are most useful for estimating
the concentration of THM precursors in the raw water or for measuring the effectiveness of a
treatment change under fixed standard conditions. A second standard method using a 3-day
reaction time has also been used. Additionally, a Uniform Formation Conditions (UFC) test is
available that uses a contact time of 24 1 hour at 20.0 1.0 C, a borate buffer at pH 8.0 0.2
with a target free chlorine residual of 1.0 0.4 mg/L after 24 hours as the sample bottle to be
measured for THMs. The Formation Potential Plan can also be constructed to develop a
Simulated Distribution System Trihalomethanes (SDS-THM) study. The goal of this plan is to use
the existing treated water pH with variable chlorine doses and contact times that simulate the
chlorine levels and water age found in a distribution system. This will estimate the THM formation
occurring in the distribution system.
Another important use of Formation Potential Test plans is to use a single water source and vary
temperature, pH or chlorine contact times to meet specific treatment optimization goals. It is again
important to document all reaction conditions used in the test plan.
Examples of additional formation potential studies are:
Formation potential studies on settled waters from jar tests to evaluate the effectiveness of
coagulant dosage on removing organics responsible for THM formation.
Formation potential studies based on chlorine dosage rates and location of

chlorine application.
Formation potential studies on the rate of THM formation at variable locations within the
treatment stream.

Page 1216
Formation potential studies to correlate THM levels to UV-254, TOC or SUVA values.
Formation potential studies to study the effects of using alternative oxidants.
Formation potential procedure modification

Use the following guidelines whenever modifications are made:
Make smaller chlorine concentration additions by using a larger sample size or smaller
chlorine additions. A 237-mL bottle (contains 250 mL when filled to overflowing) is available for
low chlorine demand applications. Each 0.1 mL of chlorine standard added will add
approximately 0.5 mg/L of chlorine. Substitute 250 mL for 125 mL in Equation 1. A lower
concentration Chlorine Standard Solution, 5075 mg/L as Cl2 is available for testing low
organic waters.
High organic waters require larger additions of chlorine. Use 0.5 mL, 1.0 mL, 1.5 mL, etc., to
spike the bottles in steps 4 and 7 in the test procedure.
Wrap sample bottles made of clear colorless glass in foil to protect the sample from light, or be
kept in the dark during the contact time.
Sample pH can be modified or standardized by adding a fixed amount of a pH buffer solution

to each bottle. Prepare a reagent blank bottle using organic free water. Add the same amount
of buffer to this blank and carry the blank through the procedure. This will check the formation
potential (if any) that was added by the buffer. Subtract the formation potential of the blank
from the sample THM values.

Collect samples in glass bottles sealed with TFE-lined screw caps. Use only freshly collected
samples and process immediately. If this is not possible, store samples a 4 C and analyze as
soon as possible. Significant sample degradation can occur in unpreserved samples within
24 hours. Add one drop of thiosulfate solution per 125 mL sample bottle if the samples are
chlorinated and cannot be analyzed immediately.

Glassware used in this test must be chlorine demand-free. Treat all glassware with a dilute
solution of chlorine bleach prepared by adding 0.5 mL of commercial bleach to 1 liter of water.
Alternatively, the sample bottles may be treated by adding 2.0 mL of the Chlorine Dosing Solution
to each 125-mL bottle and filling to overflowing with deionized water. Soak glassware in this
solution for at least one hour. After soaking, rinse the glassware with copious amounts of chlorine
demand-free water before filling with sample.
Summary of method
Organic matter present in drinking water source waters reacts with chlorine to form chlorinated
organic species, some of which may be trihalomethanes or other regulated disinfection
by-products. The potential of various source or treated waters to form disinfection by-products can
be determined by adding chlorine and controlling dosage rate, pH, temperature and contact time.
The THMs formed under these user-defined conditions are determined using the
THM Plus method.

Page 1217

Required reagents
Description
Catalog number
Chlorine Dosing Solution Ampules, 11901310 mg/L as Cl2, 10-mL ampules, 16/pkg
2504810
Chloroform, 10 ppm ampule, 7/pkg
2756707
1407099
THM Plus Reagent Set
2790800
Water, Organic Free, 500 mL
2641549
Required apparatus
Description
Catalog number
714424
Caps, Black, PP Teflon liner, 12/pkg
2401812
Cell Holder assembly, TTHM
4788000
2764700
Hot Plate/Stirrer, 7 x 7 inch, 110 V
2881600
Sample cells, 10 mL w/caps
2427606
Stir Bar, Teflon-coated, 2.22 x 0.48 cm
4531500

Description
Ampule Breaker, for
Catalog number
Voluette
Ampules
2196800
714441
1409895
1407995
89868
Buffer Solution, pH 7.0, Demand Free, 500 mL
2155353
2166706
Chlorine Standard Solution Ampule, 5075 mg/L as Cl2, 10 mL, 16/pkg
1426810
DPD Free Chlorine AccuVacs, 25/pkg
2502025
2105599
DPD Free Chlorine Swiftest Dispenser w/reagents
2802300
Free Chlorine, Swiftest Dispenser Reagent (refill)
2105560

Page 1218

Description
Catalog number
Incubator, Model 205, 110 V, 0 to 40 C
2616200
Labels, PolyPaper, 1.5 x 3 " 120/pkg
2091502
Pipettes, Wiretrol, 50100 L
2568905
Sension 2 Portable pH/ISE Meter w/electrode
5172510
Sodium Hydroxide Standard Solution, 0.100N, 500 mL
19153
Sodium Thiosulfate Solution, 0.1 N, 100 mL
32332
Standard Methods Handbook

Sulfuric Acid Standard Solution, 0.100 N, 500 mL
2270800
20253
Thermometer, Double Scale, 20 to 110 C (0 to 230 F)
2095911
1970001
Tips for TenSette Pipet, 0.1 to 1.0 mL, 50/pkg
2185696
Tweezers
1428200

Page 1219

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Volatile acids, 8196
Volatile Acids
DOC316.53.01144
Esterification Method1
Method 8196
(27 to 2800 mg/L)
Reagent solution
Scope and Application: For digestor sludges.

1
Adapted from The Analyst, 87, 949 (1962).
Test preparation


Instrument
Sample cell
Cell orientation
DR 6000
2401906 and 2495402

DR 5000
2401906 and 2495402
DR 3900
2401906 and 2495402
DR 3800, DR 2800, DR 2700
2401906 and 2495402

Description
Quantity
Centrifuge
Centrifuge Tubes and Caps
Ethylene Glycol
3 mL
Ferric Chloride-Sulfuric Acid Solution
20 mL
Funnel and Filter Paper
Hot Plate
Hydroxylamine Hydrochloride Solution, 100-g/L
1 mL
Pipet Filler
Pipet, 2 mL1
Pipet, volumetric, Class A, 0.50-mL1

Pipet, volumetric, Class
A,10-mL1
Sample Cells, 10-20-25 mL

Water Bath and Rack
Water, deionized
1
1
2
4 mL
0.4 mL
1
20.5 mL

1
The TenSette Pipet can be used in place of individual pipets in this procedure.
Volatile Acids
Page 1221
Volatile Acids
Esterification Method
Stored Programs
770 Volatile Acids
Start
1. Select the test.
water into a dry 25-mL
sample cell.
3. Filter or centrifuge
10 mL of sample.
Centrifuging is faster than
filtration.
4. Prepared Sample:
Pipet 0.5 mL of the filtrate
or supernatant into a
second dry 25-mL sample
cell.
5. Pipet 1.5 mL of
ethylene glycol into each
6. Pipet 0.2 mL of 19.2 N

Solution into each cell.
Swirl to mix.
7. Insert both cells into a

boiling water bath.

timer.
Alternatively, the cells may

be boiled in a 500-mL
beaker.
period will begin.
9. When the timer

expires, cool the solutions
to 25 C (until the cell feels
cold) with cool water bath.
10. Using a pipet filler,

pipet 0.5 mL of
Hydroxylamine
Hydrochloride Solution
into each cell. Swirl to mix.
11. Using a pipet filler,

pipet 2.0 mL of 4.5 N
Sodium Hydroxide
Standard Solution into
12. Add 10 mL of Ferric

Chloride Sulfuric Acid
Solution to each cell. Swirl
to mix.
for orientation.
Volatile Acids
Page 1222
Volatile Acids
Esterification Method (continued)
13. Add 10 mL of
deionized water to each
cell. Cap and invert to mix.
14. Transfer 10 mL of the

blank solution from the
round 25-mL cell to a
clean dry square sample
cell.
15. Transfer 10-mL of the

sample solution from the
round 25-mL cell to a
clean dry square sample
cell.
16. Immediately start the

instrument timer.
Another three-minute
During this time, complete
steps 17 and 18.
Zero
17. Blot each sample cell

dry. Immediately insert the

0 mg/L HOAC
Read

the cell holder.

mg/L HOAC.
Analyze as soon as possible after collection.
Samples can be stored for up to 24 hours by cooling to 4 C (40 F) or below.
Accuracy check
Volatile Acid Voluette Ampule Standard, 62,500-mg/L as acetic acid
Ampule breaker
TenSette Pipets and tips
Three 25-mL mixing cylinders
Volatile Acids
Page 1223
Volatile Acids
ADDITIONS. Default values for standard concentration, sample volume, and spike volumes
can be accepted or edited. After values are accepted, the unspiked sample reading will
appear in the top row. See the user manual for more information.
1. Prepare a 500-mg/L volatile acid standard solution as follows. Pipet 4.00 mL of Volatile Acid
Standard Solution, 62,500-mg/L, into a 500-mL volumetric flask. Dilute to the mark with
deionized water. Prepare this solution daily.
2. Use the 500-mg/L Volatile Acid Standard Solution in place of the sample. Follow the volatile
acid test procedure.
3. To adjust the calibration curve using the reading obtained with the 500-mg/L standard solution,
navigate to Standard Adjust in the software OPTIONS>MORE>STANDARD ADJUST.
Method performance
Program
Standard
770
1350 mg/L as acetic acid

(HOAC)
Precision
Distribution
Sensitivity
12181482 mg/L HOAC
27 mg/L as HOAC
Summary of method
The volatile acids test is designed specifically for determining volatile acids in digestor sludges.
The method is based on esterification of the carboxylic acids present in the sample and
subsequent determination of the esters by the ferric hydroxamate reaction. All volatile acids
present are reported as their equivalent mg/L as acetic acid. Test results are measured at 495 nm.
Volatile Acids
Page 1224
Volatile Acids

Required reagents
Description
Quantity/Test
Unit
(1) Ethylene Glycol
3 mL
1000 mL
203953
(2) Ferric Chloride-Sulfuric Acid Solution
20 mL
1000 mL
204253
Volatile Acid Reagent Set (90 tests), includes:
Catalog number
2244700
(1) Hydroxylamine Hydrochloride Solution, 100-g/L
1 mL
100 mL
81842
(1) Sodium Hydroxide Standard Solution, 4.5 N
4 mL
1000 mL
204053
(1) Sulfuric Acid Standard Solution, 19.2 N

Water, deionized
0.4 mL
1000 mL
203832
20.5 mL
4L
27256
Quantity
Unit
Catalog number
2676500
Required apparatus
Description
Centrifuge, 115 VAC, 60 Hz.
each
Centrifuge Tubes, 15-mL
10/pkg
2278739
Centrifuge Tube Caps
20/pkg
2585220
each
50838
Filter Paper, folded, 12.5-cm
100/pkg
189457
Funnel, poly, 65-mm
each
108367
Hot Plate, 7-inch digital, 120 VAC
each
2881500
Hot Plate, 7-inch digital, 240 VAC
each
2881502
each
1465100
each
53236
each
1451534
1451538
each
Cell holder assembly
each
4788000
Evaporating dish, 125 mm x 65 mm
each
2764700
2/pkg
2495402
Description
Volatile Acids Standard Solution, 10-mL
Voluette
Ampule, 62,500-mg/L as HOAC
Unit
Catalog number
16/pkg
14270-10
each
2196800
Unit
Catalog number
189640

Description
each
Water Bath and Rack
each
195555
each
1970010
250/pkg
2199725
50/pkg
2199796

each
1970001
50/pkg
2185696
Volatile Acids
Page 1225
Volatile Acids
Description
Unit
Catalog number
1000/pkg
2185628
each
1457449
Finger cots
2/pkg
1464702

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Volatile Acids, DT, 8218
Volatile Acids
DOC316.53.001182
Sodium Hydroxide Method
Method 8218
100 to 2400 mg/L CH3COOH
Digital Titrator
Scope and Application: For wastewater
Test preparation

Distill the sample according to the Volatile Acids Procedure, Sample Distillation instructions in the General Purpose
Distillation Apparatus Set or use the distillation procedure described in Standard Methods for the Examination of Water and
Wastewater.
The final result has been adjusted to give the correct answer based on a 70% correction factor. For higher recoveries, use
the esterification method.

Description
Quantity
Volatile Acid Reagent Set
Deionized Water
varies
Digital Titrator
Graduated Cylinder
Sodium Hydroxide Method
See the
Volume multipliers
table
distillate.

tube into a 0.9274 N
Sodium Hydroxide titration
cartridge.
titrator body.

the tip.
4. Select the distillate

to the expected volatile
acids concentration in
acetic acid from the
Volume multipliers table.
Volatile Acids
Page 1227
Volatile Acids
Sodium Hydroxide Method (continued)
cylinder, transfer the
distillate volume into a
flask and dilute to
approximately the 150-mL
mark with deionized water.

one Phenolphthalein
and swirl to mix.

swirl while titrating with
sodium hydroxide until a
light pink color appears.
digits required.
8. Calculate:
Digits Required x
Digits Multiplier =
mg/L Volatile Acids
(as acetic acid, CH3COOH)

Range (mg/L as CH3COOH)
Volume (mL)
Digit Multiplier
100400
150
200800
75
6002400
25
Summary of method
A sample acidified with sulfuric acid is distilled and the distillate is then titrated to the
phenolphthalein end point with sodium hydroxide standard.
Volatile Acids
Page 1228
Volatile Acids

Required reagents
Description
Unit
Volatile Acid Reagent Set, includes:

Catalog number
2460200
100/pkg
94299
each
1484201
4L
27256
Description
Unit
Catalog number
each
50846
Digital Titrator
each
1690001
50546

Water, deionized
Required apparatus
each
Cylinder, Graduated 100 mL, TD White
each
50842
Cylinder Graduated, 25 mL TD White
each
50840
Delivery Tube, 180 hook
5/pkg
1720500
Volatile Acids
Page 1229

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Volatile Acids, BT, 8291
Volatile Acids
DOC316.53.01163
Method 8291
100 to 2400 mg/L as CH3COOH
Buret Titration

1
Test preparation

The sample must be distilled before this test can be started. Follow the distillation procedure that is given in the distillation
manual or the distillation procedure that is given in Standard Methods for the Examination of Water and Wastewater.
This method recovers approximately 65 to 95% of the volatile acids in the sample, but 70% is the accepted correction factor.
The final result has been adjusted to give the correct answer based on a 70% correction factor. For higher recoveries, use
the esterification method.

Description
Quantity
1 bottle
Graduated cylinder
Buret titration
See
Table 1
1. Distill the sample and

collect 150 mL of distillate.
Fully mix the distillate.
2. Select a sample

Sodium Hydroxide
Standard Solution.
4. Use a graduated
cylinder to measure the
selected volume of
distillate. Add the sample
to a 250-mL Erlenmeyer
flask.
Volatile Acids
Page 1231
Volatile Acids


one Phenolphthalein
Swirl to mix.

until a light pink color
forms and persists for 30
seconds.

the Range-specific
information to calculate
the concentration:
mL titrant x 86 x multiplier
= mg/L as CH3COOH
was titrated and 3 mL of
titrant was used to reach
the endpoint. The
concentration is 3 x 86 x 6
= 1550 mg/L CH3COOH

Range (mg/L as CH3COOH)
Sample volume (mL)
Multiplier
100400
150
200800
75
6002400
25

Collect samples in clean plastic or glass bottles. Analyze the sample as soon as possible after
collection. Samples can be stored for up to 24 hours by cooling to 4 C (39 F) or below. Warm to
room temperature before starting the test.
Summary of method
A sample acidified with sulfuric acid is distilled and the distillate titrated to the phenolphthalein end
point with sodium hydroxide. The volume of titrant that is necessary to reach the end point is
proportional to the volatile acids concentration. The results are in mg/L as acetic acid (CH3COOH).
Volatile Acids
Page 1232
Volatile Acids

Required reagents
Description
Quantity/Test
Unit
Catalog number
1 pillow
100/pkg
94299
varies
1L
19153
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
2636541
Buret Clamp, double
each
32800
each
50546
50840

each
each
50841
each
50842
Support Stand
each
56300
Bottles, sampling, poly, 250-mL
each
2087076
50846
Volatile Acids
Page 1233

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Zinc, 8009
Zinc
DOC316.53.01145
USEPA1 Zincon Method2
Method 8009
(0.01 to 3.00 mg/L)
Powder Pillows
Scope and Application: For water and wastewater. Digestion is required for a total zinc analysis (see Digestion).
1
USEPA approved for wastewater analyses 3500 Zn B: Federal Register, 45(105) 36166 (May 29, 1980).
Test preparation

The Instrument-specific information table displays information that may vary from instrument to
instrument. Select the spectrophotometer from the instrument column on the left. Read across to
the spectrophotometer.

Powder pillows
Instrument
Sample cell
Cell orientation
DR 6000
2495402
DR 5000
2495402
DR 3900
2495402
DR 3800, DR 2800, DR 2700
2495402

Use only glass-stoppered mixing cylinders in this procedure.
Wash glassware with 1:1 HCl1 and rinse with deionized water before use.
Use a plastic dropper in step 6 of this procedure. Droppers with rubber bulbs may contaminate the reagent.
ZincoVer 5 reagent contains potassium cyanide. Cyanide solutions are regulated as hazardous waste by the Federal
RCRA. Cyanide should be collected for disposal as a reactive (D003) waste. Be sure that cyanide solutions are stored in a
caustic solution with pH >11 to prevent release of hydrogen cyanide gas. Refer to the current MSDS for handling and
disposal information.
The Pour-Thru Cell cannot be used with this test.
1

Description
Cyclohexanone
Quantity
0.5 mL
ZincoVer 5 Reagent Powder Pillow
Zinc
Page 1235
Zinc
Zincon method
Stored Programs
780 Zinc
Start
1. Select the test.

required (the Instrumentspecific information table).

of sample.

one ZincoVer 5 Reagent
mixing cylinder. Stopper.

for orientation.
Pour 10 mL of the solution
into a sample cell.
The sample should be

orange. If the sample is
brown or blue, the zinc
concentration is too high
or an interfering metal is
present. Dilute the sample
and repeat the test.
6. Prepared sample:
Use a plastic dropper to
add 0.5 mL of
cyclohexanone to the
remaining solution in the
mixing cylinder.

timer. A 30-second
During the reaction period,
stopper the mixing cylinder
and vigorously shake the
prepared sample.
The sample will be
reddish-orange, brown, or
blue, depending on the
zinc concentration.
Zinc
Page 1236

dissolve the powder
completely. Inconsistent
readings may result if all
the particles are not
dissolved.

timer.
this reaction period,
complete step 9.
Zinc
Zincon method
Zero

sample solution from the
mixing cylinder into a
second sample cell.
10. When the timer

holder.

0.00 mg/L Zn
Read

the cell holder.
READ the results in
mg/L Zn.
Interferences
Interference level
Aluminum
Greater than 6 mg/L
Cadmium
Copper
Greater than 5 mg/L
Iron (ferric)
Greater than 7 mg/L
Manganese
Greater than 5 mg/L
Nickel
Greater than 5 mg/L
Organic Material
Large amounts may interfere. Pretreat the sample with a mild

digestion.
Highly buffered or extreme sample pH

require sample pretreatment. Adjust pH to 45.
Samples containing AMP cause a negative interference.
Digest the sample to eliminate this interference (follow the
total phosphorus hot plate digestion, Method 8190).
Amino-tri(methylene phosphonic acid) (AMP)
Important Note: Be sure to adjust the pH of the

sample after the digestion to pH 45 with sodium
hydroxide before the zinc analysis. Correct the pH level
for volume changes.

Collect samples in acid-cleaned plastic or glass bottles. If prompt analysis is impossible, preserve
the sample by adjusting to pH 2 or less with nitric acid (about 2 mL per liter). Preserved samples
may be stored up to six months at room temperature.
Before analysis, adjust the pH to 45 with 5.0 N Sodium Hydroxide. Do not exceed pH 5 as zinc
may precipitate. Correct the test result for volume additions.
Accuracy check
Zinc
Page 1237
Zinc
Zinc Voluette Ampule Standard, 25 mg/L Zn
Ampule breaker
TenSette Pipet 0.1 - 1.0 mL and tips
25-mL mixing cylinders
2. Select OPTIONS>MORE>STANDARD ADDITIONS from the instrument menu.
3. Press OK to accept the default values for standard concentration, sample volume, and spike
volumes. Press EDIT to change these values. After values are accepted, the unspiked sample
reading will appear in the top row.
100 mg/L zinc standard solution
10.00 mL Class A pipet
1. Prepare a 1.00-mg/L zinc standard solution as follows. Pipet 10.00 mL of Zinc Standard
Solution, 100-mg/L, into a 1000-mL volumetric flask. Dilute to the mark with deionized water.
2. Follow the zinc procedure.
3. To adjust the calibration curve using the reading obtained with the 1.00-mg/L standard
solution, navigate to Standard Adjust in the software (OPTIONS>(MORE)>STANDARD ADJUST).
Digestion
A sample digestion is required before an analysis for total zinc can be started. A digestion will
make sure that all zinc compounds in the sample are in a chemical form that will be measured.
Complete the following steps to digest the sample.
Note: The following procedure is the USEPA mild digestion. See the Water Analysis Guide for more
digestion procedures.
1. If nitric acid has not been added to the sample previously, add 5 mL of concentrated nitric acid
to one liter of sample (use a glass serological pipet and pipet filler). If the sample was acidified
at collection, add 3 mL of nitric acid to one liter of sample.
2. Transfer 100 mL of acidified sample to a 250-mL Erlenmeyer flask.
Zinc
Page 1238
Zinc
3. Add 5 mL of 1:1 hydrochloric acid*.
4. Heat the sample on a hot plate* at 95 C (203 F) until 15-20 mL remain. Make sure the
sample does not boil.
5. Filter the cooled sample with 0.45 m filter to remove any insoluble material.
6. Adjust the pH of the digested sample to pH 45 with 5.0 N sodium hydroxide. See Sample
collection, preservation and storage for instructions.
7. Quantitatively transfer the sample to a 100-mL volumetric flask and dilute to the mark with
deionized water.
Method performance
Program
Standard
Precision
Distribution
Sensitivity
780
1.00 mg/L Zn
0.971.03 mg/L Zn
0.013 mg/L Zn
Summary of method
Zinc and other metals in the sample are complexed with cyanide. Adding cyclohexanone causes a
selective release of zinc. The zinc reacts with 2-carboxy-2'-hydroxy-5'-sulfoformazyl benzene
(zincon) indicator to form a blue-colored species. The blue color is masked by the brown color
from the excess indicator. The intensity of the blue color is proportional to the amount of zinc
present. Test results are measured at 620 nm.
Zinc
Page 1239
Zinc

Required reagents
Description
Quantity/Test
Unit
Catalog number
2429300
0.5 mL
100 mL MDB
1403332
100/pkg
2106669
Zinc Reagent Set, 20-mL sample size, includes:

Cyclohexanone
ZincoVer 5 Reagent Powder Pillows
Required apparatus
Description
Quantity
Unit
Catalog number
each
2088640
Sample cell, 10 mL, square, matched pair
2/pkg
2495402
Description
Unit
Catalog number
Water, deionized
4L
27256
100 mL
237842
Zinc Standard Solution, 10-mg/L Voluette Ampule, 25-mL as Zn
16/pkg
1424610
Zinc Standard Solution, 1000-mg/L
100 mL
1417742
Unit
Catalog number
Zinc Standard Solution, 100-mg/L

Description
each
50546
Hot Plate, 120 V
each
1206701
Hydrochloric Acid 6.0 N, 1:1
500 mL
88449
Nitric Acid, concentrated, ACS
500 mL
15249
50 mL SCDB
245026
Sodium Hydroxide 5.0 N

Tensette Pipet, 0.11.0
Tips for Tensette Pipet 1970001
each
1970001
50/pkg
2185696
Ampule Breaker
each
2196800
each
1451538
Pipet, Filter, Safety bulb
each
1465100
each
1457453
100/pkg
1353000
Filter paper 0.45 m

pH paper test strips, 3.05.5 pH range
Filtration apparatus, glass

15 roll
37333
each
234000
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Microbiology
Page 1241
Page 1242
Bacteria Test Guidelines
DOC316.53.01188
All tests for bacteria use a nutritional broth or agar and incubation at a specific temperature to grow
the target organism. Sterile equipment and careful handling techniques are necessary to prevent
contamination of the sample.
Methods for bacteria testing

The following descriptions give a general overview of the different methods for bacteria tests.
Presence/absence (P/A)the sample is added to a bottle containing broth and incubated. A

color change indicates the presence of the target bacteria.
Most Probable Number (MPN)the sample is diluted and added to a series of tubes
containing broth. The tubes are incubated and then examined for the presence of gas.
Membrane Filtration (MF)the sample is filtered and the filter is placed on a pad containing
growth media. After incubation, the filter is examined for the growth of the target organism.
Plate count agarthe sample is mixed with an agar in a large petri dish and incubated. After
incubation, the agar is examined for bacteria colonies. This test is usually used for total or
heterotrophic bacteria.
Presumptive and confirmation tests

Two tests are necessary for most methods, a presumptive test and a confirmation test.
Presumptive testuses growth media that facilitates the growth of the target organism. A
positive result is an indication of the target organism but can include a false positive result.
The P/A, MPN and MF methods are presumptive tests.
Confirmation testuses media that is more selective for the target organism and sometimes
uses a higher incubation temperature. Some media, such as the m-ColiBlue24 broth used
with the MF method, is selective enough for the target organism (E. coli) that no confirmation
test is required.
Techniques for microbiological tests

Good laboratory technique is essential for microbiological tests. To make sure the results are
reliable, collect and preserve samples carefully. Use high-quality laboratory equipment and readyto-use media to save time and minimize errors.
Prepare the work area
Start the incubator while preparing other materials. Set the incubator to the temperature
required by the specific procedure (usually 35 0.5 C for total coliforms and enterococci and
44.5 0.2 C for fecal coliforms).
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or
dilute iodine solution. Wash hands thoroughly with soap and water.
Mark each sample container with the sample number, dilution, date and other necessary
information. Avoid contaminating the inside of the sample container in any way.
Use presterilized Whirl-Pak bags or bottles for sample collection. If the sample has been
disinfected, use bags or bottles that contain a dechlorinating agent.

Page 1243
Prepare sample containers

To collect samples, use any of the following: sterilized plastic bags, sterilized disposable bottles,
autoclavable glass or plastic bottles.
Sterilized plastic bags or disposable bottles
Presterilized plastic bags and bottles are available with or without dechlorinating agent. The
bottles are available with a 100-mL fill-to line.
Dechlorinating reagent should be used with potable or chlorinated water samples. It is not
necessary for unchlorinated or non-potable water samples. However, dechlorinating reagent will
not interfere with unchlorinated samples so, for simplicity, plastic bags containing dechlorinating
reagent may be used for all samples.
Autoclavable glass or plastic bottles:
Glass or plastic bottles (125-mL size) may be used instead of sterilized plastic bags or disposable
bottles. These containers should be prepared as follows:
1. Wash in hot water and detergent.
2. Thoroughly rinse with hot tap water, followed by a distilled water rinse to make sure that all
detergent is removed.
3. If dechlorinating agent is needed (for chlorinated, potable water), add the contents of one
Dechlorinating Reagent Powder Pillow for each 125-mL of container volume. (A 250-mL
sample container will require two powder pillows.)
4. Steam sterilize glass and autoclavable plastic containers at 121 C (250 F) for 15 minutes.
Glass sample containers may be sterilized by hot air at 170 C (338 F) for one hour.
5. Store sterile containers, tightly capped, in a clean environment until needed.
Autoclave option for sterilization

Use presterilized laboratory equipment and media to save time and minimize errors. When
numerous samples must be run on a routine basis, sterilization of non-disposable materials with
an autoclave may be preferable.
Procedure
1. Wash sample bottles, pipets, petri dishes, filter holder with stopper and graduated cylinder (if
needed) with hot water and detergent.
2. Rinse several times with tap water and then with demineralized water. Dry thoroughly.
3. Prepare all equipment for the autoclave as follows:
Loosely thread caps on sample bottles and cover caps and bottle necks with foil or paper.
Cover the openings of graduated cylinders with foil or paper.
Insert the filter funnel base into an autoclavable rubber stopper that will fit the filter flask.
Wrap the two parts of the filter funnel assembly separately in heavy wrapping paper and
seal with masking tape.
Wrap petri dishes (borosilicate glass) in paper or place in aluminum or stainless cans.
4. Sterilize equipment in an autoclave at 121 C (250 F) for 15 minutes. Borosilicate glass items
may be sterilized with dry heat at 170 C (338 F) for a minimum of 1 hour.

Page 1244
Collect and preserve samples

Collect a sufficient volume of sample for analysis (usually a minimum of 100 mL of sample). World
Health Organization guidelines prescribe 200 mL per sample while Standard Methods for the
Examination of Water and Wastewater prescribes 100 mL per sample. Avoid sample
contamination.
No dechlorination is necessary if the sample is added directly to the growth medium on site.
Otherwise, treat the samples to destroy chlorine residual. Sodium thiosulfate that has been
sterilized within the collection vessel is used to remove chlorine residual. Transport for analysis
immediately after collection.
Analyze as soon as possible after collection. Allow no more than 6 hours to elapse between
collection and examination for non-potable water samples and 30 hours for potable water
samples. For best results, maintain the sample at or below 10 C, but do not freeze. Failure to
properly collect and transport samples will cause inaccurate results.
Collect at least 100 mL of sample in a presterilized plastic bag or bottle or in a sterile glass or
plastic bottle. Do not fill sample containers completely. Maintain at least 2.5 cm (approximately one
inch) of air space to allow adequate space for mixing the sample prior to analysis.
Faucets, spigots, hydrants or pumps
Collect representative samples by allowing the water to run from a faucet, hydrant or pump at a
moderate rate for 2 to 3 minutes before sampling. Adjust the flow rate before sample collection to
avoid splashing during collection. Do not adjust the rate of flow while collecting the sample. Avoid
valves, spigots and faucets that swivel or leak or those with attachments such as aerators and
screens or remove the attachments prior to sample collection.
Handle the sample containers carefully. Open the sample containers just prior to collection and
close immediately following collection. Do not lay the lid or cap down. Do not touch the lip or inside
surface of the container. Do not rinse the containers before use. Label the sample containers
immediately and analyze promptly.
Rivers, lakes and reservoirs
When sampling a river, lake or reservoir, do not sample near the edge or bank. Remove the cap,
grasp the sample container near the bottom and plunge the container, mouth down, into the water
in order to exclude any surface scum. Fill the container entirely under water by positioning the
mouth into the current or, in non-flowing water, by tilting the container slightly and allowing it to fill
slowly. Do not rinse the container before use. Label the sample containers immediately and
analyze promptly.
Dilute non-potable samples

Non-potable water samples must be diluted to a level at which the bacteria can be measured.
Procedure
1. Wash hands.
2. Open a bottle of sterile Buffered Dilution Water.
3. Invert the sample container in a Belt to Ear motion, approximately 25 times for 30 seconds.
4. Use a sterile pipet to add 11 mL of sample into the dilution water bottle.
5. Put the cap on the dilution water bottle and invert the sample container in a Belt to Ear motion
25 times for 30 seconds. This is a 10-fold or 10x dilution (sample is diluted by a factor of 10).
6. Add 11 mL of the 10x dilution to another dilution bottle and mix well (100x dilution).
7. Add 11 mL of the 100x dilution to a third bottle and mix well (1000x dilution).
8. Continue to make dilutions until the necessary dilution level has been reached.

Page 1245
Dispose of bacteria cultures

To safely dispose of bacterial cultures left in the broth tubes, use one of the following methods:
Bleach
Sterilize used test containers with household bleach. Add 12 mL of the bleach to each test tube.
Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave
Place used test tubes in a contaminated-items bag or a biohazard bag to prevent leakage into the
autoclave. Autoclave the used test tubes in the unsealed bag at 121 C for 30 minutes at 15
pounds pressure. When cool, seal the bag, place it in another garbage bag and tie tightly.
The use of indicator organisms in water tests

Many serious diseases, such as typhoid fever and dysentery, can be traced directly to pathogenic
microorganisms in polluted water. These disease-producing organisms are discharged in fecal
wastes and are difficult to detect in water supplies. People may come in contact with these
pathogens in drinking water or in recreational waters such as swimming pools, rivers, streams and
bathing beaches.
Testing directly for bacterial pathogens is impractical for many reasons, not the least of which is
the need for lengthy and involved test procedures. It has become customary to use indicator
organisms such as coliform bacteria instead. Indicator organisms are usually not pathogenic and
are present when pathogens are present and absent when pathogens are absent. Indicator
organisms are usually of fecal origin as well.
No one organism or group of organisms satisfies all of the criteria for an indicator. For example, in
temperate climates total coliform bacteria are commonly used as indicator organisms in potable
water supplies. In many tropical climates, however, indigenous Escherichia coli (E. coli) bacteria
are present in pristine water sources where no fecal contamination exists, yet they will produce
positive results in total coliform tests.
In such cases, other bacteria, known to be associated with fecal contamination, can be used as
indicator organisms in place of the coliforms. Hydrogen sulfide-producing bacteria have been
shown to be associated with the presence of fecal contamination and total coliform bacteria and
they may be used as an indicator organism in place of coliforms.
Total coliform tests are used for potable water supplies. Fecal coliform tests are used on untreated
(non-potable) water, wastewater, bathing water and swimming water.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Membrane Filtration Guidelines
DOC316.53.01190
The Membrane Filtration (MF) method is used to estimate bacterial populations in water that is low
in turbidity. This method is especially useful for large sample volumes or for many daily tests.
Overview
The following basic steps are necessary for a membrane filtration test.
1. Non-potable water samples are diluted.
2. The sample or dilution is filtered through a membrane filter that retains the bacteria.
3. The filter is put in a petri dish on an absorbent pad that contains a nutritional broth or agar that
is selective for the growth of a specific organism.
4. The petri dish containing the filter and pad is incubated for 24 hours at a specific temperature.
5. After incubation, the colonies that have grown are identified and counted.
Sample collection and sterilization

Refer to Bacteria Test Guidelines for instructions on sample collection and equipment sterilization.

Samples that contain a high level of bacteria must be diluted so that the bacteria that grows on the
filter is at a density that can be measured. Table 372 and Table 373 list recommended sample
volumes for various types of samples.
Select a sample size to give 20 to 200 colony-forming units (CFU) per filter. The ideal sample
volume for non-potable water or wastewater yields 2080 coliform colonies per filter. For finished,
potable water, the volume to be filtered will be 100 mL.
When the sample volume is less than 20 mL (diluted or undiluted), add 10 mL of sterile dilution
water to the filter funnel before vacuum is applied. The dilution water will help to distribute the
bacteria uniformly across the membrane filter.
Table 372 Sample volume by sample typetotal coliform test 1

Sample type
100 mL
Drinking water
10 mL
Swimming pools
Wells, springs
Lakes, reservoirs
1 mL
0.1 mL
0.01 mL
0.001 mL
0.0001 mL
Water supply intake
Bathing beaches
River water
Chlorinated sewage
Raw sewage
1
50 mL
Standard Methods for the Examination of Water and Wastewater, 19th ed., Table 9222:I, page 956.

Page 1247
Table 373 Sample volume by sample typefecal coliform test1

100 mL
50 mL
Lakes, reservoirs
Wells, springs
1 mL
0.1 mL
0.01 mL
0.001 mL
Water supply intake
Natural bathing waters
Sewage treatment plant,

secondary effluent
10 mL
Farm ponds, rivers
Storm water run-off
Raw municipal sewage
Feedlot run-off
Standard Methods for the Examination of Water and Wastewater, 19th ed., Table 9222:III, pages 961.
Sample dilution
Non-potable water samples must be diluted to a level at which the bacteria can be measured. The
ideal sample volume for total coliform testing yields approximately 20 to 80 coliform colonies and
not more than 200 colonies of all types per filter. Ideal sample volumes for fecal coliform testing
yield approximately 20 to 60 coliform colonies per filter. Analyze three different sample volumes
when the coliform number is uncertain.
Procedure
1. Wash hands.
3. Invert the sample container in a waist-to-ear motion, approximately 25 times (for 30 seconds).
5. Put the cap on the dilution water bottle. Invert the bottle in a waist-to-ear motion,
approximately 25 times (for 30 seconds). This is a 10-fold or 10x dilution (sample is diluted by
a factor of 10).
8. Continue to make dilutions until the necessary dilution level has been reached.
Dilution series
d. 10-mL sample: Transfer 11 mL of sample into 99 mL of sterile Buffered Dilution Water.
e. 1-mL sample: Transfer 11 mL of the 10-mL dilution from step d into 99 mL of sterile
Buffered Dilution Water.
f.
0.1-mL sample: Transfer 11 mL of the 1-mL dilution from step e into 99 mL of sterile
g. 0.01-mL sample: Transfer 11 mL of the 0.1-mL dilution from step f into 99 mL of sterile
h. 0.001-mL sample: Transfer 11 mL of the 0.01-mL dilution from step g into 99 mL of sterile

Page 1248

i.
0.0001-mL sample: Transfer 11 mL of the 0.001-mL dilution from step h into 99 mL of

sterile Buffered Dilution Water.
Field filtration apparatus

The field filtration apparatus consists of disposable funnels, a portable funnel base (vacuum
support) and hand pump for convenient filtration in the field. A portable incubator can be used for
incubation or for transport to the laboratory.
1. Flame sterilize the top surface of the stainless steel field vacuum support.
2. Attach the syringe tip to the vacuum support tubing.
3. Using sterile forceps, place a membrane filter, grid side up, on the center of the vacuum
support.
Note: To sterilize forceps, dip forceps in alcohol and flame in an alcohol burner. Cool before use.
4. Remove a funnel (base first) from the package.

5. Place the funnel onto the vacuum support. Do not touch the inside of the funnel. Push evenly
on the funnels upper rim to snap it on the vacuum support.
6. Pour the sample into the funnel.
7. Use the hand pump to draw the sample through the filter apparatus.
Note: See specific procedures for the sample volume required.
8. Remove the funnel.

9. Press the lever on the vacuum support stem to lift the membrane filter from the vacuum
support surface.
10. Use sterile forceps to remove the membrane filter.
11. Place the membrane filter into a prepared petri dish and incubate at the specified temperature.
12. Disconnect the syringe tip from the vacuum support tubing. Dispose of the liquid in the
syringe.
13. Follow step 2 through step 12 to filter remaining samples.
Accuracy check
Positive and negative controls are important. Pseudomonas aeruginosa is recommended as a
negative control, and a Escherichia coli is recommended as a positive control for total and fecal
coliforms. Escherichia coli is recommended as a negative control and Enterococcus faecalis is
recommended as a positive control for the enterococci. Escherichia coli is recommended as a
negative control and Pseudomonas aeruginosa is recommended as a positive control for
pseudomonas.
Note: Potable water samples from municipal treatment facilities should be negative for total coliforms and
fecal coliforms.

Page 1249

Report test results as the number of colonies per 100 mL of sample.
Single filter test
Use the following equation to calculate the result from a single membrane filter. Note that
mL sample in the equation refers to the actual sample volume and not the diluted volume.
Bacterial colonies counted
Bacterial colonies per 100 mL = ------------------------------------------------------------------------- 100
mL of sample
Indistinct coloniesIf growth covers the entire filtration area of the membrane or a portion of
it, and colonies are not discrete, report the test results as Confluent growth with or without
coliforms.
High colony densityIf the total number of colonies exceeds 200 per membrane or the
colonies are too indistinct for accurate counting, report the results as Too numerous to count
(TNTC).
In either case, run a new sample using a dilution that will give about 50 to 200 colonies of all types.
When testing non-potable water, if no filter meets the desired minimum colony count, use the
equation under Multiple filter test to calculate the test result.
Multiple filter test
Use the following equation to calculate the result from multiple membrane filters such as duplicate
samples or multiple dilutions of the same sample.
Sum of colonies in all samples
Bacterial colonies per 100 mL = ----------------------------------------------------------------------------------------------------- 100
Sum of volumes (in mL) of all samples
Prepared broth and agar

Prepared broth or agar is ready to use and is available in broth ampules or agar plates. The
ampules or agar plates contain enough medium for one test. Prepared media is shipped with a
Certificate of Analysis and has an expiration date printed on the label.
The ampules are available in glass or plastic. Open the ampule and pour the broth on the
absorbent pad in a petri dish. Open the plastic ampules by unscrewing the top of the ampule.
Open the glass ampules with an ampule breaker.
Refer to Prepared media for membrane filtration for a list of prepared media that is available for
microbiological tests.

Page 1250
m-HPC
m-Green YM
m-FC with
Rosolic Acid
m-FC
m-Endo
m-EI
m-ColiBlue24
Broth in plastic ampules
2811415
Prepared agar plate

2812450
2428320
2428350
2428550
2428520
2373250
Broth in glass ampules
2811515
2373220
Prepared agar plate
Broth in 100 mL glass

bottle
2373550
2373542
2811615
2373520
Prepared agar plate

Heterotrophic
Bacteria
Yeast and Mold
Fecal Coliform
Bacteria
Fecal Coliform
Bacteria
Total Coliform
Bacteria
48 hours at
35 0.5 C
48 hours at
35 0.5 C
24 hours at
44.5 0.2
24 hours at
44.5 0.2 C
24 hours at
35 0.5 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
Drinking water
Ground water
Surface water
Recreational water
Drinking water
Beverages
Ground water
Surface water
Recreational water
Wastewater
Drinking water
Ground water
Surface water
Recreational water
Sample
Standard Method 18th

9215 A, D
40CFR 141.21 (f) (6)

(i) and Standard
Method 18th 9221 D
40CFR 141.21 (f) (5),

9222 D and Federal
Register V 68; #139 (7/
21/2003)
Drinking water
Beverages
Ground water
Surface water
Recreational water
Beverages
Ground water
Surface water
Recreational water
Wastewater
Ground water
Surface water
Recreational water
Wastewater

Drinking water
9222 A, B and Federal
Beverages
Register V 68; #139 (7/
Ground water
21/2003)
Surface water
Recreational water
Enterococci
2811715
Prepared agar plate
Does not apply to Agar

plate
40CFR 141.21 (f) (3)

footnote (11) and
40CFR 136.3
2608450
3 months at
28 C
1 year at
28 C
1 year at
28 C
2608442
24 hours at
41 0.5 C
24 hours at
35 0.5 C
Approval Citations
Shelf life
Broth in 100-mL glass

bottle
Total Coliform
and Escherichia
coli Bacteria
48 hours at
35 0.5 C
Incubation
2805215
2608420
KF-Streptococcus
Selectivity
Enterococci
Cat. No.
2812750
Prepared agar plate
Description
Media
Table 374 Prepared media for membrane filtration

Page 1251 of 1254

Page 1252 of 1254
R2A
Pseudomonas
Nutrient Agar/MUG
m-TSB/USP
m-TGE with TTC
m-TGE
m-TEC, modified
Prepared agar plate
Agar Tubes/2 tests per

tube
Prepared agar plate

tube
Prepared agar plate
m-TEC
2812350
2814215
2724106
2812250
2812115
2437306
2812650
2428450
2428420
2373850
2373820
2811815
Cat. No.
2561106
Description
tube
Media
Stressed
Heterotrophic
Bacteria
Pseudomonas
Escherichia coli
(confirmation)
Heterotrophic
Bacteria
Heterotrophic
Bacteria
Heterotrophic
Bacteria
Escherichia coli
Selectivity
At least
72 hours at
35 0.5 C
At least
72 hours at
35 0.5 C
(7 days
maximum)
24 hours at
35 0.5 C
4 hours at
35 0.5 C
24 hours at
35 0.5 C
24 hours at
35 0.5 C
24 hours at
35 0.5 C
2 hours at
35 0.5 C
then
22 hours at
44.5 0.2 C
Incubation
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C
1 year at
28 C

9215 A, D
(applies to m-TEC and

Modified m-TEC)
Approval Citations
Federal Register V 68;
#139 (7/21/2003)
Shelf life
Table 374 Prepared media for membrane filtration
Drinking water
Beverages
Drinking water
Beverages
Ground water
Surface water
Recreational water
Drinking water
Beverages
Ground water
Surface water
Recreational water
Drinking water
Beverages
Ground water
Surface water
Recreational water
Drinking water
Beverages
Ground water
Surface water
Recreational water
Drinking water
Beverages
Ground water
Surface water
Recreational water
Recreational water
Sample
Dehydrated media
Refer to the Dehydrated media and reagents table for a list of dehydrated media that can be
prepared. The media must be measured, mixed with water and sterilized before use.
Dehydrated media and reagents
Description
Unit
Catalog number
Brain Heart Infusion Agar
500 g
2405634
Brain Heart Infusion Broth
500 g
2815534
2g
2815035
Magenta GIcA (5-bromo-6-chloro-3indoyl-beta-D-glucuronide)

M-E Agar
100 g
2281226
M-E Agar
500 g
2281234
m-TEC Agar, dehydrated
100 g
2281126
Nalidixic Acid
25 g
2407124
Oxidase Reagent
0.5 mL
2622500
Tryptic Soy Agar
100 g
2565926
Tryptic Soy Broth, ampules
50/pkg
2812650
Enrichment technique for total coliforms

Stressed coliforms require an enrichment technique, such as the one using Lauryl Tryptose (LT)
Broth Ampules described here, to get complete recovery with the MF Method. Consult your local
or state authorities about approved test methods for your application.
Procedure
1. Place a sterile absorbent pad in the lid of a sterile petri dish.
2. Saturate the pad with one LT Broth Ampule. Pour off any excess liquid.
3. Filter the sample through a membrane filter.
4. Place the membrane filter onto the saturated pad.
5. Incubate the filter without inverting the petri dish for 1.5 to 2 hours at
35 0.5 C in a relative humidity of at least 90 percent.
6. Remove the petri dish from the incubator and open it.
7. Place a sterile absorbent pad in the bottom half of the petri dish.
8. Pour the contents of one m-Endo Broth Ampule into the bottom half of the petri dish.
9. Carefully remove the filter from the lid and roll it onto the new pad to avoid trapping air
between the filter and the nutrient pad.
10. Discard the old pad (the enrichment pad saturated with LT Broth). Replace the culture dish lid.
11. Invert the culture dish and incubate at 35 0.5 C for 20 to 22 hours.
12. After incubation, use an illuminated magnifier or a 10 to 15X microscope to count the colonies
with a greenish-gold metallic sheen.

Page 1253

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
MF-Bacteria, Pre-poured agar plate methods
Bacteria
DOC316.53.01189
Membrane Filtration Method
Pre-poured Agar Plate
Scope and Application: water and wastewater
Test preparation

Prepared Agar Plates contain prepared media. The plates are ready to use without the need for further preparation and
contain enough medium for one test.
The shelf life of the prepared agar plates varies from 3 months to 1 year (see the Table of Specifications). Plates are shipped
with a Certificate of Analysis and have an expiration date printed on the label.
To sterilize forceps, dip them in alcohol and flame in an alcohol or Bunsen burner. Let the forceps cool before use.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution. Wash
hands thoroughly with soap and water.
Agar plate membrane filtration
1. Set up the membrane

filter apparatus.
With sterile forceps, place
a membrane filter, grid
side up, into the assembly.
2. Invert the sample for

30 seconds to mix. Pour
funnel. Apply vacuum and
filter the sample1. Rinse
the funnel walls with 20 to
30 mL of sterile Buffered
Dilution Water. Apply
vacuum. Repeatedly rinse
the funnel walls two more
times.
3. Turn off the vacuum

and lift off the funnel top.
Using sterile forceps,
prepared agar.
4. With a slight rolling

motion, place the filter, grid
side up, on the agar.
Check for trapped air
under the filter and make
sure the filter touches the
entire surface of the agar.
Replace the lid on the
petri dish.
Bacteria
Page 1255
Bacteria
Agar plate membrane filtration (continued)
5. Invert the petri dish

and incubate. Refer to the
Table of Specifications for
temperature and time.
1
6. Remove the petri dish

from the incubator and
count the colonies using a
10 to 15X stereoscopic
microscope.
Release vacuum once dry so that the filter does not dry out and tear.

Report the density as number of colonies per 100 mL of sample. Use samples that produce about
50 and not more than 200 colonies per membrane to compute density.
Use Equation A to calculate density. Note that mL sample refers to actual sample volume and not
the volume of the dilution.
Equation A Density on a single membrane filter.
Colonies counted
Colonies per 100 mL = ------------------------------------------------- 100
mL sample filtered
If growth covers the entire filtration area of the membrane or a portion of it, ad colonies are not
discrete, report the results as Confluent Growth.
If the total number of colonies exceeds 200 per membrane or the colonies are too indistinct for
accurate counting, report the results as Too Numerous To Count (TNTC).
In either case, a new sample must be run using a dilution that will give about 50 and not more than
200 colonies.
When testing nonpotable water, if no filter meets the desired minimum colony count, the average
density can be calculated with Equation B.
Equation B Average density for duplicates, multiple dilutions or more than one filter/
sample.
Colonies per 100 mL = ----------------------------------------------------------------------------------------------------- 100
Bacteria
Page 1256
Bacteria
Specifications
Table 375 Table of Specifications
Prepared
Agar Plate
Incubation
Sensitivity
Selectivity
Shelf Life
1 year, 28 C
m-HPC
24 hours at 35 0.5 C
1 CFU/ 100 mL
Heterotrophic Bacteria
m-Endo
1 CFU/ 100 mL
Total Coliform Bacteria
1 year, 28 C
m-FC
24 hours at 44.5 0.2 C
1 CFU/ 100 mL
Fecal Coliform Bacteria
1 year, 28 C
m-ColiBlue24
1 CFU/ 100 mL
Total Coliforms and E. coli
1 year, 28 C
m-EI
24 hours at 44.5 0.5 C
1 CFU/ 100 mL
Enterococci
3 months, 28 C
Modified m-TEC
2 hours at 35 0.5 C
then 22 hours at 44.5 C
1 CFU/ 100 mL
E. coli
1 year, 28 C
Nutrient Agar/
MUG
1 CFU/ 100 mL
E. coli (confirmation)
1 year, 28 C

Required media and reagents
Description
Unit
Catalog number
m-HPC
15/pkg
2811415
m-Endo
15/pkg
2811615
m-FC
15/pkg
2811515
Agar plates, pre-poured:
m-ColiBlue24
15/pkg
2805215
m-EI
15/pkg
2811715
m-TEC modified
15/pkg
2811815
Nutrient Agar/MUG
15/pkg
2182115
Bacteria
Page 1257
Bacteria
Required apparatus
Description
Unit
Aspirator, water
Bags, Whirl-Pak1 with dechlorinating agent, 180-mL
each
213102
100/pkg
2075333
1469600
Counter, hand tally
each
Filter Holder, magnetic coupling
each
1352900
Filter Funnel Manifold, aluminum, 3-place
each
2486100
Filtering Flask, 1000-mL

Filters, Membrane, 47-mm, 0.45-m, gridded, sterile, Pall Gelman
each
54653
200/pkg
1353001
Forceps, stainless steel
each
2141100
Incubator, Culture, 120 VAC, 50/60 Hz
each
2619200
each
2619202
Incubator, Water Bath, 120 VAC, 50/60 Hz
each
2616300
each
2616302
Microscope, compound, 10X (15X available)
each
2942500
Pipet, serological, 1011 mL, sterile, disposable
25/pkg
209798
1428300
6/pkg
211908
3.7 m (12 ft.)
55919
Pump, Vacuum, hand-operated

Tubing, rubber, 0.8 cm (5/16 in.) ID
1
Catalog number
Whirl-Pak is a registered trademark of Nasco, Inc.
Optional apparatus
Description
Unit
Catalog number
Autoclave, Automatic, 120 VAC, 50/60 Hz
each
2898600
Bottles, sample, glass, with cap, 118-mL
3/pkg
2163103
Bottles, sample, sterilized, 100-mL fill-to line, disposable
12/pkg
2495012
2495050
50/pkg
Bottles, sample, sterilized, 100-mL fill-to line, disposable with dechlorinating agent
12/pkg
2599112
50/pkg
2599150
Filter Unit, sterile, disposable with gridded membrane (use with 2656700)
12/pkg
2656600
each
2586200
Filtration Support (for field use), stainless steel

Funnels, Push-Fit and membrane filters (use with 2586200)
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
50842
Incubator, portable, 12 VDC
each
2569900
Incubator, Water Bath, with gable cover, 110 VAC, 50/60 Hz
each
2616300
Incubator, Water Bath, with gable cover, 220 VAC, 50/60 Hz
each
2616302
Magnifier, illuminated, 2.5X and 5X,
each
2585400
2585300
Magnifier, illuminated, 10X, portable
each
Marker, laboratory
each
2092000
Microscope, Compound, 10X (15X available)
each
2942500
1000/pkg
1491800
each
2613200
Pad, absorbent, with dispenser

Pen, colony counter (Felt-tip pen attached to a counter, that marks, beeps and registers
accumulative count on an LCD display)
Bacteria
Page 1258
Bacteria
Optional apparatus (continued)
Description
Unit
Catalog number
Pump, Vacuum/Pressure, 115 VAC, 60 Hz
each
2824800
Pump, Vacuum/Pressure, 230 VAC, 50 Hz
each
2824802
Syringe, 140-mL, polypropylene (use with 2586200)
each
2586100
Wicks, replacement (use with 2087760)
10/pkg
2097810
Isopropyl alcohol
500 mL
1445949
Alcohol burner
each
2087742
Battery eliminator
each
2580400
Bags, Whirl-Pak, without dechlor 207 mL
100/pkg
2233199
10/pkg
1437297
Rechargeable battery pack, for portable incubator 12V DC / 115 V AC adapter
each
2580300
230V AC Rechargeable batter pack adapter (for 2580300)
each
2595902
Sterilization Indicator,
Sterikon
15/pkg
2811115
Sterikon
100/pkg
2811199
Unit
Catalog number
100/pkg
1436369
Optional media and reagents

Description
Dechlorinating Reagent Powder Pillows
Dilution Water, buffered, sterile, 99-mL bottles
25/pkg
1430598
Peptone Powder Pillows, 1-g
30/pkg
2142964
Potassium Dihydrogen Phosphate and Magnesium Chloride Powder

Pillows for buffered dilution water (25 of each)
50/pkg
2143166
Tryptic Soy Agar
100 g
2565926
Bacteria
Page 1259

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsTotal, Fecal and E. coli, MF, m-endo, 8074
ColiformsTotal, Fecal and E. coli

DOC316.53.001224
USEPA Membrane Filtration Method1
Method 8074
m-Endo
Scope and Application: For potable water, nonpotable water, recreation water and wastewater
1
Adapted from Standard Methods for the Examination of Water and Wastewater, 9222 B and 9221 B.
Introduction
The Membrane Filtration (MF) method is a fast way to estimate bacterial populations in water. The
MF method is especially useful when evaluating large sample volumes or performing many
coliform tests daily.
Method
In the initial step, an appropriate sample volume passes through a membrane filter with a pore size
small enough (0.45 micron) to retain the bacteria present. The filter is placed on an absorbent pad
(in a petri dish) saturated with a culture medium that is selective for coliform growth. The petri dish
containing the filter and pad is incubated, upside down, for 24 hours at the appropriate
temperature. After incubation, the colonies that have grown are identified and counted using a lowpower microscope.
PourRite Ampules contain prepared selective media. This eliminates the measuring, mixing, and
autoclaving needed when preparing dehydrated media. The ampules are designed with a large,
unrestrictive opening that allows media to pour out easily. Each ampule contains enough medium
for one test.
Test preparation

When the sample is less than 20 mL (diluted or undiluted), add 10 mL of sterile dilution water to the filter funnel before
applying the vacuum. This aids in distributing the bacteria evenly across the entire filter surface.
The volume of sample to be filtered will vary with the sample type. Select a maximum sample size to give 20 to 200
colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for coliform testing yields
2080 coliform colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.
If using PourRite ampules, allow the media to warm to room temperature before opening.
Potable water must not contain any coliform bacteria. Do not dilute potable water samples.
Prepared m-Endo agar plates may be used instead of m-Endo broth.
Potable water procedures

To test potable water with the MF Method, examine a 100-mL sample for total coliforms by
incubating a filter at 35 0.5 C for 2224 hours on m-Endo Broth. Coliforms ferment lactose in the
medium and produce an acid-aldehyde complex. This complex combines with Schiffs Reagent
(also in the medium) to form an iridescent green coating over the colonies. When magnified 10 to
15 times, coliforms appear as dark red colonies with a greenish-gold sheen.
For replacement items, see m-Endo Broth for presumptive total coliforms.

Page 1261

Presumptive test for total coliforms (m-Endo), method 8074
1. Place a sterile
absorbent pad in a sterile
petri dish using sterilized
forceps. Replace the lid.
Do not touch the pad or
the inside of the petri dish.
To sterilize forceps, dip
forceps in alcohol and
flame in an alcohol or
Bunsen burner. Let
forceps cool before use.
2. Invert an m-Endo
Broth PourRite Ampule 2
to 3 times to mix the broth.
Use the ampule breaker
to break open the ampule.
Carefully pour the
contents evenly over the
absorbent pad. Replace
the petri dish lid. Repeat
steps 1 and 2 for each
petri dish being prepared.
3. Set up the Membrane

Filter Assembly. Use
sterilized forceps to place
Alternatively, a sterile
disposable filter unit may
be used.
Release the vacuum when

the filter is dry to prevent
damage to the filter.
For ease of use, petri

dishes with pads are
available.

Using sterilized forceps,
transfer the filter
immediately to the
previously prepared
petri dish.

filter the sample. Release
the vacuum. Rinse the
funnel walls with 20 to
30 mL of sterile buffered
dilution water. Apply
vacuum. Repeat rinsing
step 2 more times.

motion, center the filter,
grid side up, on the
absorbent pad. Check for
air trapped under the filter
and make sure the filter
touches the entire pad.
Replace the petri dish lid.

Page 1262

and incubate at
35 0.5 C for 2224
hours.
8. After incubating, use a

10 to 15X microscope to
count the red colonies that
have a greenish-gold
metallic sheen.
The sheen may extend
over the entire colony, or it
may be localized to the
edge or to the center.

Presumptive test for total coliforms (m-Endo), method 8074 (continued)
9. Record the results of

the test. See Interpreting
and Reporting Results
Depending on the test protocol, confirm positive results.

To confirm total coliforms, follow Confirmation of total coliforms (LT and BGB),
method 8074.
To confirm fecal coliforms, follow Confirmation of fecal coliforms (EC medium),
method 8074.
To confirm E. coli, follow Confirmation of E. coli (EC or EC/MUG), method 8074.
Confirmation of total coliforms (Lauryl Tryptose and Brilliant Green Bile)

For potable water samples, confirm typical colonies to ensure they are coliforms. (Confirm sheen
colonies, up to a maximum of five.) Inoculate parallel tubes of Lauryl Tryptose (LT) single-strength
(SS) Broth and Brilliant Green Bile (BGB) Broth by transferring growth from each colony. Growth
and gas production in both tubes verifies that the suspect organisms are coliforms. Most Probable
Number (MPN) coliform tubes are ideal for this purpose.
Use the swabbing technique for fecal coliforms or E. coli:
When determining only the presence or absence of total coliforms
When inoculating EC or EC/MUG media
Inoculate in this order:

1. EC or EC/MUG
2. LT SS Broth
3. BGB
For replacement items, see Confirmation of total coliforms (brilliant green bile broth and lauryl
tryptose broth).

Page 1263

Confirmation of total coliforms (LT and BGB), method 8074
1. Sterilize an inoculating
needle, or use a sterile,
disposable inoculating
needle.
To sterilize an inoculating
needle, heat to red hot in
an alcohol or Bunsen
burner. Let the needle cool
before use.
5. After 24 2 hours,
check the inner vials for
growth and gas bubbles.
Growth (turbidity) and gas
bubbles in both the LT and
BGB Broth tubes verify
that the colonies are
coliforms. If one or both
tubes do not show gas,
continue incubating both
tubes for an additional
24 hours
2. Touch the needle to

the coliform (sheen)
colony grown on m-Endo
plate. Transfer to a singlestrength Lauryl Tryptose
(LT) Broth tube.
3. Again touch the same

coliform colony with the
needle. Transfer to a
Brilliant Green Bile (BGB)
Broth tube.
6. If no gas is present in
the LT Broth tube after
48 hours, the colony is not
a coliform and additional
testing is unnecessary.
Confirm positive results. If

growth and gas are
produced in the LT Broth
tube but not in the BGB
Broth tube, inoculate
another BGB tube from the
gas-positive LT Broth tube.
Incubate this BGB Broth
tube and check for growth
and gas after 24 hours
and/or after 48 hours. If
growth and gas are
produced within
48 3 hours, the colony is
confirmed as coliform.
Record the results of the

test. See Interpreting and
Reporting Results

Page 1264
4. Invert both tubes to

eliminate any air bubbles
trapped in the inner vials.
Incubate the tubes at
35 0.5 C. After one
hour, invert the tubes to
remove trapped air in the
inner vial, then continue
incubation.
Confirmation of fecal coliforms (EC medium)

Analyze total-coliform-positive potable water samples for the presence of fecal coliform or E. coli.
Confirm fecal coliforms from a membrane filter positive for total coliforms by swabbing the
membrane with a sterile cotton swab and inoculating a tube of EC Medium Broth. Growth and gas
production in the EC Medium confirms the presence of fecal coliforms.
For replacement items, see Confirmation of fecal coliforms (EC medium broth).
Confirmation of fecal coliforms (EC medium), method 8074
1. Use a sterile cotton

swab or inoculating loop to
swab the entire surface of
the total coliform-positive
membrane filter (colonies
grown on m-Endo Broth).
2. Swirl the cotton swab

in an EC Medium Broth
tube to transfer the
colonies collected from the
filter. Remove the cotton
swab from the medium.
Use the same cotton swab
to transfer colonies from
the same petri dish to
other broth media if
desired.
3. Invert the tubes to

trapped in the inner vial.
Incubate the tube at
44.5 0.2 C. After one
hour, Invert the tubes to
inner vial and continue
incubation.
check the inner vial for gas
bubbles. Growth
and gas bubbles in the EC
Medium Broth tube
confirm the presence of
fecal coliforms.

and Reporting Results.

Page 1265
Confirmation of E. coli (EC or EC/MUG)

Potable water samples that test positive for total coliforms may be analyzed for the presence of
E. coli in lieu of fecal coliforms. Use either EC medium or EC with MUG broth to confirm the
presence of E. coli on a membrane filter positive for total coliforms.
For replacement items, see E. coli confirmation with EC/MUG and E. coli confirmation with nutrient
agar.
Confirmation of E. coli (EC or EC/MUG), method 8074
1. Use a sterile cotton

swab or inoculating loop to
swab the entire surface of
the membrane filter that is
positive for total coliforms
(colonies grown on mEndo Broth).
2. Swirl the cotton swab

in an EC/MUG Broth tube
to transfer the colonies
collected from the filter.
Remove the cotton swab
from the medium.
Use the same cotton swab
to transfer colonies from
the same petri dish to
other broth media if
desired.

trapped in the inner vial.
Incubate the tube at
44.5 0.2 C. After one
incubation.
use a long-wave UV lamp
to check the tube for
fluorescence. Growth and
fluorescence indicate the
presence of E. coli.
Some glass will autofluoresce. Use Hach brand
MPN tubes for best
results.
Do not look directly
through the MPN tube into
the UV lamp. View the
tube at 90 from the lamp.
Examine the tubes in a
darkened area.
Have a fluorescentpositive and a fluorescentnegative tube, both with
turbidity, to compare with
the sample tube.
Alternatively use an E. coli
presence standard.

Page 1266

Confirmation of E. coli (EC or EC/MUG), method 8074 (continued)

Confirmation of E. coli (Nutrient Agar/MUG), method 8074
1. Heat a beaker of
water, or a water bath, but
do not allow it to boil.
2. Loosen the cap on

one or more NA/MUG
nutrient agar tubes. Place
the tubes into hot water.
When the agar melts,
carefully remove the tubes
from the water with tongs.
Pre-poured agar plates
can also be used.
3. Using sterile
technique, pour half of the
contents of the tube into a
sterile 47-mm petri dish.
Immediately replace petri
dish lid and allow agar to
solidify undisturbed.
4. Use sterilized forceps

to lift the membrane filter
with total coliform colonies
off the m-Endo absorbent
pad.
Bunsen burner. Let

Page 1267

Confirmation of E. coli (Nutrient Agar/MUG), method 8074 (continued)
5. Immediately transfer
the membrane filter to the
petri dish containing NA/
MUG. With a slight rolling
grid side up, on the agar.
Check for air trapped
sure the entire filter
touches the agar. Replace
the petri dish lid.
6. Invert the petri dish.

Incubate for 4 hours at
35 0.5 C.
7. Using a long-wave UV
lamp, examine the
colonies for fluorescence.
Fluorescence indicates the
presence of E. coli.

Make sure to examine the

petri dish in a darkened
area.
Some UV lamps do not
use the correct wattage
and can give false results.
Be sure to use the UV
lamps and replacement
bulbs that are specified in
Consumables and
replacement items.
Interpreting and Reporting Results

Report coliform density as the number of colonies per 100 mL of sample. For total coliforms, use
samples that produce 20 to 80 coliform colonies, and not more than 200 colonies of all types, per
membrane to compute coliform density. For fecal coliform testing, samples should produce 20 to
60 fecal coliform colonies.
Use Equation A to calculate coliform density. Note that mL sample refers to actual sample
volume, and not volume of the dilution.
Equation AColiform density on a single membrane filter
Coliform colonies counted
Coliform colonies per 100 mL = --------------------------------------------------------------------- 100
mL of sample filtered
If growth covers the entire filtration area of the membrane, or a portion of it, and colonies are
not discrete, report results as Confluent Growth With or Without Coliforms.
If the total number of colonies (coliforms plus non-coliforms) exceeds 200 per membrane or
the colonies are too indistinct for accurate counting, report the results as Too Numerous To
Count (TNTC).
In either case, run a new sample using a dilution that will give about 50 coliform colonies and not
more than 200 colonies of all types.
When testing nonpotable water, if no filter meets the desired minimum colony count, calculate the
average coliform density with Equation B.

Page 1268

Equation BAverage coliform density for 1) duplicates, 2) multiple dilutions, or 3) more
than one filter/sample
Coliform colonies per 100 mL = ----------------------------------------------------------------------------------------------------- 100
Controls:
negative control and Escherichia coli as a positive control. Use the AQUA QC-STIK Device for
quality control procedures. Instructions for use come with each AQUA QC-STIK Device.
Potable water samples from municipal treatment facilities should be negative for total coliforms
and fecal coliforms.

m-Endo Broth for presumptive total coliforms
Description
Unit
Catalog number
m-Endo prepared agar plates
15/pkg
2811615
m-Endo Broth Ampules, plastic
50/pkg
2373550
m-Endo Broth, 100 mL glass bottle
1 each
2373542
m-Endo Broth PourRite Ampules, glass (for total coliform presumptive)
20/pkg
2373520
Dilution Water, buffered, sterile, 99 mL
25/pkg
1430598
Required apparatus
Description
Alcohol Burner
Ampule Breaker, PourRite
Counter, hand tally
Unit
Catalog number
2087742
each
2484600
1469600
Dish, Petri, with pad, 47-mm, sterile, disposable, Gelman
100/pkg
1471799
Dish, Petri, with pad, 47-mm, sterile, disposable, Millipore
150/pkg
2936300
Filter Holder, magnetic coupling (use with 24861-00)
1352900
Filters, Membrane, 47-mm, 0.45-m, gridded, sterile, Gelman
200/pkg
1353001
Filters, Membrane, 47-mm, 0.45-m, gridded, sterile, Millipore
150/pkg
2936100
54653
2141100
each
2619200
each
2619202
25/pkg
2749125
2942500
Loop, inoculating, disposable

Microscope, compound
Pump, vacuum/pressure, portable, 115 VAC, 60 Hz
each
2824800
each
2824802
6/pkg
211908
3.7 m (12 ft)
56019

Tubing, rubber, 0.8 cm (5/16 in.) ID

Page 1269
Optional media, reagents and apparatus

Description
Catalog number
2595902
Adapter for rechargeable battery pack, 230 VAC (for 2580300)
each
Aspirator, water
each
213102
Autoclave, 120 VAC, 50/60 Hz
each
2898600
Bag, for contaminated items
200/pkg
2463300
Bags, Whirl-Pak, without dechlorinating agent, 207 mL
100/pkg
2233199
10/pkg
1437297
Bags, Whirl-Pak, with dechlorinating agent, 180 mL
100/pkg
2075333
Battery eliminator
each
2580400
Battery pack, rechargeable, for portable incubator 12 VDC
each
2580300
Bottle, sample, sterilized, 100-mL, disposable with dechlorinating agent
12/pkg
2599112
50/pkg
2599150
Bottle, sample, sterilized, 100-mL, disposable
12/pkg
2495012
50/pkg
2495050
100/pkg
1436369
1485299
Dish, Petri, 47-mm, sterile, disposable
100/pkg
500/pkg
1485200
12/pkg
2656600
each
2586200

72/pkg
2586300
each
2569900
Incubator, water bath, 120 VAC, 50/60 Hz
each
2616300
each
2616302
500 mL
1445949
1000/pkg
1491800
50/pkg
2143166
each
1428300
Isopropyl alcohol
Powder Pillows for buffered dilution water (25 of
each)1
Pump, hand vacuum

Sterilization Indicator, Sterikon
15/pkg
2811115
100/pkg
2811199
each
2586100

1
Unit
Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water and
autoclave (sterilize) to prepare American Public Health Association buffered dilution water.
Confirmation of total coliforms (brilliant green bile broth and lauryl tryptose broth)
Note: Many of the confirmation products are also listed under the m-Endo presumptive products.

Description
Unit
Catalog number
Brilliant Green Bile Broth Tubes (for total coliform confirmation)
15/pkg
32215
Lauryl Tryptose Broth Tubes, single-strength (for total coliform confirmation)
15/pkg
2162315
Unit
Catalog number
2087742
Required apparatus
Description
Alcohol Burner

Page 1270

Description
Unit
Catalog number
each
2619200
25/pkg
2749125
Description
Unit
Catalog number
each
2484600
Burner, Bunsen
each
2162700
Inoculating Needle, disposable
25/pkg
2748925
Lauryl Tryptose Broth Ampules, sterile (for enrichment technique)
20/pkg
1472520
Rack, coliform tube

Wicks, replacement (used with Alcohol Burner 20877-42)
each
221500
10/pkg
2097810
Confirmation of fecal coliforms (EC medium broth)


Description
EC Medium Broth Tubes (for fecal coliform confirmation)
Unit
Catalog number
15/pkg
1410415
Unit
Catalog number
Required apparatus
Description
2141100
each
2619200
25/pkg
2748925
25/pkg
2749125
Swabs, cotton, sterile (for confirmation)
100/pkg
2554300

Description
Unit
Catalog number
2569900
each
2619202
Incubator, 12-Well Dri-Bath, 115/230 VAC, 50/60 Hz with North American style plug
each
2281400
Incubator, 12-Well Dri-Bath, 115/230 VAC, 50/60 Hz with European style plug
each
2281402
each
2616300
each
2616302
2112100
each
221500
Inoculating Loop, nichrome wire, with handle

Rack, coliform tube

Page 1271

E. coli confirmation with EC/MUG

Description
EC Medium with MUG Broth Tubes (for E. coli confirmation)
Unit
Catalog number
15/pkg
2471515
Unit
Catalog number
Required apparatus
Description
2141100
Lamp, long-wave, ultraviolet, 115 VAC, 60 Hz
2184300
Lamp, long-wave, ultraviolet, 230 VAC, 50/60 Hz

Swabs, cotton, sterile (for confirmation)
2184302
100/pkg
2554300

Description
Unit
Ecoli Fluorescence standard
each
2361100
50/pkg
2463200
2569900
Germicidal Cloths
Catalog number
each
2619200
each
2619202

25/pkg
2748925
each
2281400
Incubator, 12-Well Dri-Bath, 115/230 VAC, 50/60 Hz with European style plug
each
2281402
each
2616300
each
2616302
Inoculating Loop, nichrome wire, with handle
2112100
Lamp, long-wave, ultraviolet, portable, 4 watt
2415200
25/pkg
2749125
each
221500

Rack, coliform tube

Page 1272

E. coli confirmation with nutrient agar

Description
Unit
Catalog number
Nutrient Agar with MUG prepared plates
15/pkg
2182115
Nutrient Agar with MUG Tubes, 2 tests/tube (for E. coli confirmation)
6/pkg
2437306
Unit
Catalog number
Required apparatus
Description
100/pkg
1471799
150/pkg
2936300
200/pkg
1353001
150/pkg
2936100
2141100
each
2619200
each
2619202
2184300
2184302

Description
Beaker, 600 mL
Unit
Catalog number
50052
each
2281400
2415200

Page 1273

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsFecal, MF, m-FC and m-FC/RA 8074
ColiformsFecal
USEPA Membrane Filtration Method
DOC316.53.001209
Method 80741
m-FC and m-FC/RA
Scope and Application: For potable water, nonpotable water, recreation water and wastewater.
1
USEPA approved 9222 D.
Introduction
The Membrane Filtration (MF) method is a fast way to estimate bacterial populations in water. The
MF method is especially useful when evaluating large sample volumes or performing many
coliform tests daily.
Method
In the initial step, an appropriate sample volume passes through a membrane filter with a pore size
small enough (0.45 micron) to retain the bacteria present. The filter is placed on an agar plate
prepared with a culture medium that is selective for coliform growth. The petri dish is incubated,
upside down, for 24 hours at the appropriate temperature. After incubation, the colonies that have
grown are identified and counted using a low-power microscope.
PourRite Ampules contain prepared selective media. This eliminates the measuring, mixing, and
autoclaving needed when preparing dehydrated media. The ampules are designed with a large,
unrestrictive opening that allows media to pour out easily. Each ampule contains enough medium
for one test.
Test preparation

Nonpotable waters procedures

Wastewater, river, bathing, and other nonpotable waters usually are tested for fecal coliforms. In
testing for fecal coliforms, a special medium and an elevated incubation temperature inhibit growth
of nonfecal coliforms. Fecal coliforms growing on the membrane form an acid that reacts with an
aniline dye in the medium, producing a blue color.
Use m-FC Broth with Rosolic Acid to increase specificity when high levels of non-coliform bacteria
may be present, unless all the organisms in the sample are stressed or injured.
ColiformsFecal
Page 1275
ColiformsFecal
Confirmation of fecal coliforms (m-FC or m-FC/RA), method 8074
1. Place a sterile
forceps. Replace the petri
dish lid.
Bunsen burner. Let
Petri dishes with pads are
available.

Use sterile forceps to
transfer the membrane
filter to the previously
prepared petri dish.
2. Invert an m-FC Broth

PourRite Ampule 2 to 3
times to mix the broth. Use
the ampule breaker to
open an ampule. Carefully
pour the contents evenly
onto the absorbent pad.

Use m-FC Broth with

Rosolic Acid to increase
specificity when high
levels of non-coliform
bacteria may be present,
unless the organisms are
stressed or injured.

and make sure the entire
filter touches the pad.


and incubate at
44.5 0.2 C for
24 2 hours.
To eliminate environmental
Klebsiella from the fecal
coliform population elevate
the temperature to
45.0 0.2 C.
Alternatively, a water bath
with rack may be used for
incubation by placing the
petri dishes into a sealed
bag.
ColiformsFecal
Page 1276
4. Prepare the necessary

dilutions to obtain the
proper sample size. Invert
the sample for 30 seconds
to mix. Pour sample into
the funnel. Apply vacuum
and filter the sample.
Rinse the funnel walls with
20 to 30 mL of sterile
buffered dilution water.
Apply vacuum. Repeat
rinsing step, two more
times.
8. After incubating, count

the blue colonies using a
10 to 15X microscope.
ColiformsFecal
Confirmation of fecal coliforms (m-FC or m-FC/RA), method 8074 (continued)
9. Record the results of the test. See Interpreting and

reporting results.
To verify results, follow Verifying fecal coliforms, method
8074
Confirmation of total coliforms (Lauryl Tryptose and Brilliant Green Bile)

For potable water samples, confirm typical colonies to ensure they are coliforms. (Confirm sheen
colonies, up to a maximum of five.) Inoculate parallel tubes of Lauryl Tryptose (LT) Single Strength
(SS) Broth and Brilliant Green Bile (BGB) Broth by transferring growth from each colony. Growth
and gas production in both tubes verifies that the suspect organisms are coliforms. Most Probable
Number (MPN) coliform tubes are ideal for this purpose.
Use the swabbing technique for fecal coliforms or E. coli:
When determining only the presence or absence of total coliforms
When inoculating EC or EC/MUG media
Inoculate in this order:

1. EC or EC/MUG
2. LT SS Broth
3. BGB
ColiformsFecal
Page 1277
ColiformsFecal
Confirmation of total coliforms (LT and BGB), method 8074
needle.
burner. Let the needle cool
before use.
check the inner vials for
growth and gas bubbles.
Growth (turbidity) and gas
bubbles in both the LT and
BGB Broth tubes verify
that the colonies are
coliforms. If one or both
tubes do not show gas,
continue incubating both
tubes for an additional
24 hours
ColiformsFecal
Page 1278
2. Touch the needle to

the coliform (sheen)
colony grown on m-Endo
Broth. Transfer to a singlestrength Lauryl Tryptose
(LT) Broth tube.
3. Again touch the same

coliform colony with the
needle. Transfer to a
Broth tube.
6. If no gas is present in
the LT Broth tube after
48 hours, the colony is not
a coliform and additional
testing is unnecessary.
Confirm positive results. If

growth and gas are
produced in the LT Broth
tube but not in the BGB
Broth tube, inoculate
another BGB tube from the
gas-positive LT Broth tube.
Incubate this BGB Broth
tube and check for growth
and gas after 24 hours
and/or after 48 hours. If
growth and gas are
produced within
48 3 hours, the colony is
confirmed as coliform.

reporting results
4. Invert both tubes to

trapped in the inner vials.
35 0.5 C. After one
inner vial, then continue
incubation.
ColiformsFecal
Verifying fecal coliforms, method 8074
needle.
burner flame. Let the
needle cool before use.
2. Touch the needle to a

typical blue colony and
transfer to a Lauryl
Tryptose (LT) Broth tube.
Repeat steps 1 and 3 for
each test being verified.
Steps 3 and 4 can be
performed simultaneously
if multiple incubators are
available.

eliminate air trapped
inside the inner vials.
35 0.5 C. After one
incubation. Check tubes
for growth and gas
production at 24 hours.
If no change has occurred,
continue incubation for
another 24 hours.

eliminate air trapped
inside the inner vials.
Incubate the EC Medium
tubes at 44.5 0.2 C for
24 2 hours. After one
inner vial.
If growth and gas are not

produced in 48 3 hours,
the colony was not
coliform. If growth and gas
are produced in
48 3 hours, use a sterile
loop to inoculate one
EC Medium Broth tube
from each gas-positive LT
Broth tube.
5. Growth and gas production at 44.5 C within

24 2 hours confirms the presence of fecal coliforms.
Record the results of the test. See Interpreting and
reporting results.
ColiformsFecal
Page 1279
ColiformsFecal

Count (TNTC).
Controls:

Confirmation of fecal coliforms (m-FC or m-FC/RA)
Description
Unit
Catalog number
2811515
m-FC prepared agar plates
15/pkg
m-FC Broth Ampules, plastic
50/pkg
2373250
m-FC w/Rosolic Acid Broth Ampules, plastic
50/pkg
2428550
m-FC Broth PourRite Ampules (for fecal coliform presumptive)
20/pkg
2373220
m-FC with Rosolic Acid Broth PourRite Ampules (fecal coliform presumptive)
20/pkg
2428520
ColiformsFecal
Page 1280
ColiformsFecal
Required apparatus
Description
Unit
each
2484600
1469600
Counter, hand tally
Catalog number
100/pkg
1471799
150/pkg
2936300
1352900
Filter Funnel Manifold, aluminum, 3-place (use with 13529-00)
2486100
200/pkg
1353001
150/pkg
2936100
54653
2141100
Incubator, Culture, low profile, 110 VAC, 50/60 Hz
each
2619200
each
2619202
25/pkg
2748925
25/pkg
2749125
each
2942500

Description
Unit
Catalog number
100/pkg
2233199
each
2616300
each
2616302
Confirmation of total coliforms (brilliant green bile broth and lauryl tryptose broth)
Description
Unit
Catalog number
Brilliant Green Bile Broth Tubes (for total coliform confirmation)
15/pkg
32215
Lauryl Tryptose Broth Ampules, sterile (for enrichment technique)
20/pkg
1472520
Lauryl Tryptose Broth Tubes, single-strength (for total coliform confirmation)
15/pkg
2162315
Unit
Catalog number
2087742
Required apparatus
Description
Alcohol Burner
each
2484600
Burner, Bunsen
each
2162700
each
2619200
each
2619202
Isopropyl alcohol
500 mL
1445949
25/pkg
2749125
1000/pkg
1491800
ColiformsFecal
Page 1281
ColiformsFecal

Description
Unit
each
2595902
2087742
Alcohol Burner
Catalog number
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
100/pkg
2075333
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
100/pkg
1436369
100/pkg
1485299
500/pkg
1485200
each
2486100
12/pkg
2656600
each
2586200

72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900
each
2824800
each
2824802
6/pkg
211908
Tubing, rubber, 0.8 cm ID
3.7 m (12 ft)
56019
Sterikon
15/pkg
2811115
100/pkg
2811199
each
2586100
10/pkg
2097810

Wicks, replacement, for alcohol burner 2087742

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsE. coli, MF, m-TEC, 8367
ColiformsE. coli
DOC316.53.001210
Method 83671
m-TEC
1
USEPA accepted.
Test preparation

m-TEC Agar confirmation for presumptive E. coli test

The m-TEC method detects E. coli in recreational fresh water samples with a two step process.
First, membrane filters are incubated on m-TEC Agar for 2 hours at 35 C to resuscitate injured
organisms. The thermotolerant organisms are then selected by fermentation of lactose at an
elevated temperature of 44.5 C. The second step uses a substrate medium containing urea to
distinguish urease-negative E. coli from other thermotolerant coliforms that hydrolyze urea. Yellow
or yellow-brown urease-negative colonies are positive for E. coli.
Presumptive E. coli test (m-TEC Agar), method 8367
1. Heat a beaker of water

or water bath but do not
allow it to boil.
2. Place m-TEC Agar

tubes into hot water. When
agar melts, carefully
remove tubes from hot
water with tongs.
3. Using sterile
technique, pour half of the
contents of the tube into a
sterile, 47-mm petri dish.
Immediately replace the
petri dish lid and allow
agar to solidify
undisturbed.

Filter Assembly. Using
sterilized forceps, place a
membrane filter, grid side
up, into the assembly.
forceps into alcohol and
Bunsen burner. Let
ColiformsE. coli
Page 1283
ColiformsE. coli

Apply vacuum. Repeat the
rinsing step, two more
times.
ColiformsE. coli
Page 1284

prepared petri dish.

side up, on the agar.
the petri dish lid.

Incubate at 35 0.5 C for
2 hours and then at
44.5 0.2 C for 22 hours.
ColiformsE. coli
9. To confirm, use
sterilized forceps to
transfer the filter to a pad
saturated with at least
2 mL of urea substrate.
Preparing the urea
substrate:
10. After 15 minutes,

count yellow or yellowbrown colonies by using a
10 to 15X microscope. The
presence of yellow or
yellow-brown colonies
confirms E. coli.

and reporting results.
a. Dissolve 2.0 g of urea in

100 mL of deionized water.
b. Add 10 mg of phenol
red sodium indicator to the
urea solution.
c. Use 0.02 N sulfuric acid
to adjust the pH to
between 3 and 4. The
solution will turn yellow.
d. Store solution at 2 to
8 C. Use within one
week.

Count (TNTC).
ColiformsE. coli
Page 1285
ColiformsE. coli
Controls:
negative control and Escherichia coli as a positive control.

Description
Unit
m-TEC Agar Tubes, 2 tests/tube (for E. coli determination)
6/pkg
2561106
5g
2563922
Phenol Red Sodium Salt
Catalog number
100 mL
20342
Urea Reagent, ACS
100 g
1123726
Required apparatus
Description
Counter, hand tally
Unit
Catalog number
each
1469600
25/pkg
1430598
100/pkg
1471799
150/pkg
2936300
each
1352900

200/pkg
1353001
150/pkg
2936100
each
54653
each
2141100
each
2619200
each
2619202
Microscope, Compound
each
2942500
each
2824800
each
2824802
6/pkg
211908
3.7 m (12 ft)
56019
ColiformsE. coli
Page 1286
ColiformsE. coli

Description
Unit
each
2595902
2087742
Alcohol Burner
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
Bunsen burner with tubing

each
2162700
100/pkg
1436369
100/pkg
1485299
500/pkg
1485200
Filter Funnel Manifold, aluminum, 3-place (use with 1352900)

each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
500 mL
1445949
m-ColiBlue24 Broth, 100 mL glass bottle
1 each
2608442
1000/pkg
1491800
50/pkg
2143166
each
1428300

each)1
Pump, hand vacuum

15/pkg
2811115
100/pkg
2811199

1
Catalog number
each
2586100
2097810
ColiformsE. coli
Page 1287

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsE. coli, MF, mod m-TEC, 8367
ColiformsE. coli
DOC316.53.001211
Method 83671
modified m-TEC
1
USEPA accepted
Test preparation

Modified m-TEC prepared Agar Plates for E. coli in recreational waters

A revised single-step method uses modified m-TEC Agar to detect E. coli in recreational fresh
water samples. The modified m-TEC Agar contains an enzymatic substrate which is cleaved to
produce colonies. Red or magenta colored colonies specifically represent the presence of E. coli.
Confirmation steps are not required with this method.
ColiformsE. coli
Page 1289
ColiformsE. coli
Modified m-TEC for E. coli in recreational waters, method 8367

Filter Assembly. Refer to
Introduction to Coliforms.
place a membrane filter,
grid side up, into the
assembly.
Bunsen burner. Let

dilutions to obtain 2080
E. coli colonies on the
membranes.
Invert the sample for 30
seconds to mix.

filter to the modified mTEC prepared agar plate.
Pour the sample into the

filter the sample. Rinse the
step, two more times.

Incubate at 35 0.5 C
for 2 hours, then
at 44.5 0.2 C for
22 hours.
ColiformsE. coli
Page 1290
6. Count the number of

red or magenta colonies
by using a 10 to 15X
microscope.
7. The presence of these

colonies confirms E. coli.
reporting results.

side up, on the agar plate.
the petri dish lid.
ColiformsE. coli

Count (TNTC).
Controls:
ColiformsE. coli
Page 1291
ColiformsE. coli

Description
Unit
Catalog number
15/pkg
2811815
Description
Unit
Catalog number
Counter, hand tally
each
1469600
25/pkg
1430598
m-TEC, modified, prepared agar plates, 47-mm
Required apparatus

100/pkg
1471799
150/pkg
2936300
each
1352900

200/pkg
1353001
150/pkg
2936100
each
54653
each
2141100
each
2619200
each
2619202
each
2942500
each
2824800
each
2824802
6/pkg
211908
3.7 m (12 ft)
56019
Description
Unit
Catalog number
each
2595902
2087742
Alcohol Burner
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
each
2162700
ColiformsE. coli
Page 1292
ColiformsE. coli
Optional media, reagents and apparatus (continued)
Description
Catalog number
1436369
100/pkg
100/pkg
1485299
500/pkg
1485200

each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
Powder Pillows for buffered dilution water (25 of each)1
Pump, hand vacuum
each
2616300
500 mL
1445949
1 each
2608442
1000/pkg
1491800
50/pkg
2143166
each
1428300
15/pkg
2811115
Sterikon
100/pkg
2811199

1
Unit
each
2586100
2097810
ColiformsE. coli
Page 1293

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsTotal and E. coli, MF, m-ColiBlue, 10029
ColiformsTotal and E. coli

DOC316.53.001213
Method 100291
m-ColiBlue24
1
USEPA approved.
Test preparation

m-ColiBlue24 Broth PourRite Ampules

The m-ColiBlue24 Broth can be used to analyze drinking water, bottled water, beverages; surface,
well, and groundwater, waste water, recreational waters and process water for ultrapure, chemical
processing and pharmaceutical applications.

Page 1295

Simultaneous total coliform and E. coli screening, method 10029

to place a sterile,
petri dish. Replace the lid
on the dish.
To sterilize the forceps, dip
them in alcohol and flame
in an alcohol or Bunsen
burner. Let the forceps
cool before use.
2. Invert ampules two or

three times to mix broth.
Break open an ampule of
m-ColiBlue24 Broth using
an ampule breaker. Pour
the contents evenly over
the absorbent pad.

Page 1296

Filter Apparatus. With
sterile forceps, place a
be used.

100 mL of sample or
diluted sample into the
vacuum. Rinse again two
more times.

Simultaneous total coliform and E. coli screening, method 10029 (continued)

Using sterile forceps,
previously prepared petri
dish.

side up, on the absorbent
pad. Check for trapped air
entire pad. Replace the
petri dish lid.

and incubate at
35 0.5 C for 24 hours.
8. Remove the petri dish

from the incubator and
examine the filters for
colony growth. Colonies
are typically readily visible;
however, a microscope or
other 1015X magnifier
may be useful. Red and
blue colonies indicate total
coliforms and blue
colonies specifically
indicate E. coli.
Sometimes only the center
of a colony will show color.
Therefore, a colony with
any amount of red color
should be counted as red
and a colony with any
amount of blue should be
counted as a blue colony.
Red colonies may vary in
color intensity. Blue
colonies may appear blue
to purple. Count all the red
and blue colonies as total
coliforms. Count all the
blue to purple colonies as
E. coli.
Optional testing of red colonies

The m-ColiBlue24 Broth is formulated so that coliforms other than E. coli grow as red colonies.
The percentage of red colonies that are false positives (non-coliforms) is comparable to the
percentage of sheen colonies grown on m-Endo Broth that are false positives (non-coliforms);
therefore, confirmation is not required.
A few varieties of the non-coliform bacteria Pseudomonas, Vibrio, and Aeromonas spp. may grow
on m-ColiBlue24 Broth and form red colonies. Such bacteria can be readily distinguished from
total coliforms by the oxidase test. Pseudomonas, Vibrio, and Aeromonas spp. are oxidasepositive. Total coliforms and Escherichia coli are oxidase-negative. If your sample contains high
levels of interfering bacteria, you can perform an oxidase test to confirm which red colonies are
total coliforms.

Page 1297

Two oxidase procedures are provided. Count the red and blue colonies on the m-ColiBlue24 Broth
membrane filter before starting the oxidase test.
Oxidase method 1
This method enables you to conveniently and rapidly evaluate membrane filters that have
numerous colonies. Use this method after 24 hours of incubation on m-ColiBlue24 Broth.
Research* shows that the oxidase test cannot be performed on media that undergoes acidification
during bacterial growth. The m-ColiBlue24 Broth is formulated so that the medium does not
undergo such acidification. Consequently, many colonies can be simultaneously tested for their
oxidase reaction using the following procedure.
1. Remove the lid from the petri dish containing the m-ColiBlue24 Broth membrane filter, invert
the lid, and place it on the bench top.
Controls: Positive and negative controls are important. Pseudomonas aeruginosa is
recommended for positive controls and Escherichia coli for negative controls. Use Aqua
QC-Stiks for quality control procedures.
2. Drop approximately 0.5 mL of Difco SpotTest Oxidase Reagent into the center of the
inverted lid.
3. Using sterile forceps, transfer the membrane filter from the pad and place the filter upright in
the inverted lid.
4. Within 10 to 15 seconds, the oxidase reagent will soak into the filter and cause the oxidasepositive colonies to turn purple. This purple color may be visible in the colony itself or adjacent
to the colony. Oxidase-negative colonies will retain the red color they developed when
incubated on m-ColiBlue24 Broth.
5. After the initial 10 to 15 second reaction time, start counting the red colonies that turn purple.
Count individual colonies by using a microscope with
1015X magnification
Note: To simplify colony counting place a spare lid on the lid containing the oxidase reagent and
membrane filter. Use a felt-tip pen to mark the lid as you identify the purple colonies. After 30
seconds, you can count marks that indicate purple (oxidase-positive) colonies.
6. Stop counting 30 seconds after initial 10 to 15 second reaction time, because

oxidase-negative colonies will start to develop a purple color.
Note: Bacteria are not killed with this procedure, so colonies may be selected for streaking and for
additional testing.
Colonies that are blue after the initial 24-hour incubation on m-ColiBlue24 Broth are almost always
E. coli and do not need confirmation with the oxidase procedure.
Oxidase method 2
This method is the official oxidase test described in Standard Methods for the Examination of
Water and Wastewater, 18th edition, 1992.
1. Select red colonies from an m-ColiBlue24 Broth membrane filter and streak onto Tryptic
Soy Agar.
2. Incubate Tryptic Soy Agar plates at 35 C (95 F) for 1824 hours or until isolated colonies are
obtained.
* A.H. Havelaar et al. 1980. False-negative oxidase reaction as a result of medium acidification. Antonie van Leeuwenhoek.
46, 301-312. L.K. Hunt et al. 1981. Role of pH in oxidase variability of Aeromonas hydrophila. Journal of Clinical Microbiology.
13: 1054-1059.

Page 1298

Controls: Positive and negative controls are important. Pseudomonas aeruginosa is
recommended for positive and Escherichia coli for negative controls. Use Aqua QC-Stiks*
for quality control procedures.
3. Saturate a piece of filter paper with Difco SpotTest Oxidase Reagent. (This reagent contains a
stabilized solution of N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride.)
Note: Alternatively, oxidase reagent can be dropped directly onto colonies growing on Tryptic Soy Agar.
Oxidase-positive colonies will turn from pink to purple.
4. Using a sterile nichrome inoculating needle, transfer cellular material from an isolated Tryptic
Soy Agar colony to the moist filter paper.
Note: Do not use iron or other reactive needles for inoculation, because they will cause false-positive
results. Wooden applicator sticks work well.
5. Oxidase-negative colonies will not react with the reagent, but oxidase-positive colonies will
cause the reagent to turn dark purple within 10 seconds.
6. Oxidase-negative colonies should be counted as total coliform bacteria.

Count (TNTC).
Controls:
* Aqua QC-Stiks is a trademark of MicroBiologics.

Page 1299

Description
Unit
Catalog number
m-ColiBlue24 Broth Ampules, glass
20/pkg
2608420
m-ColiBlue24
Broth Ampules, plastic
50/pkg
2608450
m-ColiBlue24
prepared agar plates
15/pkg
2805215
Description
Unit
Catalog number
each
2484600
Required apparatus

Counter, hand tally
100/pkg
2075333
each
1469600
1430598
25/pkg
100/pkg
1471799
150/pkg
2936300
each
1352900
each
2486100
200/pkg
1353001
150/pkg
2936100
each
54653
each
2141100
each
2619200
each
2619202
each
2942500
each
2824800
each
2824802
6/pkg
211908
3.7 m (12 ft)
56019
Description
Unit
Catalog number
each
2595902
2087742
Tubing, rubber, 0.8 cm
(5/16
in.) ID
Alcohol Burner
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112

Page 1300

Description
Unit
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050

each
2162700
100/pkg
1436369
100/pkg
1485299
500/pkg
1485200

each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
Pump, hand vacuum
each
2616300
500 mL
1445949
1 each
2608442
1000/pkg
1491800
50/pkg
2143166
each
1428300
15/pkg
2811115
Sterikon
100/pkg
2811199

1
Catalog number
each
2586100
2097810

Page 1301

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Enterococci, MF, m-EI, 1600
Enterococci
DOC316.53.001212
Proposed Method 1600
M-EI
Test preparation

colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for bacteria testing yields
2080 colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.
m-EI prepared Agar Plates for Enterococci test in recreational waters

Enterococci are used as an indicator organism for recreational water quality evaluation. The m-EI
Agar specifically detects enterococci in 24 hours after enzymatic cleavage of a substrate
(indoxyl--D-glucoside) which results in formation of colonies surrounded by a blue halo.
Enterococci
Page 1303
Enterococci
m-EI for Enterococci, proposed method 1600

Filter Assembly. Using
sterilized forceps, place a
Bunsen burner. Let

Apply vacuum. Rinse
again two more times.

prepared agar plate.

side up, on the prepared
m-EI agar plate. Check for
and make sure the entire
filter touches the agar.


Incubate at 41 0.5 C for
24 hours.
6. Count any colonies

(regardless of color) with a
blue halo. Use a 10 to 15X
microscope.
7. Record these colonies

as enterococci. See
Interpreting and reporting
results.
Optional verification of Enterococci

Several media can be used to verify the presence of enterococci grown on m-EI Agar.
Gram-positive cocci, such as enterococci, will grow and hydrolyze esculin on Bile Esculin Agar,
which results in the formation of a black or brown precipitate. Enterococci can also be confirmed
by their ability to grow in Brain Heart Infusion Broth at an elevated incubation temperature of 45 C
and in Brain Heart Infusion Broth containing 6.5% NaCl.
Enterococci
Page 1304
Enterococci
Brain and heart infusion broth for optional detection
1. Use a sterile
inoculating needle to
transfer cells from the
centers of at least 10 wellisolated typical colonies
into a Brain Heart Infusion
Broth (BHIB) tube and
onto a Brain Heart Infusion
Agar (BHIA) slant.
2. Incubate broth tubes

for 24 hours and agar
slants for 48 hours at
35 0.5 C.
3. After 24 hours,
transfer a loopful of broth
from the BHIB tube to
each of the following
media and incubate for
48 hours:
4. After 48 hours
observe all media for
growth and apply a Gram
stain to the growth from
each BHIA slant.
Bile Esculin Agar (BEA)

and incubate at
35 0.5 C.
BHIB and incubate at
45 0.5 C.
BHIB with 6.5% NaCl and
incubate at 35 0.5 C.
5. Gram-positive cocci that grow and hydrolyze

esculin on BEA (i.e. produce a black or brown
precipitate) and grow in BHIB and BHIB with salt are
verified as Enterococci.
Record the results of the test. See Interpreting and
reporting results
Enterococci
Page 1305
Enterococci

Report enterococci density as the number of colonies per 100 mL of sample. Use samples that
produce 20 to 80 enterococci colonies and not more than 200 colonies of all types per membrane.
Use Equation A to calculate enterococci density. Note that mL sample refers to actual sample
volume and not volume of the dilution.
Equation AEnterococci density on a single membrane filter
Colonies counted
Colonies per 100 mL = ------------------------------------------------------- 100
If growth covers the entire filtration area of the membrane or a portion of it and colonies are not
discrete, report results as Confluent Growth With or Without Enterococci.
In either case, run a new sample using a dilution that will give about 50 enterococci colonies and
not more than 200 colonies of all types.
average enterococci density with Equation B.
Equation BAverage enterococci density for 1) duplicates, 2) multiple dilutions or 3) more
Colonies per 100 mL = ----------------------------------------------------------------------------------------------------- 100

Description
m-EI prepared agar plates, 47-mm
Enterococci
Page 1306
Unit
Catalog number
15/pkg
2811715
Enterococci
Required apparatus
Description
Counter, hand tally
Unit
Catalog number
1469600
25/pkg
1430598
100/pkg
1471799
150/pkg
2936300
1352900

200/pkg
1353001
150/pkg
2936100
54653
2141100
each
2619200
each
2619202
25/pkg
2749125
each
2942500
each
2824800
each
2824802
6/pkg
211908
3.7 m (12 ft)
56019
Description
Unit
Catalog number
each
2595902
2087742
Alcohol Burner
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
Bile Esculin Agar
500 g
2815634
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
2405634
Brain Heart Infusion Agar
500 g
Brain Heart Infusion Broth
500 g
2815534
each
2162700
100/pkg
1436369
100/pkg
1485299
500/pkg
1485200
each
2486100
12/pkg
2656600

Enterococci
Page 1307
Enterococci
Description
Catalog number
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
25/pkg
2748925
each
2569900

Incubator, 12-well Dri-Bath, 120 VAC 50/60 Hz
Isopropyl alcohol
Pump, hand vacuum
each
2281400
500 mL
1445949
1 each
2608442
1000/pkg
1491800
50/pkg
2143166
each
1428300
15/pkg
2811115
Sterikon
100/pkg
2811199

1
Unit
each
2586100
2097810

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria MF, m-TGE with TTC, 8242
DOC316.53.01195
Method 8242
m-TGE Broth with TTC Indicator
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method is a way to estimate bacterial
populations in water. Since no single medium can satisfy the growth requirements of all bacteria,
several types of media are offered for detecting heterotophic bacteria in water. The m-HPC
medium, available in both the broth and agar formats, is a high-nutrient medium used to
enumerate heterotrophs in treated potable water samples. The m-TGE broth, originally developed
for use with dairy products, is now commonly used to determine bacterial counts in water by
membrane filtration. The m-TGE broth with TTC contains a redox dye, triphenyltetrazolium
chloride, which colors the colonies red, thus enhancing their visibility. The m-TSB/USP broth is a
general purpose medium which was designed to conform with the formula specified in the
USEPAs Code of Federal Regulations (21 CFR) for sterility testing of pharmaceutical products.
Convenient packaging
Hachs m-TGE and m-TGE Broth with TTC come prepared and packaged in glass or plastic
ampules. Prepared ampules eliminate measuring, mixing and autoclaving steps necessary for
preparing dehydrated medium. Break off the top of the ampule and pour the medium onto an
absorbent pad in a petri dish.
Each ampule contains enough selective medium for one test. Packaged medium, when stored
under prescribed conditions, has a shelf-life of one year. Ampules are shipped with a Certificate of
Analysis and have an expiration date printed on the label.
If using PourRite ampules, all the media should be warm to room temperature before opening.
Page 1309
m-TGE broth with TTC indicator
1. Place a sterile,
petri dish (use sterile
forceps to do this) and
replace the lid or use
a sterile petri dish
with pad.
2. Open an ampule of
m-TGE with TTC Indicator
and carefully pour the
absorbent pad.

sterile forceps to place a
membrane filter in the
assembly, making sure the
grid side is up.
Alternatively, a sterile,
be used.
Be careful not to touch the

pad or the inside of the
petri dish.
For ease of use,
petri dishes with pads
are available.

Remove the membrane
filter, using sterile forceps.
Still using the forceps,
transfer the filter
immediately to the
previously prepared
petri dish.
1

absorbent pad. Be sure air
is not trapped under the
filter and that the filter
touches the pad at all
points. Replace the petri
dish lid.
Release the vacuum once dry so that the filter does not dry out or tear.
Page 1310
7. Label the petri dish

with the sample number,
dilution and date. Invert
the petri dish and incubate
at 35 0.5 C for 24 hours.
4. Mix the water sample

by inverting for 30
seconds. Filter the
appropriate volume
through the sterile 47-mm,
0.45-m, gridded
membrane filter, under
partial vacuum1. Rinse the
funnel, using 20 to 30 mL
portions of sterile buffered
vacuum. Rinse the funnel
again for two more times.
8. Remove the dish from

the incubator. Count
colonies on membrane
filters using a 10 to 15X
microscope.
Colonies grown with mTGE with TTC Indicator
appear pink to red aid
visibility.

Optimal colony density per filter is 20 to 200. Report all colonies counted as colony forming-units
(CFU)/mL. Include in the report the method used, the incubation temperature and time and the
medium. For example: 98 CFU/mL, membrane filter method, 35 C, 24 hours, m-TGE Broth.
Follow these counting and recording guidelines, based on the number of colonies (on the average)
per square:
1 to 2 or fewer colonies per square:
Count all of the colonies on the filter and divide the results by the volume of original sample used.
Example: if there are 122 colonies on the filter and the volume of original sample used was 10 mL,
compute results as follows:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
3 to 10 colonies per squareCount all colonies in 10 representative squares and divide by 10 to

obtain an average number of colonies per square. Multiply this number by 100 and divide by the
volume of original sample used. Example: if you calculated an average of 8 colonies per square
and the volume of original sample used was 0.1 mL, compute results as follows:
8 colonies/square x 100
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample

volume of original sample used. Example: if there are an average of 17 colonies per square and
the volume of original sample used was 0.1 mL, compute results
colonies/square 100----------------------------------------------------------------as follows: 17
= 17,000 CFU/mL
0.1 mL sample
More than 20 colonies per squareIf there are more than 20 colonies per square, record the
count as >2000 divided by the volume of original sample used.
ExampleIf the original sample volume was 0.01 mL, results would be >2000/0.01 or
>200000 CFU/mL.
Report averaged counts as estimated CFU/mL. Make estimated counts only when there are
discrete, separated colonies without spreaders.
Summary of method
In the initial step, an appropriate sample volume is passed through a membrane filter with a
pore size small enough (0.45 microns) to retain the bacteria present. The filter is placed either
on an absorbent pad (in a petri dish) saturated with a culture medium or on an agar medium
that is selective for heterotrophic bacteria growth. The petri dish containing the filter and pad is
incubated, upside down, for 24 to 48 hours, depending on the medium used, at the
appropriate temperature. After incubation, the colonies which have developed are identified
and counted by using a low-power microscope. The MF method is especially useful for testing
drinking water because large volumes of sample can be analyzed in a short time.
Page 1311

Description
Unit
Catalog number
m-TGE Broth with TTC PourRite Ampules
20/pkg
2428420
Dilution Water, Buffered, sterile, 99 mL1
25/pkg
1430598
Description
Unit
Catalog number
Buffered Dilution Water is prepared with magnesium chloride and potassium dihydrogen phosphate
Required apparatus
Alcohol Burner
each
2087742
each
2484600
Aspirator
each
213102
100/pkg
2075333
each
1469600
Bags, Whirl-Pak with dechlorinating reagent, sterile, 180-mL

Counter, hand tally
100/pkg
1471799
each
1352900
each
2486100
each
54653
each
2141100
50/pkg
2463200
Filter Holder, magnetic coupling (use with 2486100)
Germicidal Cloths
each
50842
each
2619200
each
2619202
each
2616300

Membrane Filters, 0.45-m, gridded, sterile, Gelman
each
2616302
200/pkg
1353001
Microscope Compound
each
2942500
each
1428300
Stopper, Rubber, one hole, No. 8
6/pkg
211908
Tubing, Rubber, 0.8 cm (5/16") ID, 3.7 m (12 ft.)
each
56019
Petri dish with pad, 47-mm, Millipore
150/pkg
2936300
Millipore membrane Filters, 0.45-m, gridded, sterile
150/pkg
2936100
Isopropyl alcohol
500 mL
1445949
1000/pkg
1491800
Pump, Vacuum/Pressure, Portable, 115 V 60 Hz
each
2824800
each
2824802
Page 1312

Description
Unit
Catalog number
100/pkg
1436369
Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for buffered
dilution water (25 of each)1
50/pkg
2143166
Sodium Thiosulfate, ACS grade
454 g
46001

Description
Unit
Aspirator, water
each
Catalog number
213102
Replacement wicks for 2087742
each
2097810
15/pkg
2811115
100/pkg
2811199
100/pkg
2233199
10/pkg
1437297
each
2898600

12/pkg
2495012
50/pkg
2495050
12/pkg
2599112
50/pkg
2599150
each
2162700
each
2569900
12/pkg
2656600
each
2586200
72/pkg
2586300
Petri dish, 47 mm, sterile, disposable
500/pkg
1485200
100/pkg
1485299
each
2586100

Rechargeable battery pack, for portable incubator 12 V DC / 115 V AC adapter
each
2580300
230 V AC Rechargeable batter pack adapter (for 2580300)
each
2595902
Battery eliminator
each
2580400
Page 1313

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria MF, m-TGE Broth, 8242
DOC316.53.01194
Method 8242
m-TGE Broth
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method is a fast, simple way to estimate
bacterial populations in water. Since no single medium can satisfy the growth requirements of all
bacteria, several types of media are offered for detecting heterotophic bacteria in water. The mHPC medium, available in both the broth and agar formats, is a high-nutrient medium used to
preparing dehydrated medium. Simply break off the top of the ampule and pour the medium onto
an absorbent pad in a petri dish.
If using PourRite ampules, all the media should be warm to room temperature before opening.
Page 1315
m-TGE broth
1. Place a sterile,
with pad.
m-HPC and carefully pour
the absorbent pad.

grid side is up.
be used.

petri dish.
For ease of use,
are available.

by inverting for 30
seconds. Filter the
appropriate volume
0.45-m, gridded
Alternatively, a prepoured
m-HPC agar plate may be
used.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
1

dish lid.
Release the vacuum once dry so that the filter does not dry out or tear.
Page 1316


the incubator. Count the
clear to cream colored
microscope.

per square:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample

--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample

= 17,000 CFU/mL
0.1 mL sample
>200000 CFU/mL.
Summary of method
Page 1317

Description
Unit
Catalog number
m-TGE Broth PourRite Ampules
50/pkg
2373850
25/pkg
1430598
Description
Unit
Catalog number
Alcohol Burner
each
2087742
each
2484600
100/pkg
2075333
Required apparatus

Counter, hand tally
each
1469600
100/pkg
1471799
each
1352900
each
2486100
each
54653
each
2141100
50/pkg
2463200
Germicidal Cloths
each
50842
each
2619200
2619202
each
each
2616200
each
2616202
Gelman Membrane Filters, 0.45-m, gridded, sterile

Pipets, serological, 1011 mL, sterile, disposable
200/pkg
1353001
each
2942500
25/pkg
209798
each
1428300
6/pkg
211908
each
56019
150/pkg
2936300
150/pkg
2936100
Isopropyl alcohol
500 mL
1445949
1000/pkg
1491800
each
2824800
each
2824802
Page 1318

Description
Unit
Aspirator, water
each
213102
each
2097810
100/pkg
1436369
Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for

buffered dilution water (25 of each)1
50/pkg
2143166
15/pkg
2811115
100/pkg
2811199
100/pkg
2233199
10/pkg
1437297
each
2898600

12/pkg
2495012
50/pkg
2495050
12/pkg
2599112
50/pkg
2599150
each
2569900

12/pkg
2656600
each
2586200
72/pkg
2586300
500/pkg
1485200
100/pkg
1485299
Catalog number
each
2586100
each
2580300
each
2595902
Battery eliminator
each
2580400
Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water. Autoclave
(sterilize) to prepare the American Public Health Association buffered dilution water.
Page 1319

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria MF, m-HPC, 8242
DOC316.53.01192
Method 8242
m-HPC, m-TSP-USP, m-TGE Broth or

m-TGE Broth with TTC Indicator
m-HPC
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method* is a fast, simple way to estimate
enumerate heterotrophs in treated potable water samples. The m-TGE broth originally developed
preparing dehydrated medium. Simply break off the top of the ampule and pour the medium onto
an absorbent pad in a petri dish.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution.
Wash hands thoroughly with soap and water.
* Method 8242
Page 1321
m-HPC media
1. Place a sterile,
with pad.
m-HPC and carefully pour
the absorbent pad.

grid side is up.
be used.

petri dish.
For ease of use,
are available.

by inverting for 30
seconds. Filter the
appropriate volume
0.45-m, gridded
two more times.
Alternatively, a prepoured
used.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
1

dish lid.
Release vacuum once dry so that the filter does not dry out and tear.
Page 1322


colonies on the membrane
microscope.

per square:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample

--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample

= 17,000 CFU/mL
0.1 mL sample
>200000 CFU/mL.
Summary of method
Page 1323

Description
Unit
Catalog number
25/pkg
1430598
m-HPC Agar Plates
15/pkg
2811415
m-HPC Broth Ampules, plastic, 2-mL
50/pkg
2812450
Unit
Catalog number
each
2087742
Buffered Dilution Water is prepared with magnesium chloride and potassium dihydrogen phosphate.
Required apparatus
Description
Alcohol Burner
Counter, hand tally
100/pkg
2075333
each
1469600
100/pkg
1471799
each
1352900
each
2486100
each
54653
each
2141100
50/pkg
2463200
Germicidal Cloths
each
50842
each
2619200
each
2619202
each
2616300

Gelman, Membrane Filters, 0.45-m, gridded, sterile
each
2616302
200/pkg
1353001
each
2942500
25/pkg
209798
each
1428300
6/pkg
211908
each
56019
150/pkg
2936300
150/pkg
2936100
Pump, Vacuum/Pressure, Portable, 115v, 60 Hz
each
2824800
Pump, Vacuum/Pressure, Portable, 220v, 50 Hz
each
2824802
Page 1324

Description
Catalog number
100/pkg
1436369
Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for buffered
dilution water (25 of each)1
50/pkg
2143166
Sodium Thiosulfate, ACS grade
454 g
46001
Aspirator, water 1445949 - Isopropyl alcohol, 500 mL
each
213102
Sterilization Indicator, Sterikon,
15/pkg
2811115
100/pkg
2811199
100/pkg
2233199
10/pkg
1437297
each
2898600

12/pkg
2495012
50/pkg
2495050
12/pkg
2599112
50/pkg
2599150
each
2569900
12/pkg
2656600

Unit
each
2586200
72/pkg
2586300
500/pkg
1485200
100/pkg
1485299
each
2586100
each
2580300
each
2595902
Battery eliminator
each
2580400
1000/pkg
1491800
each
2097810
Page 1325

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria MF, m-TSB-USP, 8242
DOC316.53.01193
Method 8242
m-TSB-USP
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method is a fast, simple way to estimate
preparing dehydrated medium. Break off the top of the ampule and pour the medium on an
absorbent pad in a petri dish.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution. .
Page 1327
m-TSB-USP media
1. Place a sterile,
with pad.
m-TSB-USP and carefully
pour the contents evenly
over the absorbent pad.

grid side is up.
be used.

petri dish.
For ease of use,
are available.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
1

dish lid.
Release vacuum once dry so that the filter does not dry out and tear
Page 1328


by inverting for 30
seconds. Filter the
appropriate volume
0.45-m, gridded

microscope.

per square:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample

--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample

= 17,000 CFU/mL
0.1 mL sample
>200000 CFU/mL.
Summary of method
Page 1329

Description
Unit
Catalog number
m-TSB/USP Broth Ampules, plastic, 2-mL
50/pkg
2812650
25/pkg
1430598
Unit
Catalog number
each
2087742
Required apparatus
Description
Alcohol Burner
Counter, hand tally
100/pkg
2075333
each
1469600
100/pkg
1471799
each
1352900
each
2486100
each
54653
each
2141100
50/pkg
2463200
Germicidal Cloths
each
50842
each
2619200
each
2619202
each
2616300

Gelman Membrane Filters, 0.45-m, gridded, sterile
Microscope Compound
each
2616302
200/pkg
1353001
each
2942500
25/pkg
209798
each
1428300
6/pkg
211908
each
56019
150/pkg
2936300
150/pkg
2936100
Isopropyl alcohol
500 mL
1445949
1000/pkg
1491800
each
2824800
each
2824802
Page 1330

Description
Unit
Aspirator, water
each
213102
each
2097810
100/pkg
1436369
Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for

buffered dilution water (25 of each)1
50/pkg
2143166
15/pkg
2811115
100/pkg
2811199
100/pkg
2233199
10/pkg
1437297
each
2898600

12/pkg
2495012
50/pkg
2495050
12/pkg
2599112
50/pkg
2599150
each
2569900

12/pkg
2656600
each
2586200
72/pkg
2586300
500/pkg
1485200
100/pkg
1485299
Catalog number
each
2586100
each
2580300
each
2595902
Battery eliminator
each
2580400
Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water. Autoclave
(sterilize) to prepare the American Public Health Association buffered dilution water.
Page 1331

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Pseudomonas, MF, 8026
Pseudomonas
DOC316.53.001214
Methods 8026
Pseudomonas Broth
Test preparation

colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for bacteria testing yields
2080 colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.
Pseudomonas Broth for detection of Pseudomonas species

Some species of pseudomonads are opportunistic pathogens that can inhabit recreational waters
such as swimming pools and hot tubs. Pseudomonas Broth is formulated to isolate species of
pseudomonas. Pseudomonas Broth contains components that promote the growth of the pigment
pyocyanin, which differentiates Pseudomonas aeruginosa from other species by forming a blue or
blue-green color. Other species of pseudomonas grow on this medium without the colony
color formation.
Pseudomonas
Page 1333
Pseudomonas
Pseudomonas broth, method 8026
1. Place a sterile
forceps. Replace the petri
dish lid.
Bunsen burner. Let

transfer the filter
immediately to the
previously prepared
petri dish.
Pseudomonas
Page 1334
2. Invert an ampule
containing Pseudomonas
Broth 2 to 3 times to mix.
Twist the cap to break it
open. Carefully pour the
contents evenly onto the
the petri dish lid. Repeat
steps 1 and 2 for each
petri dish.

be used.

vacuum. Rinse again two
more times.

and make sure that the
filter touches the entire
pad. Replace the petri dish
lid.

and incubate at
35 0.5 C for 2224
hours.
8. After incubating, count

the colonies using an
illuminated magnifier or a
10 to 15X microscope.
Pseudomonas
Pseudomonas broth, method 8026 (continued)
9. Colonies present on
the filter after incubation
are pseudomonas
species. Colonies with a
blue-green, green or
yellow-green color are
Pseudomonas
aeruginosa.
reporting results.

Report pseudomonas density as the number of colonies per 100 mL of sample. Use samples that
produce 20 to 80 pseudomonas colonies and not more than 200 colonies of all types, per
membrane to compute pseudomonas density.
Use Equation A to calculate pseudomonas density. Note that mL sample refers to actual sample
volume and not volume of the dilution.
Equation APseudomonas density on a single membrane filter
If growth covers the entire filtration area of the membrane or a portion of it and colonies are not
discrete, report results as Confluent Growth With or Without Pseudomonas.
In either case, run a new sample using a dilution that will give about 50 pseudomonas colonies
and not more than 200 colonies of all types.
average pseudomonas density with Equation B.
Equation BAverage pseudomonas density for 1) duplicates, 2) multiple dilutions or 3)
more than one filter/sample
Colonies per 100 mL = ----------------------------------------------------------------------------------------------------- 100
Pseudomonas
Page 1335
Pseudomonas

Description
Unit
Catalog number
Pseudomonas Broth Ampules, plastic
50/pkg
2812250
25/pkg
1430598
Unit
Catalog number
1469600
Required apparatus
Description
Counter, hand tally
100/pkg
1471799
150/pkg
2936300

Filters, Membrane, 47-mm, 0.45-m, gridded, sterile,
Gelman
each
1352900
200/pkg
1353001
150/pkg
2936100
each
54653
each
2141100
2619200
each
each
2619202
each
2942500
Pump, hand vacuum
each
1428300
each
2824800
each
2824802
6/pkg
211908
3.7 m (12 ft)
56019

Description
Unit
each
2595902
2087742
Alcohol Burner
Catalog number
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
100/pkg
2075333
Battery eliminator
each
2580400
each
2580300

Bags,
Whirl-Pak,
with dechlorinating agent, 180 mL
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
each
2162700
Pseudomonas
Page 1336
Pseudomonas
Description
Unit
Catalog number
100/pkg
1436369
100/pkg
1485299
500/pkg
1485200
each
2486100
12/pkg
2656600
each
2586200

72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
1445949
1000/pkg
1491800
2143166
50/pkg
15/pkg
2811115
100/pkg
2811199

1
500 mL
each
2586100
2097810
Pseudomonas
Page 1337

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
MPN Dilution Guidelines
DOC316.53.01231
Non-potable water samples must be diluted so that a MPN test will have three consecutive
dilutions that contain both positive and negative tubes.
If all of the tubes in a MPN test are positive, the sample must be diluted several more times and
the test must be repeated. If all of the tubes in a MPN test are negative, the sample was diluted too
many times. Repeat the test with less serial dilutions.
Sterile buffered dilution water

Use dilution water that is buffered to a neutral pH and sterilized for microbiological testing. Hach
dilution water is recommended for dilution of most non-potable and wastewater samples. This
solution is packaged in 99 mL bottles. Each bottle contains 99 mL of sterile buffered dilution water.
When 11 mL of sample is added to a 99-mL bottle of dilution water, the sample is diluted by a
factor of 10 (10-fold or 10x dilution). Be sure to mix the bottles thoroughly before and after the
sample is added. The dilution factor of an undiluted sample is 1.
Serial sample dilutions

Complete the following procedure to make serial dilutions of the sample. Refer to Serial dilutions
for non-potable water, MPN test for an illustrated dilution procedure.
Procedure
1. Wash hands.
3. Invert the sample container vigorously for 30 seconds, approximately 25 times, using a waistto-ear range of motion.
5. Put the cap on the dilution water bottle and invert (for 30 seconds) 25 times. This is a 10-fold
or 10x dilution (sample is diluted by a factor of 10).
8. Continue to make dilutions until there are three bottles that contain the dilutions listed under
Serial dilutions for non-potable water, MPN test.
Note: Shaking the sample too vigorously will injure or stress the organisms.

Page 1339
Serial dilutions for non-potable water, MPN test

Swimming pool water, chlorinatedLowest dilution factor = 1
A
Undiluted
Undiluted
sample
B
Dilution of 10x
pipet 11 mL
Inoculate 5 tubes
99 mL
dilution
water
C
Dilution of 100x
pipet 11 mL
Inoculate 5 tubes
99 mL
dilution
water
Inoculate 5 tubes
Bathing beach water; lake water, unpolluted river waterLowest dilution factor = 10
B
Dilution of 10x
Prepared
as B
above
C
Dilution of 100x
pipet 11 mL
Inoculate 5 tubes
99 mL
dilution
water
D
Dilution of 1000x
pipet 11 mL
Inoculate 5 tubes
99 mL
dilution
water
Inoculate 5 tubes
Final effluent, chlorinatedLowest dilution factor = 100

C
Dilution of 100x
Prepared
as C
above
Inoculate 5 tubes

Page 1340
D
Dilution of 1000x
pipet 11 mL
99 mL
dilution
water
Inoculate 5 tubes
E
Dilution of 10,000x
pipet 11 mL
99 mL
dilution
water
Inoculate 5 tubes

Serial dilutions for non-potable water, MPN test (continued)
River water, pollutedLowest dilution factor = 1,000
D
Dilution of 1000x
Prepared
as D
above
E
Dilution of 10,000x
pipet 11 mL
Inoculate 5 tubes
99 mL
dilution
water
F
Dilution of 100,000x
99 mL
dilution
water
pipet 11 mL
Inoculate 5 tubes
Inoculate 5 tubes
Storm water, unchlorinated final effluentLowest dilution factor = 10,000

E
Dilution of 10,000x
F
Prepared
as E
above
99 mL
dilution
water
pipet 11 mL
Inoculate 5 tubes
G
Dilution of 1,000,000x
pipet 11 mL
Inoculate 5 tubes
99 mL
dilution
water
Inoculate 5 tubes
Raw sewageLowest dilution factor = 100,000

F
G
H
Prepared
as F
above
99 mL
dilution
water
99 mL
dilution
water
Inoculate 5 tubes
pipet 11 mL
Inoculate 5 tubes
pipet 11 mL
Inoculate 5 tubes

Page 1341

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Coliforms-Fecal, MPN, method 8368
ColiformsFecal
DOC316.53.01215
USEPA1 A-1 Medium
Method 8368
Most Probable Number (MPN) Method
Scope and Application: For non-potable water and wastewater.

1
Most Probable Number Method 8368, A-1 Medium, is USEPA-accepted for testing non-potable waters. Method 8368 meets or exceeds the
specification criteria stated in Standard Methods for the Examination of Water and Wastewater, 18th edition, 9221 E. Fecal Coliform
Procedure. USEPA Manual for the Certification of Laboratories Analyzing Drinking Water states 5.5.3. A-1 medium may be used as an
alternative to EC Medium to enumerate fecal coliforms in source water, in accordance with the Surface Water Treatment Rule. A-1 Medium
must not be used for drinking water samples.

Make sure that all materials used for containing or transferring samples are sterile.
The bottles of dilution water contain 99 mL of sterile buffered dilution water. When 11 mL of sample is added to a 99-mL
bottle of dilution water, the sample is diluted by a factor of 10 (10-fold or 10x dilution). Be sure to mix the bottles thoroughly
before and after the sample is added.
Refer to Sample Dilution in the Introduction to Bacteria section to find the number of dilutions that must be made for the
sample type. For example if Class A sludge, add a 100X sample dilution to five tubes, a 1000X sample dilution to five tubes
and a 10,000X sample dilution to five tubes. If the coliform density is not known, add five different dilutions to 5 sets of MPN
tubes.
If all the tubes are positive, dilute the sample several more times and repeat the test. If all tubes are negative, the sample
was diluted too many times. Repeat the test with less serial dilutions.
If more than three dilutions were made, use three consecutive dilutions that contain both positive and negative tubes.
The dilution factor for an undiluted sample is 1.
No confirmation is needed when using A-1 Medium.

Description
A-1 Medium Broth Tubes
Dilution Water, buffered, 99-mL, sterile
Quantity
15
3 bottles
Incubator
Pipet, serological, 10-11 mL, sterile
Pipet filler
Coliform tube rack
ColiformsFecal
Page 1343
ColiformsFecal
Fecal Coliforms, A-1 Medium Broth, method 8368
1. Wash thoroughly with

soap and water.
seconds, approximately 25
times, to make sure it is
well-mixed.
2. Using sterile buffered

dilution water, prepare at
least 3 serial dilutions of
the sample.
Refer to Sample dilution
for instructions.

15 tubes of A-1 Medium
Broth. Use a sterile pipet
to transfer 10 mL of the
first dilution into 5 tubes.
Use a sterile pipet to
transfer 10 mL of the
second dilution into
5 tubes. Use a sterile pipet
to transfer 10 mL of the
third dilution into the
remaining 5 tubes.
4. Replace the screw cap

on each tube immediately
after the sample is added.
Invert and swirl the tube
several times to
thoroughly mix the sample
with the nutrient medium.
After the last inversion,
make sure that the inner
vial is full of liquid with no
air bubbles.
Do not touch the open end

of the tubes or the inside
of the caps.
5. Place the tubes in the

incubator at a temperature
of 35 0.5 C .
Any bubbles that form in
the inner vials during this
period are not from
bacteria. Be sure to
remove the bubbles by
inverting the tubes. Make
sure there are no bubbles
and then carefully return
the tubes to an upright
position. Return the tubes
to the incubator.
ColiformsFecal
Page 1344
6. After 3 hours, invert

the tubes to remove
trapped air in the inner
vials. Loosen the caps
slightly before returning
the tubes to the incubator.
Increase the temperature
to 44.5 0.2 C and
incubate for an additional
21 hours. The tubes must
be kept upright for the rest
of the test.
7. After 24 2 hours, tap

each tube gently and
examine the inner vials for
gas. If the inner vials
contain gas bubbles, the
test is positive for fecal
coliform bacteria. Count
the number of tubes that
contain gas.
If none of the tubes
contain gas, the test is
negative for fecal coliform
bacteria.
8. Find the MPN index

per 100 mL from the MPN
index table.
Multiply the MPN index by
the dilution factor in the
first set of tubes (lowest
dilution used in the test).
(MPN index x lowest
dilution factor = fecal
coliform bacteria per 100
mL sample)
See the Example
calculation.
ColiformsFecal
Sample dilution
Complete the following procedure to make serial dilutions of the sample. Refer to the Dilution
guidelines by sample type to find the number of times the sample must be diluted. Use the three
dilutions from the Dilution guidelines by sample type for the test (step 3 of the fecal coliform
procedure).
Procedure
1. Wash hands.
3. Invert the sample container for 30 seconds, approximately 25 times.
5. Put the cap on the dilution water bottle and invert the sample (for 30 seconds) 25 times. This is
a 10-fold or 10x dilution (sample is diluted by a factor of 10).
8. Continue to make dilutions until there are three bottles that contain the dilutions listed in the
Dilution guidelines by sample type.
Table 376 Dilution guidelines by sample type

Sample type
Dilution 1
Dilution 2
undiluted (1x)
10x
100x
Bathing beach water
10x
100x
1000x
Lake water
10x
100x
1000x
Unpolluted river water
10x
100x
1000x
Final effluent, chlorinated
100x
1000x
10,000x
Swimming pool water, chlorinated
River water, polluted

Storm water
Dilution 3
1000x
10,000x
100,000x
10,000x
100,000x
1,000,000x
Unchlorinated final effluent
10,000x
100,000x
1,000,000x
Raw sewage
100,000x
1,000,000x
10,000,000x
Example calculation
A sample was diluted into 3 different buffered dilution bottles. The dilutions were 10-fold (10x),
100-fold (100x) and 1000-fold (1000x). 5 MPN tubes were filled from each dilution (15 tubes total).
The first set of tubes (10x) had four tubes with gas, the second set (100x) had 2 tubes with gas
and the third set (1000x) had 1 tube with gas.
1. Find the MPN index for the three sets of tubes from the MPN index table.
2. Multiply the MPN index by the lowest dilution factor.
The MPN index from the MPN index table table for 4, 2 and 1 positive tubes is 26. The coliform
result for the sample is 26 x 10 = 260 coliforms per 100 mL of sample.
ColiformsFecal
Page 1345
ColiformsFecal
Table 377 MPN index table

Number of positive tubes
First dilution
set
Second
dilution set
Third
dilution set
MPN
index per
100 mL
First dilution
set
Second
dilution set
Third dilution
set
MPN
index per
100 mL
<2
26
27
33
34
23
30
40
30
50
60
50
70
90
80
12
110
140
11
170
11
130
14
170
14
220
17
280
13
350
17
240
17
300
21
500
26
900
22
1600
1600
Collect at least 100 mL of sample in sterilized Whirl-Pak bags, sterilized disposable bottles or
autoclaved glass or plastic bottles.
Do not fill sample containers completely. Leave at least 2.5 cm (approximately 1 inch) of air
space to allow adequate space for mixing the sample prior to analysis.
Make sure that the samples are representative of the sample source. Fill sample containers
from a tank or reservoir entirely under water.
Start the analysis as soon as possible after collection. Allow no more than 6 hours to elapse
after collection. If the test cannot be started immediately, cool the sample to below 10 C. Do
not freeze. Failure to properly collect and transport samples will cause inaccurate results.
ColiformsFecal
Page 1346
ColiformsFecal
Controls
quality control procedures. Instructions for use come with each AQUA QC-STIK Device. Potable
water samples from municipal treatment facilities should be negative for total and fecal coliforms.
Bacteria disposal
Bleach
Sterilize used test containers with household bleach. Add 12 mL of the bleach to each test
container. Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave
Place used test containers in a contaminated-items bag or a biohazard bag to prevent leakage into
the autoclave. Autoclave the used test containers in the unsealed bag at 121 C for 30 minutes at
15 pounds pressure. When cool, seal the bag, place it in another garbage bag and tie tightly.
Summary of method
The Most Probable Number (MPN) method (also referred to as the Multiple Tube Fermentation
Technique) uses screw-capped tubes containing sterile broth medium. The tubes contain an
inverted inner vial (Durham tube) for gas collection. Sample is diluted, added to the tubes and
incubated. If coliforms are present, gas is produced and is trapped in the inner vial.
The number of tubes that form gas is used to estimate the number of coliform organisms in the
sample. The MPN method allows for the analysis of highly turbid samples by dilution prior to
analysis. No filtering is necessary.

Description
Unit
Catalog number
A-1 Medium Broth Tubes
15/pkg
2560915
Dilution Water, buffered, 99-mL, sterile1
25/pkg
1430598
Required apparatus
Description
Bags,
Whirl-Pak,
without dechlorinating agent, 207-mL
Unit
Catalog number
100/pkg
2233199
Incubator, 12-well Dri-Bath, 120 VAC, 50/60 Hz
each
2281400
each
2619200
Pipet, serological, 10-11 mL, sterile, disposable

Pipet safety bulb
25/pkg
209798
each
1465100
ColiformsFecal
Page 1347
ColiformsFecal

Description
1
each)1
Unit
Catalog number
50/pkg
2143166
Optional apparatus
Description
Unit
Catalog number
each
2595902
Bags for contaminated items
200/pkg
2463300
Bags, Whirl-Pak, with dechlorinating agent, 180-mL
100/pkg
2075333
Bags, Whirl-Pak, without dechlorinating agent, 207-mL
500/pkg
2233100
Battery eliminator
each
2580400
each
2580300
Bottle, polysulfone, autoclavable (use for buffered dilution water)
12/pkg
2245300
Bottle, sample, sterilized, 100-mL fill-to line, disposable
12/pkg
2495012
50/pkg
2495050
Bottle, sample, sterilized, 100-mL fill-to line, disposable with dechlorinating agent
50/pkg
2599150
100/pkg
1436369
Germicidal Cloths
50/pkg
2463200
each
2569900
Incubator, Portable, 12 VDC

Marker, Laboratory
each
2092000
Pipet, Serological, 1-mL, sterile, disposable, individually wrapped
50/pkg
2092835
50/pkg
2092628
each
1970010

Pipet tips, sterile, individually wrapped
200/pkg
2558996
Pipet Aid, 110 VAC Recharger, 4 replacement filters (UL, CSA approved)
each
2551701
Rack, Coliform Tube
each
221500

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsTotal and E. Coli, MPN, method 8091
ColiformsTotal and E. Coli
DOC316.53.01218
Lauryl Tryptose with MUG Broth1
Method 8091
Scope and Application: For potable and non-potable water.

1
Based on publication by Peter C.S. Feng and Paul A. Hartman Fluorogenic Assays for Immediate Confirmation of Escherichia coli. Applied
and Environmental Microbiology, Vol. 43, No. 6, pp. 1320-1329, 1982. This method is not USEPA accepted.

Make sure that all materials used for containing or transferring samples are sterile.
Fluorescence without gas production indicates an anaerogenic (non-gas-producing) strain(s) of E. coli.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray, or dilute iodine solution. Wash

Description
Lauryl Tryptose with MUG Broth Tubes
Dilution Water, buffered, 99-mL, sterile (for non-potable water samples only)
Quantity
515
3 bottles
Coliform tube rack
Incubator
Pipet, serological, 10-11 mL, sterile
Pipet filler

Page 1349

Potable water test for coliformstotal and E. coli, Lauryl Tryptose with MUG Broth

soap and water.
well-mixed.

5 or 10 tubes of Lauryl
Tryptose with MUG Broth
one at a time. Use a sterile
pipet to transfer 10 mL of
sample into each of the
tubes.
of the caps.
5. After one hour, invert

the tubes to remove
vials.
the inner vials during the
first hour are not from
bacteria. Remove the
bubbles by inverting the
tubes. Make sure there are
no bubbles and then
carefully return the tubes
to an upright position.
Loosen the caps slightly
before returning the tubes
to the incubator. Continue
incubation. The tubes
must be kept upright for
the rest of the test.
6. After 24 (2) hours,

tap each tube gently and
gas. If the broth is cloudy
and the inner vials contain
gas bubbles, coliform
bacteria are likely present.
The presence of gas in
any amount is an
indication of coliform
bacteria.
negative for total coliform
bacteria.
If tubes are cloudy but
have no gas bubbles,
check the tubes for
fluorescence (step 7).

Page 1350

Invert the tube several
times to thoroughly mix the
sample with the nutrient
medium. After the last
inversion, make sure the
inner vial is full of liquid
with no air bubbles.

of 35 (0.5) C .
7. Use a longwave
ultraviolet (UV) lamp to
check the tubes for
fluorescence. Examine the
tubes in a dark area.

use a longwave ultraviolet
(UV) lamp to check the
tubes for fluorescence.
dark area.
If the solution shows

fluorescence, the test is
positive for E. coli.
If the tube does not
fluoresce, return the tubes
to the incubator and
examine again after a total
of 48 (3) hours.
Compare the fluorescence
of the sample tubes to a
tube containing a known
E. coli culture to make a
positive confirmation.

If there is no fluorescence,
the test is negative for
E. coli.
Refer to Potable water
MPN results to find the
MPN of the sample.

Non-potable water test for coliformstotal and E. coli, Lauryl Tryptose with MUG Broth

soap and water.
times, to make sure that it
is well-mixed.

the sample.
for instructions.

15 tubes of Lauryl
Tryptose with MUG Broth.
Use a sterile pipet to
transfer 10 mL of the first
dilution into 5 tubes. Use a
sterile pipet to transfer
10 mL of the second
10 mL of the third dilution
into the remaining 5 tubes.


of the caps.

Page 1351

Non-potable water test for coliformstotal and E. coli, Lauryl Tryptose with MUG Broth
(continued)

of 35 0.5 C .
After one hour, invert the
tubes to remove trapped
air in the inner vials.
to the incubator.
Continue incubation. The
tubes must be kept upright
for the rest of the test.

any amount is an
bacteria.
negative for total coliform
bacteria.
If tubes are cloudy but
have no gas bubbles,
check the tubes for
fluorescence (step 7).
7. Use a longwave
ultraviolet (UV) lamp to
check the tubes for
fluorescence. Examine the
tubes in a dark area.
If the tube does not
fluoresce, return the tubes
to the incubator and
examine again after a total
of 48 (3) hours.

dark area.
If there is no fluorescence,
the test is negative for
E. coli.
Refer to Non-potable
water MPN results to find
the MPN of the sample.
Sample dilution
dilutions from the Dilution guidelines by sample type for the test.
Procedure
1. Wash hands.

Page 1352

Sample type
Dilution 1
Dilution 2
undiluted (1x)
10x
100x
Bathing beach water
10x
100x
1000x
Lake water
10x
100x
1000x
10x
100x
1000x
100x
1000x
10,000x

Storm water
Dilution 3
1000x
10,000x
100,000x
10,000x
100,000x
1,000,000x
10,000x
100,000x
1,000,000x
Raw sewage
100,000x
1,000,000x
10,000,000x
Potable water MPN results

Use the number of positive tubes to find the MPN per 100 mL from the MPN table for 10 tubes.
Example: 6 of the 10 tubes showed a positive response. The MPN per 100 mL is 9.2.
Table 379 MPN table for 10 tubes1
MPN per 100 mL
< 1.1
1.1
2.2
3.6
5.1
6.9
9.2
12.0
16.1
23.0
10
> 23.0
Table is for undiluted samples, 10 mL per tube. Values are 95 percent confidence limits.
5 broth tubes can be used in place of 10 tubes and the MPN table for 5 tubes can be used.
MPN per 100 mL
< 2.2
2.2
5.1
9.2
16.0
> 16.0

Page 1353
Non-potable water MPN results

2. Multiply the MPN index by the lowest dilution factor.

First dilution
set
Second
dilution set
Third
dilution set
MPN
index per
100 mL
<2
26
27
33
34
23
30
40
30
50
60
50
70
90
80
12
110
140
11
170
11
130
14
170
14
220
17
280
13
350
17
240
17
300
21
500
26
900
22
1600
1600

Page 1354
First dilution
set
Second
dilution set
Third dilution
set
MPN
index per
100 mL
Controls
Bacteria disposal
Bleach
Sterilize used test containers with household bleach. Add 12 mL of the bleach to each test
container. Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave
Place used test containers in a contaminated-items bag or a biohazard bag to prevent leakage into
the autoclave. Autoclave the used test containers in the unsealed bag at 121 C for 30 minutes at
15 pounds pressure. When cool, seal the bag, place it in another garbage bag, and tie tightly.
Summary of method
inverted inner vial (Durham tube) for gas collection. Sample is diluted, added to the tubes and
incubated. If coliforms are present, gas is produced and is trapped in the inner vial.
The number of tubes that form gas is used to estimate the number of coliform organisms in the
sample. The MPN method allows for the analysis of highly turbid samples by dilution prior to
analysis. No filtering is necessary.
The Lauryl Tryptose with MUG Broth will detect total coliforms and E. coli. The results are
comparable to the traditional MPN fecal coliform tests, but obtained in far less time. No transfer
from presumptive to confirmed medium is necessary with the LT/MUG method. The LT/MUG
medium contains lauryl tryptose broth and 4-methylumbelliferyl--D-glucuronide (MUG), a
fluorogenic reagent. Tubes positive for E. coli will fluoresce when the incubated tubes are
examined under long-wave UV light.

Page 1355

Description
Unit
Catalog number
Lauryl Tryptose with MUG Broth Tubes
15/pkg
2182115
25/pkg
1430598
Required apparatus
Description
Bags,
Whirl-Pak,
with dechlorinating agent, 180-mL
Unit
Catalog number
100/pkg
2075333
each
2281400
each
2619200
each
2619202
each
2184300
each
2184302

25/pkg
209798
each
1465100
Description
Unit
Catalog number
each
2595902
each
2898600
2463300
Pipet safety bulb
200/pkg
100/pkg
1437297
500/pkg
2233100
Battery eliminator
each
2580400
each
2580300
Brilliant Green Bile (BGB) Broth Tubes
15/pkg
32215
Bottle, polysulfone, autoclavable (use for buffered dilution water)
12/pkg
2245300
12/pkg
2495012
50/pkg
2495050
Bottle, sample, sterilized, 100-mL fill-to line, disposable w/dechlorinating agent
50/pkg
2599150
100/pkg
1436369
Ecoli Fluorescence standard

Germicidal Cloths
each
2361100
50/pkg
2463200
Incubator, Portable, 12 VDC
each
2569900
Marker, Laboratory
each
2092000
50/pkg
2092835
50/pkg
2092628
each
1970010
Pipet,
TenSette,
1.010.0 mL

Pipet Aid, 110 VAC Recharger, 4 replacement filters (UL, CSA approved)

Page 1356
200/pkg
2558996
each
2551701

Description
Rack, Coliform Tube
Unit
Catalog number
50/pkg
2143166
each
221500
15/pkg
2811115
100/pkg
2811199

Page 1357

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsTotal, Fecal and E. coli, MPN, method 8001A
ColiformsTotal, Fecal and E. Coli

USEPA1 Lauryl Tryptose Broth presumptive test with BGB, EC
Medium and EC/MUG confirmation
DOC316.53.01217
Method 8001A

Scope and Application: For non-potable water and wastewater.
1
Most Probable Number Method 8001A for wastewater is USEPA-accepted. Method 8001A meets or exceeds the specification criteria stated
in Standard Methods for the Examination of Water and Wastewater, 19th edition, 9221 Multiple-Tube Fermentation Technique for Members of
the Coliform Group.

Make sure that all the materials that are used for containing or transferring samples are sterile.
Use the Lauryl Tryptose Broth for the presumptive test. If the presumptive test is positive, use Brilliant Green Bile (BGB)
broth to confirm if the sample has total coliforms, EC Medium for fecal coliforms or EC Medium with MUG for E. coli. The
confirmation test is used to eliminate false-positive results that can occur with the presumptive test.
For USEPA reporting, the confirmation tubes must be inoculated by an inoculation loop. Cap transfer is not allowed.
To sterilize an inoculating needle, heat the needle to red hot in an alcohol or Bunsen burner. Let the needle cool before use.

Description
Lauryl Tryptose Broth Tubes
Quantity
15
varies
EC Medium Broth Tubes
varies
EC Medium with MUG Broth Tubes

varies
3 or more bottles
Incubator
Alcohol burner
Inoculating loop
Pipet, serological, 1011 mL, sterile
Pipet filler
Coliform tube rack

Page 1359

Presumptive test for coliform bacteria (Lauryl Tryptose Broth)

soap and water.
times, to make sure that it
is well-mixed.

the sample.
for instructions.

15 tubes of Lauryl
Tryptose Broth one at a
time. Use a sterile pipet to
transfer 10 mL of the first
10 mL of the second
10 mL of the third dilution
into the remaining 5 tubes.


of the caps.
Positive
Confirm
Results

of 35 (0.5) C .
bacteria.

the tubes to remove
vials.
Remove the bubbles by
inverting the tubes. Make
sure there are no bubbles
and then carefully return
the tubes to an upright
position.

Page 1360

If no gas can be seen,
return the tubes to the
incubator and examine
again after a total of
48 (3) hours.
any amount is an
bacteria.

tubes that contain gas in
the inner vial.
Complete a confirmation
test for all tubes that
contain gas. The
confirmation test will
confirm whether total
coliforms, fecal coliforms
or E.Coli are present in the
sample.
negative for coliform
bacteria.

Confirmation test for total coliforms (Brilliant Green Bile Broth)
1. From each positive LT

Broth tube, inoculate a
Broth tube. Use a sterile,
disposable loop or a
flame-sterilized, nichrome
wire.
2. Replace the screw

cap on each tube
immediately. Invert the
BGB tubes to remove
vials.
Put the loop into the

positive Lauryl Tryptose
tube and then into a BGB
Broth tube, making sure
not to touch the rim of
either tube.
3. Place the BGB tubes

in the incubator at a
temperature of
35 (0.5) C.

the tubes to remove
vials.
tubes. Make sure the
bubbles are gone and then

gas. If the inner vial
contains gas bubbles, the
test is positive for total
coliform bacteria.
48 (3) hours.

contain gas.
7. Find the MPN index per 100 mL from the MPN

index table.
Multiply the MPN index by the dilution factor in the first
set of tubes (lowest dilution used in the test).
(MPN index x lowest dilution factor = total coliform
bacteria per 100 mL sample)
See the Example calculation.

negative for coliforms.

Page 1361

Confirmation test for fecal coliforms (EC Medium)

Broth tube, inoculate an
EC Medium Broth tube.
Use a sterile, disposable
loop or a flame-sterilized,
nichrome wire.
2. Replace the screw

cap on each tube
immediately. Invert the EC
Medium Broth tubes to
inner vials.
3. Place the EC Medium

Broth tubes in the
of 44.5 (0.2) C.

tube and then into a EC
Medium tube, making sure
either tube.

the tubes to remove
vials.

contain gas.
6. Find the MPN index per 100 mL from the MPN

index table.
Multiply the MPN index by the dilution factor in the first
set of tubes (lowest dilution used in the test).
(MPN index x lowest dilution factor = fecal coliform
bacteria per 100 mL sample)
See the Example calculation.

negative for fecal coliforms.

Page 1362

Confirmation test for E. coli (EC Medium with MUG)

EC Medium with MUG
Broth tube.
nichrome wire.
Medium with MUG tube,
making sure not to touch
the rim of either tube.
Replace the screw cap on
each tube immediately.
Invert to mix.

with MUG tubes in the
of 44.5 (0.2) C for
24 (2) hours.

dark area.

tubes that show
fluorescence.

Multiply the MPN index by

the dilution factor in the
first set of tubes (lowest
dilution used in the test).

positive for E. coli. If there
is no fluorescence, the test
is negative for E. coli.
Find the MPN index per

100 mL from the MPN
index table.
(MPN index x lowest

dilution factor = E. coli
bacteria per 100 mL
sample)
See the Example
calculation.
Note: If more than 6 hours will elapse after collection, refer to the preservation and storage requirements
in Standard Methods for the Examination of Water and Wastewater.
Bacteria disposal
Bleach
Sterilize used test tubes with household bleach. Add 12 mL of the bleach to each test tube. Allow
10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.

Page 1363

Autoclave
Sample dilution
dilutions from the Dilution guidelines by sample type for the test (step 3 of the Presumptive test for
coliform bacteria (Lauryl Tryptose Broth)).
Procedure
1. Wash hands.

Sample type
Dilution 1
Dilution 2
undiluted (1x)
10x
100x
Bathing beach water
10x
100x
1000x
Lake water
10x
100x
1000x
10x
100x
1000x
100x
1000x
10,000x

Storm water
Dilution 3
1000x
10,000x
100,000x
10,000x
100,000x
1,000,000x
10,000x
100,000x
1,000,000x
Raw sewage
100,000x
1,000,000x
10,000,000x
Example calculation
2. Multiply the MPN index by the lowest dilution factor (e.g. 10 for a 10x dilution).
Page 1364

Dilution 1
Dilution 2
Dilution 3
MPN
index per
100 mL

Dilution 1
Dilution 2
Dilution 3
MPN
index per
100 mL
<2
26
27
33
34
23
30
40
30
50
60
50
70
90
80
12
110
140
11
170
11
130
14
170
14
220
17
280
13
350
17
240
17
300
21
500
26
900
22
1600
1600
Controls
water samples from municipal treatment facilities should be negative for total and fecal coliforms.
Summary of method
inverted inner vial (Durham tube) for gas collection. Sample is added to the tubes and incubated. If
coliforms are present, gas is produced and is trapped in the inner vial. The number of tubes that
form gas is used to estimate the number of coliform organisms in the sample. When the EC
Medium with MUG broth is used, fluorescence under a longwave UV lamp confirms the presence
of E. coli.
Page 1365

Description
Unit
Catalog number
15/pkg
2101415
15/pkg
32215
15/pkg
1410415
15/pkg
2471515
25/pkg
1430598
Required apparatus
Description
Unit
Catalog number
Alcohol Burner
each
2087742
100/pkg
2233199
each
2281400
each
2619200
each
2619202
Inoculating Loop, nichrome wire
each
2112100
each
2184300
each
2184302
Bags,
Whirl-Pak,
without dechlorinating agent, 207-mL

25/pkg
209798
each
1465100
Description
Unit
Catalog number
each
2595902
each
2898600
2463300
Pipet safety bulb
200/pkg
100/pkg
2075333
500/pkg
2233100
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
E. coli Fluorescence standard

Germicidal Cloths
each
2361100
50/pkg
2463200
each
2569900
Inoculating Loops, sterile, disposable
25/pkg
2749125
Isopropyl alcohol
500 mL
1445949
each
2415200

Page 1366

Description
Marker, laboratory
Catalog number
each
2092000
Pipet, serological, 1-mL, sterile, disposable, individually wrapped
50/pkg
2092835
50/pkg
2092628
each
1970010

Pipet Aid, 110 VAC recharger, 4 replacement filters (UL, CSA approved)
Rack, coliform tube
200/pkg
2558996
each
2551701
50/pkg
2143166
each
221500
15/pkg
2811115
100/pkg
2811199

1
Unit
2097810

Page 1367

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ColiformsTotal, Fecal and E. coli, MPN, method 8001

USEPA1 Lauryl Tryptose Broth presumptive test with BGB, EC
Medium and EC/MUG confirmation
DOC316.53.01216
Method 8001

Scope and Application: For potable water.
1
Most Probable Number Method 8001 for potable water is USEPA-accepted. Method 8001 meets or exceeds the specification criteria stated in
Standard Methods for the Examination of Water and Wastewater, 19th edition, 9221 Multiple-Tube Fermentation Technique for Members of
the Coliform Group. For potable water, confirm fecal coliforms with EC Medium Broth as cited in 40 CFR Part 141.21, Subpart (F)(5); or
confirm E. coli with EC/MUG Medium Broth as cited in 40 CFR Part 141.21, Subpart (F)(6)(i).

Make sure that all materials that are used for containing or transferring samples are sterile.
Use the Lauryl Tryptose Broth for the presumptive test. If the presumptive test is positive, use Brilliant Green Bile (BGB)
broth to confirm if the sample has total coliforms, then EC Medium for fecal coliforms or EC Medium with MUG for E. coli.
Potable water should not contain any coliform bacteria. Samples should not be diluted.
For USEPA reporting, the confirmation tubes must be inoculated by an inoculation loop. Cap transfer is not allowed.
To sterilize an inoculating needle, heat the needle to red hot in an alcohol or Bunsen burner. Let the needle cool before use.
If all 10 tubes (for a 10-tube MPN test) of the confirmed coliform test are negative, the sample is accepted as meeting
bacterial standards. To make sure that sample results are interpreted in accordance with appropriate standards and
regulations, contact the local, county, state or federal regulatory agency.
If the test will not be used for USEPA reporting, 5 broth tubes can be used in place of 10 tubes. Use the MPN table for 5
tubes to find the result of the 5-tube test. The 5-tube test cannot be used for USEPA reporting.

Description
Quantity
10
varies
varies
varies
Incubator
Alcohol burner
Inoculating loop
Pipet, serological, 1011 mL, sterile
Pipet filler
Coliform tube rack

Page 1369

Presumptive test for coliform bacteria (Lauryl Tryptose Broth)

soap and water.
well-mixed.

10 tubes of Lauryl
Tryptose Broth one at a
time. Use a sterile pipet to
transfer 10 mL of sample
into each of the tubes.
of the caps.
3. Replace and tighten

the screw cap on each
tube immediately after the
sample is added. Invert
and swirl the tube several
Positive
Confirm
Results

the tubes to remove
tubes. Make sure there are
no bubbles and then

48 (3) hours.
any amount is an
bacteria.
Count the number of tubes
that contain gas in the
inner vial.

Page 1370
7. Complete a
confirmation test for all
tubes that contain gas.
The confirmation test will
confirm whether total
coliforms, fecal coliforms
or E.Coli are present in the
sample.
The confirmation test is
used to eliminate falsepositive results that can
occur with the presumptive
test.
bacteria.

of 35 (0.5) C .

Confirmation test for total coliforms (Brilliant Green Bile Broth)

Broth tube, inoculate a
Broth tube. Use a sterile,
disposable loop or a
flame-sterilized, nichrome
wire.
tube and then into a BGB
Broth tube, making sure
either tube.

tube immediately. Invert
and swirl the BGB tubes to
inner vials.
3. Place the BGB tubes

in the incubator at a
temperature of
35 (0.5) C.

the tubes to remove
If Durham tube vials are

used, after the last
inversion, make sure that
the inner tube does not
contain any air bubbles.

Positive
Confirm
Results

coliform bacteria.

coliform bacteria.

48 (3) hours.

bacteria.

the inner vial.
Find the MPN of the
sample (total coliform
bacteria per 100 mL
sample) from the MPN
table for 10 tubes.
8. If the test is positive

for total coliform bacteria,
complete a confirmation
test for fecal coliform or
E.Coli bacteria (USEPA
requirement).

Page 1371

Confirmation test for fecal coliforms (EC Medium)

EC Medium Broth tube.
nichrome wire.
Medium tube, making sure
either tube.

coliform bacteria.
negative for fecal coliform
bacteria.

tube immediately. Invert
and swirl the EC Medium
Broth tubes to remove
vials.

the inner vial.
Find the MPN of the
sample (fecal coliform
bacteria per 100 mL
sample) from the MPN
table for 10 tubes.

Page 1372

Broth tubes in the
of 44.5 (0.2) C.

the tubes to remove

Confirmation test for E. coli (EC Medium with MUG)

EC Medium with MUG
Broth tube.
nichrome wire.
Medium with MUG tube,
making sure not to touch
the rim of either tube.
Replace and tighten the
screw cap on each tube
immediately. Invert and
swirl to mix.

with MUG tubes in the
of 44.5 (0.2) C for
24 (2) hours.

dark area.

tubes that show
fluorescence.
Find the MPN of the
sample (E. coli bacteria
per 100 mL sample) from
the MPN table for 10
tubes.

positive for E. coli. If there
is no fluorescence, the test
is negative for E. coli.


Page 1373
MPN table
Use the number of positive tubes to find the MPN per 100 mL from the MPN table for 10 tubes.
Example: 6 of the 10 tubes showed a positive response. The MPN per 100 mL is 9.2.
MPN per 100 mL
< 1.1
1.1
2.2
3.6
5.1
6.9
9.2
12.0
16.1
23.0
10
> 23.0
If the test will not be used for USEPA reporting, 5 broth tubes can be used in place of 10 tubes and
the MPN table for 5 tubes can be used. The 5-tube test cannot be used for USEPA reporting.
MPN per 100 mL
< 2.2
2.2
5.1
9.2
16.0
> 16.0
Table is for undiluted samples, 10 mL per tube. Values are 95 percent confidence limits. The MPN table for 5 tubes
cannot be used for USEPA reporting.
Bacteria disposal
Bleach
Sterilize used test tubes with household bleach. Add 12 mL of the bleach to each test tube. Allow
10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave

Page 1374
Summary of method
inverted inner vial (Durham tube) for gas collection. Sample is added to the tubes and incubated. If
coliforms are present, gas is produced and is trapped in the inner vial. The number of tubes that
form gas is used to estimate the number of coliform organisms in the sample. When the EC
Medium with MUG broth is used, fluorescence under a longwave UV lamp confirms the presence
of E. coli.

Description
Unit
Catalog number
2101415
Lauryl Tryptose Broth tubes
15/pkg
Brilliant Green Bile (BGB) Broth tubes
15/pkg
32215
EC Medium Broth tubes
15/pkg
1410415
EC Medium with MUG Broth tubes (without Durham tubes)
15/pkg
2471515
EC Medium with MUG Broth tubes (with Durham tubes)
15/pkg
2282415
Required apparatus
Description
Unit
Catalog number
Alcohol Burner
each
2087742
100/pkg
2075333
each
2281400
Inoculating Loop, nichrome wire
each
2112100
each
2184300
each
2184302

Pipet safety bulb
25/pkg
209798
each
1465100
Unit
Catalog number

Description
100/pkg
1436369
25/pkg
1430598
50/pkg
2143166

Page 1375
Optional apparatus
Description
Unit
Catalog number
2463300
200/pkg
100/pkg
2233199
500/pkg
2233100
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
each
2162700
E. coli Fluorescence standard
each
2361100
50/pkg
2463200
Inoculating Loops, sterile, disposable
25/pkg
2749125
Isopropyl alcohol
500 mL
1445949
each
2415200
Germicidal Cloths

Marker, laboratory
each
2092000
50/pkg
2092835
50/pkg
2092628
each
1970010

200/pkg
2558996
Pipet Aid, 110 VAC recharger, 4 replacement filters (UL, CSA approved)
each
2551701
Rack, coliform tube
each

221500
2097810
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Bacteria, Hydrogen Sulfide Producing, MPN, 10032
Bacteria, Hydrogen Sulfide

Producing
DOC316.53.01196
Method 10032
Pathoscreen Medium
Scope and Application: For the detection of Salmonella, Citrobacter, Proteus, Edwardsiella and Klebsiella (some
spp.) in drinking water, surface water and recreational water
Test preparation
PathoScreen Medium
PathoScreen Medium detects the presence of hydrogen sulfide-producing bacteria including
Salmonella, Citrobacter, Proteus, Edwardsiella and some species of Klebsiella. The sterilized
powder medium is a reliable medium for monitoring drinking water systems in developing tropical
countries, in remote field locations and in disaster or emergency situations.
PathoScreen Medium is dehydrated, sterilized and packaged in powder pillows. Powder pillows
are available for both Presence/Absence (P/A) and Most Probable Number (MPN) testing. Each
powder pillow contains enough medium for one test. The medium is shipped with a Certificate of
Analysis and has an expiration date printed on the label.
For MPN testing, add one MPN Pillow to a 20-mL sample. For MPN testing, inoculate a set of five
tubes.
Conducting MPN tests with PathoScreen Medium

The MPN method can be used for drinking water, as well as marine and fresh recreational waters,
swimming pools, lakes, shellfish-growing waters, heavily polluted waters and wastewater. For
water that is heavily contaminated, use the multiple tube decimal dilution procedure.
Incubate samples 2448 hours between 2535 C, 7795 F. (30 C, 80 F is considered optimal.)
PathoScreen Medium has a detection sensitivity of 1 CFU/100 mL.
Bacteria, Hydrogen Sulfide Producing

Page 1377

PathoScreen medium MPN pillows, method 10032
1. Wash hands
thoroughly with soap and
water.

five sterile tubes one at a
time and pipet 20 mL of
sample into each of the
tubes with a sterile pipet.
Use aseptic technique to
avoid contaminating the
tubes or the caps.
3. Swab the end of a

PathoScreen Medium
MPN Pillow with alcohol
and aseptically cut it open
with clippers. Add pillow
contents to the 20 mL
sample.
4. Cap and swirl each

tube and immediately.
Invert the tubes a few
sample with the medium.
The sample will be yellow
in color.
5. Place the tubes in a

location with a constant
temperature of 2535 C
for 2448 hours.
6. If an incubator is
available, incubate the
sample at 30 0.5 C for
2448 hours.
7. Note the reaction after

24 hours of incubation.
(See the MPN Results
table.)
8. Record results. (See

the Five-tube MPN values
table.)
Dispose of all completed

tests appropriately. Refer
to the MSDS and local
regulations for proper
disposal.
If the temperature varies

significantly, continue to
incubate negative tubes
for an additional day.
Table 386 MPN Results

Hydrogen sulfide producing bacteria
Test Results
Positive
Color changes from yellow to black
Black precipitate forms
Negative
X
No color change

Page 1378
Follow-up
Incubate additional 1224 hours and reevaluate. If there is no color change,

record as negative.

Using statistical methods it is possible to estimate the number of organisms from any combination
of positive and negative test results. The MPN values in the Five-tube MPN values table are based
on 20 mL of undiluted sample in each of five tubes. If the sample is diluted, multiply the result by
the dilution factor.
Example 1:
Five tubes of undiluted sample are inoculated. Positive results are obtained from three of the five
tubes. The result obtained from the Five-tube MPN values table is 4.6.
Example 2:
A river water sample is collected and diluted. A dilution factor of 10,000 is prepared and five tubes
are inoculated. Positive results are obtained from two of the five tubes. The result obtained from
two of the five tubes. The result obtained from the Five-tube MPN values table is 2.6. This result is
multiplied by 10,000 and a result of 26,000 is recorded.
Table 387 Five-tube MPN values

Positive Tubes
MPN/100 mL
<1.1
1.1
2.6
4.6
8.0
>8.0

Required reagents
Description
PathoScreen Medium, MPN Pillows, 20-mL sample
Unit
Catalog number
50/pkg
2610796
Dilution watermedia, reagents and apparatus

Description
Unit
Catalog number
Buffered Dilution Water, sterile, 99-mL1
25/pkg
1430598
100/pkg
1436369
Pipet, sterile, disposable, 11-mL
25/pkg
209798
Pipet, sterile, disposable, individually wrapped, 10-mL
50/pkg
2092628
Pipet, sterile, disposable, 10-mL
50/pkg
2092828
each
2551701
Pipet Filler, portable, with recharger (UL, CSA approved), 110 VAC
1

Page 1379
Required apparatus
Description
Unit
Catalog number
Autoclave, Automatic, 120 VAC
each
2898600
Clippers, large
each
2065800
Contaminated Items Bags
200/pkg
2463300
Germicidal Cloth
50/pkg
2463200
Incubator, Culture, 120 VAC
each
2619200
each
2619202
10/pkg
1497054
each
2497903
100/pkg
2075333
MPN Vials
Rack for coliform tubes
Sampling Containers
Sampling Bags, Whirl-Pak with dechlorinating agent, 180-mL
Sampling Bottles, autoclavable
6/pkg
2324333
48/pkg
2324373
Sampling Bottles, sterilized, 100-mL fill-to line
12/pkg
2495012
50/pkg
2495050
Sampling Bottles, sterilized, 100-mL fill-to line, with dechlorinating agent
12/pkg
2599112
50/pkg
2599150

Description
Unit
Bottle, polysulfone, autoclavable (for preparing buffered dilution water)

Magnesium Chloride and Potassium Dihydrogen Phosphate Powder Pillows
Safety bulb
Sterikon
Catalog number
12/pkg
2245300
25 of each
2143166
30/pkg
2142964
each
1465100
15/pkg
2811115
100/pkg
2811199
100/pkg
2233199
10/pkg
1437297
each
2281400
Incubator, 12-well, Dri Bath

Pipet, TenSette 1.010.0 mL

each
1970010
200/pkg
2558996
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Coliforms-Total, Fecal and E. coli, P/A, 8319 and 8364
Coliforms
DOC316.53.01191
Presence/Absence
Methods 8319 and 8364
P/A Broth (8319)

P/A Broth w/ MUG (8364)
Test preparation

Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray, or dilute iodine solution
Introduction
Both P/A Broth and P/A Broth with MUG come packaged in disposable bottles and in glass
ampules. The medium in both bottles and ampules is concentrated and must be diluted with sterile
water 5:1. The medium is sterilized by membrane filtration to prevent degradation.
P/A Broth ampules, method 8319
sample in a sterile sample
container. Use aseptic
technique to avoid
contaminating the sample
and sample container.
2. Add sample to the fillto line of a sample bottle.

Sample may be added
from a sterile container, or
directly from a faucet or
spigot.

one P/A Broth ampule to
the 100 mL of sample.
4. Incubate the sample at

35 0.5 C for
2448 hours.
Coliforms
Page 1381
Coliforms
P/A Broth ampules, method 8319
Confirm
Positive
Confirm
Samples
Positive
Samples

See Interpret and report
results on page 1383.
6. Confirm presumptive
positive samples by
inoculating the appropriate
media directly from
positive
P/A broth cultures. See the
Confirmation Table on
page 1383.
Dispose of all
completed tests
7. Dispose of all
completed tests
appropriately.
Dispose of all completed
tests
P/A broth with MUG, method 8364
container. Use aseptic
technique to avoid
contaminating the sample
and sample container.
Coliforms
Page 1382
2. Add sample to the fillto line of a Broth

Disposable Bottle.
Sample may be added
from a sterile container, or
directly from a faucet or
spigot.
3. Incubate the sample at

35 0.5 C for
2448 hours.

See Interpret and report
results.
Coliforms
P/A broth with MUG, method 8364 (continued)
Confirm Positive
Samples
Dispose of all
completed tests
5. Confirm presumptive
positive samples by
inoculating the appropriate
media directly from
positive P/A broth cultures.
See the Confirmation
Table.
6. Dispose of all
completed tests
appropriately.
Interpret and report results

Reactions using P/A Broth
Color change from reddish purple to yellow or yellow brownrecord the test as presumptive
positive for total coliform bacteria.
No color changeincubate for an additional 24 hours and recheck the sample for
color change.
If after 24 hours of incubation, the color changes from reddish purple to yellow or yellow
brownrecord the test as presumptive positive for total coliform bacteria.
If after 48 3 hours of incubation, the sample still appears reddish purplerecord the test as
negative for total coliform bacteria.
Reactions using P/A Broth with MUG
Examine the sample under long-wave UV light; if the sample fluorescesrecord the test as
Confirm positive samples

Inoculum from incubated presence/absence samples are used to confirm the presence of total
coliforms, fecal coliforms or E. coli in the original water sample. Use a sterile inoculating loop to
transfer sample to an appropriate confirmation medium.
Table 388 Confirmation Table

Bacteria
Confirmation Medium
Incubation
Incubator
Positive Result
Total Coliform
Brilliant Green Bile Broth

(322-15)
24 to 48 hours.
35 0.5 C
Culture (2619200 or -02)

or 12-well Dri-Bath
(2281400)
Gas/Turbidity
Fecal Coliform
EC Medium Tubes
(14104-15)
24 hours
44.5 0.2 C
12-well Dri-Bath
(2281400)
Gas/Turbidity
E. coli
EC Medium with MUG Tubes

(24715-15)
24 hours
44.5 0.2 C
12-well Dri-Bath
(2281400)
Fluorescence
Coliforms
Page 1383
Coliforms
Controls
water samples from municipal treatment facilities should be negative for total coliforms and fecal
coliforms.
Completed test disposal

Active bacterial cultures grown during incubation must be disposed of safely. Use either Bleach or
an Autoclave to neutralize bacteria.
Bleach
1. Sterilize used test containers with a 10% bleach solution.
2. Add approximately 12 mL of bleach to each test containerand allow to stand for 10 to 15
minutes.
3. Pour the liquid down the drain. Dispose of the test tubes with the normal garbage.
Autoclave
Test containers must be placed in a bag before autoclaving to prevent leakage of solution into the
autoclave.
1. Place used test tubes in a contaminated-items bag or a biohazard bag and seal tightly.
1. Autoclave the used test tubes in the bag at 121 C for 15 minutes at 15 pounds of pressure.
2. Place the bag of test tubes in a separate garbage bag and tie tightly.
3. Once the test tubes are sterile, dispose of them with the normal garbage.
Summary of method 8319

P/A Broth is ideal for screening drinking water samples for total coliforms. The method is a simple
modification of the multiple-tube method. It uses lactose and lauryl tryptose broths with bromcresol
purple, which detects acidity formed during lactose fermentation by the bacteria.
Simply combine 100 mL of sample and P/A Broth, incubate for 24 hours and check for a color
change. A yellow color indicates the presence of total coliforms.
Summary of method 8364

P/A Broth with MUG allows simultaneous detection of total coliform bacteria and E. coli. In addition
to the lactose and lauryl tryptose broths with bromcresol purple, this medium contains MUG
reagent (4-methylumbelliferyl-b-D-glucuronide). The MUG reagent produces a fluorogenic product
when hydrolyzed by glucuronidase (an enzyme specific to E. coli). MUG detects non gasproducing (anaerogenic) strains of E. coli and works well when competitive organisms are present.
Combine 100 mL of sample and P/A Broth with MUG, incubate for 24 hours and check for a color
change and fluorescence. A yellow color indicates the presence of total coliforms. To detect E. coli,
examine samples under a long-wave ultraviolet (UV) light. Fluorescence indicates the presence of
E. coli.
Coliforms
Page 1384
Coliforms

Description
Unit
Catalog number
P/A Broth Ampules

P/A Broth Ampules
25/pkg
2494925
P/A Broth with MUG Ampules
25/pkg
2495525
P/A Broth Disposable Bottles
12/pkg
2323212
P/A Broth Disposable Bottles
50/pkg
2323250
P/A Broth with MUG Disposable Bottles
12/pkg
2401612
P/A Broth with MUG Disposable Bottles
50/pkg
2401650
Unit
Catalog number
12/pkg
2495012
P/A Broth in Disposable Bottles
Required apparatus
Description
Bottles, sample, presterilized, 100-mL fill-to line
Incubator, Culture, low profile, 110/120 VAC, 50/60 Hz
each
2619200
each
2619202
Lamp, handheld, long-wave, ultraviolet, 115 VAC, 60 Hz
each
2184300
each
2184302
Description
Unit
Catalog number
each
2898600
Optional apparatus
200/pkg
2463300
Bags, Whirl-Pak1, with dechlorinating agent
100/pkg
2075333
50/pkg
2495050
Bottles, sample, presterilized, with dechlorinating agent, 100-mL fill-to line
12/pkg
2599112
Bottles, sample, presterilized, with dechlorinating agent, 100-mL fill-to line
12/pkg
2599150
Lamp, long-wave, ultraviolet, portable
each
2415200
Incubator, Dri-Bath, 12-Well, 120V
each
2281400
100/pkg
2233199
Bags,
Whirl-Pak1,
without dechlor 207 mL
Bags, Whirl-Pak1, without dechlor 720 mL

Dechlorinating reagent, powder pillows
Inoculating loop, nichrome V wire
10/pkg
1437297
100/pkg)
1436369
each
2112100
Inoculating loop, disposable
25/pkg
2749125
Isopropyl alcohol
500 mL
1445949
each
2087742
Alcohol burner
each
2097810
each
2569900
Battery eliminator
each
2580400
Coliforms
Page 1385
Coliforms
Optional apparatus
Description
Unit
Catalog number
each
2580300
rach
2595902
Unit
Catalog number
12/pkg
2495012
Optional media
Description
each
2619200
each
2619202
each
2184300
each
2184302

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Bacteria, Hydrogen Sulfide Producing, P/A 8506
Bacteria, Hydrogen Sulfide

Producing
DOC316.53.01197
Presence/Absence (P/A) Method
Methods 8506
Pathoscreen Medium
Scope and Application: For the detection of Salmonella, Citrobacter, Proteus, Edwardsiella and Klebsiella (some
spp.) in drinking water, surface water and recreational water
Test preparation
PathoScreen Medium
PathoScreen Medium detects the presence of hydrogen sulfide-producing bacteria including
Salmonella, Citrobacter, Proteus, Edwardsiella and some species of Klebsiella. The sterilized
powder medium is a reliable medium for monitoring drinking water systems in developing tropical
countries, in remote field locations and in disaster or emergency situations.
PathoScreen Medium is dehydrated, sterilized and packaged in powder pillows. Powder pillows
are available for both Presence/Absence (P/A) and Most Probable Number (MPN) testing. Each
powder pillow contains enough medium for one test. The medium is shipped with a Certificate of
Analysis and has an expiration date printed on the label.
For P/A testing, add one P/A powder pillow to a 100-mL sample.
Incubate samples 2448 hours between 2535 C, 7795 F. (30 C, 80 F is considered optimal.)
PathoScreen Medium has a detection sensitivity of 1 CFU/100 mL.

Page 1387
PathoScreen medium P/A pillows, method 8506
1. Wash hands
thoroughly with soap and
water.

container.
3. Swab the end of the

PathoScreen Medium
P/A Pillow with alcohol and
aseptically cut it open with
clippers. Add pillow
contents to the 100 mL
sample.
4. Place the bottle in a

location with a constant
temperature between
2535 C for 2448 hours.
If an incubator is available,
incubate the sample at 30
0.5 C for 24 to 48 hours.
6. Record the results.

Dispose of all completed tests appropriately. Refer to a
(See the P/A results table.) current MSDS and local regulations.
If the temperature varies

significantly, incubation
may be extended an
additional day.
Table 389 P/A results

Hydrogen sulfide producing bacteria
Test Results
Positive
Color changes from yellow to black
Black precipitate forms
No color change

Page 1388
Negative
Follow-up
Incubate additional 1224 hours and re-evaluate. If there is no

color change, record as negative.

Required reagents
Description
PathoScreen Medium, P/A Pillows, 100-mL sample
Unit
Catalog number
50/pkg
2610696
Dilution watermedia, reagents and apparatus

Description
Unit
Catalog number
12/pkg
2245300
99-mL1
25/pkg
1430598
100/pkg
1436369
Buffered Dilution Water, sterile,
Required apparatus
Description
Unit
Catalog number
Autoclave, Automatic, 120 VAC
each
2898600
Clippers, large
each
2065800
Contaminated Items Bags
200/pkg
2463300
Germicidal Cloth
50/pkg
2463200
each
2619200
each
2619202
100/pkg
2075333
Sampling Containers
Sampling Bags, Whirl-Pak with dechlorinating agent, 180-mL
6/pkg
2324333
48/pkg
2324373
12/pkg
2495012
50/pkg
2495050
12/pkg
2599112
50/pkg
2599150

Description
Magnesium Chloride and Potassium Dihydrogen Phosphate Powder Pillows
Unit
Catalog number
12/pkg
2245300
25 of each
2143166
2142964
30/pkg
15/pkg
2811115
100/pkg
2811199
100/pkg
2233199
10/pkg
1437297

Page 1389

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria, m-HPC, 8242
DOC316.53.01225
Pour Plate Method
Method 8242
m-HPC
Test preparation
Introduction
The Pour Plate Method, also known as the standard plate count, is simple to perform and is
commonly used to determine heterotrophic bacteria density. This method does, however, have
disadvantages that limit recovery of the maximum number of organisms. Tempered medium at
4446 C (111115 F) may cause heat shock to stressed bacteria and the nutritionally rich
medium may decrease recovery of starved bacteria.
The standard plate count attempts to provide a standardized means of determining the density of
aerobic and facultatively anaerobic heterotrophic bacteria in water. Bacteria occur singly or in
pairs, chains, clusters or packets, and no single method, growth medium, or set of physical
conditions can satisfy the physiological requirements of all bacteria in a water sample. However,
the heterotrophic plate count is a good measure of water treatment plant efficiency, aftergrowth in
transmission lines, and the general bacterial composition of source water.
See the Introduction to Bacteria for more information about preparing sample containers and collecting and
preserving samples.
To sterilize the forceps, dip them in alcohol and flame in an alcohol or Bunsen burner. Let the forceps cool before use.
Limit the number of samples to be plated at any one time so that no more than 20 minutes (preferably 10 minutes) elapse
between the dilution of the first sample and the pouring of the last plate.
To save time, start the incubator before preparing the other materials. Set the incubator for the temperature required in the
procedure (usually 35 0.5 C).
Mark each pour plate, membrane filtration petri dish, or other sample container with the sample number, dilution, date, and
any other necessary information. Take care not to contaminate the inside of the sample container in any way.
Page 1391
Pour plate procedure for heterotrophic bacteria m-HPC, method 8242

to place a sterile,
on the dish. Do not touch
the pad or the inside of
the petri dish.
cool before use.
Alternatively, a prepared
used.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
Page 1392

Open an ampule of
m-HPC Broth, using an
ampule breaker if
necessary. Pour the
the petri dish lid.
be used.

30 seconds, approximately
25 times, to make sure it is
well-mixed. Filter the
appropriate volume
through the sterile 47 mm,
0.45m, gridded
membrane filter. Apply
vacuum and filter the
sample. Rinse the funnel
walls three times with
2030 mL of sterile


filters using a 1015X
microscope.

up, in the assembly.
For broth prepared from

dehydrated medium, pipet
approximately 2.0 mL of
broth onto the pad using a
sterile pipet. Drain excess
medium from the petri dish
and replace the lid.

petri dish lid.
Bacterial colonies grown

on m-HPC medium appear
clear to cream in color.
Diluting the Sample

The pour plate method requires use of 1 mL, 0.1 mL, and 0.01 mL or 0.001 mL of sample. The
difficulty measuring and working with the two smaller volumes, 0.01 and 0.001 mL, require the use
of sample dilutions. These dilutions are prepared by pipetting 1 mL of undiluted sample into 99 mL
of buffered dilution water. Diluting the sample allows 1 mL of diluted sample to be used instead of
0.01 mL of undiluted sample, and 0.1 mL of diluted sample instead of 0.001 mL of undiluted
sample.
Selecting Sample Volumes/Dilutions

Select the sample volumes or dilutions to be used so that the total number of colonies on a plate
will be between 30 and 300. For most potable water samples, plates suitable for counting will be
obtained by plating 1 mL of undiluted sample, 0.1 mL of undiluted sample and 1 mL of diluted
sample (which equals 0.01 mL of undiluted sample). In examining sewage or turbid water, do not
measure a 0.1-mL inoculum of the original undiluted sample, but do prepare an
appropriate dilution.
Counting, Computing and Reporting Results

(CFU)/mL. Include in the report the method used, the incubation temperature and time, and
the medium.
For example: 98 CFU/L, mL, 35 C, 24 hours, m-HPC broth.
1 to 2, or fewer colonies per square Count all of the colonies on the filter, and divide the
results by the volume of original sample used.
For example, if there are 122 colonies on the filter, and the volume of original sample used was 10
mL, compute results as follows:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
3 to 10 colonies per square Count all colonies in 10 representative squares and divide by 10
to obtain an average number of colonies per square. Multiply this number by 100 and divide by the
volume of original sample used.
For example, if you calculated an average of 8 colonies per square, and the volume of original
sample used was 0.1 mL, compute results as follows:
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
10 to 20 colonies per square Count all colonies in 5 representative squares and divide by 5 to
For example: if there are an average of 17 colonies per square, and the volume of original sample
used was 0.1 mL, compute results as follows:
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
Page 1393
count as > 2000 divided by the volume of original sample used.
For example, if the original volume of sample used were 0.01 mL, results would be > 2000/0.01 or
> 200,000 CFU/mL.
Note: Report averaged counts as estimated CFU/mL. Make estimated counts only when there are discrete,
separated colonies without spreaders.

Description
Unit
Catalog number
Dilution Water, Buffered, sterile, 99-mL
25/pkg
1430598
m-HPC Agar Plates
15/pkg
2811415
m-HPC Broth Ampules, plastic, 2-mL
50/pkg
2812450
Unit
Catalog number
1000/pkg
1491800
Required apparatus
Description
Absorbent Pads with dispenser, sterile, Gelman
each
2484600
100/pkg
2075333
each
1352900
each
54649
Forceps
each
2141100
Incubator, Culture, low profile, 110 VAC
each
2619200
each
2619202
Membrane filters, 0.45-m, gridded, sterile, Gelman
200/pkg
1353001
Membrane filters, 0.45-m, gridded, sterile, Millipore
150/pkg
2936100
each
2942500
Whirl-Pak Bags with declorinating agent, sterile, 180-mL
Petri Dish, polystyrene, sterile, disposable, without pad
100/pkg
1485299
Petri Dish, polystyrene, sterile, disposable, w/pad, Gelman
100/pkg
1471799
Petri Dish, polystyrene, sterile, disposable, w/pad, Millipore
150/pkg
2936300
each
2824800
Pump, vacuum, 220/230 VAC, Continental European Plug
each
2824802
Rubber Stopper, one hole, No. 8
6/pkg
211908
Rubber Tubing, 3.6-m
each
56019
25/pkg
209798
Pump, vacuum, 110/115 VAC
Pipets, Serological, 1011 mL, sterile, disposable

Description
Unit
Catalog number
each
2595902
Alcohol Burner
each
2087742
Aspirator, water
each
213102
each
2898600
Page 1394
Description
Unit
Catalog number
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
Counter, hand tally

each
1469600
100/pkg
1436369
each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
Microscope, Stereo Binocular
Pump, hand vacuum
500 mL
1445949
each
2942600
each
1428300
15/pkg
2811115
Sterikon
100/pkg
2811199
2097810
Page 1395

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria, m-TGE with TTC, 8242
DOC316.53.01227
Pour Plate Method
Method 8242
m-TGE with TTC
Test preparation
Introduction
preserving samples.
Page 1397
Pour plate procedure for heterotrophic bacteria m-TGE with TTC, method 8242

to place a sterile,
the petri dish.
cool before use.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
Page 1398

Open an ampule of
m-TGE with TTC or use an
ampule breaker if
necessary. Pour the
the petri dish lid.
be used.

appropriate volume
0.45m, gridded
2030 mL of sterile


microscope.



petri dish lid.

on m-TGE with TTC
medium appear
red to aid visibility.
Diluting the Sample

sample.


the medium.
For example: 98 CFU/L, mL, 35 C, 24 hours, m-TGE with TTC broth.
1 to 2, or fewer colonies per squareCount all of the colonies on the filter, and divide the results
by the volume of original sample used.
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample

------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
Page 1399
> 200,000 CFU/mL.

Description
Unit
Catalog number
25/pkg
1430598
m-TGE with TTC PourRite Ampules, glass, 2-mL
20/pkg
2428420
Unit
Catalog number
1000/pkg
1491800
Required apparatus
Description
each
2484600
100/pkg
2075333
each
1352900
each
54649
Forceps
each
2141100
each
2619200
each
2619202
200/pkg
1353001
150/pkg
2936100
each
2942500
100/pkg
1485299
100/pkg
1471799
150/pkg
2936300
each
2824800
each
2824802
6/pkg
211908
each
56019
25/pkg
209798

Description
Unit
Catalog number
each
2595902
Alcohol Burner
each
2087742
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
Page 1400
Description
Unit
Catalog number
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
Counter, hand tally

each
1469600
100/pkg
1436369
each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
Pump, hand vacuum
500 mL
1445949
each
2942600
each
1428300
15/pkg
2811115
Sterikon
100/pkg
2811199
2097810
Page 1401

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria, m-TGE, 8242
DOC316.53.01226
Pour Plate Method
Method 8242
m-TGE
Test preparation
Introduction
preserving samples.
Page 1403
Pour plate procedure for heterotrophic bacteria m-TGE, method 8242

to place a sterile,
the petri dish.
cool before use.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
Page 1404

Open an ampule of
m-TGE or use an ampule
breaker if necessary. Pour
the absorbent pad.
be used.

appropriate volume
0.45m, gridded
2030 mL of sterile


microscope.



petri dish lid.

on m-TGE medium appear
clear to cream in color.
Diluting the Sample

sample.


the medium.
For example: 98 CFU/L, mL, 35 C, 24 hours, m-TGE broth.
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
Page 1405
More than 20 colonies per square If there are more than 20 colonies per square, record the
> 200,000 CFU/mL.

Description
Unit
Catalog number
25/pkg
1430598
m-TGE PourRite Ampules, glass, 2-mL
20/pkg
2373820
Unit
Catalog number
1000/pkg
1491800
Required apparatus
Description
each
2484600
100/pkg
2075333
each
1352900
each
54649
Forceps
each
2141100
each
2619200
each
2619202
200/pkg
1353001
150/pkg
2936100
each
2942500
100/pkg
1485299
100/pkg
1471799
150/pkg
2936300
each
2824800
each
2824802
6/pkg
211908
each
56019
25/pkg
209798

Description
Unit
Catalog number
each
2595902
Alcohol Burner
each
2087742
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
Page 1406
Description
Unit
Catalog number
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
Counter, hand tally

each
1469600
100/pkg
1436369
each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
Pump, hand vacuum
500 mL
1445949
each
2942600
each
1428300
15/pkg
2811115
Sterikon
100/pkg
2811199
2097810
Page 1407

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria, m-TSB/USP, 8242
DOC316.53.01228
Pour Plate Method
Method 8242
m-TSB/USP
Test preparation
Introduction
preserving samples.
Page 1409
Pour plate procedure for heterotrophic bacteria m-TSB/USP, method 8242

to place a sterile,
the petri dish.
cool before use.

Remove the membrane
transfer the filter
immediately to the
previously prepared
petri dish.
Page 1410

Open an ampule of
m-TSB/USP or use an
ampule breaker if
necessary. Pour the
the petri dish lid.
be used.

appropriate volume
0.45m, gridded
2030 mL of sterile


microscope.



petri dish lid.

on m-TSB/USP medium
appear clear to cream in
color.
Diluting the Sample

sample.


the medium.
For example: 98 CFU/L, mL, 35 C, 24 hours, m-TSB/USP broth.
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
More than 20 colonies per square If there are more than 20 colonies per square, record the
> 200,000 CFU/mL.
Page 1411

Description
Unit
Catalog number
25/pkg
1430598
m-TSB/USP Broth Ampules, plastic, 2-mL
20/pkg
2812650
Unit
Catalog number
1000/pkg
1491800
Required apparatus
Description
each
2484600
100/pkg
2075333
each
1352900
each
54649
Forceps
each
2141100
each
2619200
each
2619202
200/pkg
1353001
150/pkg
2936100
each
2942500
100/pkg
1485299
100/pkg
1471799
150/pkg
2936300
each
2824800
each
2824802
6/pkg
211908
each
56019
25/pkg
209798

Description
Unit
Catalog number
each
2595902
Alcohol Burner
each
2087742
Aspirator, water
each
213102
each
2898600
200/pkg
2463300
100/pkg
2233199
10/pkg
1437297
Battery eliminator
each
2580400
each
2580300
12/pkg
2599112
50/pkg
2599150
Page 1412
Description
Unit
Catalog number
12/pkg
2495012
50/pkg
2495050
Counter, hand tally

each
1469600
100/pkg
1436369
each
2486100
12/pkg
2656600
each
2586200
72/pkg
2586300
Germicidal Cloths
50/pkg
2463200
each
2569900

Isopropyl alcohol
Pump, hand vacuum
500 mL
1445949
each
2942600
each
1428300
15/pkg
2811115
100/pkg
2811199
2097810
Page 1413

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Heterotrophic Bacteria, Pour Plate, 8241
DOC316.53.01229
Pour Plate Method
Method 8241
Plate Count Agar1

1
This method meets or exceeds the specification criteria stated in Standard Methods for the Examination of Water and Wastewater,
19th edition, Method 9215 B. Pour Plate Method.
Test preparation

preserving samples.
Introduction
Page 1415
Pour plate procedure for heterotrophic bacteria, method 8241
1. Melt the sterile solid

agar medium by placing a
tube of Plate Count Agar
in a beaker of boiling
water. (Each tube contains
enough medium for two
plates.)
Avoid prolonged exposure
to unnecessarily high
temperatures during and
after melting.
When the medium
is melted in two or more
batches, use all of each
batch in order of melting,
provided the contents
remain fully melted.
Discard any melted agar
that contains precipitate.
Loosen caps before
heating to make it easier
to pour just after the media
has melted.
Page 1416
2. Keep the melted

medium in a water bath,
between 4446 C, until
used.
Do not depend on the
sense of touch to indicate
the proper temperature
when pouring the agar.
Place a thermometer in
the water bath to ensure
correct temperature
rangeto avoid
contamination, the
thermometer should be in
a separate container of
melted agar or water, not
in the agar tubes to be
plated.
3. Pipet the appropriate

amount of undiluted or
diluted sample (1 mL or
0.1 mLsee Diluting the
Sample) into the sterile
petri dish. Prepare at least
two plates for each
different volume of
undiluted or diluted
sample used.
Thoroughly mix all
undiluted and diluted
samples by inverting about
25 complete up-and- down
(or back-and-forth)
movements. Or, use a
mechanical shaker to
shake samples or dilutions
for 15 seconds.
Lift the lid of the petri dish
just high enough to insert
the pipet. Hold the pipet at
a 45 angle, with the tip
just touching the bottom of
the dish. Allow enough
time (at least
24 seconds) for the pipet
to drain.
4. Pour at least 10 to
12 mL of liquefied medium
( of the contents of Plate
Count Agar tube) into the
dish by gently lifting the
cover just high enough to
pour.
Avoid spilling the medium
on the outside of the
container or on the inside
of the dish lid when
pouring. Replace the lid
when finished.
Pour plate procedure for heterotrophic bacteria, method 8241 (continued)
5. Mix the melted

medium thoroughly with
the sample in the petri dish
by swirling in a figure-eight
motion with the petri dish
on the bench top.
Do not invert the petri dish
to mix.
6. Place the plates on a

level surface and let them
solidify. This generally
takes 10 minutes.
7. Invert the plates, place

them in a plastic bag, and
seal the bag. Place the
bag in an incubator that
has been prewarmed to
35 C.
8. Incubate the plates for

48 3 hours at
35 0.5 C.
During incubation,
maintain humidity within
the incubator so that
plates will not have
moisture weight loss
greater than 15%. A pan of
water placed at the bottom
of the incubator may be
sufficient. For incubation
in non-humidified
incubators, make certain
that the plastic bags are
tightly sealed.
9. Using a Quebec
Colony Counter, count all
colonies on the plates
promptly after incubation.
Reporting Results.
Page 1417
Diluting the Sample

sample.
Note: Standard microbiological procedures require at least 2 tests per sample.
undiluted
sample
pipet 1 mL
petri dish
1
petri dish
2
Figure 17 Dilution factor 1, 1-mL sample volume
undiluted
sample
pipet 0.1mL
petri dish
1
Figure 18 Dilution factor 10, 0.1-mL sample volume
Page 1418
petri dish
2
undiluted
sample
pipet 1 mL
99 mL
sample
blank
pipet 1 mL
petri dish
1
petri dish
2
undiluted
sample
pipet 1 mL
99 mL
sample
blank
pipet 0.1 mL
petri dish
1
petri dish
2

Interpreting and Reporting Results

Count all colonies on selected plates promptly after incubation. If count must be delayed
temporarily, store plates at 510 C for no more than 24 hours, but avoid routine delays.
Quebec Colony Counters feature a built-in grid to simplify counting. The easiest way to count
colonies is to follow a back and forth pattern, moving down the grid. See the Colony counting
technique figure.
Page 1419
Figure 21 Colony counting technique

Report all counts as colony-forming units (CFU)/mL. Include in the report the method used, the
incubation temperature and time, and the medium. For example, 75 CFU/mL, pour plate method,
35 C/48 hours, plate count agar.
Generally, results are obtained by averaging the number of colonies on all plates from the same
undiluted or diluted sample volume, and multiplying by a dilution (described below). In this case,
results should be rounded to two significant digits to avoid creating false precision. For three-digit
results, raise the middle digit if the last digit is 5 or greater. Retain the middle digit if the last digit is
4 or smaller. The last digit will be zero.
For example, 143 would become 140, 255 would become 260. Two digit numbers require no
rounding.
Be familiar with the following terms before counting and reporting results:
Average number of colonies/plate The average number of colonies per plate is derived by
dividing the total number of colonies on all plates that were inoculated with the same sample
volume or dilution volume, and dividing that sum by the number of plates used.
For example, if two plates were each inoculated with 1 mL of diluted sample, and there were 89
colonies on one plate and 103 on the other, then the average number of colonies/plate would be:
89
colonies + 103 colonies
---------------------------------------------------------------------= 96 colonies
2 plates
Colony-forming units (CFU)/mL This is the unit used for reporting bacterial density. To derive
the number of CFU/mL, multiply the average number of colonies/plate by the dilution factor of
the incubated sample.
Note: In some instances where a large number of colonies are observed, the average number of
colonies/plate is obtained by adding colonies counted only in a specified number of squares on each
plate.
Dilution factor The dilution factor is the reciprocal of the volume of original, undiluted sample
plated, and is used to standardize the results according to the sample volume.
For example, if 1 mL of original sample was used, the dilution factor is 1.
If 0.1 mL of original sample was used, the dilution factor is 10. The dilution factor for 1 mL of
diluted sample (0.01 mL of original sample) is 100, and the dilution factor for 0.1 mL of diluted
sample (0.001 mL of original sample) is 1000.
Representative colony distributionWhen counting colonies in a specified number of squares
(as seen through the colony counter), count those squares that appear to have an average
number of colonies. Avoid counting squares that have many less or many more colonies than most
of the other squares on the plate.
SpreadersSpreaders are colonies of bacteria which grow in such a way that they appear to be
spread across the plate. See the Spreader growth figure.
Page 1420
Figure 22 Spreader growth

It is preferable when counting and recording results, to consider plates having between 30 and 300
colonies. However, this is not always the case, so when counting and recording colonies, choose
the situation that best describes
your results.
If spreaders are encountered on the plates selected, count colonies on representative portions
only when the colonies are well distributed in
spreader-free areas, and the area covered by the spreaders does not exceed
one-half of the plate area.
When spreading colonies must be counted, count each of the following types as one colony:
1. A chain of colonies that appears to be caused by disintegration of a bacterial clump as agar
and sample were mixed.
2. A spreader that develops as a film of growth between the agar and the bottom of the petri dish.
3. A colony that forms in a film of water at the edge or over the agar surface.
Count as individual colonies the similar-appearing colonies growing in close proximity but not
touching, provided that the distance between them is at least equal to the diameter of the smallest
colony.
Also count as individual colonies those colonies which are touching, but are different in
appearance, such as morphology or color.
To obtain results, multiply the average number of colonies/plate by the dilution factor. Report
counts as CFU/mL.
If plates have excessive spreader growth, report as spreaders (Spr). When plates are
uncountable because of missed dilution, accidental dropping, or contamination, or the control
plates indicate that the medium or other material or labware was contaminated, report as
laboratory accident (LA).
No colonies If plates from all dilutions of any sample have no colonies, report the count as less
than one (<1) times the dilution factor for the largest volume of original sample used.
For example, if no colonies develop using 0.1 mL of original sample, report the count as less than
10 (<10) estimated CFU/mL.
Less than 30 colonies/plateOrdinarily, no more than 1.0 mL of sample is plated. Therefore,
when the total number of colonies developing from 1.0 mL is less than 30, record the number of
colonies as CFU/mL.
30 to 300 colonies/plateCompute bacterial count per mL by multiplying the average number of
colonies/plate by the dilution factor. Report counts as CFU/mL.
Page 1421
For example, 0.1 mL of undiluted sample was used to inoculate two plates. After incubation, the
plates had colony counts of 115 and 145. The CFU/mL value is computed as follows:
115 colonies + 145 colonies
-------------------------------------------------------------------------- 10 (dilution factor) = 1300 CFU/mL
2 plates
Greater than 300 colonies/plate If no plate has 30 to 300 colonies, and one or more plates
have more than 300 colonies, use the plates having a count closest to 300 colonies. Compute the
count by multiplying the average number of colonies/plate by the dilution factor, and report as
estimated CFU/mL.
Far more than 300 colonies/plate If there are greater than 300 colonies/plate,
do not report the result as too numerous to count (TNTC). Instead, follow guidelines for reporting
if there are less than 10 colonies/cm2:
Less than 10 colonies/cm2 If there are fewer than 10 colonies per cm2 (one square as seen
through the colony counter), then count colonies in 13 squares having representative colony
distribution.
a. Select seven consecutive squares horizontally across the plate, and six consecutive
squares vertically, being careful not to count a square more than once. See the Colony
counting, > 300 colonies figure.
b. Add the number of colonies in each square.
c. Multiply this sum by 4.38 when the plate area is 57 cm2 (disposable plastic plates).
d. Multiply the sum by 5 when the plate area is 65 cm2 (glass plates). To determine colonyforming units (CFU)/mL, compute the average number of colonies/plate and multiply the
result by the dilution factor. Report as estimated CFU/mL.
Figure 23 Colony counting, > 300 colonies

Note: One thousand is the dilution factor for 0.1 mL of diluted sample. This is the sample volume that should
be used when bacterial counts are this high.
More than 10 colonies/cm2When there are more than 10 colonies per cm2 (one square as
seen through the colony counter), count colonies in four squares having representative colony
distribution. Add the number of colonies in these four squares, and divide the sum by 4, to get the
average number of
colonies/square. Multiply this number by 57 when the plate area is 57 cm2 (disposable plastic
plates). Multiply this number by 65 when the plate area is 65 cm2 (glass plates). To determine
CFU/mL, compute the average number of colonies/plate and multiply the result by 1000 (see
Note below). Report as estimated CFU/mL.
Page 1422
Avoiding Errors
Avoid inaccuracies in counting due to damaged or dirty optics that impair vision, or due to failure to
recognize colonies. Be careful not to contaminate plates due to improper handling. Laboratory
workers who cannot duplicate their own counts on the same plate within 5%, and counts of other
analysts within 10%, should discover the cause and correct such disagreements.

Description
Unit
Catalog number
100/pkg
1436369
25/pkg
1430598
Plate Count Agar Tubes
20/pkg
2406720
Unit
Catalog number
2075333
Required apparatus
Description
Bags, Whirl-Pak1 with dechlorinating agent, sterile, 180-mL
100/pkg
Bags, Whirl-Pak without dechlorinating agent, sterile, 205-mL
100/pkg
2233199
Bags, Whirl-Pak without dechlorinating agent, sterile, 710-mL
10/pkg
1437297
Beaker, 250-mL
each
50046
Bottle, sample, glass, with cap, 118-mL
3/pkg
2163103
Clamp, Test Tube
each
63400
Colony Counter, Quebec, 110 VAC, 60 Hz
each
2252100
Colony Counter, Quebec, 220 VAC, 50 Hz
each
2252102
Counter, hand tally
each
1469600
Dish, Petri, 100 x 15 mm, sterile, disposable
20/pkg
2178901
Dish, Petri, 100 x 15 mm, sterile, disposable
500/pkg
2178900
Germicidal Cloths
50/pkg
2463200
each
2881500
Hot Plate, 120 VAC, 50/60 Hz

Hot Plate, 240 VAC, 50/60 Hz
each
2881502
Incubator, Culture, Low-Profile, 120 VAC, 50/60 Hz
each
2619200
Incubator, Culture, Low-Profile, 220 VAC, 50/60 Hz
each
2619202
50/pkg
2092835
25/pkg
209798
each
2635702
Thermometer -20 to 110 C, non-mercury

1
Page 1423

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Total Aerobic Bacteria, Yeasts and Molds, Paddle testers
Total Aerobic Bacteria, Yeasts

and Molds
DOC316.53.01223
Total Aerobic Bacteria (Amber)/Yeast and Mold (Red)

Total Aerobic Bacteria (Amber)/Total Coliform (Red)
Total Aerobic Bacteria (Amber)/Disinfection Control (Purple)
Paddle Testers
Scope and Application: For detection of contamination of equipment that contacts food, cosmetics or
pharmaceuticals, boiler and cooling tower water, laboratory surfaces requiring aseptic conditions and kitchen
surfaces in restaurants and hotels
Test preparation

To ensure accurate results, read carefully before proceeding.
Paddle testers have a 12-month shelf life from date of manufacture.
Paddle Testers are most suitable for semi-quantitative screening. Monitoring with Paddle Testers over time will provide a
good indication of how the microbial load is changing at a given site.
For greater accuracy when interpreting results, count the colonies with the aid of the molded-in grid.
Paddle testers in liquid
1. Remove the paddle

from the vial. Do not touch
the media.
2. Dip the paddle into a

liquid sample. Immerse all
the growth media on the
paddle into the liquid.

from the liquid and allow
excess liquid to drip off.
Do not shake. Return the
paddle to the vial and
screw on the cap.
4. Incubate the
Paddle Tester.
See the Incubation of
Paddle Testers for
incubation times.
Total Aerobic Bacteria, Yeasts and Molds

Page 1425

Paddle testers in liquid (continued)
5. Refer to the Bacterial

colony density and Yeast
and mold colony density
tables for results.
Sterilize the paddles
before disposal. (See
Disposing of Paddles
table.)
Paddle testers on solid and flat surfaces

from the vial. Do not touch
the media.
2. Press one side of the

paddle to the surface to be
tested.

Page 1426
3. Turn the paddle over

and press the other side to
a new area of the test
surface. Return the paddle
to the vial and screw on
the cap.
4. Incubate the
Paddle Tester.
See the Incubation of
Paddle Testers table for
incubation times.

Paddle testers on solid and flat surfaces (continued)
5. Refer to the Bacterial

colony density and Yeast
and mold colony density
tables for results.
Sterilize the paddles
before disposal. (See
Disposing of Paddles.)
Table 390 Incubation of Paddle Testers

Incubation of Paddle Testers
Incubation Temperature
Examine at:
Yeast and Mold
25 to 30 C
Bacteria: 24 to 48 hours
Yeast and Mold: 48 hours and up to 120 hours (5 days)
Total Coliform
35 to 37 C
24 to 48 hours
Disinfection Control
35 to 37 C
24 to 48 hours
Disposing of Paddles
To sterilize Paddle Testers before disposal:
Autoclave for 15 minutes at 121 C (250 F) at 103 kPa (15 psi) or
Pour 10 mL of household bleach (5.25% NaOCl) into the vial and allow it to sit for a minimum
of 30 minutes.
Interpreting the level of Contamination for total bacteria or total coliform bacteria
Identify the slide that most closely matches the sample. Use the density value above the slide.
Table 391 Bacterial colony density

100 (102)
1000 (103)
10,000 (104)
100,000 (105)
1,000,000 (106)
10,000,000 (107)

Page 1427

Table 391 Bacterial colony density
Interpreting the level of contamination for yeast and mold
Table 392 Yeast and mold colony density

10 (101)
100 (102)
1000 (103)
10,000 (104)
100,000 (105)

Required reagents and media
Description
Unit
Catalog number
Total Aerobic Bacteria/Yeast and Mold Paddle Testers
10/pkg
2610810
Total Aerobic Bacteria/Total Coliform Paddle Testers
10/pkg
2610910
Total Aerobic Bacteria/Disinfection Control Paddle Testers
10/pkg
2619510
Description
Unit
Catalog number
each
2619200
each
2619202
Required apparatus

Page 1428

Description
Unit
Catalog number
each
2595902
each
2898600
200/pkg
2463300

Battery eliminator
each
2580400
each
2580300
Beaker, 100-mL
each
50042H
12/pkg
2599112
50/pkg
2599150
12/pkg
2495012
50/pkg
2495050
100/pkg
1436369
Germicidal Cloths
50/pkg
2463200
each
2569900

15/pkg
2811115
100/pkg
2811199

Page 1429

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Electrochemistry
Page 1431
Page 1432
Conductivity, 8160
Conductivity
DOC316.53.01199
USEPA1 Direct Measurement Method2
Method 8160
(0.01 S/cm to 200.00 mS/cm)
Conductivity Meter

1
USEPA accepted for reporting for Standard method 2510-B
Procedure is equivalent to Standard Method 2510-B for wastewater.
Test preparation

Meter
Standard probe
Rugged probe1
HQ40d
CDC40101
CDC40103
CDC40105
CDC40110
CDC40115
CDC40130
HQ30d
CDC40101
CDC40103
CDC40105
CDC40110
CDC40115
CDC40130
HQ14d
CDC40101
CDC40103
CDC40105
CDC40110
CDC40115
CDC40130
sension 5
5197500
5197503
sension 7
5197500
5197503
Designed for field use.
Conductivity
Page 1433
Conductivity

Analyze samples as soon as possible after collection. However, samples may be stored at least 24 hours by cooling to 4 C
(39 F) or below (all storage temperatures have changed to 0 to 6 C as per the EPA MUR, March 2007). When measuring
solutions that are not at the reference temperature, the meter automatically adjusts the conductivity value to the reference
temperature from 20 or 25 C.
Water samples containing oils, grease, or fats will coat the electrode and affect the accuracy of the readings. If this occurs,
clean the probe with a strong detergent solution, then thoroughly rinse with deionized water.
Mineral build-up on the probe can be removed with a diluted 1:1 Hydrochloric Acid Solution. Refer to the meter user manual.
Calibration instructions are given in the operation section of the meter manual. For most accurate results calibrate before
use, or check the accuracy of the meter with a known conductivity standard.
Measurement errors can occur if the appropriate temperature correction value is not chosen. Refer to the Temperature
correction table for typical correction values.

Description
One of the following meter/sensor combinations:
Quantity
1
HQd meter and conductivity IntelliCAL probe

sension meter and conductivity electrode
One of the following Hach standards:

NaCl Standard Solution, 180 10 S/cm
NaCl Standard Solution, 18 10 mS/cm
Radiometer Analytical Certified Conductivity Standards:

KCl, 1 Demal, 111.3 mS/cm 0.5% at 25 C
500 mL
KCl, 0.1 Demal, 12.85 mS/cm 0.35% at 25 C
500 mL
KCl, 0.01 Demal, 1408 S/cm 0.5% at 25 C
500 mL
NaCl, 0.05%, 1015 S/cm 0.5% at 25 C
500 mL
KCl Conductivity Standards:

0.1 Molar KCl, 12.88 mS/cm at 25 C
500 mL
0.01 Molar KCl, 1413 S/cm at 25 C
500 mL
500 mL
Beaker, poly, 100-mL

Conductivity
Page 1434
Conductivity
Conductivity
1. Refer to the operation

section of the meter
manual to prepare the
conductivity electrode and
meter.
The meter will select the
range automatically.
For most accurate results,
calibrate the meter before
use or check the accuracy
of the meter with a known
conductivity standard.
Refer to the meter user
manual for calibration and
measurement options.
2. Laboratory tests:
Immerse the probe in a
beaker containing the
sample solution. Move the
probe up and down and
tap it on the beaker to
remove bubbles from the
electrode.
3. Turn the meter on.

Make sure that the meter
is set to measure
conductivity.
4. Rinse the probe

thoroughly with deionized
water after each
measurement.
To display other units such
as TDS, salinity or
resistivity (HQd only), refer
to the meter user manual.
Field tests: Immerse the

probe in the sample
solution. Move the probe
up and down to remove
bubbles from the
electrode.
The vent holes should be
completely submerged.
Conversions
The Unit conversion table provides equations for converting the conductivity readings to other
units of measure.
Table 394 Unit conversion

From
To
Use this Equation
mS/cm
S/cm
mS/cm x 1000
S/cm
mS/cm
S/cm x 0.001
S/cm
mhos/cm
S/cm x 1
mS/cm
mmhos/cm
mS/cm x 1
S/cm
mg/L TDS
S/cm x 0.51
g/L TDS
mg/L TDS
g/L TDS x 1000
mS/cm
g/L TDS
mS/cm x 0.5
mg/L TDS
g/L TDS
mg/L TDS x 0.001
mg/L TDS
gpg TDS
mg/L TDS x 0.05842
g/L TDS
gpg TDS
g/L TDS x 58.42
S/cm
ohms cm
1,000,000 S/cm
mS/cm
ohms cm
1,000 mS/cm
TDS is an empirically-derived value from the conductivity measurement. A value of 0.5 is selected here for simplicity and suitability to a wide
variety of waters. The sension 5 uses a more complex algorithm, based on additional factors, such as temperature, to determine TDS.
Conductivity
Page 1435
Conductivity
The Temperature correction table shows typical temperature correction values for selected
solutions using the linear temperature correction option.
Table 395 Temperature correction

Solution
Percent per C
Ultrapure Water
4.55
Salt (NaCl)
2.125
NaOH
1.72
Dilute Ammonia
1.8810
10% HCl
1.325
5% Sulfuric Acid
0.9698
Interferences
When measuring conductivity, the following items should be considered in order to ensure
accurate results:
If measuring very low levels of conductivity, protect the sample from atmospheric gases
(carbon dioxide, ammonia). These gases dissolve readily in water and may cause a rapid
change in conductivity. To minimize these effects, boil the sample, then place in a covered
container, such as a Low Ionic Strength (LIS) chamber for cooling.
To remove the conductivity with hydroxide ions, neutralize by adding 4 drops of

Phenolphthalein Indicator Solution to 50 mL of sample, then adding Gallic Acid Solution, dropwise, until the pink color completely disappears.
Accuracy check
Pour a Sodium Chloride Standard Solution (with a conductivity value in the same range as the
sample) into a beaker. Perform the conductivity measurements as described above. The
conductivity reading should be the same (within accuracy limits) as listed on the Standard Solution
label if the meter is calibrated correctly. Calibration can be performed using this solution. Refer to
the meter user manual.
Method performance
The accuracy of a conductivity measurement is dependent on many factors associated with the
overall system, including the meter, meter settings, choice of electrode and conductivity standards
being used during calibration. Refer to the appropriate electrode, meter manual and standard
certificate of analysis to help determine system performance.
Summary of method
Electrolytic conductivity is the capacity of ions in a solution to carry electrical current and is the
reciprocal of the solution resistivity. Current is carried by inorganic dissolved solids (e.g., chloride,
nitrate, sulfate, and phosphate anions) and cations (e.g., sodium, calcium, magnesium, iron, and
aluminum). Organic material like oils, phenols, alcohols, and sugars do not carry electrical current
well and thus do not have enough conductivity for a useful estimate of concentration.
Measuring conductivity is done by measuring the resistance occurring in an area of the test
solution defined by the probes physical design. Voltage is applied between the two electrodes
immersed in the solution, and the voltage drop caused by the resistance of the solution is used to
calculate conductivity per centimeter. The basic unit of measure for conductivity is the Siemen
(or mho), the reciprocal of the ohm in the resistance measurement. Because ranges normally
found in aqueous solutions are small, milliSiemens/cm (103 S or S/cm) and microSiemens/cm
(106 S or S/cm) are most commonly used.
Conductivity
Page 1436
Conductivity

Required apparatus
Description
Quantity
Unit
Catalog number
Select one meter and probe combination:

HQ40d Meter
each
HQ40d53000000
HQ30d Meter
each
HQ30d53000000
HQ14d Meter
each
HQ14d53000000
IntelliCAL Conductivity Probe, standard, with 1m cable
each
CDC40101
IntelliCAL Conductivity Probe, standard, with 3m cable
each
CDC40103
IntelliCAL Conductivity Probe, rugged, with 5m cable
each
CDC40105
each
CDC40110
each
CDC40115
IntelliCAL Conductivity Probe, rugged, with 30 m cable
each
CDC40130
sension 5
each
5180000
sension 7
each
5450000
sension meters and probes. Select one meter and probe

combination:
Conductivity probe, with 1 m cable
each
5197500
Conductivity probe, with 3 m cable
each
5197503
Unit
Catalog number
Sodium Chloride Standard Solution, 180 10 mS/cm, 90 1 mg/L TDS
100 mL
2307542
100 mL
1440042
100 mL
210542
Sodium Chloride Standard Solution, 18,000 50 mS/cm, 9000 25 mg/L TDS
100 mL
2307442
S51M001
Description
Hach, NaCl Conductivity Standards:
Radiometer Analytical, Certified Conductivity Standards:

KCl, 1 Demal, 111.3 mS/cm 0.5% at 25 C
500 mL
KCl, 0.1 Demal, 12.85 mS/cm 0.35% at 25 C
500 mL
S51M002
KCl, 0.01 Demal, 1408 S/cm 0.5% at 25 C
500 mL
S51M003
NaCl, 0.05%, 1015 S/cm 0.5% at 25 C
500 mL
S51M004
KCl Conductivity Standards:

0.1 Molar KCl, 12.88 mS/cm at 25 C
500 mL
C20C250
500 mL
C20C270
500 mL
C20C280
Conductivity
Page 1437
Conductivity

Description
Unit
Beaker, poly, 100-mL

Gallic Acid Solution
Hydrochloric Acid Solution, 1:1
Catalog number
each
108042
50 mL SCDB
1442326
500 mL
88449
Low Ionic Strength Chamber (LIS)
each
5189900
Phenolphthalein Indicator Solution
15 mL SCDB
16236
Wash Bottle, 125-mL

Water, deionized

each
62014
4L
27256
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Fluoride, acid solutions
Fluoride in Acid Solutions
DOC316.53.01236
Direct Measurement ISE Method
Method 8323
0.1 to 100.0 mg/L F
ISE Electrode
Scope and Application: For industrial waters (solutions below a pH of 5)
Test preparation


Meter
Electrode
sension 4 meters
5192800
sension 2 meters1
5192800
The user must construct the calibration curve with the sension 2 meter.

Refer to the meter user manual for meter operation. Refer to electrode manual for electrode maintenance and care.
Prepare the electrode. Refer to Electrode assembly and Condition the electrode in this procedure.
In solutions below a pH of 5, hydrogen ion complexes some of the fluoride ions, forming HF or HF2 , which the electrode will
not detect. To free the complexed fluoride, adjust the pH of the solution so it is weakly acidic to weakly basic before analysis.
Do not use a strong base (such as sodium hydroxide) for adjustment since the amount of base added will vary from sample
to sample. Using a strong base can also change the ionic strength of the sample and alter measurement accuracy.
Dilution of samples and standards with a large excess of sodium acetate will buffer the pH and help adjust the total ionic
strength of samples and standards to the same level.

Description
Quantity
Fluoride ISA buffer pillows (TISAB)
1 mL
Sodium acetate, ACS
varies
Fluoride standard solutions:

10.00-mg/L F or
varies
2.00-mg/L F or
varies
100.0-mg/L F
varies
Potassium Chloride Reference Electrolyte Gel Cartridges

Water, deionized
1
varies

Page 1439

Description
Quantity
Tensette pipet, 1.010.0 mL or
Class A 1000 mL volumetric
Platinum Series Fluoride Combination Electrode, BNC
sension2 Portable pH/ISE Meter.
OR
sension4 Laboratory pH/ISE Meter
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm)
Stirrer, electromagnetic with stand and stir bar
Fluoride in Acid Solutions method
1. Prepare a 15%
sodium acetate solution by
dissolving 150 g of reagent
grade sodium acetate in
1000 mL of deionized
water. Prepare enough
solution to dilute all the
samples and standards.

Page 1440
2. Using deionized water,

prepare a background
solution containing all the
sample matrix
components except
fluoride. Use this solution
to prepare the standards.
If a standard prepared with
background solution gives
the same reading (after
sodium acetate dilution) as
standard prepared with
pure sodium acetate, it is
not necessary to prepare a
background solution.
3. For calibration,
prepare fluoride standards
by adding fluoride to the
background solution.
Prepare fresh standards
every two weeks if
standards are less than 10
mg/L F.
When testing with a direct
reading fluoride meter, use
two standards. When
testing with a pH/mV
meter, use three
standards.
4. Dilute each standard

10:1 with sodium acetate
solution (9 parts sodium
acetate solution to 1 part
standard) to prepare the
buffered solution. Mix
thoroughly.
For example, measure
3 mL of standard (with
background solution) and
dilute it with 27 mL of
sodium acetate solution.

Fluoride in Acid Solutions method (continued)

one Fluoride Total Ionic
Strength Adjustment
Buffer (TISAB) Powder
Pillow to 25 mL of the
prepared buffered sample.
Stir to dissolve.
6. Turn the meter on. Set

the electrode type to BNC.
Set the units to mg/L.
Press CAL.
manual for details.
7. Place the beaker with

the 1-mg/L prepared
buffered standard on a
magnetic stirrer. Stir at a
moderate rate.
Stabilizing...
Repeat 710
9. Edit the display to

show the concentration of
the standard.
10. The display will show

Stabilizing until the
measurement is complete.

the 10-mg/L and 100 mg/L
standards.
Press ENTER and accept

the concentration.
Remove the electrode

from the standard solution
Rinse with deionized water
and blot dry.
After the last standard is

measured, store the
calibration in the meter.
manual for details.
13. Dilute each unknown

10:1 with sodium acetate
solution before
measurement.
14. Place a stir bar in the

beaker and set the beaker
on a magnetic stirrer. Stir
at a moderate speed.
15. Rinse the electrode

blot dry.
8. Place the electrode

into the standard.

into 50-mL beakers to
measure 25 mL of
prepared buffered sample.
For example, measure
3 mL of sample and dilute
it with 27 mL of sodium
acetate solution. Pour
25 mL of this solution into
a 50-mL beaker.

into the sample. When the
reading is stable, record
the value.
Page 1441
Electrode assembly
1. Remove the cap from the electrolyte cartridge.
2. Visually inspect the Luer tip of the electrolyte cartridge. If air is present, rotate the feed-screw
counter-clockwise until gel expels the air and fills the tip.
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body (Figure 24).
Figure 24 Attach the outlet tube

4. Place the dispenser unit over the electrolyte cartridge. Screw the dispenser unit onto the
electrode body until reaching the stop. Do not over tighten.
5. Dispense the electrolyte gel by pressing the pump button. Repeat this procedure until gel is
visible at the reference outlet (Figure 25).
Figure 25 Dispense the electrolyte gel

6. Rinse the electrode with deionized water. Do not scratch the crystal.
7. To remove an empty cartridge, unscrew the dispenser unit and rotate the cartridge
counterclockwise while gently pulling it out of the electrode.

Page 1442

8. Connect the BNC connector of the electrode to the BNC connector on the meter (Figure 26).
Figure 26 BNC connector

Note: One BNC and one 5-pin connector are on the back of the meter. Choose the BNC for the fluoride
electrode. Disconnect the pH electrode from the 5-pin connector when using the BNC connector.
Condition the electrode

Condition and store the electrode in 1 mg/L Fluoride standard storage with Ionic Strength Adjuster
for 15 to 30 minutes.
For electrode storage procedures, refer to the Fluoride Electrode Instruction Manual.
Clean the Lanthanum Fluoride Crystal

It may be necessary to clean the LaF crystal on the sensing tip of the probe if it becomes covered
with organic film or buildup.
1. Put a small amount of fluoride toothpaste on a soft toothbrush or cloth.
2. Gently rub the LaF crystal with the toothpaste using a circular motion. Rub until the film is
removed.
3. Thoroughly rinse the probe with deionized water and blot dry. Verify the crystal is clean. If not,
repeat cleaning and rinsing until it is clean.
4. If the crystal becomes contaminated by oil, grease, or fingerprints, soak for a few minutes in
isopropyl alcohol then rinse with deionized water.
Interferences
Interference level
Cations
Do not interfere
Cl, Br, SO42, HCO3,

PO43, acetate
Do not interfere
Interferes: refer to pH Effects. Some ions, such as CO32 or PO43, make the sample more
OH (Hydroxyl ions)
basic, which increases OH interference, but do not directly interfere with the electrode
operation.
CO32 or PO43
Can make the sample more basic and increase OH

Page 1443
pH Effects
In solutions with a pH below 5, hydrogen ion complexes some of the fluoride ions, forming the
undissociated acid HF and the ion HF2. Figure 27 shows the proportion of free fluoride ion in acid
solutions.
If the background ionic strength is high and constant in comparison with the ion being measured,
the activity coefficient is constant and activity is directly proportional to ion concentration. Total
ionic strength adjustor is added to standards and samples to make the background ionic strength
high, decomplex fluoride, and adjust the solution pH to 5.05.5.
Figure 27 Ratio of free F in acid solutions
Collect samples in plastic bottles.
Samples may be stored up to 28 days.
Accuracy check
To verify measurement accuracy, perform a standard addition spike on the sample. The spike
should roughly double the measured concentration without significantly diluting the sample.
To perform a standard addition sample:
1. Use the Spike volumes table to determine the concentration and volume of standard to spike
the sample. The volume of sample transferred must be accurate.
2. Add the amount and concentration specified in the Spike volumes table to the sample.
3. After adding the standard, proceed with the calculations. Results from 90-110% recovery are
considered acceptable. Calculate percent recovery as follows:
100 ( X s X u )
% Recovery = ---------------------------------K
Where:
Xs = measured value for spiked sample in mg/L
Xu = measured value for unspiked sample adjusted for dilution by the spike, in mg/L
K = known value of the spike in the sample in mg/L
Calculations
1.
Xi Vu
X u = ----------------Vu + V

Page 1444

Where:
Xi = measured value of unspiked sample in mg/L
Vu = volume of separate unspiked portion in mL
V = volume of spike in mL
2.
CV
K = ----------------Vu + V
Where:
C = concentration of standard used in spike in mg/L
Vu = volume of separate portion before spike in mL
100 ( X X )
K
s
u
3. Final calculation plugging in Xu and K: % Recovery = ----------------------------------
Example:
A sample was analyzed and read 5.0 mg/L F. As directed in the Spike volumes table, a 1.0-mL
spike of 100-mg/L F standard was added to another 25-mL sample, giving a final standard
addition result of 8.75 mg/L.
Calculate the percent recovery as follows:
1.
5.0 mg/L 25 mL
X u = ---------------------------------------------- = 4.81 mg/L
25 mL + 1 mL
2.
100 mg/L 1 mL
K = --------------------------------------------- = 3.85 mg/L
25 mL + 1 mL
3.
100 ( X s X u )
100 ( 8.75 4.81 )
%R = ---------------------------------------= -------------------------------------------------- = 102.3 % Recovery
K
3.85
Table 398 Spike volumes

Measured Sample Concentration
(mg/L)
Measured Sample Volume

(mL)
Standard Concentration
(mg/L)
Standard Volume
(mL)
0.10.6
25
100
0.1
0.61.0
25
100
0.2
1.01.5
25
100
0.3
1.53.0
25
100
0.5
36
25
100
1.0
2.0
610
25
100
1015
25
100
3.0
1525
25
1000
0.5
2535
25
1000
0.7
3550
25
1000
1.0
50100
25
1000
2.0
Summary of method
The fluoride electrode consists of a sensing Lanthanum Fluoride element bonded into an epoxy
body. When the sensing element contacts fluoride ions in a solution, a potential develops across
the sensing element. The potential is proportional to the level of fluoride ions present. The
potential is measured against a constant reference potential with a pH/mV meter or ISE meter.

Page 1445

Required reagents
Description
Quantity/Test
Unit
Catalog number
Fluoride Standard Solutions:

1.00-mg/L F or
varies
500 mL
29149
10.00-mg/L F or
varies
500 mL
40520
100.0-mg/L F
varies
500 mL
35949
Potassium Chloride Reference Electrolyte

varies
2/pkg
2546902
Sodium Acetate, ACS
Gel Cartridges
varies
454 g
17801H
Water, deionized
varies
4L
27256
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
108041
each
62011
each
108140
Fluoride Combination Electrode, BNC, w/ filling solution
each
5192800
sension 2 Portable pH/ISE Meter
each
5172500
sension 4 Laboratory pH/ISE Meter
each
5177500
Stir Bar, 7/8 x 3/16 in. (22.2 x 4.8 cm)
each
4531500
OR

Stirrer, electromagnetic, 115 VAC, with stand and stir bar
each
4530001
each
4530002
Tensette pipet 1.010.0 mL
each
1970010
Class A, 25 mL volumetric pipet
each
1451540
Safety bulb pipet filler
each
1418900
Class A, 1000 mL volumetric flask
each
1457453
Description
Unit
Catalog number
Electrode Washer
each
2704700
Pipet, TenSette, 0.1 to 1. 0 mL
each
1970001
50/pkg
2185696
Optional apparatus
Pipet Tips, for 19700-01 TenSette Pipet

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Fluoride, drinking water, 8323
Fluoride in Drinking Water
DOC316.53.01237
USEPA1 Direct Measurement ISE Method

0.1 to 10.0 mg/L
Method 8323
Powder Pillow or TISAB Solution
Scope and Application: Drinking water

1
USEPA equivalent method
Test preparation


Meter
Electrode
sension 4 meters
5192800
sension 2 meters1
5192800
The user must construct the calibration curve with the sension 2 meter.

For USEPA reporting, replace the standards in step 1 with a 0.5-mg/L, 1.0-mg/L and 2.0-mg/L fluoride standard solution to
calibrate the electrode.
Prepare the electrode. Refer to Electrode assembly and Condition the electrode in this procedure.

Description
Fluoride ISA buffer pillows (TISAB)
Quantity
1
OR
Fluoride ISA solution, concentrated (TISAB)
5.0 mL
Sodium acetate, ACS
varies
Fluoride standard solutions:

1.00-mg/L F
varies
2.00-mg/L F or
varies
0.5 mg/L F (USEPA)
varies
Potassium chloride reference electrolyte gel cartridges
varies
Water, deionized
varies

Page 1447

Description
Quantity
3 or 4 (USEPA)
Platinum series fluoride combination electrode, BNC
sension2 Portable pH/ISE Meter. or sension4 Laboratory pH/ISE Meter
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm)
2 or 3 (USEPA)
Fluoride in drinking water, powder pillows method
1. In 50-mL beakers,
prepare two 25-mL
standard solutions of
1-mg/L and 10-mg/L F.

one Fluoride Total Ionic
Strength Adjustment
Buffer (TISAB) Powder
Pillow to each standard.
Stir to dissolve.
Standard

into the standard.

Page 1448

Press CAL.

the 1-mg/L standard on a
moderate rate.

manual for details.
Stabilizing...
Repeat 47

the standard.


the 10-mg/L and 0.5 mg/L
(USEPA) standard.
Press ENTER to accept the

concentration.

and blot dry.

measured, store the
manual for details.

Fluoride in drinking water, powder pillows method (continued)
sample to a 50-mL beaker.
Add a stir bar to the
beaker. Place the beaker
on a magnetic stirrer and
stir at a moderate rate.
10. Remove the electrode

from the standard solution.
Rinse it with deionized
water and blot dry. Place it
into the sample.

one TISAB Powder Pillow.
Stir to dissolve.
12. After the

measurement is stable,
record or store the
measurement value.
The sample should be the

same temperature as the
standards, 1 C.
Repeat 912
13. Repeat steps 9

through 12 for each
sample.

after reading the last
sample. Rinse the
electrode. Store in a
fluoride standard of similar
concentration to the
sample that will be
analyzed next.
Refer to the electrode
manual for more
information on electrode
storage.

Page 1449

Fluoride in drinking water, liquid TISAB solution method
prepare two 25-mL
standard solutions of
1-mg/L and 10-mg/L F.
2. Add the contents of 5

mL concentrated Fluoride
Total Ionic Strength
Adjustment Buffer (TISAB)
per 25 mL of standard. Stir
to mix.
Standard

into the standard.

Page 1450

Press CAL.

the 1-mg/L standard on a
moderate rate.

manual for details.
Stabilizing...

the standard.

Accept the concentration.

and blot dry.
Repeat 47

the 10-mg/L standard.
measured, store the
manual for details.

Fluoride in drinking water, liquid TISAB solution method (continued)
sample to a 50-mL beaker.
Add a stir bar to the
beaker. Place the beaker

water and blot dry. Place it
into the sample.
11. Add 5 mL
concentrated liquid TISAB
to the sample. Stir to mix.
12. After the

record or store the
measurement value.
The sample should be the

standards, 1 C.
Repeat 912
13. Repeat steps 9

through 12 for each
sample.

after reading the last
sample. Rinse the
electrode. Store in a
fluoride standard of similar
sample that will be
analyzed next.
manual for more
storage.

Page 1451
Electrode assembly
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body (Figure 28).

visible at the reference outlet (Figure 29).

6. Rinse the electrode with deionized water. Do not scratch the crystal.
7. To remove an empty cartridge, unscrew the dispenser unit and rotate the cartridge
counterclockwise while gently pulling it out of the electrode.

Page 1452

8. Connect the BNC connector of the electrode to the BNC connector on the meter (Figure 30).

Note: One BNC and one 5-pin connector are on the back of the meter. Choose the BNC for the fluoride
electrode. Disconnect the pH electrode from the 5-pin connector when using the BNC connector.
Condition the electrode

Condition and store the electrode in 1 mg/L Fluoride standard storage with Ionic Strength Adjuster
for 15 to 30 minutes.
For electrode storage procedures, refer to the Fluoride Electrode Instruction Manual.
Clean the Lanthanum Fluoride Crystal

It may be necessary to clean the LaF crystal on the sensing tip of the probe if it becomes covered
with organic film or buildup.
1. 1. Put a small amount of fluoride toothpaste on a soft toothbrush or cloth.
2. Gently rub the LaF crystal with the toothpaste using a circular motion. Rub until the film is
removed.
3. Thoroughly rinse the probe with deionized water and blot dry. Verify the crystal is clean. If not,
repeat cleaning and rinsing until it is clean.
4. If the crystal becomes contaminated by oil, grease, or fingerprints, soak for a few minutes in
isopropyl alcohol then rinse with deionized water.
Interferences
Interference level
Cations
Do not interfere
Cl, Br, SO42, HCO3,

PO43, acetate
Do not interfere
OH (Hydroxyl ions)
Interferes: refer to
CO32 or PO43
Make the sample more basic and increase OH

Page 1453
pH Effects
In solutions with a pH below 5, hydrogen ion complexes some of the fluoride ions, forming the
undissociated acid HF and the ion HF2. Figure 31 shows the proportion of free fluoride ion in acid
solutions.
If the background ionic strength is high and constant in comparison with the ion being measured,
the activity coefficient is constant and activity is directly proportional to ion concentration. Total
ionic strength adjustor is added to standards and samples to make the background ionic strength
high, decomplex fluoride, and adjust the solution pH to 5.05.5.
Figure 31 Ratio of free F in acid solutions
Collect samples in plastic bottles.
Samples may be stored up to 28 days.
Accuracy check
Checking electrode response
To verify measurement accuracy, measure the electrode potential of two fluoride standard
solutions that are one decade apart in concentration. For example, use 1-mg/L and 10-mg/L
standards to bracket an expected sample concentration of 3 mg/L. The two standards should have
mV potentials that are 58 3 mV apart at 25 C. Both solutions must be greater than 0.2 mg/L F.
Checking calibration accuracy
To verify calibration accuracy, measure the concentration of a known standard (e.g., 2.00 mg/L)
within the calibration range.
Checking the accuracy of the sample reading
To verify sample measurement accuracy, add a spike of standard fluoride solution with a
TenSette or volumetric pipet. Use the Spike Volumes table and the formulas in.
Table 401 Spike Volumes

Measured sample
Concentration
Volume & Concentration of F Standard to Add

CxV
V
0.61 mg/L
0.3 mL of...
...100-mg/L
30
12 mg/L
0.5 mL of...
...100-mg/L
50
36 mg/L
1.0 mL of...
...10-mg/L
100

Page 1454

Percent recovery
To calculate the percent recovery (only applicable if sample volume is 25 mL):
M = S 25 + C V
M
E = ----------------25 + V
A
R = ---- 100%
E
Where:
M = calculated mass of fluoride present after the spike (micrograms)
S = mg/L of F in sample (before spike)
C = concentration of standard used for spiking (mg/L)
V = spike volume from the Spike Volumes table (mL)
E = expected concentration after spiking (mg/L)
R = percent recovery (should be 95100%)
A = actual reading on meter after spike (mg/L F)
Method performance
Instrument
Standard
Precision
95% Confidence Limits of Distribution
sension 4
1.6 mg/L
1.5951.605 mg/L
sension 2
Summary of method
The fluoride electrode consists of a sensing Lanthanum Fluoride element bonded into an epoxy
body. When the sensing element contacts fluoride ions in a solution, a potential develops across
the sensing element. The potential is proportional to the level of fluoride ions present. The
potential is measured against a constant reference potential with a pH/mV meter or ISE meter.

Required reagents
Description
Fluoride ISA buffer pillows
Quantity/Test
Unit
Catalog number
100/pkg
258999
5 mL
3.78 L
2829017
29149
OR
Fluoride ISA solution
Fluoride Standard Solutions:
1.00-mg/L
varies
500 mL
2.00-mg/L
varies
500 mL
40520
10.0-mg/L
varies
500 mL
35949
Potassium Chloride Reference Electrolyte Gel Cartridges
varies
2/pkg
2546902
Water, deionized
varies
4L
27256

Page 1455
Required apparatus
Description
Quantity/Test
Unit
Catalog number
108041
each
each
62011
each
108140
Fluoride Combination Electrode, BNC, w/ filling solution
each
5192800
each
5172500
each
5177500
each
4531500
OR
Stir Bar,
7/8
3/16
in. (22.2 x 4.8 cm)

each
4530001
each
4530002
Description
Unit
Catalog number
Electrode Washer
each
2704700
Pipet, TenSette, 0.1 to 1. 0 mL
each
1970001
50/pkg
2185696
Optional apparatus
Pipet Tips, for 19700-01 TenSette Pipet

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate, 8358
Nitrate
DOC316.53.01238

0.1 to 100.0 mg/L NO3N
Method 8358
Powder Pillow or TISAB Solution
Scope and Application: Water and wastewater
Test preparation


Meter
Electrode
sension 4 meters
5192000
sension 2 meters
5192000

Prepare the electrode. Refer to Electrode assembly and Nitrate half-cell preparation in this procedure.
When using the electrode for the first time, condition the reference electrode for eight hours in a 100 mg/L nitrate-nitrogen
standard
Temperature variation causes inaccurate measurements. Calibration and sample measurements should be made at the
same temperature 1 C.
The stable sample reading is the nitrate-nitrogen (NO3N) concentration expressed as elemental nitrogen (N). The results
can be expressed as mg/L nitrate (NO3) by multiplying the results by 4.4.

Description
Ammonium sulfate reference electrolyte gel cartridge
Nitrate half-cell internal filling solution
Nitrate ISA powder pillows
Quantity
1
0.5 mL
1 per sample or standard
OR
Nitrate ISA solution
25 mL per sample or standard
Nitrate-Nitrogen standard solutions:

1-mg/L as NO3N
25 mL
10-mg/L as NO3N
25 mL
Nitrate
Page 1457
Nitrate
Description
Quantity
100-mg/L as NO3N
25 mL
Water, deionized
varies
Combination Nitrate Electrode, BNC
pH/ISE meter, sension2 (portable) or sension4 (laboratory)
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm)
TenSette pipet, 1.010.0 mL
OR
Membrane Tip, for nitrate electrode
Nitrate-Nitrogen, powder pillow ISA method
prepare three 50-mL
standard solutions of 1, 10
and 100 mg/L
NO3N.

to pipet 25 mL of standard
into each beaker. Add the
contents of one Ionic
Strength Adjustor (ISA)
powder pillow into each
beaker to make the
buffered standard.
Stir to dissolve.
Nitrate
Page 1458

Press CAL.
manual for details.
4. Add a stir bar to each

beaker. Put the beaker
with the 1-mg/L buffered
standard on a magnetic
stirrer. Stir at a moderate
rate.
Starting with the lowest
concentration standard
reduces carry-over
contamination and gives
optimal electrode
response.
Nitrate
Nitrate-Nitrogen, powder pillow ISA method (continued)
Repeat 47
5. Put the electrode into

the 1.0 mg/L buffered
standard.

the standard.
concentration.

water and blot dry.

25.0 mL of sample into a
100-mL beaker.
7. When the
measurement stabilizes,
remove the electrode from
the standard solution
and blot dry.

buffered standards.

one ISA powder pillow into
the beaker with the sample
to make the buffered
sample. Stir to dissolve.

buffered sample. Put the
buffered sample on a
stirrer and stir at a
moderate rate. Put the
electrode into the buffered
sample.
A white precipitate will

form if chloride or other
ions are present. This will
not harm the electrode or
interfere with the analysis.

measured, store the
manual for details.
Nitrate
Page 1459
Nitrate
Nitrate-Nitrogen, powder pillow ISA method (continued)
13. Press READ. When

the measurement
stabilizes, record or store
the value.

after reading the sample.
Rinse the electrode. Store
in a nitrate standard
(without ISA) of similar
sample that will be
analyzed next.
manual for more
storage.
Nitrate-Nitrogen, liquid ISA method
prepare three 50-mL
standard solutions of 1, 10
and 100 mg/L
NO3N.
Nitrate
Page 1460

to pipet 25 mL of standard
into each beaker. Pipet
25 mL of liquid Ionic
Strength Adjustor (ISA)
into each beaker to make
the buffered solution.

Press CAL.
manual for details.
4. Add a stir bar to each

beaker. Put the beaker
with the 1-mg/L standard
on a magnetic stirrer. Stir
at a moderate rate.
Starting with the lowest
concentration standard
reduces carry-over
contamination and gives
optimal electrode
response.
Nitrate
Nitrate-Nitrogen, liquid ISA method (continued)
Repeat 47

the 1.0 mg/L buffered
standard.

the standard.
the concentration.

from the buffered
standard. Rinse it with
deionized water and blot
dry.

25.0 mL of sample into a
100-mL beaker.
7. When the
remove the electrode from
the standard solution
and blot dry.

buffered standard.
11. Pipet 25.0 mL of liquid

ISA into the beaker with
the sample.

buffered sample. Put the
buffered sample on a
moderate rate. Put the
electrode into the buffered
sample.

ions are present. This will
not harm the electrode or
interfere with the analysis.

measured, store the
manual for details.
Nitrate
Page 1461
Nitrate
13. Press READ when the

measurement stabilizes to
record or store the value.

after reading the sample.
Rinse the electrode. Store
in a nitrate standard of
similar concentration to
the sample that will be
analyzed next.
manual for more
storage.
Nitrate half-cell preparation

1. Condition the nitrate membrane tip by immersing the membrane tip in beaker with 100 mg/L
NO3N solution for approximately one hour prior to use. When using the electrode for the first
time, condition the reference electrode for eight hours in a 100 mg/L nitratenitrogen standard.
2. Clean all gel from the electrode tip and glass stem. Rinse with Deionized water and dry
completely with a soft paper towel.
3. Use a soft paper towel soaked with isopropyl or rubbing alcohol to wipe the glass stem. Dry
the stem with a dry, soft paper towel. Make sure that alcohol does not get into the glass stem.
4. Use the provided syringe to fill the membrane to with the electrode filling solution. Fill with
solution to the top of the tip. Alternatively, fill the glass stem as shown in the electrode manual.
Do not fill the stem completely or the pressure will cause the membrane tip to fall off.
5. Use a gentle, twisting action to carefully slide a clean, dry nitrate membrane tip over the glass
stem until the end of the stem rests midway through the Nitrate Membrane Tip and some
resistance is met. Leave a 1/8-inch gap between the tip and the electrode body.
6. Shake the electrode as if shaking down mercury in a thermometer to make sure the Nitrate
Electrode Filling Solution contacts the end of the nitrate membrane tip. Verify that air bubbles
are not present in the tip.
Electrode assembly
Nitrate
Page 1462
Nitrate
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body (Attach the outlet
tube).

visible at the reference outlet (Dispense the electrolyte gel). If readings become erratic make
sure that the electrolyte gel is completely purged through the reference line.

6. Rinse the electrode with deionized water. Do not scrub the electrode tip.
7. Connect the BNC connector of the electrode to the BNC connector on the meter (BNC
connector).
Nitrate
Page 1463
Nitrate
Interferences
For the nitrate electrode, the major interferences include perchlorate, iodide, nitrite, bromide and
chloride. The addition of Nitrate ISA will eliminate most of these interferences. The highest level of
chloride the ISA can accommodate is 40 mg/L Cl. Concentrations greater than 40 mg/L Cl in the
sample will cause false high nitrate values. For more information on selectivity coefficients without
ISA, refer to the Electrode Characteristics section of the electrode manual.
Collect samples in clean glass or plastic bottles. Start nitrate analysis promptly after sampling.
If storage is necessary, store for up to 24 hours at 4 C or lower (06 C as per USEPA MUR
March 2007).
For longer storage periods, adjust sample pH to 2 or less with 2 mL sulfuric acid per liter of
sample. Sample refrigeration is still necessary. When sample is preserved with acid, NO3 and
NO2 cannot be determined.
Before testing a preserved sample, warm to room temperature, then neutralize to

approximately pH 7 with 5.0 N Sodium Hydroxide Standard Solution.
Do not use mercury compounds as preservatives.
Accuracy check
To verify electrode response, measure the electrode potential (in mV) of two NitrateNitrogen
standard solutions one decade apart in concentration, bracketing the expected sample
concentration. For example, use 10 and 100 mg/L NitrateNitrogen Standard Solutions to bracket
an expected sample concentration of 30 mg/L. The two solutions should have potentials (in mV)
that are 58 3 mV apart at 25 C. Both solutions must be above 5 mg/L NO3N.
To verify calibration accuracy, measure the concentration of a known standard within the
calibration range.
To verify sample measurement accuracy, add a spike of standard NO3N solution with a
TenSette or volumetric pipet. Use the Spike volumes table and the formulas in Percent recovery.
Table 403 Spike volumes

Measured sample
Concentration
Volume & Concentration of F Standard to Add

CxV
V
12 mg/L
0.5 mL of
100 mg/L
50
36 mg/L
1.0 mL of
100 mg/L
100
715 mg/L
0.3 mL of
1000 mg/L
300
1530 mg/L
0.5 mL of
1000 mg/L
500
Nitrate
Page 1464
Nitrate
Percent recovery
To calculate the percent recovery (only applicable if sample volume is 25 mL):
M = S 25 + C V
M
E = ----------------25 + V
A
R = ---- 100%
E
Where:
M = calculated mass of nitrate as nitrogen present after the spike (micrograms)
S = mg/L of NO3N in sample (before spike)
V = spike volume from the Spike volumes table (mL)
A = actual reading on meter after spike (mg/L NO3N)
Method performance
Instrument
Standard
Precision
sension 41
5.0 mg/L
4.565.44 mg/L
sension 21
1
With a default stabilization criteria of 0.5 mV/min.
Summary of method
Nitrate ions are selectively absorbed by the ISE membrane, establishing a potential (voltage) that
is proportional to the concentration of nitrate in the sample. This potential is compared to the
constant potential of a reference electrode by measuring the potential of known standard. A
calibration curve can be constructed to determine the concentration of nitrate in unknown samples.
The solvent-polymer membrane is a nitrate ion-exchanger in an inert polyvinyl chloride (PVC)
plastic matrix. The nitrate electrode has an internal silver/silver chloride element, which
establishes a fixed potential when in contact with the internal filling solution. The ion selective
membrane undergoes ion exchange with nitrate in the sample, creating a potential across the
membrane which varies with the amount of nitrate ion in the sample. This potential will decrease
by about 58 mV for every tenfold increase in nitrate concentration in the linear operating range
at 25 C.
Nitrate
Page 1465
Nitrate

Required reagents
Description
Nitrate ISA powder pillows
Nitrate half-cell filling solution
Quantity/Test
Unit
100/pkg
Catalog number
258999
varies
50 mL
4456369
Nitrate-Nitrogen Standard Solutions:

1-mg/L
varies
500 mL
29149
10-mg/L
varies
500 mL
35949
100-mg/L
varies
500 mL
35949
1000-mg/L
varies
500 mL
35949
Ammonium Sulfate Reference Electrolyte Gel Cartridge
varies
2/pkg
2597102
Water, deionized
varies
4L
27256
Required apparatus
Description
Quantity/Test
Unit
Catalog number
each
108041
each
62011
each
108140
Nitrate Combination Electrode, Platinum series, BNC
each
5192000
each
5172500
OR
each
5177500
Stir Bar, 7/8 x 3/16 in. (22.2 x 4.8 cm)
each
4531500

each
4530001
each
4530002
each
1970001
OR
Class A 25 mL volumetric pipet
each
1451540
each
1465100
Membrane Tips, for nitrate electrode (replacement)
each
4613300
Nitrate
Page 1466
Nitrate
Optional reagents
Description
Nitrate Ionic Strength Adjustor, powder
Unit
Catalog number
454 g
4456301
100 mL
245032
Sulfuric Acid, ACS
500 mL
97949
Unit
Catalog number
50/pkg
2185696
Optional apparatus
Description
Pipet Tips, for 197001 TenSette Pipet
Scoop, measuring, 0.5 gram
each
90700
Scoop, measuring, 0.2 gram
each
63800
Nitrate
Page 1467

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrate, drinking water, 8359
Nitrate
DOC316.53.01239
Method 8359
0.04 to 4.00 mg/L NO3N
TISAB Solution
Scope and Application: Drinking water
Test preparation


Meter
sension 4 meters1
1
Electrode
5192000
Only sension 4 meters can be used for this analysis.

When using the electrode for the first time, condition the reference electrode for eight hours in a 100 mg/L nitratenitrogen
standard. After initial conditioning (before the first use or after long-term storage), condition the electrode for 60 minutes only,
unless the electrode is stored dry for longer than four weeks. If the electrode is used daily, store the electrode in a nitrate
standard having a concentration similar to the measured samples.
The low range nitrate test requires several conditioning steps and careful attention to temperature. Read through the
procedure steps and the Sample collection, preservation and storage section before proceeding. The 2-hour conditioning
step must be completed before the calibration step.
Sample concentration for this test must be 3.0 mg/L NO3N or less. Use a test strip to determine nitrate concentration . If
the electrode is conditioned to a low nitrate-nitrogen concentration (as in this procedure), putting it in a concentrated
NO3N sample (10 mg/L or higher) will swamp the electrode with nitrate ion and the 2-hour conditioning step must
be repeated.
Note A: If the electrode is conditioned properly, the mV potential should not change more than 0.1 mV every five minutes in
step 7. It may be necessary to press the electrolyte dispenser button again to stabilize the potential reading. If the potential
drifts one direction more than 0.2 mV every 5 minutes, leave the electrodes in the 150-mL beaker until the drift has slowed. If
the drift is higher than 0.1 mV per minute, the electrode should be conditioned longer in 100 mL of 0.040 mg/L standard. Do
not put ISA into the standard.
Prepare the electrode. Refer to Electrode assembly and Nitrate half-cell preparation in this procedure.
Use identical amounts of ionic strength adjustor (ISA) in the standard beaker and in sample measurements. Use liquid ISA.
Temperature variation causes inaccurate measurements. Calibration and sample measurements should be made at the
same temperature 1 C.
The stable sample reading is the nitrate-nitrogen (NO3N) concentration expressed as elemental nitrogen (N). The results
can be expressed as mg/L nitrate (NO3) by multiplying the results by 4.4.
Nitrate
Page 1469
Nitrate

Description
Quantity
Ammonium sulfate reference electrolyte gel cartridge

Nitrate half-cell internal filling solution
Nitrate ISA solution
1
0.5 mL
25 mL per sample or standard
Nitrate Electrode Membrane Tip (replacement)
Nitrate-Nitrogen standard solutions:

10-mg/L as NO3N
100-mg/L as NO3
Water, deionized
25 mL
25 mL
varies
Combination Nitrate Electrode, BNC
sension4 (laboratory)
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm)
Thermometer, Digital
100 mL plastic volumetric flask
Nitrate
Page 1470
Nitrate
Nitrate-Nitrogen, liquid ISA method
1. Condition the
electrode in 100 mg/L
NO3N standard for one
hour (refer to Nitrate halfcell preparation and
Electrode Assembly in this
procedure). Then
condition the electrode in
0.010
mg/L NO3N for at least 2
hours.

to pipet 5.0 mL of of nitrate
liquid ISA into a plastic
Bring to the 100-mL mark
The deionized water must
be at room temperature.
3. Pour the solution from

the flask into a 150-mL
beaker.
Add a large stir bar (50.8 x
7.9 mm) to the beaker,
place the beaker on an
electromagnetic stirrer and
begin stirring at a
moderate rate.

from the 0.010 mg/L
nitratenitrogen standard
and rinse it well with
deionized water. Place the
electrode in the 150-mL
beaker, submerging the tip
below the solution surface.
To make 100 mL of
0.010 mg/L NO3N, use a
TenSette Pipet to deliver
0.10 mL of 10 mg/L
NO3N into a 100 mL
volumetric flask and dilute
to the mark. Mix well.

to add 0.4 mL of 10 mg/L
nitratenitrogen standard
solution to the solution in
the beaker (this makes
100 mL of 0.04 mg/L
nitratenitrogen).
Allow the electrode to
condition for 30 minutes in
this solution before
proceeding.

Press SETUP, set the
electrode type to BNC and
scroll to Stabilizing.
7. Press ISE MV until the

mV potential shows on the
display. Refer to Before
starting the test: Note A.
Press ENTER and edit the

display to show 0.1 mg/
min. Accept the value and
EXIT the setup menu.
After the measurement is

stable, record the potential
value.
8. Press ISE MV to toggle

to concentration units.
Press CAL and scroll to
mg/L. Press ENTER and
accept the concentration
units.
Measure the temperature

of the standard (C) with a
lab grade thermometer.
Record the temperature.
Nitrate
Page 1471
Nitrate
Repeat step 10

the solution in the 150 mL
beaker (0.040 mg/L).
Refer to the Low level
nitrate calibration table.
concentration.
10. When the

pipet the corresponding
additional volume from the
Low level nitrate
calibration table. Wait the
amount of time specified
for step 2 in the table to
allow the membrane to
respond.
Enter the concentration in
mg/L (0.080 mg/L). Accept
the concentration.
If Error 2 occurs, use
0.08 mg/L as the first
standard for this
calibration. Then try
conditioning in 0.04 mg/L
NO3N solution for
subsequent attempts.
Nitrate
Page 1472
11. Repeat step 10,

adding the additional
volumes of 10 mg/L and
100 mg/L NO3N
standard from the Low
level nitrate calibration
table until all seven
standards have been
measured. Store the
calibration and return to
measurement mode.

from the last standard,
rinse well with deionized
water, and blot dry.
Save the solution in the
150-mL beaker with the
3.84 mg/L NO3N for
later calibration checks.
Nitrate
13. Accurately pipet 5 mL

of nitrate liquid ISA into a
Class A 100-mL
volumetric flask. Bring to
the mark with the sample
being measured.
The sample must be at the
standard solution in the
150-mL beaker used to
perform the calibration.
ions that react with silver
are present in solution.
This will not hurt the
electrode or interfere with
the analysis.
14. Pour this 100 mL of

beaker. Add a magnetic
stir bar, place the beaker
on an electromagnetic
stirrer. Stir at a
moderate rate.

the sample and press the
electrolyte dispenser
button once.
16. Wait 510 minutes to

allow electrode to
condition to the low level
nitrate in solution.
Read the nitrate
concentration from the
display after it stabilizes.
This is the sample nitrate
concentration expressed
as elemental nitrogen
(NO3N ).
Note: Record this value.
Between uses, the electrode
can be stored in the sample
(if not an extreme pH). See
electrode manual for details.
Calibration
Make sure to measure millivolt potentials of all nitrate standards at the same temperature 0.5 C.
The sample and standards must be measured at the same temperature, 1 C. This procedure
keeps temperature error to a minimum by using a spiked additions method of calibration. Even so,
care should be taken to use the 100-mg/L NO3N standard at room temperature.
Use a water bath slightly above room temperature (25 C) to equilibrate the standard temperature
and sample temperature before measuring mV potentials. Use a laboratory-grade thermometer to
monitor the temperature. A one degree centigrade difference may result in as much as a 0.4 mV
inaccuracy. This temperature variation will, in turn, decrease accuracy of concentration
measurements.
Nitrate
Page 1473
Nitrate
Table 405 Low level nitrate calibration table
Volume 100 mg/L NO3N
standard added
Concentration
Step
Volume 10 mg/L NO3N

standard added
mg/L
Time
0.4 mL
0.040
30 min.
0.4 mL
0.080
10 min.
1.2 mL
0.20
5 min.
0.2 mL
0.40
5 min.
0.4 mL
0.80
5 min.
1.2 mL
1.96
5 min.
2.0 mL
3.84
5 min.
Nitrate half-cell preparation

1. Condition the nitrate membrane tip by immersing the membrane tip in beaker with 100 mg/L
NO3N solution for approximately one hour prior to use.When using th electrode for the first
time, condition the reference electrode for eight hours in a 100 mg/L nitratenitrogen standard.
2. Clean all gel from the electrode tip and glass stem. Rinse with Deionized water and dry
completely with a soft paper towel.
3. Use a soft paper towel soaked in isopropyl or rubbing alcohol to wipe the glass stem. Dry the
stem with a dry, soft paper towel. Make sure that alcohol does not get in the glass stem.
4. Use the provided syringe to fill the membrane to with the electrode filling solution. Fill with
solution to the top of the tip. Alternatively, fill the glass stem as shown in the electrode manual.
Do not fill the stem completely or the pressure will cause the membrane tip to fall off.
5. Use a gentle, twisting action to carefully slide a clean, dry nitrate membrane tip over the glass
stem until the end of the stem rests midway through the Nitrate Membrane Tip and some
resistance is met. Leave a 1/8-inch gap between the tip and the electrode body.
6. Shake the electrode as if shaking down mercury in a thermometer to make sure the Nitrate
Electrode Filling Solution contacts the end of the nitrate membrane tip. Verify that air bubbles
are not present in the tip.
Electrode assembly
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body.
visible at the reference outlet. If readings become erratic make sure that the electrolyte gel is
completely purged through the reference line.
6. Rinse the electrode with deionized water. Do not scrub the electrode tip.
7. Connect the BNC connector of the electrode to the BNC connector on the meter.
Nitrate
Page 1474
Nitrate
Interferences
For the nitrate electrode, the major interferences include perchlorate, iodide, nitrite, bromide and
chloride. The addition of Nitrate ISA will eliminate most of these interferences. The highest level of
chloride the ISA can accommodate is 40 mg/L Cl. Concentrations greater than 40 mg/L Cl in the
sample will cause false high nitrate values. For more information on selectivity coefficients without
ISA, refer to the Electrode Characteristics section of the electrode manual.
Start nitrate measurements promptly after sampling. If storage is necessary, store for up to 24
hours at 4 C or lower (as per the USEPA MUR in March 2007, the storage criteria changed
from 4 C to 06 C).
Do not adjust sample pH. This method is for low ionic strength use only. Adjusting the pH
with acid will change the ionic strength and make this method unusable.
Accuracy check
To verify electrode response at these low levels of nitrate, the millivolt potential should decrease
upon each addition of 100 mg/L NO3-N. Using the Low Level Nitrate-Nitrogen procedure, at least
a 5.0 mV drop should be observed from step 1 to step 2 (0.040 mg/L to 0.080 mg/L NO3--N). Each
additional spike should decrease the mV reading substantially from the previous change. If this is
not the case, check the purity of the standard. If this is not the problem, use a new membrane.
1. Pipet 1 mL liquid Nitrate ISA into a 100-mL volumetric flask.
2. Fill the 100-mL volumetric flask to the mark with 1.0 mg/L NO3N. Pour this solution in a
100-mL beaker and add a stir bar.
3. Put the beaker on an electromagnetic stirrer and measure the concentration of the solution
with the calibrated electrode. The measurement should be 1.0 mg/L 0.1 mg/L.
Note: The beaker containing the 3.0 mg/L NO3N standard used in the calibration may also be used as a
check on the calibration. It should read close to 3.0 mg/L NO3N 0.1 mg/L.
Nitrate
Page 1475
Nitrate
To verify sample measurement accuracy, add a spike of standard NO3N solution with a
TenSette or volumetric pipet. Use the Spike volumes of 100-mg/L standard table and the
formulas in Percent recovery.
Table 406 Spike volumes of 100-mg/L standard

Measured sample Concentration
NO3N
Volume of 100 ppm Standard to

Add
CxV
10
0.04 to 0.3
0.1 mL
0.30 to 0.60
0.3 mL
30
0.6 to 0.9
0.5 mL
50
0.9 to 3.0
1.0 mL
100
Percent recovery
To calculate the percent recovery:
M = S 100 + ( C V )
M
E = -------------------100 + V
A
R = ---- 100%
E
Where:
M = calculated mass of nitrate as nitrogen present after the spike (micrograms)
S = mg/L of NO3N in sample (before spike)
V = spike volume from the Spike volumes of 100-mg/L standard table (mL)
A = actual reading on meter after spike (mg/L NO3N)
Method performance
Instrument
Standard
Precision
sension 41
0.1 mg/L
0.0950.1005 mg/L
With a stabilization criteria of 0.1 mV/min
Nitrate
Page 1476
Nitrate
Summary of method
Nitrate ions are selectively absorbed by the ISE membrane, establishing a potential (voltage) that
is proportional to the concentration of nitrate in the sample. This potential is compared to the
constant potential of a reference electrode by measuring the potential of known standard. A
calibration curve can be constructed to determine the concentration of nitrate in unknown samples.
The solvent-polymer membrane is a nitrate ion-exchanger in an inert polyvinyl chloride (PVC)
plastic matrix. The nitrate electrode has an internal silver/silver chloride element, which
establishes a fixed potential when in contact with the internal filling solution. The ion selective
membrane undergoes ion exchange with nitrate in the sample, creating a potential across the
membrane which varies with the amount of nitrate ion in the sample. This potential will decrease
by about 58 mV for every tenfold increase in nitrate concentration in the linear operating range
at 25 C.

Required reagents
Description
Quantity/Test
Unit
Catalog number
Ammonium Sulfate Reference Electrolyte Gel Cartridge
varies
2/pkg
2597102
Nitrate Electrode Membrane Tips
varies
16/pkg
4613300
Nitrate-Nitrogen Standard Solutions 10 mg/L as NO3N
varies
500 mL
30749
Nitrate ISA liquid
varies
500 mL
2488349
varies
50 mL
4456369
varies
500 mL
194749
Nitrate test strips
varies
25/pkg
2745425
Water, deionized
varies
4L
27256
Description
Unit
Catalog number
each
108044
each
62011
Pipet, TenSette, 0.11.0 m
each
1970001
Flask, volumetric, poly, 100 mL
each
1406042
Thermometer, Digital
each
2630600
Flask, volumetric, 100 mL, polypropylene
each
1406041
Nitrate Combination Electrode, Platinum series, BNC
each
5192000
each
5177500
each
4531500
each
2095355
Nitrate half-cell filling solution

Nitrate-Nitrogen Standard Solution 100 mg/L NO3
Required apparatus
Stir Bar,
7/8
3/16
in. (22.2 x 4.8 cm)
Stir Bar, 11/8 x 3/10 in. (28.6 x 7.9 cm)

each
4530001
each
4530002
Nitrate Electrode Membrane Tips (replacements)
6/pkg
4613300
Nitrate
Page 1477
Nitrate
Optional reagents
Description
Nitrate Ionic Strength Adjustor, powder
Nitrate Nitrogen Standard Solutions 1 mg/L as NO3N
Unit
Catalog number
454 g
4456301
500 mL
204649
Unit
Catalog number
Optional apparatus
Description
Beaker, 100 mL, polypropylene
each
108042
50/pkg
2185696
Water Bath, circulating
each
2616300
Pipet, Mohr, 2.00 mL, glass
each
2093636

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen Ammonia ISE, 10001
Nitrogen, Ammonia
DOC316.53.01235
USEPA1 Direct Measurement ISE Method2
Method 10001
0.1 to 10.0 mg/L NH3-N
ISE Electrode

1
USEPA Accepted for reporting wastewater analyses
Adapted from Standard Methods for the Examination of Water and Wastewater, 20th Edition, Method 4500NH3E (with distillation). Manual
distillation may not be required if comparability data on representative samples in company files show the distillation is not necessary. Manual
distillation will be required to resolve any controversies.
Test preparation


Meter
Electrode
sension 4 meters
5192700
sension 2 meters
5192700

Refer to the meter manual for meter operation. Refer to electrode manual for electrode maintenance and care.
Prepare the electrode. Refer to New electrodes or electrodes stored more than 7 days and Electrodes stored 1 to 7 days for
more information.
After every hour of continuous use, place the electrode in the storage solution for 10 minutes to thoroughly recondition.
Check with a 10 mg/L NH3N standard for accuracy and calibrate if necessary.
At high pH, ammonia solutions lose ammonia to the atmosphere, lowering the concentration. It is important to take
measurements as soon as possible after the solution is basic. For most wastewater samples, 1 mL of 10 N NaOH (or
equivalent ISA) is sufficient to increase the pH above 11. If in doubt, check the pH with pH paper and add additional NaOH in
0.1 mL increments until the pH exceeds 11.
Nitrogen, Ammonia
Page 1479
Nitrogen, Ammonia
Description
Quantity
Ammonia electrode filling solution
varies
Ammonia electrode storage solution
20 mL
Ammonia nitrogen standard, 100 mg/L NH3N
varies
Water, deionized
100 mL
Ammonia ISA solution
2 mL per 100 mL sample
Ammonia electrode, combination BNC
Wash bottle
TenSette pipet, 1.010.0 mL
sension 4 laboratory pH/ISE meter or sension 2 portable pH/SE meter
Stir bar, 22.2 x 4.76 mm (7/16 x 3/16 in.)
Stirrer, electromagnetic, with stand and stir bar
Nitrogen, Ammonia in wastewater method

Place it in the Ammonia
ISE storage solution with
the ammonia membrane
sensor module on to
condition for at least
15 minutes.
Nitrogen, Ammonia
Page 1480
2. During the
conditioning period
prepare three standards.
Make a 10-mg/L NH3N
standard by pipeting 25
mL of 100-mg/L NH3N
Standard into a 250-mL
volumetric flask. Dilute to
the mark with ammonia free deionized water,
stopper and thoroughly
mix.
3. Prepare a 1.0-mg/L
NH3N standard by
pipeting 25 mL of the
10-mg/L standard into a
Dilute to the mark with
ammonia-free
deionized water, stopper
and thoroughly mix.
4. Prepare a 0.1-mg/L
NH3N standard by
pipeting 25 mL of the
1.0-mg/L NH3N standard
into a 250-mL volumetric
flask. Dilute to the mark
with ammonia -free
deionized water, stopper
and thoroughly mix.
Nitrogen, Ammonia
Nitrogen, Ammonia in wastewater method (continued)
5. Connect the Ammonia

ISE to the BNC connector
on the pH/ISE meter.
Verify that BNC is selected
in Setup 1 of the
Setup menu.

Press ISE/MV until the
display shows mg/L or
other chosen
concentration units.
7. Press CAL. Use the

ARROW keys to select the
desired units. Press
ENTER and accept
the units.

0.1-mg/L NH3N standard
to a 150-mL beaker. Add a
stir bar to the beaker. Put
the beaker on a magnetic
moderate rate.
Stabilizing...

from the storage solution.
water and blot dry. Put the
electrode into the 0.1-mg/L
NH3N standard.
Make sure no air bubbles
are trapped under the tip
of the electrode.
10. Pipet 2.0 mL of

Ammonia ISA Solution into
the standard. Immediately
proceed to the next step.
11. Press READ. The

display will show the value
from the previous
calibration. Accept the
numerical value or use the
number keys to change
the value to match the
concentration of the
standard, then press
ENTER to accept the
change.

Stabilizing... until the
reading is stable. The
display will show
Standard 2 and _ _ _ _ or
the value of standard 2
from the previous
calibration.
Nitrogen, Ammonia
Page 1481
Nitrogen, Ammonia

Place it in the storage
solution for one minute.
Repeat steps 813 for
substituting the 1.0- and
10-mg/L standards.
14. After the last standard

is measured, press EXIT.

Store?. Press ENTER to
store the calibration or
EXIT to leave the
calibration mode without
storing the calibration
values.
16. Press REVIEW. Use

the Up arrow key to scroll
to the second slope value.
It should be 57 3 mV/
decade. If the slope is not
57 3 mV/decade,
recalibrate the electrode. If
the slope is still incorrect
after recalibration, replace
the ammonia membrane
sensor module.
Press EXIT to return to
measurement mode.

from the last standard.
water and place it in the
storage solution.
Nitrogen, Ammonia
Page 1482
18. Transfer 100 mL of

sample to a 150-mL
beaker. Add a stir bar to
the beaker. Put the beaker

and blot dry. Put the
electrode into the sample.
20. Pipet 2.0 mL of

Ammonia ISA solution into
the sample and proceed
immediately to the next
step.
Nitrogen, Ammonia
Stabilizing...
21. Press READ. The

display will show
reading is stable. Record
or store the measurement
value.
Repeat steps 1721 for
other samples.
Stabilization times will take
longer for lower
concentrations. A slow
downward drift in
concentration indicates
probable loss of ammonia
to the atmosphere. Record
the highest value that is
stable.
Calibration
Prepare ammonia standard working solutions of 10.0, 1.0 and 0.1 mg/L ammonia nitrogen from a
100-mg/L stock solution. Prepare the standards daily before use. Higher or lower concentration
ranges (0.051400 mg/L NH3N) can be obtained by calibrating the meter with different
standard solutions.
Electrode preparation
New electrodes or electrodes stored more than 7 days
Before using a new Ammonia Electrode or an electrode that has been stored dry, remove the
protective cap from the end.
1. Unscrew the top cap. Carefully remove the internal glass electrode from the outer body. A
white membrane is mounted at the tip of the outer body.
2. Fill the outer body with 3.5 mL of Internal Fill Solution.
3. Rinse the internal glass electrode with deionoized water. Blot dry. Return the electrode to the
filled outer body. Make sure that the key pin at the top of the internal glass electrode is seated
in the slot at the top of the outer body.
4. Reinstall the threaded top cap onto the top of the ammonia electrode body. Finger-tighten the
cap until snug. Do not over-tighten.
Nitrogen, Ammonia
Page 1483
Nitrogen, Ammonia
5. Hold the fully assembled electrode securely by one end and shake the electrode with an
abrupt downward motion (like shaking the mercury down in a thermometer) to remove
bubbles.
6. Place the assembled electrode into the Ammonia Electrode Storage Solution or 1000 mg/L
Ammonia Standard for at least 60 minutes.
Electrodes stored 1 to 7 days
Keep the electrode in 1000 mg/L ammonia standard without Ionic Strength Adjustor (ISA).
Alternatively, keep the electrode in the Ammonia Electrode Storage Solution.
Never let the membrane dry out. Cover the storage beaker and electrode body with Parafilm
to prevent solution evaporation.
Electrodes stored between samples

Place the electrode in Ammonia Electrode Storage Solution for at least one minute to initialize the
electrode for the next measurement.
Interferences
Distillation prior to ammonia analysis removes all inorganic interferences that complex ammonia.

Interference level
Amines
Volatile low molecular weight gives a positive interference
Mercury
Complexes with ammonia
Silver
Collect samples in glass or plastic containers of convenient size. Clean new bottles by
washing with deionized or distilled water. Fill the sample bottle completely and stopper
immediately. Analyze the sample as soon as possible.
Ammonia may be lost more quickly from samples at temperatures above 50 C, so it is

important to collect samples at less than 40 C or use a cooling coil between the bottle and
sampling point if necessary.
If chlorine is present, treat the sample immediately with sodium thiosulfate. Add one drop of
0.1 N Sodium Thiosulfate Standard Solution for each 0.3 mg of chlorine present in a one
liter sample.
If prompt analysis is not possible, preserve the sample with 0.8 mL of concentrated sulfuric
acid per liter. Use a sension pH meter to be sure the pH of the preserved sample is between
1.5 and 2. Some wastewater samples may require more sulfuric acid to achieve this pH. Store
the sample at 4 C. Samples preserved in this manner may be stored up to 28 days.
Before analysis, neutralize the sample to pH 7 with 5 N sodium hydroxide. Do not let the pH go
above 10. Correct the test results for the volume addition.
Do not use mercuric chloride as a preservative because ammonia complexes with

mercuric ions.
Nitrogen, Ammonia
Page 1484
Nitrogen, Ammonia
Accuracy check
1. Use the Spike volumes for standard additions table to determine the concentration and volume
of standard to spike the sample. The volume of sample transferred must be accurate.
2. Add the amount and concentration specified in the Spike volumes for standard additions table
to the 100 mL of sample.
3. After adding the standard, proceed with the calculations. Results from 90-110% recovery are
typically considered acceptable. Calculate percent recovery as follows:
100 ( X s X u )
% Recovery = ---------------------------------K
Where:
Calculations
1.
Xi Vu
X u = ----------------Vu + V
Where:
2.
CV
K = ----------------Vu + V
Where:
100 ( X X )
K
s
u
Nitrogen, Ammonia
Page 1485
Nitrogen, Ammonia
Example:
A sample was analyzed and read 5.0 mg/L NH3N. As directed in the Spike volumes for standard
additions table, a 4.0-mL spike of 100-mg/L NH3N standard was added to another 100-mL
sample, giving a final standard addition result of 8.75 mg/L.
1.
5.0 mg/L 100 mL

X u = ------------------------------------------------- = 4.81 mg/L
100 mL + 4 mL
2.
100 mg/L 4 mL
K = -------------------------------------------- = 3.85 mg/L
100 mL + 4 mL
3.
100 ( X s X u )
100 ( 8.75 4.81 )
%R = ---------------------------------------= -------------------------------------------------- = 102.3 % Recovery
K
3.85
Table 409 Spike volumes for standard additions

(mg/L)

(mL)
(mg/L)
Standard Volume
(mL)
0.10.3
100
100
0.2
0.30.5
100
100
0.4
0.50.7
100
100
0.6
0.70.9
100
100
0.8
0.91.1
100
100
1.0
1.03.0
100
100
2.0
3.06.0
100
100
4.0
6.010.0
100
100
8.0
Method performance
Instrument
Standard
Precision
sension 41
0.80 mg/L
0.780.82 mg/L
sension
1
21
With a default stabilization criteria of 0.5 mV/min.
Summary of method
The ammonia electrode measures ammonia gas or ammonium ions in aqueous solutions that
have been converted to gas by the addition of a strong base. The electrode is a complete
electrochemical cell consisting of a glass pH electrode and a reference electrode.
The gas-permeable membrane separates the sample from a thin layer of electrolyte that is
pressed between the pH bulb and the membrane. At high pH, ammonium ion is converted to
ammonia gas.
The gas diffuses through the membrane and causes a pH change in the thin layer of electrolyte.
The potential across the pH glass changes as a result of the pH change and the electrode
measures the change in potential. The measured pH change is proportional to the ammonia
concentration in the solution.
Nitrogen, Ammonia
Page 1486
Nitrogen, Ammonia

Required reagents
Description
Quantity/Test
Unit
Catalog number
Ammonia Electrode Filling Solution
varies
60 mL
4447226
Ammonia Electrode Storage Solution
20 mL
500 mL
2541249
Ammonia Nitrogen Standard, 100 mg/L NH3-N
100 mL
500 mL
2406549
2 mL/100 mL
sample
500 mL
2824349
100 mL
4L
27256
Ammonia ISA Solution

Water, deionized
Required apparatus
Description
Quantity/Test
Unit
Catalog number
Ammonia Electrode
each
5192700
each
108044
Bottle, wash, 500 mL
each
62011
each
1457446
sension 4 Laboratory pH/ISE Meter or sension 2 pH/ISE (portable)
each
5177500
each
4531500
each
1970010
varies
50/pkg
2199796
Class A 25 mL volumetric pipet
each
1451540
each
1418900
Stirrer, electromagnetic 115 V, with stand and stir bar
each
4530001
each
4530002
Description
Unit
Catalog number
Ammonia Nitrogen Standard Solution 1000 mg/L NH3-N
1L
2354153
Pipet tips for 1970010 TenSette Pipet
Optional reagents
pH Paper, pH 9.0-12.0
5 rolls/pkg
38533
500 mL
97949
Nitrogen, Ammonia
Page 1487
Nitrogen, Ammonia
Optional apparatus
Description
Unit
Catalog number
Air Gap Assembly
each
5025300
Ammonia Electrode Membrane Modules
4/pkg
5192711
Cylinder, graduated, glass
100 mL
50842
Electrode Washer
each
2704700
each
1451535
each
1970001
50/pkg
2185696
each
5172500


HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Nitrogen Ammonia ISE, 10002
Nitrogen, Ammonia
DOC316.53.01234
USEPA1 Known Addition ISE Method2
Method 10002
0.8 mg/L NH3-N
ISE Electrode

1
USEPA Accepted for reporting wastewater analyses
Adapted from Standard Methods for the Examination of Water and Wastewater, 20th Edition, Method 4500NH3E (with distillation). Manual
distillation is not required if comparability data on representative samples in company files show the distillation is not necessary. Manual
distillation will be required to resolve any controversies.
Test preparation


Meter
sension 4 meters
Electrode
5192700

Refer to the meter manual for meter operation. Refer to electrode manual for electrode maintenance and care.
Prepare the electrode:
Refer to the Distillation Apparatus manual to perform sample distillation, if necessary.
Prepare spiking solutions according to the Nitrogen and Ammonia in the Wastewater section.
When there is a linear relationship between concentration and response, the known addition method can be used to
measure occasional samples because calibration is not required. The sample concentration must be known within a factor of
three, in order to get accurate measurements, because the concentration of ammonia in the sample must be approximately
doubled by the standard addition.
Nitrogen, Ammonia
Page 1489
Nitrogen, Ammonia
Description
Quantity
Ammonia electrode filling solution
varies
Ammonia electrode storage solution
20 mL
Ammonia nitrogen standard, 1000 mg/L NH3N
varies
Ammonia ISA solution
2 mL per 100 mL sample
Ammonia electrode, combination BNC
Wash bottle
TenSette
pipet, 1.010.0 mL
sension 4 laboratory pH/ISE meter
Stir bar, 22.2 x 4.76 mm (7/16 x 3/16 in.)
Select on based on available voltage:

Stirrer, electromagnetic, 115 V, with stand and stir bar
Stirrer, electromagnetic, 230 V, with stand and stir bar
Nitrogen, Ammonia known addition method
1. Accurately transfer
100 mL of sample to a
150-mL beaker using a
volumetric pipet or
graduated cylinder. Add a
stir bar to the beaker.
Nitrogen, Ammonia
Page 1490
2. Stir at a constant and

moderate rate on a
magnetic stirrer to improve
response time and
accuracy.

to add 1.0 mL of 10 N
NaOH solution into the
sample.

water and blot dry. Put the
electrode in the sample.
Make sure that no air
bubbles are trapped under
the tip of the electrode.
Remove bubbles by lightly
tapping the electrode or by
tilting the electrode to 20.
Nitrogen, Ammonia
Nitrogen, Ammonia known addition method (continued)

Press STD ADDN. Use the
ARROW keys to select the
required units. Accept the
units.

the slope value for the last
calibration (the default is
59.2 mV). Accept the
numerical value or change
the slope value.
mL, Sample, ?
Stabilizing...
7. The meter will prompt

for the sample volume
(in mL). Enter the sample
volume and press ENTER
to accept the volume.

baseline reading is stable.
The meter will then prompt
for the standard volume.
11. Add the volume of a

known standard (listed in
Table 4) to the beaker and
proceed as quickly as
possible through the rest
of the procedure.
12. Enter the

standard used (for
example, 1000 mg/L).
Standard
9. Enter the volume of

standard to be used (for
example, 1.0 mL). Press
ENTER and accept the
volume.
10. Obtain the standard

concentration and volume
from Table 4 on page 48
after estimating the
sample concentration.

the concentration.
Nitrogen, Ammonia
Page 1491
Nitrogen, Ammonia
Nitrogen, Ammonia known addition method (continued)
Sample +
Standard
20 mg/L

Sample+Standard and
reading is stable.
14. The meter will

calculate and display the
adjusted value for the
original sample in mg/L.
Record or store this value.
STANDARD ADDITONS
when data is recalled for
standard additions.
Nitrogen ammonia in wastewater

Known addition is also a convenient check on the results of direct measurement. Because an
accurate measurement requires that the concentration approximately double as a result of the
addition, the approximate sample concentration must be known within a factor of three. Make a
spiking solution before beginning the procedure.
1. Use the Spiking solutions table to determine how to dilute a 1000 mg/L NH3-N stock solution
to use as a spiking solution.
2. Pipet the appropriate amount of 1000-mg/L NH3-N standard into a 100-mL volumetric flask.
3. Dilute to the mark with ammonia-free water.
4. Determine the slope before performing standard additions of the sample.
a. Use the 100 mg/L and 1000-mg/L NH3-N stock solutions to determine the slope.
b. Check the electrode occasionally to determine if it is functioning properly and to determine
its exact slope value. The frequency of this operation depends on the harshness of
the sample.
Table 411 Spiking solutions

Expected Sample Concentration (mg/L)
mL of 1000-mg/L NH3-N
0.84.0
20 mg/L
2.57.5
50 mg/L
515
10
100 mg/L
1250
25
250 mg/L
2575
50
500 mg/L
50150
100
1000 mg/L
Nitrogen, Ammonia
Page 1492
Nitrogen, Ammonia
Electrode preparation
Before using a new Ammonia Electrode or an electrode that has been stored dry, remove the
protective cap from the end.
1. Unscrew the top cap. Carefully remove the internal glass electrode from the outer body. A
white membrane is mounted at the tip of the outer body.
2. Fill the outer body with 3.5 mL of Internal Fill Solution.
3. Rinse the internal glass electrode with deionoized water. Blot dry. Return the electrode to the
filled outer body. Make sure that the key pin at the top of the internal glass electrode is seated
in the slot at the top of the outer body.
4. Reinstall the threaded top cap onto the top of the ammonia electrode body. Finger-tighten the
cap until snug. Do not over-tighten.
5. Hold the fully assembled electrode securely by one end and shake with an abrupt downward
motion (like shaking the mercury down in a thermometer) to remove bubbles.
6. Place the assembled electrode into the Ammonia Electrode Storage Solution or 1000 mg/L
Ammonia Standard for at least 60 minutes.
Keep the electrode in 1000 mg/L ammonia standard without Ionic Strength Adjustor (ISA).
Alternatively, keep the electrode in the Ammonia Electrode Storage Solution.
Never let the membrane dry out. Cover the storage beaker and electrode body with Parafilm
to prevent solution evaporation.
Electrodes stored between samples

Place the electrode in Ammonia Electrode Storage Solution for at least one minute. This
reinitializes the electrode for the next measurement.
Interferences
Distillation prior to ammonia analysis removes all inorganic interferences that complex ammonia.

Interference level
Amines
Volatile low molecular weight gives a positive interference
Mercury
Silver
Collect samples in glass or plastic containers of convenient size. Clean new bottles by
washing with deionized or distilled water. Fill the sample bottle completely and stopper
immediately. Analyze the sample as soon as possible.
Ammonia may be lost more quickly from samples at temperatures above 50 C, so it is

important to collect samples at less than 40 C or use a cooling coil between the bottle and
sampling point if necessary.
If chlorine is present, treat the sample immediately with sodium thiosulfate. Add one drop of
0.1 N Sodium Thiosulfate Standard Solution for each 0.3 mg of chlorine present in a one
liter sample.
Nitrogen, Ammonia
Page 1493
Nitrogen, Ammonia
If prompt analysis is not possible, preserve the sample with 0.8 mL of concentrated sulfuric
acid per liter. Use a sension pH meter to be sure the pH of the preserved sample is between
1.5 and 2. Some wastewater samples may require more sulfuric acid to achieve this pH. Store
the sample at 4 C. Samples preserved in this manner may be stored up to 28 days.
Before analysis, neutralize the sample to pH 7 with 5 N sodium hydroxide. Do not let the pH go
above 10. Correct the test results for the volume addition.
Do not use mercuric chloride as a preservative because ammonia complexes with

mercuric ions.
Accuracy check
1. Use the Spike volumes for known additions table to determine the concentration and volume
of standard to spike the sample. The volume of sample transferred must be accurate.
2. Add the amount and concentration specified in the Spike volumes for known additions table to
the sample while performing the standard addition method on the sample. Do not allow the
sample to stand too long before spiking or ammonia will be lost to the atmosphere. T
3. After adding the standard, proceed with the calculations. Results from 90110% recovery are
typically considered acceptable. Calculate percent recovery as follows:
100 ( X s X u )
% Recovery = ---------------------------------K
Where:
Calculations
1.
Xi Vu
X u = ----------------Vu + V
Where:
2.
CV
K = ----------------Vu + V
Where:
100 ( X X )
K
s
u
Example:
Nitrogen, Ammonia
Page 1494
Nitrogen, Ammonia
A sample was analyzed and read 5.0 mg/L NH3N. As directed in the Spike volumes for known
additions table, a 4.0-mL spike of 100-mg/L NH3N standard was added to another 100-mL
sample, giving a final standard addition result of 8.75 mg/L.
1.
5.0 mg/L 100 mL

X u = ------------------------------------------------- = 4.81 mg/L
100 mL + 4 mL
2.
100 mg/L 4 mL
K = -------------------------------------------- = 3.85 mg/L
100 mL + 4 mL
3.
100 ( X s X u )
100 ( 8.75 4.81 )
%R = ---------------------------------------= -------------------------------------------------- = 102.3 % Recovery
K
3.85
Table 413 Spike volumes for known additions

(mg/L)

(mL)
(mg/L)
Standard Volume
(mL)
0.81.0
100
100
1.0
13
100
100
2.0
36
100
100
4.0
69
100
100
8.0
912
100
100
10.0
1220
100
1000
2.0
2040
100
1000
4.0
4060
100
1000
6.0
6075
100
1000
8.0
Method performance
Instrument
Standard
Precision
sension 4
5.00 mg/L
4.815.19 mg/L
sension 2
4.815.19 mg/L
Summary of method
The ammonia electrode measures ammonia gas or ammonium ions in aqueous solutions that
have been converted to gas by the addition of a strong base. The electrode is a complete
electrochemical cell consisting of a glass pH electrode and a reference electrode.
The gas-permeable membrane separates the sample from a thin layer of electrolyte that is
pressed between the pH bulb and the membrane. At high pH, ammonium ion is converted to
ammonia gas.
The gas diffuses through the membrane and causes a pH change in the thin layer of electrolyte.
The potential across the pH glass changes as a result of the pH change and the electrode
measures the change in potential. The measured pH change is proportional to the ammonia
concentration in the solution.
Nitrogen, Ammonia
Page 1495
Nitrogen, Ammonia

Required reagents
Description
Quantity/Test
Unit
Catalog number
Ammonia Electrode Filling Solution
3 mL
50 mL
4447226
Ammonia Electrode Storage Solution
5 mL
500 mL
2541249
Ammonia Nitrogen Standard, 1000 mg/L NH3-N
varies
1L
2354153
Sodium Hydroxide Solution, 10 N
10 mL
500 mL
2545049
Water, deionized
100 mL
4L
27256
Quantity/Test
Unit
Catalog number
Required apparatus
Description
Ammonia Electrode
each
5192700
each
108044
Bottle, wash, 500 mL
each
62011
each
50842
each
5177500
each
4531500
each
1970010
varies
50/pkg
2199796

each
4530001
each
4530002
Unit
Catalog number
5 rolls/pkg
38533
Optional reagents
Description
pH Paper, pH 9.0-12.0
500 mL
97949
5.0 N NaOH
1000 mL
245053
Description
Unit
Catalog number
Ammonia Electrode Membrane Modules
4/pkg
5192711
Electrode Washer
each
2704700
each
1451535
each
1451538
each
1451542
50/pkg
2185696
Optional apparatus

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Sodium ISE, 8359
Sodium
DOC316.53.01240

10 to 2000 g/L
Method 8359
Na+
ISE Electrode
Scope and Application: Drinking water and process water application
Test preparation


Meter
sension 4 meters1
1
Electrode
5192000
Only sension 4 meters can be used for this analysis.

Before the test, prepare a 10 mg/L Na+ standard:
1. Use a Class A pipet to dispense 10 mL of 1000 mg/L Na+ standard into a 1.0 L volumetric flask.
This procedure requires several conditioning steps. Read through all the procedure steps before proceeding.
This test is for low range analysis only. If the electrode is conditioned to a low sodium concentration (as in this procedure),
putting it in a concentrated Na+ (10 mg/L or higher) will swamp the electrode with sodium ion and the overnight conditioning
step must be repeated.
Measure millivolt potentials of all sodium standards at the same temperature 0.5 C. Samples and standards must be
measured at the same temperature, 1 C. Use room temperature 100 mg/L Na+ standard. This procedure keeps
temperature error to a minimum by using a spiked additions method of calibration.
Note A: If the electrode is conditioned properly, the mV potential should not change more than 0.1 mV every minute in step
10. If excessive drift occurs leave the electrode in the 600-mL beaker until the drift slows. If the electrode drifts 10 mV or
more in the positive direction (for example, from 175 to 165 mV), repeat steps 510, but do not dispense electrolyte gel
again as directed in step 7.

Description
Quantity
Potassium chloride electrolyte gel cartridge
pH/ISE meter, sension4 (laboratory)
Sodium ISE Electrode, BNC
Sodium ISA powder pillows
Sodium
Page 1497
Sodium
Description
Quantity
Sodium standard solutions:

100 mg/L Na+
1000 mg/L Na+
Water, deionized
varies
Flask, 100-mL, volumetric
Pipet, TenSette, 0.1 to 1.0 mL, plus tips
Stir Bar,
7/8
3/16
in. (22.2 x 4.8 cm)
Sodium ISE, powder pillow method
1. Install the Potassium

Chloride Electrolyte Gel
Cartridge in the Platinum
Series Combination
Sodium Electrode.
2. Connect the
combination sodium
electrode to the meter.
Ensure the electrode has
been conditioned for at
least 8 hours in Sodium
Electrode Storage Solution
before its initial use.
3. Prime the electrode by

pushing the dispenser
button until gel comes out
of the reference junction.
Rinse excess gel from the
tip and the outlet.
4. Condition the
electrode in sodium
electrode storage solution
for a minimum of 1 hour
before use. Then,
condition the electrode in
0.10 mg/L sodium for at
least 8 hours.
To make 100 mL of 0.10
mg/L Na+ standard, use a
TenSette Pipet to put
0.10 mL of 100 mg/L Na+
into a 100-mL volumetric
flask and dilute to the
mark. Mix well.
Sodium
Page 1498
Sodium
Sodium ISE, powder pillow method (continued)
5. Accurately
measure 400 mL of
plastic 500-mL graduated
cylinder.
The deionized water must
be at room temperature.
6. Pour the water in the

cylinder into a 600-mL
plastic beaker. Add the
contents of one Sodium
Ionic Strength Adjustor
powder pillow to
the solution.
Add a large stir bar (50.8 x
7.9 mm) to the beaker.
Place the beaker on an
electromagnetic stirrer and
begin stirring at a
moderate rate.

Press SETUP and scroll to
Stabilizing.
Press ENTER and edit the
display to show 0.1 mg/
min. Accept the value and
EXIT the setup menu.
10. Press ISE MV until the

mV potential shows on the
display. Refer to Before
starting the test: Note A.
After the measurement is
stable, record the potential
value.
Measure the temperature
of the standard (C) with a
lab grade thermometer.
Record the temperature.

from the 0.10 mg/L sodium
standard, dispense gel
and rinse excess gel away
Place the electrode in the
600-mL beaker,
submerging the tip below
the solution surface.

to add 0.4 mL of 10 mg/L
Sodium Standard Solution
to the solution in the
beaker. Refer to the Low
level sodium calibration
table. (This makes 400 mL
of 0.010 mg/L or 10 g/L
sodium.)
Allow the electrode
to condition for 15 minutes
in this solution
before proceeding.
11. Press ISE MV to toggle

to concentration units.
Press CAL and scroll to
g/L. Press ENTER to
accept the concentration
units.

the solution in the 600 mL
beaker (10 g/L). Refer to
the Low level sodium
calibration table.
concentration.
Sodium
Page 1499
Sodium
Repeat step 13
13. When the

pipet the corresponding
additional volume from the
10-mg/L Na+ standard in
the Low level sodium
calibration table. Wait the
amount of time specified
for step 2 in the table to
allow the membrane to
respond.
Enter the concentration in
g/L (20 g/L). Press
ENTER to accept the
concentration.
Sodium
Page 1500
14. Repeat step 13,

adding the additional
volumes of 10 mg/L and
100 mg/L Na+ standard
from the Low level sodium
calibration table until all
seven standards have
been measured. Store the
calibration and return to
measurement mode.

from the last standard,
rinse well with deionized
water and blot dry.

400 mL of sample into a
500-mL graduated
cylinder.
Save the solution in the

600-mL beaker with the
2.00 mg/L Na+ for later
calibration checks.
The sample must be at the

standard solution in the
600-mL beaker used to
perform the calibration.
Sodium
17. Pour this 400 mL of

stir bar, place the beaker
rate.

one Sodium Ionic Strength
Adjustor Powder Pillow.

into the sample.
20. When the

record the sample
concentration value.
Wait 10 minutes to allow

electrode to condition to
the low level sodium in
solution.
If the sodium
concentration is more than
the 10 to 2000 g/L
calibration range, press
the ISE mV key. If the mV
reading is greater than the
mV at 2000 g/L Na+,
analyze the sample using
the procedure for Potable,
Ground and Irrigation
Water (refer to the Sodium
electrode manual). If the
mV reading is less than
the mV reading of the 10
g/L standard, there is less
than 10 g/L Na+ in the
sample.
If the electrode is placed in

a sample of high sodium
concentration, errors may
result and reconditioning
to lower sodium levels will
be required.
Calibration
Use a water bath slightly above room temperature (25 C) to equilibrate the standard temperature
and sample temperature before measuring mV potentials. Use a laboratory-grade thermometer to
monitor the temperature. A one degree centigrade difference may result in as much as a 0.4 mV
inaccuracy. This temperature variation will, in turn, decrease accuracy of concentration
measurements.
Table 415 Low level sodium calibration

Step
Volume 10 mg/L Na+ standard

added
Volume 100 mg/L Na+

standard added
Concentration
g/L
Time
0.4 mL
10
until mV stabilizes
0.4 mL
20
15 min.
1.2 mL
50
15 min.
Sodium
Page 1501
Sodium
Table 415 Low level sodium calibration
Volume 10 mg/L Na+ standard
added
Concentration
Volume 100 mg/L Na+

standard added
g/L
Time
0.2 mL
100
10 min.
0.4 mL
200
10 min.
1.2 mL
495
10 min.
2.0 mL
1000
10 min.
4.0 mL
2000
10 min.
Step
Interferences
The Sodium ISA is formulated to remove most interferences. Silver is a major interference.

Analyze immediately after sampling. If immediate analysis is impossible, cool samples to 4 C and
analyze within six hours
Low ionic strength conditioning

The conditioning steps in the section apply to measurements in samples containing less than
1.0 mg/L Na+.
Initial use
The sensing half-cell is packaged and shipped dry from the factory with a dry cap over the glass
membrane tip.
1. Remove the cap and install the gel cartridge.
2. Click the reference electrolyte dispenser until gel emerges from the tip.
3. Rinse the electrode with a small stream of sample delivered through a disposable plastic
Pasteur pipette or with deionized water from a wash bottle.
4. Place the tip in Sodium Electrode Storage Solution or in 1 M sodium chloride solution and soak
for at least one hour. Condition the electrode in 0.10 mg/L sodium solution for at least 8 hours
before calibrating using the Low Range Sodium Method. Refer to the electrode user manual
for more information.
Sodium
Page 1502
Sodium
Between uses
Between uses, in intervals of up to a few hours, the electrode can be stored in the sample (if not an
extreme pH) or in a neutral low-ionic-strength solution such as tap water. Before measuring a new
sample, refresh the reference electrolyte gel by clicking the dispenser several times. Carefully
rinse the electrode to prevent contaminating the sample.
Accuracy check
Electrode response
To verify electrode response at these low levels of sodium, the millivolt potential should increase
upon each addition of 100 mg/L Na+. Using the Low Level Sodium procedure, at least a 4.0 mV
increase should be observed from step 1 to step 2 (10 g/L to 20 g/L Na+). Each additional spike
should increase the mV reading substantially from the previous change. If this is not the case,
check the purity of the standard used. If this is not the problem, the electrode is probably not
conditioned for low sodium levels.
Calibration accuracy
1. Fill the 500-mL graduated cylinder to the 400-mL mark with deionized water.
2. Pour this solution in a 600-mL beaker and add a stir bar.
3. Pipet 0.2 mL of 100 mg/L Sodium Standard into the 600-mL beaker.
4. Add the contents of one Sodium Ionic Strength Adjustor powder pillow and place on an
electromagnetic stirrer.
5. Rinse the calibrated electrode before placing in solution. Measure the concentration of the
solution. The reading should be approximately 50 g/L.
Note: The beaker with the 2000 g/L Na+ standard used in the calibration may be used as a check on the
calibration. It should read close to 2000 g/L.
Accuracy of the sample measurement

To verify sample measurement accuracy, add a spike of standard solution with a TenSette or
volumetric pipet. Use the Spike volumes of 100-mg/L standard table and the formulas in Percent
recovery.
Table 416 Spike volumes of 100-mg/L standard

Measured sample Concentration
Na+ (g/L)
Volume of 100 ppm Standard to

Add
CxV
10 to 49
0.1 mL
10
50 to 99
0.2 mL
20
100 to 299
0.4 mL
40
300 to 599
1.2 mL
120
600 to 2000
2.0 mL
200
Sodium
Page 1503
Sodium
Percent recovery
To calculate the percent recovery:
M = S 400 + ( C V )
M
E = -------------------400 + V
A
R = ---- 100%
E
Where:
M = calculated mass of sodium present after the spike (micrograms)
S = mg/L of Na+ in sample (before spike)
V = spike volume from the Spike volumes of 100-mg/L standard table (mL)
A = actual reading on meter after spike
Method performance
Instrument
Standard
Precision
sension 41
25 g/L
21.3428.66 g/L
With a stabilization criteria of 0.1 mV/min
Sodium
Page 1504
Sodium

Required reagents
Description
Quantity/Test
Unit
Catalog number
Potassium Chloride Reference Electrolyte Gel Cartridge
varies
3/pkg
.2546902
Sodium Ionic Strength Adjustor Powder Pillows
varies
100/pkg
4451569
Sodium Standard Solutions 100 mg/L as Na+
varies
1000 mL
2318153
varies
500 mL
1474949
varies
4L
27256
Description
Unit
Catalog number
each
108052
each
62011
Cylinder, graduated, 500 mL, polypropylene
each
108149
Sodium Standard Solutions 1000 mg/L as
Na+
Water, deionized
Required apparatus
each
1970001
50/pkg
2185696
each
5177500
Sodium Combination Electrode, Platinum series, BNC
each
5192500
Stir Bar, 7/8 x 3/16 in. (22.2 x 4.8 cm)
each
4531500
each
2095355
each
4530001
each
4530002
Description
Unit
Catalog number
Sodium Ionic Strength Adjustor (ISA), powder
454 g
4451501
Description
Unit
Catalog number
each
1406041
each
1406042
Water Bath, circulating
each
2616300
Stir Bar,
11/8
3/10
in. (28.6 x 7.9 cm)
Optional reagents
Optional apparatus
Sodium
Page 1505

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen, dissolved, 8157
Oxygen, Dissolved
DOC316.53.01241
Direct Measurement Method

0 to 20 mg/L or 0 to 200% saturation
Method 8157
Clark-type Amperometric Sensor
Scope and Application: For water, wastewater and process water applications
Test preparation


Meter
Electrode
sension 5 meters
5197000, 5197003 or 5197015
sension 8 meters
5197000, 5197003 or 5197015

Assemble the probe. Refer to the Probe assembly section in this procedure.
Refer to the sension meter manual for instructions on polarizing and zeroing the meter.
Dissolved oxygen probes are continuously polarized when they are connected to the meter. A steady reading will not be
seen for 30 to 50 minutes when the probe electrolyte is new or when the probe has been unplugged for more than one hour.
Interrupted connections of less than one hour will require 5 to 25 minutes before a stable reading is observed.
While not in use, the probe should be stored with the Calibration and Storage Chamber attached to the end of the probe. The
sponge inside the Chamber should be kept moist.
Keep the DO probe at a uniform temperature. When holding the probe, do not touch the metallic button on the side of the
probe. The button is a temperature sensor. An inaccurate calibration will result if the temperature of the thermistor is different
from the probe membrane.
The displayed % saturation is based on a meter calculation for the equilibrium dissolved oxygen concentration. The
calculation uses the sample temperature, salinity, barometric pressure, altitude and measured concentration in mg/L values.
Changing the entries in setups 4, 5 or 6 will change the displayed mg/L or % saturation. Refer to the sension meter manual
for more information.

Description
Quantity
Dissolved oxygen probe
sension 6 or 8 meter
Filling solution, dissolved oxygen
2 mL
Calibration and storage chamber
Oxygen, Dissolved
Page 1507
Oxygen, Dissolved
Description
Quantity
Membrane for dissolved oxygen probe
Beaker, polypropylene, 100-, 250-, 400- or 600-mL
Dissolved oxygen, amperometric sensor method
1. At least one hour

before measurement,
polarize the probe by
connecting it to the meter.
2. Zero the Dissolved

Oxygen meter before
calibration when
measuring dissolved
oxygen levels less than
1 mg/L or 10% saturation.
Refer to Zeroing the
probe.
3. Secure the probe

cable to the calibration and
storage chamber by
wrapping cable through
the bottom of the chamber
lid before filling with water.
4. Prepare the calibration

and storage chamber by
holding it under water and
squeezing it a couple of
times to pull water into the
lower chamber through the
inlet. Alternately, open the
bottom of the chamber and
insert a damp sponge.
New sponges will be
compressed. Add water to
expand them. Avoid
completely filling the lower
chamber with water.
Oxygen, Dissolved
Page 1508
Oxygen, Dissolved
Dissolved oxygen, amperometric sensor method (continued)
5. Insert the DO probe

into the calibration and
storage chamber. The
probe tip must not be
flooded with water or be
holding a drop of water on
the membrane.
6. Allow at least ten

minutes for the
atmosphere in the
chamber to reach a steady
state.
7. Press CAL. Edit the

barometric pressure.
Press ENTER and enter
the current altitude and
press ENTER.
To speed up probe
stabilization, squeeze the
lower chamber a couple of
times to force water
saturated air into
the chamber.
Refer to the Adjusting

barometric pressure and
altitude table.
9. Press ENTER. The

display will show 100%.
10. Add the weight

assembly to the probe if
required (3- or 15-m cable
versions only).
11. If sample salinity has

been measured using an
Electrolytic Conductivity
Meter, enter the value in
parts per thousand.
Press READ. When the

calibration is complete, the
meter will return to
measurement mode.

the current value for
sample salinity ().
Set the salinity to zero
(0).
12. Insert the probe into

the sample. The probe
must be deep enough to
cover the thermistor
(metallic button) located
on the side of the probe.
Oxygen, Dissolved
Page 1509
Oxygen, Dissolved
Dissolved oxygen, amperometric sensor method (continued)
13. Agitate the probe in

the sample to dislodge air
bubbles from the sensing
area of the probe tip.
14. Stir the sample

vigorously with the probe
or use a stir stand and stir
bar (for laboratory use).
15. When the reading on

the meter stabilizes,
record or store the value in
the meter memory.
16. Change the display

from concentration in mg/L
to % saturation if %
saturation is required.

the sample.
4. When the
record the sample
concentration value.
When measuring deep

bodies of water, create
sufficient flow across the
probe tip by pulling on the
cable to move the probe
up and down (for field
use).
For high Sodium concentration samples
1. Pour the 400 mL

stir bar and put the beaker
rate.
If the electrode is in a
sample of high sodium
concentration, errors may
result and reconditioning
to lower sodium levels will
be required.
Oxygen, Dissolved
Page 1510

one Sodium Ionic Strength
Adjustor Powder Pillow.
Wait 10 minutes to allow

electrode to condition to
the low level sodium in
solution.
Oxygen, Dissolved
Barometric pressure and altitude adjustment

Table 418 Adjusting barometric pressure and altitude
Enter a new barometric pressure when the barometric pressure or the altitude of the instrument changes using one of the
methods below:
Sea level equivalent
True barometric pressure
1.
Obtain the sea level equivalent barometric pressure from

TV, radio or a local airport.
1.
Obtain the true barometric pressure from a nearby

mercury barometer.
2.
Enter this value into the meter. Refer to the Changing the
Barometric Pressure section of the meter manual.
2.
Enter this value into the meter. Refer to the Changing the
Barometric Pressure section of the meter manual.
3.
Enter the local altitude. Refer to the Adjusting the Altitude

section of the meter manual.
3.
Enter the altitude as 0 feet or meters. Refer to the

Adjusting the Altitude section of the meter manual.
Probe assembly
1. Hold the membrane module cap in a vertical position. Fill the module cap about 2/3 of the way
full with Dissolved Oxygen Electrolyte Filling Solution.
2. Hold the DO probe in a vertical position with the tip down. Gently screw the module cap onto
the tip. Electrolyte should leak out of the threads.
Note: If electrolyte does not leak out of the threads, air may remain inside the module cap. Repeat this
procedure using more filling solution.
3. Attach the DO probe cable connector to the input connector at the top of the meter. Refer to
the sension 6 Dissolved Oxygen Meter Instruction Manual for additional information.
Probe polarization
Dissolved oxygen probes are continuously polarized when they are connected to the sension
meter. A steady reading will not be seen for 30 to 50 minutes when the probe electrolyte is new or
when the probe has been unplugged for more than one hour. Interrupted connections of less than
one hour will require 5 to 25 minutes before a stable reading is observed.
While not in use, the probe should be stored with the calibration and storage chamber attached to
the end of the probe. Keep the sponge inside the chamber moist.
Zeroing the probe

Zeroing the sension 6 Dissolved Oxygen meter is necessary only when measuring dissolved
oxygen levels less than 1 mg/L or 10% saturation. A new DO probe can generate a 0.02 to 0.05
mg/L positive error in an oxygen-free (anoxic) solution. If this level of error cannot be tolerated,
zero the meter using the following procedure. Zero the meter also after replacing the sensing
membrane or changing the internal filling solution.
Procedure
1. Measure about 150 mL of sample or deionized water into a 250-mL beaker. Place a magnetic
stir bar in the beaker.
2. Add 0.25 g sodium sulfite or the contents of one Silica 3 Reagent Powder Pillow to the water.
Stir to dissolve the reagent.
3. Catalyze the reduction of dissolved oxygen by adding 0.1 mL of a 1000 mg/L Cobalt Standard
solution to the water.
Oxygen, Dissolved
Page 1511
Oxygen, Dissolved
4. Place the probe in the stirring sample for at least 10 minutes. This solution is effective for 30
minutes or more.
5. Press the CAL key. The CAL icon will appear in the upper left corner of the display.
6. Press the READ ENTER key three times to skip to the display showing 100%.
7. Press the 0 key on the keypad then press READ ENTER.
8. The meter shows Stabilizing... while the readings are taken. When the meters zero DO
criteria have been met, it will return to the read mode. The meter will not exit the zeroing
routine until the meters zero criteria have been met.
9. When the meter cannot complete the zeroing procedure, it will begin to beep and show the
faulty probe icon. If the meter does not complete the zeroing procedure and exits to the
reading mode, add additional sodium sulfite and cobalt standard solution to the stirring water.
Otherwise, press EXIT to return to one display screen at a time and leave the calibration
routine without completing the zeroing procedure.
Interferences
Oxidizing gases such as chlorine, chlorine dioxide, sulphur trioxide and bromine can react at the
cathode to produce positive interferences. Reducing gases such as hydrogen, hydrogen sulfide,
sulfur dioxide and boranes can react at the anode. After exposure to reducing gases, the user may
need to clean the anode and replace the internal filling solution and membrane cap.
Oxygen, Dissolved
Page 1512
Oxygen, Dissolved
Analyze samples in-situ, if possible.
Collect samples in 300 mL glass BOD bottles. Fill the bottles completely.
Analyze samples immediately. Do not store samples.
Accuracy check
1. Return the electrode to the calibration and storage chamber. The chamber should contain a
wet sponge or a small amount of water.
2. Allow at least 10 minutes for stabilization.
3. Enter the current barometric pressure and altitude into the meter. Refer to the meter manual.
4. The meter should display 100% saturation. If not, recalibrate the meter.
Method performance
Refer to the electrode and meter manual to determine the method performance.
Summary of method
The Dissolved Oxygen Probe is a Clark-type amperometric sensor used to measure dissolved
oxygen in aqueous solutions. It consists of an anode/cathode electrode system and potassium
chloride-based electrolyte, separated from the sample by a replaceable oxygen-permeable
Teflon membrane. At a constant temperature, the electric current varies linearly with the oxygen
concentration of the solution. A built-in thermistor provides automatic temperature compensation
when using the sension Dissolved Oxygen Meters. The unit % Dissolved Oxygen is dependant
on the temperature and salinity of the sample and the barometric pressure of the environment
where the measurement is taken.

Required reagents
Description
Unit
Catalog number
59 mL
2759126
Description
Unit
Catalog number
sension 6 meter, 115 VAC
each
5185000
sension 8 meter, 115 VAC
each
5455000
Cable, Dissolved Oxygen Probe, 1 meter
.each
.5197000
each
5197003
each
5197015
Barometer, Digital
.each
.2758400
Batteries, AA
4/pkg
1938004
BOD Accessory Kit includes funnel and spacer for Dissolved Oxygen Probe
each
5197100
Calibration Storage Chamber, Dissolved Oxygen Probe
each
5197400
Dissolved Oxygen Service Kit, includes 2 membranes, fill solution, polishing cloth,
2 sponges
each
5196800
Filling Solution, Dissolved Oxygen
Required apparatus
Oxygen, Dissolved
Page 1513
Oxygen, Dissolved
Description
Unit
Catalog number
Docking Station, external, 115 V, N. American style plug
each
5187501
Docking Station, external, 230 V, European style plug
each
5187502
Membranes, for Dissolved Oxygen Probe
2/pkg
5197300
Weight Assembly
each
5196900
Unit
Catalog number
Cobalt Standard Solution, 100 mg/L
100 mL
2150342
Silica 3 Reagent Powder Pillows (contains sodium sulfite)
100/pkg
27169
454 g
19501

Description
Sodium Sulfite

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Oxygen Demand, Biochemical, LBOD 10230

Dilution Method1
1
DOC316.53.01242
Method 10230
LBOD Measurement
Adapted from Standard Methods for the Examination of Water and Wastewater and from Klein, R.L.; Gibbs, C. Journal of Water Pollution
Control Federation, 1979, 51(9), 2257. USEPA recommended for compliance monitoring. Meets ASTM 888-05 (C)
Test preparation

The BOD test is a 5-day test. Follow all steps carefully to make sure that the test does not have to be repeated. The
IntelliCAL LBOD probe measures dissolved oxygen in a 300 mL BOD bottle.
The dilution water for this test must not have an oxygen demand or any toxins. When incubated for 5 days at 20 C, the
dissolved oxygen concentration in the dilution water must not change by more than 0.2 mg/L.
Carbonaceous BOD (CBOD) can be determined by the addition of nitrification inhibitor. A test for CBOD is recommended for
biologically treated effluents, samples seeded with biologically treated effluents and river water.

Description
BOD bottles, 300-mL, glass, with glass stoppers and plastic caps
Dilution water containing nutrient buffer and seed (see Dilution water preparation)
HQ40d or HQ30d meter with IntelliCAL LBOD101 probe
Nitrification inhibitor (for CBOD only)
Quantity
6
varies
1
1 bottle
Pipet, seriological
Incubator

Page 1515

Dilution method
select
sample
size
1. Prepare the dilution

water using a BOD
Nutrient Buffer Pillow. See
Dilution water preparation.

volumes. See Sample size
selection.
3. Stir the sample gently

with the pipet. Use the
pipet to add the minimum
sample volume to the first
BOD bottle.
4. Fill an additional BOD

bottle with dilution water
only. This will be the
dilution water blank.
Add the remaining four

sample volumes to four
more BOD bottles.
When analyzing
disinfected samples or
industrial effluents, refer to
Interferences.
5. If the test is for CBOD,

add two portions of
(approximately 0.16 g) to
each bottle.
The oxidation of nitrogen
compounds will be
prevented. Report results
as CBOD.
6. Fill each bottle to just

below the lip with dilution
water. Allow the dilution
water to flow down the
sides of the bottle to
prevent air bubbles from
becoming trapped in the
bottle.

Page 1516

trapped. Press down on
the stopper and invert the
bottles several times to
mix.
8. Probe calibration is
required before initial and
final BOD readings. Refer
to the Calibration section
of this procedure.
Be sure to measure the
DO of the blank.
An initial DO
measurement is not
necessary when the
graphical method (not for
reporting) is used for
calculation.

Dilution method (continued)
Read
9. Rinse the LBOD probe

10. Place the LBOD Probe

in the BOD Bottle
containing the sample.
Make sure there are no air
bubbles trapped under the
probe.
11. Engage the stir

paddle on the LBOD probe
by pushing the button on
the top of the probe. The
green indicator light on the
top of the probe will
illuminate when the stirrer
is running.
12. Press the key under

READ. After the
measurement has
stabilized, the dissolved
oxygen value will show on
the display.
Read
DO
13. Record the value.

Data is stored
automatically in the Data
Log when Press to Read
or Interval is selected in
the Setup Measurement
Mode. When Continuous
is selected, data will only
be stored when the
GREEN/RIGHT key under
Store is pressed.
14. Remove the LBOD

Probe from the bottle and
stopper the bottles
trapped. Add dilution water
to the lip of each BOD
Bottle to make a water
seal.
15. After five days,

measure the remaining
dissolved oxygen
bottle.
At least 1.0 mg/L DO
should be left in each
BOD bottle.
16. Calculate the BOD

value (see BOD
calculationstandard
methods or BOD
calculationgraphical
method).
Repeat Steps 914 for

each BOD bottle.
Calibration
Water-saturated air calibration
1. Fill a BOD bottle full with water (225 mL). If a 0% calibration point is required, refer to the
Sulfite correction section.
2. Put the BOD stopper in the BOD bottle and shake vigorously for about one minute to saturate
the air with water.

Page 1517

3. Remove the stopper. Put the Intellical LBOD Probe into the BOD bottle for several minutes to
reach equilibrium. Inspect the LBOD probe sensor cap surface to make sure it is dry. If the
sensor cap is wet, carefully dry the cap with a non-abrasive cloth.
4. Make sure the meter is in the measurement screen. Press CALIBRATION.
Note: For the HQ40d meter with two probes connected, make sure the meter is in the single scree
LDO101 mode.
5. Press READ. When the measurement has stabilized, the calibrated measurement will show on
the screen. The standard value will be highlighted.
6. Press DONE to view the calibration summary. The slope value is the comparison between the
latest calibration and the factory calibration expressed as a percentage.
Note: If the calibration slope does not meet the acceptance criteria, the display will show Slope out of
range. Let the probe stand in water-saturated air for several minutes. When the probe reaches
equilibrium, press READ.
7. Press STORE to accept the calibration and return to the measurement mode. The calibration
record is stored in the data log.
Note: A successful calibration will show OK in the measurement screen.
Sulfite correction
1. Fill a BOD bottle full with deionized water.
2. Add 300 mg of sodium sulfite to the bottle.
3. Add a small crystal of cobalt chloride.
4. Put the stopper in the BOD bottle and invert several times to mix the chemicals.
5. Put the LBOD probe in the bottle and engage the stirrer. This will help speed up the
calibration. When the meter reaches a stable reading, press the calibration button on
the meter.
6. After the 0% saturated message is displayed press STORE. After using sulfite, be sure to clean
the probe thoroughly.
7. To clean the sulfite off of the probe, put the LBOD probe in a BOD bottle full of water, activate
the stirrer and run for 10 minutes to remove sulfite residue.
Dilution water preparation

The dilution water must be prepared very carefully to make sure that no source of oxygen demand
or toxins are added. The water that is used to prepare the dilution water must be of very high
quality. The water must not have any organic compounds or any toxic compounds such as
chlorine, copper and mercury.
Use the following guidelines to make sure the dilution water is of high quality.
Guidelines
Use distilled water from an alkaline permanganate distillation for the best results.
Do not use deionized water from ion exchange columns. The resins in the cartridges
(especially new cartridges) will occasionally release organic materials that have an oxygen
demand. In addition, bacteria can grow on the columns and contaminate the dilution water.
Store the distilled water in clean jugs in an incubator at 20 C. Shake the jugs to saturate the
water with air or cap the jugs loosely and store for 24 hours or more.

Page 1518
A small aquarium pump or air compressor can be used to saturate the water with air. Make
sure that the air is filtered and that the filter does not grow bacteria.
Add the nutrients and seed (if necessary) to the distilled water immediately before the test.
The dissolved oxygen concentration in the dilution water must not change by more than
0.2 mg/L when incubated for 5 days at 20 C.
Procedure
1. Prepare and store the distilled water at 20 C (see Guidelines).
2. Select a BOD nutrient buffer pillow from the BOD nutrient buffer pillows table.
3. Shake the pillow to mix the contents.
4. Add the contents of the pillow to the distilled water. Cap the jug and shake vigorously for one
minute to dissolve the nutrients and to saturate the water with air.
5. If the sample is known to be low in bacteria, for example industrial waste or sewage that has
been disinfected, add 3 mL of bacterial seed to each liter of the dilution water. Use raw
sewage for the bacterial seed. Allow the sewage to stand undisturbed at 20 C for 24 to
36 hours before use. Pipet from the upper portion of the sewage. Make sure to measure the
BOD of the seed so that it can be subtracted from the BOD of the sample.
Table 419 BOD nutrient buffer pillows

Volume of dilution water to prepare
BOD nutrient buffer pillow catalog no.
300 mL (add pillow to each BOD bottle)
1416066
3 liters
1486166
4 liters
2436466
6 liters
1486266
19 liters
1486398
Note: To prepare dilution water by the conventional method, pipet 1 mL of each of the following solutions per
liter of distilled water at 20 C: Calcium Chloride Solution, Ferric Chloride Solution, Magnesium Sulfate
Solution and Phosphate Buffer Solution. Cap the bottle and shake vigorously for one minute. The
Phosphate Buffer Solution should be refrigerated to decrease the rate of biological growth. Use care with
all solutions to avoid contamination.

Make an estimation of the sample volumes that are necessary for the test. At least 2.0 mg/L of
dissolved oxygen (DO) should be consumed during the test and at least 1.0 mg/L DO should be
left in the BOD bottle.
Samples such as raw sewage will have a high BOD. Small sample volumes must be used
because large samples will consume all of the oxygen. Samples with a low BOD must use larger
sample volumes to make sure that enough oxygen is consumed to give accurate results.
The elevation of the laboratory changes the amount of oxygen that can dissolve in water (refer to
the Oxygen values at various altitudes (20 C) table). At higher elevations, the amount of oxygen
that can dissolve in water decreases, so less oxygen is available to microorganisms.
Procedure
1. Refer to the Minimum sample volume table to select the minimum sample volume. For
example, if a sewage sample is estimated to contain 300 mg/L BOD, the minimum sample
volume is 2 mL. For sewage effluent with an estimated BOD of 40 mg/L, the minimum sample
volume is 15 mL.

Page 1519

2. Refer to the Maximum sample volume table to select the maximum sample volume. At 1000
feet, with an estimated BOD of 300 mg/L, the largest sample volume is 8 mL. For a BOD of
40 mg/L the maximum volume is 60 mL (also at 1000 feet).
3. Select three other sample volumes between the minimum and maximum volumes so that
there are five sample volumes total.
Table 420 Minimum sample volume

Sample type
Estimated BOD (mg/L)
Minimum sample volume (mL)
Strong trade waste
600
Raw and settled sewage
300
200
150
120
100
Oxidized effluents
Polluted river waters
75
60
10
50
12
40
15
30
20
20
30
10
60
100
200
300
Table 421 Maximum sample volume

BOD at sea level
BOD at 1000 ft
BOD at 5000 ft
2460
2380
2032
1230
1189
1016
820
793
677
615
595
508
492
476
406
410
397
339
304
294
251
246
238
203
10
205
198
169
12
164
158
135
15
123
119
101
20
82
79
68
30
41
40
34
60
25
24
21
100
12
12
10
200
300

Page 1520
Maximum sample volume (mL)
Table 422 Oxygen values at various altitudes (20 C)

Altitude (ft)
Oxygen value (mg/L) in water saturated with air
Sea level
9.2
1000
8.9
2000
8.6
3000
8.2
4000
7.9
5000
7.6
6000
7.4
BOD calculationstandard methods

Use the Standard Methods calculation when the results must be reported to a regulatory agency.
When dilution water is not seeded:
D1 D2
BOD 5, mg/L = ------------------P
When dilution water is seeded:

( D 1 D 2 ) ( B 1 B 2 )f
BOD 5, mg/L = --------------------------------------------------------P
where:
BOD5 = BOD value from the 5-day test
D1 = DO of diluted sample immediately after preparation, in mg/L
D2 = DO of diluted sample after 5 day incubation at 20 C, in mg/L
P = Decimal volumetric fraction of sample used
B1 = DO of seed control before incubation, in mg/L
B2 = DO of seed control after incubation, in mg/L
f = ratio of seed in diluted sample to seed in seed control =
(% seed in diluted sample)/(% seed in seed control) OR
If seed material is added directly to sample or to seed control bottles:
f = (volume of seed in diluted sample)/(volume of seed in seed control)
Report results as CBOD5 if nitrification inhibitor was added.
Averaged results are acceptable if more than one sample dilution meets all of the following criteria:
The remaining DO is at least 1 mg/L
The final DO value is at least 2 mg/L lower than the initial DO value
There is no evidence of toxicity at higher sample concentrations
There are no obvious anomalies

Page 1521
BOD calculationgraphical method

Important Note: The Graphical Method cannot be used when the results must be reported to a
regulatory agency.
1. Plot the mg/L dissolved oxygen (DO) remaining in each diluted sample versus the mL sample
taken. Draw the best straight line through the plotted points. Refer to Dissolved Oxygen per
mL of Sample.
Note: An erroneous point is visually evident at this time and can be disregarded. However, at least three
points should be on the line or very close to it. For unseeded dilution water, the line should cross the
mg/L oxygen remaining scale near or below the oxygen saturation value for the altitude of the laboratory
as discussed in Dilution water preparation.
2. To calculate the BOD, use the following equation which is mathematically equivalent to the
BOD equation in Standard Methods.
mg/L BOD = (A x 300) B + C
where:
A = the slope
The slope of the line is equal to the mg/L DO consumed per mL of sample taken. Take any
point on the line and subtract the mg/L DO remaining at that point from the mg/L DO where the
line crosses the DO scale (Y intercept, mg/L DO remaining). Divide the difference by the mL of
sample at the point chosen.
300 = the volume of the BOD bottle
B = the Y intercept
This is the DO value where the line crosses the DO remaining scale. (This should be very
close to the actual dilution water blank value.)
C = the sample DO
This is the DO of the undiluted sample.
Another way to write this equation is:
mg/L BOD = (Slope x 300) Y intercept + Sample DO
Note: If the best straight line is obtained by linear regression through use of a calculator, the sign (-) of
the slope must be changed (+) before multiplying by 300.
Example:
The mg/L DO remaining was determined for a series of four dilutions of domestic sewage after five
days of incubation. Results were as follows:
mL of sample taken
mg/L DO remaining
2.0
7.50
3.0
6.75
6.0
4.50
9.0
2.25

Page 1522

The DO values were plotted versus the mL of sample taken and a straight line drawn as in
Dissolved Oxygen per mL of Sample. If a set of BOD dilutions is run correctly with a homogeneous
sample, a graph of the mg/L DO remaining versus the sample volume would result in a straight
line. The value where the line intersects the y-axis is equal to the DO content of the dilution water
after incubation, although this is not actually measured. In this case, it was equal to 9.0 mg/L and
the DO of the domestic sewage sample was assumed to be zero. If another type of sample is
used, the DO of an undiluted sample should be measured either by the Winkler titration or
potentiometrically.
The American Public Health Association formula for calculating BOD also can be written as follows
(not approved for reporting purposes):
mg/L DO remaining w/smaller sample volume mg/L DO remaining w/larger sample volume
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 300 DO D + S = mg/L BOD
mL of larger sample volume mL of smaller sample volume
Using this information in the example:

mg/L DO remaining with smaller sample volume = 7.50
mg/L DO remaining with larger sample volume = 2.25
mL of larger sample volume = 9.0
mL of smaller sample volume = 2.0
300 = volume (mL) of BOD bottle
DOD = mg/L DO of dilution water = 9.0
S = mg/L DO of sample = assumed in this case to be zero
Therefore:
7.50
2.25--------------------------- 300 9 + 0 = mg/L BOD = 216 mg/L BOD
9.0 2.0
Using the equation below:

(slope x 300) Y-Intercept + sample DO = mg/L BOD
To determine slope, arbitrarily select point A in Figure 1. At this point the mg/L DO remaining is
equal to 3.0 mg/L. The mL of sample at this point is 8 mL.The difference between the yintercept of 9.0 mg/L and 3.0 mg/L equals 6 mg/L; 6 mg/L divided by 8 mL = 0.75 mg/L per mL.
slope = 0.75 mg/L per mL
Y intercept = 9.0 mg/L
sample DO = 0 (Because the sample is domestic sewage, this is assumed to be zero.)
Therefore:
(0.75 x 300) 9.0 + 0 = mg/L BOD = 216 mg/L BOD

Page 1523
mg/L DO Remaining
y Intercept
mL of Sample
Figure 35 Dissolved Oxygen per mL of Sample
Interferences
Many chlorinated and industrial effluents require special handling to ensure reliable BOD results.
Usually, careful experimentation with the particular sample will indicate what modifications should
be made to the test procedure.
Toxins in the sample will adversely affect any microorganisms present and result in lower BODs.
To eliminate small amounts of residual chlorine, allow the sample to stand for one to two hours
at room temperature. For larger quantities, determine the amount of sodium thiosulfate to add to
the sample as follows:
c. Measure 100 mL of sample into a 250-mL Erlenmeyer flask. Using a 10-mL serological
pipet and a pipet filler, add 10 mL of 0.020 N Sulfuric Acid Standard Solution and 10 mL of
Potassium Iodide Solution, 100-g/L, to the flask.
d. Add three full droppers of Starch Indicator Solution and swirl to mix.
e. Fill a 25-mL buret with 0.025 N Sodium Thiosulfate Standard Solution and titrate the
sample from dark blue to colorless.
f.
Calculate the amount of 0.025 N Sodium Thiosulfate Standard Solution to add to the
sample:
mL titrant used x volume of remaining sample
mL 0.025 N sodium thiosulfate required = ------------------------------------------------------------------------------------------------------------------------100
g. Add the required amount of 0.025 N Sodium Thiosulfate Standard Solution to the sample.
Mix thoroughly. Wait 10 to 20 minutes before running the BOD test.

Page 1524

To eliminate the effect of phenols, heavy metals or cyanide, dilute the sample with high quality
distilled water. Alternately, the seed used in the dilution water may be acclimatized to tolerate such
materials. Acclimatize seed as follows:
a. Fill a one-gallon stainless steel or plastic container with domestic sewage and aerate for
24 hours. Allow the heavier material to settle.
b. After settling for one hour, siphon off three quarts of material and discard.
c. Fill the container with a mixture of 90% sewage and 10% wastes containing the toxic
material.
d. Aerate for 24 hours. Repeat steps b and c with increasing amounts of waste until the
container holds 100% toxic waste material.
Optimum pH for the BOD test is between 6.5 and 7.5. Adjust samples to pH 7.2 with Phosphate
Buffer Solution or 1 N Sulfuric Acid or Sodium Hydroxide Standard Solution if the pH is not in
this range.
Cold samples may be supersaturated with oxygen and will have low BOD results. Fill a one-quart
bottle about halfway with cold sample and shake vigorously for two minutes. Allow sample
temperature to reach 20 C before testing.
Accuracy check
BOD Standard Solution, Voluette Ampule, 300-mg/L, 10-mL (300-mg/L of glucose and
300-mg/L of glutamic acid)
Seeded dilution water
4 BOD bottles
1.04.0 mL Class A volumetric pipets
TenSette Pipet

2. Use a pipet to add 1.00, 2.00, 3.00 and 4.00 mL of standard into four BOD bottles.
3. Fill the bottles with seeded dilution water and measure the DO concentration.
4. Incubate the bottles at 20 C for five days.
5. Measure the DO remaining in each bottle.
6. Calculate the BOD value (refer to BOD calculationstandard methods or BOD calculation
graphical method).
7. Divide the value by two. The result for comparison with Standard Methods should be
198 ( 30.5) mg/L.
Note: The result must be divided by 2 to correspond with values reported in Standard Methods because
the Standard Methods procedure uses 150 mg/L each of glucose and glutamic acid.

Page 1525
Method performance
The following statements are true for dissolved oxygen when the measurement is below 10 mg/L
DO and the temperature is kept between 10 and 30 C for a single probe.
Instrument
Standard
Precision
Distribution
LBOD101
7.948.06 mg/L DO
7.978.03 mg/L DO
Summary of method
Biochemical Oxygen Demand (BOD) is a measurement of the oxygen requirements of municipal
and industrial wastewaters and sewage. The test results are used to calculate the effect of waste
discharges on the oxygen resources of the receiving waters. The BOD test is of limited value in
measuring the actual oxygen demand because temperature change, biological population, water
movement, sunlight, oxygen concentration and other environmental factors cannot be reproduced
accurately in the laboratory. The BOD test is of greatest value after patterns of oxygen uptake for a
specific effluent and receiving water have been established.
The BOD test is performed by incubating a sealed wastewater sample (or a prepared dilution) for
the standard five-day period and then determining the change in dissolved oxygen content. The
BOD value is then calculated from the results of the dissolved oxygen tests.

Required reagents
Description
BOD Nutrient Buffer Pillows, for 3 liters of dilution water
Quantity/Test
Unit
Catalog number
1 pillow
50/pkg
1486166
Quantity/Test
Unit
Catalog number
Required apparatus
Description
BOD Bottle, glass-stoppered, 300-mL
each
62100
BOD Bottle Cap
6/pkg
241906
each
62011
Clippers, large
each
96800
HQ40d meter
each
HQ40d
HQ30d meter
each
HQ30d
IntelliCAL LBOD probe
each
LBOD10101
each
919002
each
53237
each
53238
each
1218900
OR
Pipet, seriological:
Pipet Filler

Page 1526
Description
BOD Standard Solution, Voluette Ampule, 300-mg/L, 10-mL
Unit
Catalog number
16/pkg
1486510
Unit
Catalog number

Description
BOD Nutrient Buffer Pillows
for 300 mL of dilution water
50/pkg
1486166
50/pkg
2436466
50/pkg
1486266
25/pkg
1486398
Buffer Solution, APHA, for BOD, pH 7.2, phosphate type
1L
43153
Calcium Chloride Solution, APHA, for BOD
1L
42853
Ferric Chloride Solution, APHA, for BOD
1L
42953
Magnesium Sulfate Solution, APHA, for BOD
1L
43053
35 g
253335
Dispenser Cap, for Nitrification Inhibitor
each
45901
500 mL
1228949

Sodium Hydroxide, pellets, ACS
Up-Grade Kit, HQd Accessories, LBOD Probe
500 g
18734
100 mL MDB
104532
1L
35253
100 mL MDB
34932
1L
20353
1L
127053
each
LBOD10130
Replacement LBOD Sensor cap, with I-button
each
5838000
Replacement Stirrer assembly
5/pkg
5850800
5825800
Field Kit, includes: protective glove, 2 standard probe holders and 5 120-mL sample cups
each
Keyboard (HQ40d meter only)
each
LZV582
Citizen PD-24 USB Handy printer, 115 VAC
each
5835800
10/pkg
5818400
Color Coded Probe Clips (5 color coded sets), 5 sets

Page 1527

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
LDO, 10360
Oxygen, Dissolved
DOC316.53.01243
Method 10360
(0.1 to 20.0 mg/L or 1 to 200% saturation)
LDO Probe
Scope and Application: For water, wastewater and process water applications
Test preparation


Meter
HQd meters
Probe
LDO101

Before attaching probes to the HQd meter for the first time, set the meter time and date.
Refer to the probe instructions for probe preparation.
For probes that are continuously immersed in aqueous solutions, condition the sensor cap for 72 hours.
When an IntelliCAL probe is connected to a HQ30d or HQ40d meter, the meter automatically recognizes the measurement
parameter and is ready for use.
The IntelliCAL LDO101 probes automatically compensate for barometric pressure, elevation and temperature.
The LDO probe is calibrated at the factory. For more accurate results, manual calibration is recommended. Refer to the
Calibration section of this procedure.
Salinity affects the concentration of dissolved oxygen in the sample. To correct for salinity effects, refer to Modifying LDO
Measurement Options in the meter manual.

Description
Quantity
HQd meter
IntelliCAL LDO101 probe
Sensor cap for HQd with I-button
Shroud
BOD bottle, 300-mL or Erlenmeyer flask, 250-mL
Beaker, polypropylene (100-, 250-, 400- or 600-mL)
Oxygen, Dissolved
Page 1529
Oxygen, Dissolved
Method Name for powder pillows
1. Prepare the probe.

Refer to the probe
instructions.
2. Connect the probe to

the meter.
3. Calibrate the probe.

Refer to the Calibration
section of this procedure.
Go to step 4 for laboratory
tests. Go to step 5 for field
tests.
4. Laboratory tests:
Immerse the probe in the
beaker containing the
sample solution. Move the
probe up and down and
tap it on the beaker to
remove bubbles from the
probe.
Read
5. Field tests: Immerse

the probe directly into the
sample. Move the probe
up and down to remove
bubbles from the probe.
6. Press READ to store

data in the data log.
Calibration
The LDO probe is calibrated at the factory. For more accurate results, manual calibration
is recommended.
1. Remove the shroud from the probe body.
2. Add a small amount of water (about 1 cm ) to the bottom of narrow-neck bottle, such as a
BOD bottle.
Note: Use a wider neck bottle or flask (for example, a 250-mL Erlenmeyer flask) for the rugged probe.
3. Insert a stopper and shake vigorously for several minutes.

4. Remove the stopper. If the sensor cap surface is wet, carefully dry the cap with a
nonabrasive cloth, then put the probe in the bottle. Allow several minutes for the probe to
reach equilibrium.
Oxygen, Dissolved
Page 1530
Oxygen, Dissolved
5. Make sure the meter is in the measurement screen. Press the CALIBRATION key.
Note: For HQ40d meters with two probes attached, the display must be in the single screen
LDO101 mode.
6. Press READ. When the measurement is stable, the calibrated measurement will show on the
display. The standard value will be highlighted on the display.
7. Press DONE to view the calibration summary. The slope value is the comparison between the
latest calibration and the factory calibration expressed as a percentage.
Note: If the calibration slope does not meet the acceptance criteria, the display will show Slope out of
range. Let the probe stand in water-saturated air for several minutes. When the probe reaches
equilibrium, press READ.
8. Press STORE to accept the calibration and return to the measurement mode. The calibration
record is stored in the data log.
Note: A successful calibration will show OK in the measurement screen.
Interferences
There are no significant interferences with the LDO technology.
The IntelliCAL LDO101 probes are designed for water and wastewater applications, but can be
used for other applications. Some organic solvents may damage the sensor cap and probe body.
Analyze samples in-situ, if possible.
Collect samples in an appropriate container. Fill completely and analyze immediately.
Do not store samples.
Accuracy check
1. Return the electrode to a water-saturated air environment.
2. Allow at least 10 minutes for stabilization.
3. Read the % saturation on the right side of the measurement mode screen. The meter should
display 100% saturation. If not, allow additional time for the air to reach water saturation or
calibrate the probe.
Method performance
The following statements are true for dissolved oxygen when the temperature is kept between 10
and 30 degrees C.
Method
Standard
Precision
Distribution
Accuracy
10360
8.00 mg/L DO
7.958.05 mg/L DO
7.908.10 mg/L DO
10360
15.00 mg/L DO
14.9015.10 mg/L DO
14.8015.20 mg/L DO
Oxygen, Dissolved
Page 1531
Oxygen, Dissolved
Summary of method
The oxygen sensor is made up of a clear, oxygen impermeable hard substrate. An oxygen
sensitive luminescent dye, along with a scattering agent, is pad-printed on the substrate. A final
overlay of dark pigment is added to prevent stray light from entering the measurement cell. The
luminescent dye emits red light when exposed to blue light. The scattering agent distributes the
emitted light throughout the sensor matrix and contributes to the opacity of the sensor. Pulses from
a red LED serve as an internal reference. The duration of the luminescence is proportional to the
concentration of dissolved oxygen in the sample.

Required apparatus (select one)
Description
Quantity/Test
Unit
Catalog number
HQ40d multi-parameter meter, dual input
each
HQ40d53000000
HQ30d multi-parameter meter, single input
each
HQ30d53000000
Description
Unit
Catalog number
LDO Probe, standard, with 1 m cable
each
LDO10101
LDO Probe, standard, with 3 m cable
each
LDO10103
Required probes (select one)
LDO Probe, rugged, with 5 m cable
each
LDO10105
each
LDO10110
each
LDO10115
each
LDO10130
Description
Unit
Catalog number
AC Power Adapter for HQd meters (included w/ HQ40d)
each
5826300
BOD bottle, 300 mL
each
62100
BOD bottle, 300 mL
6/pkg
62106
Optional apparatus
Citizen PD-24 USB Handy printer, 115 VAC
each
5835800
Color Coded Probe Clips (5 color coded sets) 5 sets
10/pkg
5818400
Depth Markers for Rugged LDO probe only
10/pkg
5828610
each
2089846
Field Kit (Includes glove kit, 2 probe holders and 5 120 mL sample cups)1
each
5825800
Glove kit only for HQd meters
each
5828700
Probe Holder for HQd meter, IntelliCAL Standard probes only
each
5829400
Replacement Sensor cap w/ I-button
each
5811200
Replacement Shroud kit Rugged LDO probe
each
5825900
USB Keyboard for HQd meters (must have 5813400 & 5826300)
each
LZV582
USB/DC Adapter for HQd meters (must have 5826300, inc w/HQ40d)
each
5813400
Included with HQ40d

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
ORP, 10228
Oxidation Reduction Potential

(ORP)
DOC316.53.01244
Method 10228
(2000 mV to 2000 mV)
ORP Electrode
Scope and Application: For drinking water, wastewater and process water applications
Test preparation


Meter
Platinum-series ORP electrode
Gel-filled ORP electrode
sension 1
5193700
5193900
sension 2
5193700
5193900
sension 3
5193700
5193900
sension 4
5193700
5193900

Description
Quantity
sension pH/mV meter
Gel-filled or combination ORP electrode
Beaker, polypropylene (100-, 250-, 400- or 600-mL)
Deionized water
Oxidation Reduction Potential (ORP)

Page 1533

Oxidation reduction potential (ORP)
1. Connect the electrode

to the meter.
2. Turn on the meter.

Press PHMV to select mV.
5. Put the electrode in

the sample.
6. When the
store or record the mV and
temperature readings.
3. Rinse the electrode in

deionized water and
blot dry.
4. Platinum-series
electrodes only: Press
the dispenser button on
top of the electrode until
the electrolyte get is visible
at the reference junction.
Standard hydrogen electrode (SHE) calculation

For some applications it is customary to report oxidation reduction (redox) potential measurements
relative to the standard hydrogen electrode (SHE).
1. Select the value that corresponds to the temperature of the solution measured. Refer to the
Reference electrode potentials table.
2. Substitute the electrode potential value (C) in the equation and solve for ESHE:
E SHE = E O + C
Where:
ESHE = oxidation reduction potential of the sample relative to the SHE, following the
international sign convention.
EO = potential developed by the ORP electrode
C = potential developed by the reference electrode relative to the SHE.

Page 1534

The Reference electrode potentials table shows the potentials, C, developed by the reference
electrode relative to the standard hydrogen electrode at various temperatures.
Table 425 Reference electrode potentials

Temperature (C)
Electrode potential in mV (C)
10
221
15
216
20
213
25
208
30
204
35
200
40
196
Interferences
Many factors limit the interpretation of ORP measurements in water. These factors include
irreversible reactions, electrode poisoning, the presence of multiple redox couples, very small
exchange currents and inert redox couples. ORP measurements in the field correlate poorly with
ORP values calculated from the redox couples present. Due to these factors, the interpretation of
ORP measurements will be specific to your particular application.
Sample collection, preservation, storage and cleaning
Analyze samples immediately after collection.
For best results, minimize contact with the environment and the time between collection
and measurement.
If the electrode is not clean, remove inorganic deposits by immersing the electrode tip in roomtemperature 0.1 N HCl for 10 minutes. To remove grease, oil or other organic deposits,
immerse the tip in warm water and detergent and swirl gently. After cleaning, rinse with DI
water. Repeat Procedure A after cleaning.
Accuracy check
Checking the electrode is necessary only when there is evidence of malfunction that cannot be
traced to other causes.
Procedure A
1. Open an ampule of Light's Solution or ORP verification solution*. Pour the contents of the
ampule into a beaker.
2. Immediately place the ORP Electrode tip into the solution.
3. Verify that the potential is 475 10 mV or the specified ORP value.
Note: This potential is the standard reduction potential for Fe2+/3+ with the reference electrode potential
subtracted. The solution is 0.01 M in both Fe2+ and Fe3+.
* See Optional apparatus.

Page 1535

Procedure B
1. Prepare solution A (0.1 M potassium ferrocyanide and 0.05 M potassium ferricyanide) as
follows:
a. Weigh out 4.22 g reagent-grade K4Fe(CN)63H2O and 1.65 g reagent-grade K3Fe(CN)6.
Put the solids in a 100-mL volumetric flask.
b. Add about 50 mL deionized water and swirl to dissolve the solids.
c. Dilute to volume with deionized water.
2. Prepare solution B (0.01 M potassium ferrocyanide, 0.05 M potassium ferricyanide and 0.36 M
potassium fluoride) as follows:
a. Weigh out 0.42 g of reagent-grade K4Fe(CN)6? 3H2O, 1.65 g of reagent-grade
K3Fe(CN)6 and 3.39 g of reagent-grade KF2H2O. Put the solids in a 100-mL volumetric
flask.
b. Add about 50 mL deionized water and swirl to dissolve the solids.
c. Dilute to volume with deionized water.
3. Transfer solution A to a 150-mL beaker. Place the electrode in the solution and wait until the
reading stabilizes. The potential should be about 234 mV.
4. Rinse the electrode and repeat the measurement with solution B. The potential should be
about 66 mV greater in solution B than in solution A.
Summary of method
Redox measurements are made by determining the electron activity of a solution using an inert
indicator electrode and a reference electrode. The potential difference between the indicator
electrode and the reference electrode equals the redox potential of the system. The Gel-filled ORP
and Platinum Series ORP electrodes use a platinum indicator electrode and a silver/silver chloride
reference electrode.

Required apparatus
Description
Quantity/Test
Unit
sension 1 Portable pH/mV Meter
each
5170010
each
5172510
sension 3 Benchtop pH/mV Meter
each
5175010
sension 4 Benchtop pH/ISE Meter
each
5177510
Select one meter and probe combination:
Gel-filled Combination ORP Electrode, 5-pin
Catalog number
5193900
5193700
OR
Platinum Series Combination ORP Electrode, 5-pin

Page 1536
Optional reagents
Description
Unit
Catalog number
Light's Solution, ampules
20/pkg
26125-20
200 mV ORP Solution
500 mL
25M2A1001-115
600 mV ORP Solution
500 mL
25M2A1002-115
Hydrochloric Acid Standard Solution 0.1 N
1L
1481253
Sodium Hydrochloride Standard Solution, 0.1 N
1L
19153
Description
Unit
Catalog number
each
.108042
each
108046
each
108048
each
108052
Optional apparatus
each
108053
Digital Titrator
each
1690001
each
50543
Electrode Holder, with electromagnetic stirrer
each
4530001
Electrode Holder
each
4530000
Stir Bar, Magnetic, 22.2 x 7.9 mm
each
2095350

Page 1537

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
pH
pH
DOC316.53.01245
USEPA Electrode Method
Method 8156
pH Meter
Scope and Application: For drinking
water1,
wastewater2
and process water applications.
Based on Standard Method 4500-H+B, ASTM Method D1293-95 and USEPA Method 150.1
2 Based on Standard Method 4500-H+B, ASTM Method D1293-84(90)/(A or B) and USEPA Method 150.1
Test preparation

Meter
Standard probe
Rugged probe1
HQ40d
PHC10101, PHC10103 (gel)

PHC30101, PHC30103 (liquid)
PHC10105, PHC10110, PHC10115, PHC10130
HQ30d
PHC10101, PHC10103 (gel)

PHC10105, PHC10110, PHC10115, PHC10130
HQ11d
PHC10101, PHC10103 (gel)

PHC10105, PHC10110, PHC10115, PHC10130
sension 1
5191000 (platinum)
5193500 (gel)
5194000 (refillable)
5191500 (flat)
sension 3
5191000 (platinum)
5193500 (gel)
5194000 (refillable)
5191500 (flat)
Designed for field use.

For optimal response time, condition the electrode for several minutes in a solution comparable to the sample in terms of pH
and ionic strength.
For rugged IntelliCAL electrodes, the shroud may need to be removed before measurement and calibration.
For HQd meters, data is stored automatically when Press to Read or Interval is selected in the Setup Measurement Mode.
When Continuous is selected, data will only be stored when the key under STORE is pressed. For sension meters, the
STORE key must be pressed.
Description
Quantity
pH meter and probe combination
pH buffers (4.0, 7.0, 10.0)
Beakers/sample containers
pH
Page 1539
pH
Sample pH measurement (calibration required)
1. Refer to the operation

section of the electrode or
meter manual to prepare
the pH electrode and
meter.
2. Connect the pH
electrode to the meter.

Make sure that the meter
is set to measure to
measure pH.
4. For setup options such

as measurement
resolution, temperature
units, calibration buffer set
and other options refer to
appropriate meter or
electrode manual.
5. In three separate
beakers or appropriate
containers, prepare fresh
buffers of 4.0, 7.0 and 10.0
pH.
6. Calibrate the pH meter

and electrode as directed
in the instructions in the
meter or probe manual.
7. Rinse the electrode in

deionized water and blot
dry prior to sample
measurement. Rinse the
electrode between
measurements to
minimize contamination.
8. Put the electrode in

the sample and press
READ. For faster
response, stir at a slow to
moderate rate.
The sample pH should fall

within the pH range of the
calibration buffers. One,
two or three calibration
buffers may be used to
calibrate.
Other pH calibration
buffers sets may be used.
pH
Page 1540
Make sure that the

calibration slope is
acceptable (typically -58
3 mV per pH unit at
25 C).
pH
Sample pH measurement (calibration required) (continued)
9. When the
store or record the pH and
temperature values.
For HQd meters, data is
stored automatically when
Press to Read or Interval
is selected in the Setup
Measurement Mode.
When Continuous is
selected, data will only be
stored when the key under
STORE is pressed. For
sension meters, the
STORE key must be
pressed.
10. Store the pH electrode

in pH storage solution
when not in use. See
Sample collection,
preservation, general
storage and cleaning for
more details.
Low Ionic Strength (LIS) or high purity water measurements

Low ionic strength solutions have very low buffering capacity and readily absorb carbon dioxide
from the air. When a sample absorbs carbon dioxide from the atmosphere, carbonic acid forms.
Carbonic acid decreases the sample pH and increases conductivity, causing inaccurate readings.
One solution to this problem is to test the sample in a low volume, airtight sample chamber such
as a Low Ionic Strength (LIS) Chamber. Use refillable or platinum series electrodes for
measurement of pH in LIS or high purity waters.
Initial use
1. Before measuring an LIS sample, soak the electrode in a solution similar to the sample in ionic
strength and pH for 10 to 15 minutes.
2. Rinse the electrode with deionized water from a wash bottle.
3. Blot excess liquid with a soft paper towel.
4. Put the electrode in the sample.
pH
Page 1541
pH
Between uses
Between uses, in intervals of up to a two hours, the electrode can be stored in the sample (if the
sample is not an extreme pH), or in a neutral LIS solution such as tap water. Rinse the electrode
before use to prevent sample contamination.
Important Note: If pH electrodes are stored in LIS samples for a long period of time, the electrode
life may be shortened.
After measuring the LIS samples, put electrode back into the electrode storage solution or
3 M KCl.
Sample collection, preservation, general storage and cleaning
Analyze samples immediately, preferably in the field.
Storage of an electrode is based on how long the electrode will be stored, how quickly the
electrode needs to be used and the type of sample being measured. For general storage, use
the Hach storage solution or a 3 M Potasium Chloride (KCl) solution.
A contaminated glass bulb or fouled electrode may cause slow response times. Do not clean
the bulb too often because the bulb life may shorten.
To clean an electrode with general contamination, immerse the electrode tip in 0.1 N
hydrochloric acid (HCl). Then, immerse the electrode in 0.1 N sodium hydroxide (NaOH) and
again in 0.1 N hydrochloric acid, each for a 2-minute period. Rinse with deionized water and
soak in deionized water for at least 15 minutes.
To clean an electrode contaminated with oils and fats, immerse the electrode tip in a detergent
solution. Use a soft brush or ultrasonic bath if necessary. Avoid scratching the glass bulb.
Interferences
Acid error is negligible.
Sodium error, usually present in alkaline solutions, is low but increases at pH values higher
than pH 11.
For more detailed information, refer to the meter or electrode manual.
Accuracy check
Check electrode response
An electrode is responding properly if its calibration slope meets the slope specifications of the
electrode (typically -58 3 mV at 25 C).
Check calibration accuracy
Return the electrode to a calibration buffer and measure the pH to test the system. Rinse and
recondition the electrode before measuring subsequent samples.
Method performance
The accuracy of a pH measurement depends on many factors associated with the overall pH
system, including the pH meter, choice of electrode and pH standards or buffers used during pH
calibration. Refer to the appropriate electrode and meter manual to determine method
performance.
pH
Page 1542
pH
Summary of method
pH is a measure of the hydrogen ion activity in a solution and is defined as:
log10 aH+
Where
aH+ is the activity of the hydrogen ion.
A Combination pH Electrode responds to the hydrogen ion concentration (activity) by developing
an electrical potential at the glass/liquid interface. At a constant temperature, this potential varies
linearly with the pH of the solution being measured.
Water with relatively high conductivity typically has a fairly high buffer capacity. Slight pH changes
due to absorption of carbon dioxide are usually not significant. If the sample conductivity is not
known and high accuracy is desired, follow either the LIS or high purity water methods.

Required apparatus and reagents
Description
Quantity
Unit
Catalog number
HQ meters and probes (select one meter and probe combination)

HQ40d meter
each
HQ40d53000000
HQ30d meter
each
HQ30d53000000
HQ11d meter
each
HQ11d53000000
pH Gel Probe, standard, with 1 m cable
each
PHC10101
pH Gel Probe, standard, with 3 m cable
each
PHC10103
pH Liquid Probe, standard, with 1 m cable
each
PHC30101
pH Liquid Probe, standard, with 3 m cable
each
PHC30103
pH Gel Probe, rugged, with 5 m cable
each
PHC10105
each
PHC10110
each
PHC10115
each
PHC10130
sension 1
each
5170000
sension 3
each
5175000
Electrolyte cartridge, potassium chloride
2/pkg
2546902
Gel Filled pH electrode
each
5193500
Refillable pH electrode, platinum series electrode (5191000

as #1); flat Platinum series electrode (5195000 as #4)
each
5194000
each
5189900
sension meters and probes (select one meter and probe combination)
For LIS and high purity water measurements

Low Ionic Strength (LIS) Chamber
pH
Page 1543
pH
Description
Hach
Unit
Catalog number
Solutions1
pH Color buffer solution kit (NIST), 500 mL, includes:

pH 4.01 +/- 0.02 pH buffer (NIST)
each
2947600
500 mL
2283449
pH 7.00 +/- 0.02 pH buffer (NIST)
500 mL
2283549
pH 10.01+/- 0.02 pH buffer (NIST)
500 mL
2283649
Powder pillows1
pH 4.01 +/- 0.02 pH buffer powder pillow (NIST)
50/pkg
2226966
pH 7.00 +/- 0.02 pH buffer powder pillow (NIST)
50/pkg
2227066
pH 10.01+/- 0.02 pH buffer powder pillow (NIST)
50/pkg
2227166
Radiometer Analytical (IUPAC Series certified pH standards):

pH 1.679 0.010 at 25 C
500 mL
S11M001
pH 4.005 0.010 at 25 C
500 mL
S11M002
S11M004
pH 7.000 0.010 at 25 C
500 mL
pH 10.012 0.010 at 25 C
500 mL
S11M007
pH buffer 1.09, technical
500 mL
S11M009
500 mL
S11M010
500 mL
S11M011
pH Filling Solution (for PHC301), 3M KCl, saturated with AgCl
30 mL
2841700
pH Electrode Storage Solution
500 mL
2756549
Description
Unit
Catalog number
Sample bottle, general purpose with Screw-cap, polypropylene, 500-mL
each
2758101
Sample bottle, cleaned and certified, HDPE, suitable for EPA reporting, 500-mL
each
2758201
sension 2 meter
each
5172511
sension 4 meter
each
5177500
Refill Solution and Storage:
Larger quantities are available

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Chemical Procedures Explained
Page 1545
Page 1546
Acidity tests explained
Acidity
For water, wastewater and seawater
Digital Titration Method
Acidity is the quantitative expression of waters capacity to neutralize a strong base to a

designated pH and an indicator of how corrosive water is. Acidity can be caused by weak organic
acids, such as acetic and tannic acids, and strong mineral acids including sulfuric and hydrochloric
acids; however, the most common source of acidity in unpolluted water is carbon dioxide in the
form of carbonic acid.
Acidity is classified by the pH value of a titration end point. Acidity caused by mineral acids exhibits
a pH below 4.5. Salts of certain metals, particularly those with trivalent iron and aluminum, may
hydrolyze in water and also contribute to acidity.
Standard Methods for the Examination of Water and Wastewater (Standard Methods)
recommends titration with sodium hydroxide to an end point pH of 3.7 to determine mineral acidity.
Titrate to pH 8.3 to determine total acidity.
Acidity is commonly determined using methyl orange as a color indicator of the pH end point.
Because methyl orange undergoes a color change from red to orange at a pH of 3.7, the results of
the titration are termed Methyl Orange Acidity. Hach procedures for acidity use bromphenol blue
indicator instead of methyl orange because the methyl orange color change is difficult to detect.
The bromphenol blue indicator gives a sharp end point change from yellow to blue-violet.
Total acidity includes acidity caused by mineral acids, weak organic acids, and carbon dioxide (in
the form of carbonic acid). Acidity determined by titrating to a phenolphthalein end point pH of 8.3
corresponds to the neutralization of carbonic acid to bicarbonate. Because carbon dioxide is the
major cause of acidity in natural waters, in most cases the phenolphthalein acidity is equal to the
total acidity. Acidity tests can be performed using a pH meter to detect the end points; however,
methyl orange acidity and phenolphthalein acidity are the terms used to describe the results.
Results of the acidity tests are reported in mg/L CaCO3.
Table 427 Chemical reactions

Methyl orange acidity
Phenolphthalein acidity
2NaOH + H 2 SO 4 Na 2 SO 4
NaOH + H 2 CO 3 NaHCO 3 + H 2 O
NaOH + HCl NaCl + H 2 O
NaOH + HC2 H 3 O 2 NaC2 H 3 O 2 + H 2 O
Reactions of indicator Phenolphthein

HO
OH
O
C
Figure 36 ColorlesspH < 8.3
Acidity
Page 1547
Acidity
HO
C O
Figure 37 PinkpH > 8.3

Reactions of indicator Bromphenol blue*
Br
Br
HO
OH
Br
C
Br
O
O
S
O
Figure 38 Yellow=pH 3
Br
Br
HO
Br
Br
C
SO3
Figure 39 Blue-violet=pH 4.6
* 3,3,5,5, -Tetrabromophenolsulfonephthalein
Acidity
Page 1548
Alkalinity tests explained
Alkalinity
Titration Method
Introduction
Alkalinity is a measure of the capacity of water to neutralize acids. Alkalinity of water is due
primarily to the presence of bicarbonate, carbonate, and hydroxide ions. Salts of weak acids, such
as borates, silicates and phosphates, may also contribute. Salts of certain organic acids may
contribute to alkalinity in polluted or anaerobic water, but their contribution usually is negligible.
Bicarbonate is the major form of alkalinity. Carbonates and hydroxide may be significant when
algal activity is high and in certain industrial water and wastewater, such as boiler water.
Alkalinity is significant in the treatment processes for potable water and wastewater. The alkalinity
acts as a pH buffer in coagulation and lime-soda softening of water. In wastewater treatment,
alkalinity is an important parameter in determining the amenability of wastes to the treatment
process and control of processes such as anaerobic digestion, where bicarbonate alkalinity, total
alkalinity and any fraction contributed by volatile acid salts become considerations.
Alkalinity is expressed as phenolphthalein alkalinity or total alkalinity. Both types can be
determined by titration with a standard sulfuric acid solution to an end point pH, evidenced by the
color change of a standard indicator solution. The pH also can be determined with a pH meter.
Phenolphthalein alkalinity is determined by titration to a pH of 8.3 (the phenolphthalein end point)
and registers the total hydroxide and one half the carbonate present. Total alkalinity is determined
by titration to a pH of 4.9, 4.6, 4.5, or 4.3, depending on the amount of carbon dioxide present. The
total alkalinity includes all carbonate, bicarbonate and hydroxide alkalinity.
The following end points are recommended for determining total alkalinity in water samples of
various compositions and alkalinity concentrations.
Table 428 Determine total alkalinity

Sample Trait
End Point
Alkalinity approximately 30 mg/L
pH 4.9
pH 4.6
pH 4.3
Silicates or phosphates known present or suspected
pH 4.5
Industrial waste or complex system
pH 4.5
Routine or automated analyses
pH 4.5
Chemical reactions
Sulfuric acid (hydrochloric acid may be used) reacts with the three forms of alkalinity, converting
them to water or carbonic acid. If hydroxide is present, it reacts to form water:
2OH + H 2 SO 4 2H 2 O + SO 4
This conversion usually is complete at a pH of about 10. Phenolphthalein alkalinity is determined

by titration to an end point pH of 8.3, which corresponds to the conversion of carbonate to
bicarbonate.
2CO 3
+ H 2 SO 4 2HCO 3 + SO 4
If hydroxide is present, titration to pH 8.3 will indicate the alkalinity due to all of the hydroxide plus
one-half of the carbonate. Continued titration to pH 4.5 completes the conversion of carbonate
plus any bicarbonate present to carbonic acid. This value is termed Total Alkalinity.
Alkalinity
Page 1549
Alkalinity
2HCO 3 + H 2 SO 4 2H 2 CO 3 + SO 4
Methyl red
O
C OH
CH3
N N
N
CH3
H
Figure 40 Red=pH 4.8
O
C O
CH3
N
CH3
Figure 41 Yellow=pH 6.0
Bromcresol green
Br
Br
OH
Br
O3S
CH3
Br
CH3
Figure 42 Blue=pH 5.5
Alkalinity
Page 1550
Alkalinity
Br
Br
OH
OH
CH3
Br
Br
C
CH3
O
S
O
Figure 43 Yellow=pH 3.8
Alkalinity
Page 1551
Aluminum tests explained
Aluminum
For water
Aluminon Method
Introduction
Aluminum, the earths most abundant metal, is present in natural waters through contact with
rocks, soil and clay. Alum coagulation in water clarification systems may also contribute to the
aluminum content of treated water, although only 2050 g/L of aluminum remain in the finished
product from a well-controlled operation.
The Aluminon Method is one of the oldest and most thoroughly documented methods available for
determining aluminum in water. The AluVer 3 Aluminum Reagent used in this method is
packaged in powder pillow form, providing exceptional stability.
Chemical reactions
AluVer is an aluminon reagent in combination with a pH buffer. AluVer 3 reacts with aluminum
present in a sample to form a reddish-colored solution in direct proportion to the aluminum
concentration.
Ascorbic acid is added prior to the addition of AluVer 3 to eliminate interference due to iron. To
establish a reagent blank, the sample is split after the addition of the AluVer 3. Bleaching 3
Reagent is then added to one-half of the split sample to bleach out the color of the aluminum
aluminon complex.
COOH
COOH
O
HO
COOH
O
O
HO
+Al3+
COOH
OH
Al3+ + 3H+
COOH
OH
Aurintricarboxylic acid
Figure 44 Chemical reaction
Aluminum
Page 1552
Barium tests explained
Barium
For water, wastewater, oil-field water and
seawater
Introduction
Although barium is relatively abundant in nature, usually only trace amounts are found in water.
Barium concentrations average about 0.05 mg/L in potable waters, but may range as high as
0.9 mg/L in some natural waters. More than 1 mg/L of barium implies that the water is not suitable
for drinking and is polluted by industrial wastes. Barium and its compounds can be found in
pigments, rat poisons, fireworks, and are used in rubber making, x-ray photography and even as
weighting agents for oil well drilling.
Chemical reactions
Barium is determined by adding sulfate to the water sample to form barium sulfate, which
precipitates. These particles are held in suspension as colloids by the BariVer 4 Reagent. The
barium concentration is determined by measuring the resulting turbidity using a spectrophotometer
or colorimeter. The barium concentration is proportional to the increase in turbidity when barium
sulfate precipitates. The Hach procedure uses sodium sulfate, contained in BariVer 4 Reagent
Powder, as the source of sulfate. The BariVer 4 Method is especially useful for brines where
barium and sulfate coexist in solution and precipitation usually cannot be initiated by the simple
addition of more sulfate.
Ba
2+
+ SO 4
BaSO 4
Barium
Page 1553
Boron tests explained
Boron
For water and wastewater
Azomethine-H and Carmine Methods
Introduction
Boron normally occurs in natural waters at concentrations less than 1.0 mg/L. Boron in natural
water could be an indicator of sanitary pollution from domestic wastewater, usually in the form of
borates from laundry detergents. In water for human consumption, boron concentrations typically
should be less than 300 g/L. Large amounts of boron can affect the central nervous system;
when continually ingested over an extended period of time boron can cause a syndrome
called borism.
In the semiconductor industry, boron has been used as an indicator of ion-exchange resin
exhaustion in wafer rinsewater treatment. Boron is routinely monitored in irrigation water since
many varieties of plants are sensitive to excess boron.
Analytical colorimetric methods for boron include the Curcumin Method, the Carmine Method and
the Azomethine-H Method.
Chemical reactions
Azomethine-H method
The Azomethine-H Method involves the coupling of H-acid with an aromatic hydroxyaldehyde,
such as salicylaldehyde, due to the catalytic effect when boron is present. At neutral pH values
and a controlled temperature, the condensation reaction is completed quickly (within 15 minutes).
After product formation, the solution is adjusted to an acidic pH for optimum color measurement at
410 nm (yellow) using a colorimeter or spectrophotometer. The method is sensitive and highly
selective for the determination of dissolved boron in water.
Aromatic
Hydroxyaldehyde
H-acid
Azomethine
(a Schiff base)
CHO
HO3S
OH
HO3S
N
NH2
HO
B(OH)
OH
OH
HO3S
HO3S
Figure 45 Chemical reactions for the Azomethine-H method
Boron
Page 1554
Boron
Carmine method
In the presence of concentrated sulfuric acid, boron exists as the cation B3+. The cation
complexes to the carmine indicator causing the solution to change color from red to blue.
The blue-colored complex is read at 605 nm using a spectrophotometer, and the amount of color is
proportional to the dissolved boron concentration.
B2+
CH3
CH3
OH
CO2H
OH
HO
CO2H O
Carmine
OH
3+
CO2H
H+
OH
OH
CO2H O
OH
BoronCarmine Complex
Figure 46 Chemical reactions for the Carmine method
Boron
Page 1555
Benzo- and tolylltriazole tests explained
Benzotriazole and Tolyltriazole

For water
Ultraviolet Digestion Method
Introduction
Benzotriazole and tolyltriazole (BZT and TTA) are used extensively as corrosion inhibitors for
copper alloys. In both open and closed recirculating water cooling systems, concentrations of 2 to
10 mg/L provide effective corrosion control after initial passivation
CH3
Benzotriazole
Tolyltriazole
Figure 47 Chemical procedures
The Hach method for BZT and TTA determinations offers substantial improvements over
conventional analytical methods. Time-consuming conventional methods, such as ultraviolet (UV)
spectroscopy, liquid chromatography and potentiometric titration, require expensive equipment
and highly skilled personnel. The Hach method uses simple, inexpensive equipment and can be
performed in less than 10 minutes without loss of accuracy or precision. The analysis range is 0
15 mg/L at a wavelength of 425 nm.
Chemical reactions
This method of analysis is based on the UV-photolysis of triazole in the presence of a chemical
catalyst to form a dimer or polymer of the triazole. A stoichiometric amount of a soluble yellowcolored compound is then formed. The calibration follows Beers Law throughout the 015 mg/L
concentration range, with an estimated detection limit of approximately 0.3 mg/L
UV
N
N
(Yellow-colored complex)
Chemical
Catalyst
H
Figure 48 Chemical reaction
Benzotriazole and Tolyltriazole

Page 1556
Carbon dioxide tests explained
Carbon Dioxide
For water and seawater
Titration Method
Introduction
Carbon dioxide is present in all surface waters, generally in amounts less than 10 mg/L; however,
higher concentrations are not uncommon in ground waters. Dissolved carbon dioxide has no
harmful physiological effect on humans and is used to recarbonate water during the final stages of
water-softening processes and also to carbonate soft drinks. High concentrations of dissolved
carbon dioxide are corrosive and have been known to kill fish.
The analysis for carbon dioxide is similar to that for acidity. A water sample is titrated to a
phenolphthalein end point with Sodium Hydroxide Standard Solution. Strong mineral acids are
assumed to be absent or to be negligible in effect. Care must be taken during the analysis to
minimize the loss of carbon dioxide from the water sample as a result of aeration during collection
and swirling of the sample.
Chemical reactions
The reaction of sodium hydroxide with carbon dioxide (as carbonic acid) occurs essentially in two
steps; first a reaction from carbonic acid to bicarbonate and then to carbonate.
Because the conversion of carbon dioxide to bicarbonate is complete at pH 8.3, phenolphthalein
can be used as a color indicator for the titration. The sodium hydroxide titrant must be of high
quality and be free from sodium carbonate.
CO 2 + H 2 O H2 CO 3 (Carbonic acid)
H 2 CO 3 + NaOH NaHCO 3 + H 2 O
NaHCO 3 + NaOH Na 2 CO 3 + H 2 O
Carbon Dioxide
Page 1557
Chloramine (Mono) tests explained
Chloramine (Mono)
Indophenol Method
Introduction
Chloramination disinfection is the practice of forming inorganic chloramines in water to reduce
microbial concentrations to within acceptable limits. The chloramines; monochloramine (NH2Cl),
dichloramine (NHCl2) and nitrogen trichloride (NCl3), form when chlorine and ammonia are
combined in water. Traditionally, treated wastewater, which contains ammonia, is disinfected by
the addition of chlorine. In recent years, many drinking water facilities have converted to
chloramination to disinfect potable water. Roughly 20% of all drinking water facilities in the United
States now use chloramines as the residual disinfectant.
For the chloramination of drinking water, monochloramine is the preferred disinfectant. Formation
of dichloramine and nitrogen trichloride is avoided, since more chlorine is consumed and the
presence of these chloramines can produce odors or off-tastes.
In treated wastewater, any organic nitrogen compounds present will form organic chloramines
during chlorination. Organic chloramines, as a class, are much weaker disinfectants than the
inorganic chloramines. Chlorine overfeeds and ineffective mixing can lead to greater production of
organic chloramines, thereby diminishing the total germicidal activity.
Hach chemists have developed a method for the specific determination of monochloramine in
water. The method is based on the classic indophenol chemistry for determining ammonia. The
chemistry has been improved to increase the specificity of the method for inorganic
monochloramine in the presence of organic chloramines. In addition, the method was modified to
greatly accelerate the color development time and increase the precision of the test. The new test
has been shown to be specific for monochloramine, without interference from organic or inorganic
amines, dichloramines, free chlorine, organic chloramines, nitrites or manganese.
Chemical reactions
Monochloramine reacts specifically with a substituted phenate to form a quinone imine
intermediate. In the presence of a cyanoferrate, the intermediate couples with excess phenate to
form a green-colored indophenol. The amount of indophenol formed is proportional to
concentration of monochloramine in the sample. See the Chemical reactions figure below.
Benzoquinone Monoimine formation

NH2Cl +
OH
R
Indophenol
Formation
HN
O+
HN
OH
H2N
O
R
N
R
O
R
Figure 49 Chemical reactions1

1
R= reaction accelerating group
Chloramine (Mono)
Page 1558
Chloramine (Mono) and Free Ammonia explained
Chloramine (Mono);
Indophenol method
For determining free ammonia and monochloramine simultaneously in finished
chloraminated water
Introduction
Chloramination disinfection is the practice of forming inorganic chloramines in water to reduce
microbial concentrations to within acceptable limits. The chloraminesmonochloramine (NH2Cl),
dichloramine (NHCl2), and trichloramine (NCl3)form when chlorine and ammonia are combined
in water. In recent years, many drinking water facilities have converted from free chlorination to
chloramination to disinfect potable water. Chloramines are weaker oxidants than free chlorine and
therefore minimize the formation of harmful disinfection by-products.
A typical chloramination curve is presented in Figure 50. For the chloramination of drinking water,
monochloramine is the preferred disinfectant (Section I of the curve). This is optimized by an
approximate 5:1 ratio (by weight) of chlorine to ammonia. Adding too much chlorine leads to the
decrease of monochloramine and the formation of dichloramine and trichloramine, causing taste
and odor problems (Section II). Adding too little chlorine leaves excess unreacted or "free"
ammonia in the water which acts as a food source and can lead to nitrification and bacterial growth
in the distribution system. At the breakpoint, which is the vertical line between Sections II and III,
no monochloramine remains. Any additional chlorine added will be in the form of free chlorine.
In treated wastewater, any organic nitrogen compounds present will form organic chloramines
during chlorination. Organic chloramines, as a class, are much weaker disinfectants than the
inorganic chloramines. Chlorine overfeeds and ineffective mixing can lead to greater production of
organic chloramines, thus diminishing the total germicidal activity.
Figure 50 Breakpoint curve for chloraminated water

Page 1559
Chemical reactions
Refer to Figure 51 for the indophenol method mechanism.
Added hypochlorite combines with free ammonia to form more monochloramine (1). In the
presence of a cyanoferrate catalyst, monochloramine in the sample reacts with a substituted
phenol to form an intermediate monoimine compound (2). The intermediate couples with excess
substituted phenol to form a green-colored indophenol, which is proportional to the amount of
monochloramine present in the sample (3). Free ammonia is determined by comparing the color
intensities, with and without added hypochlorite.
Figure 51 Chemical reactions for the indophenol method

Page 1560
Chloride tests explained
Chloride
Mercuric Nitrate, Mohr Argentometric and
Mercuric Thiocyanate Methods
Introduction
Chlorides are present in all potable water supplies and in sewage, usually as a metallic salt. When
sodium is present in drinking water, chloride concentrations in excess of 250 mg/L give a salty
taste. If the chloride is present as a calcium or magnesium salt, the taste detection level may be as
high as 1000 mg/L chloride.
Chloride is essential in the human diet and passes through the digestive system unchanged,
thereby becoming one of the major components of raw sewage. The wide use of zeolite spheres in
water softeners also contributes a large amount of chloride to sewage and wastewaters.
High chloride concentrations in water are not known to have toxic effects on humans, although
large amounts may act corrosively on metal pipes and be harmful to plant life. The maximum
allowable chloride concentration of 250 mg/L in drinking water has been established for reasons of
taste rather than as a safeguard against physical hazard.
Chemical reactions
Mercuric nitrate method
Mercuric nitrate reacts selectively with all the chloride present in a sample to produce mercuric
chloride and nitrate ions. When all the chloride present in the sample has been complexed, excess
mercuric ions combine with diphenylcarbazone to form a purple-colored complex indicating the
end point. Hach procedures use Diphenylcarbazone Reagent Powder containing the indicator and
a buffer for maximum convenience and reagent stability.
Hg ( NO 3 ) 2 + 2Cl HgCl 2 + 2NO 3
Silver nitrate method (Mohr Argentometric method)

In the chloride test, using silver nitrate as the titrant and potassium chromate as the indicator,
silver nitrate first reacts selectively with the chloride present in the sample to produce insoluble
white silver chloride. After all the chloride has been precipitated, the silver nitrate then reacts with
the potassium chromate to form an orange-colored silver chromate precipitate, thereby marking
the end point of the titration. Potassium chromate indicator is combined with a buffer in Chloride 2
Indicator Powder.
AgNO 3 + K 2 CrO 4 + Cl AgCl + NO 3 + K 2 CrO 4

2AgNO 3 + K 2 CrO 4
Ag 2 CrO 4
(Orange)
+ 2KNO 3
Chloride
Page 1561
Chloride
Mercuric Thiocyanate method
Colorimetric determination of chloride by the Mercuric Thiocyanate Method involves reaction of
chloride in the sample with mercuric thiocyanate to produce mercuric chloride and free thiocyanate
ions. In the presence of Fe3+ (ferric ion), the free thiocyanate ion forms highly colored ferric
thiocyanate in proportion to the chloride concentration. Two liquid reagents have been formulated
for this test: Mercuric Thiocyanate Solution and Ferric Ion Solution.
1.
Hg ( SCN ) 2
+ 2Cl HgCl 2 + 2SCN
(Mercuric Thiocyanate)
Fe ( SCN ) 3
3+
2. Fe + 3SCN
(Red-orange)
Chloride
Page 1562
Chlorine dioxide tests explained
Chlorine Dioxide
DPD and Chlorophenol Red Methods
Introduction
Chlorine Dioxide is a deep yellow gas that is generated directly for on-site use as a bleaching
agent in industrial processes, such as the manufacture of pulp and paper. It is used increasingly
for special treatment objectives in municipal water treatment because, unlike chlorine, chlorine
dioxide does not form trihalomethanes (THMs) in reaction with certain organic compounds. Two
colorimetric methods for chlorine dioxide at low levels are used in Hach procedures. Hach also
offers a high range method that directly measures the yellow color of the chlorine dioxide gas
dissolved in the sample water.
The DPD method is an extension of the N,N-diethyl-p-phenylenediamine (DPD) method for
determining free and total chlorine. Glycine is used to eliminate chlorine interference.
The Chlorophenol Red (CPR) method reacts specifically with chlorine dioxide.
Chemical reactions
DPD Method
Chlorine dioxide reacts with the DPD (N,N-diethyl-p-phenylenediamine) Indicator Reagent (to the
extent of one-fifth of its total available chlorine content corresponding to the reduction of chlorine
dioxide to chlorite) to form a pink color. The color intensity is proportional to the ClO2 in the
sample. Chlorine interference is eliminated by adding glycine, which converts free chlorine to
chloroaminoascorbic acid, but has no effect on chlorine dioxide at the test pH.
Chlorophenol red method
Chlorophenol Red (CPR) indicator reacts specifically with chlorine dioxide with a distinct color
change; no interference is experienced form other mild oxidants, including hypochlorite, chlorite,
chromate, permanganate, ferric iron, or low levels of chloramines. One mole of CPR reacts with
two moles of chlorine dioxide to form a colorless product with a net decrease in absorbance at
570 nm. The discoloration of CPR is linear to approximately 0.6 mg/L, although concentrations to
approximately 1.0 mg/L are easily determined. The reaction of CPR with ClO2 is reproducible. No
equation for this reaction will be suggested; however, the reaction may result in the formation of an
ion-pair complex.
The reaction of CPR with chlorine dioxide is pH-sensitive. A pH of 7.0 has been suggested for the
spectrophotometric method. Hach researchers found the optimum pH for this reaction is actually
5.2. It was also determined that the sensitivity is improved if the solution is buffered to near pH 10
after the initial reaction. The reagents for this method are contained in three convenient solutions.
Reagent 1 is a buffer which adjusts the sample to the optimum pH, 5.2. Reagent 2 is a special
formulation of CPR which is added after the pH adjustment. Reagent 3 is a pH 10 buffer added
after CPR to increase sensitivity. Blanks for standardizing the spectrophotometer are prepared by
adding dechlorinating agent to a 50-mL sample, thereby destroying up to 35 mg/L of ClO2.
Chlorine Dioxide
Page 1563
Chlorine Dioxide
OH
Cl
Cl
Cl
C
Cl
C
SO3
SO3
Yellow (acid color)
Red (base color)
Figure 52 Chlorophenol Red structures
Chlorine Dioxide
Page 1564
Chlorine, free and total tests explained

DPD Method
Introduction
Chlorine is the disinfectant most frequently used for water and wastewater treatment. It was used
first for industrial applications and to control odor in wastewater, in the early 1800s. The
subsequent use of chlorine to disinfect water occurred by the mid-1800s. Industrial uses of
chlorine include applications such as bleaching paper and controlling nuisance organisms in
cooling towers.
Hydrochloric and hypochlorous acids are formed when chlorine is added to water. The disinfectant
and form causing bleaching action, is hypochlorous acid.
Cl 2 + H 2 O HCl + HOCl (Hypochlorous acid)

Depending upon variables such as pH, temperature and the amount of organic or ammonia
nitrogen, other forms of chlorine in water may include hypochlorite ions (OCl) and chloramines.
Chlorine existing in water as hypochlorous acid or the hypochlorite ion is termed free available
chlorine. Chloramines, including monochloramine (NH2Cl), dichloramine (NHCl2) and nitrogen
trichloride (NCl3) are referred to as Combined Available Chlorine. Total Chlorine refers to the sum
of free and combined available forms.
Methods for determining free, combined and total chlorine include: amperometric titration,
colorimetric DPD, titrimetric DPD and iodimetric titration. The most widely used method,
colorimetric DPD, is easy to perform, requires little apparatus, is inexpensive and adapts well to
field test situations. DPD (N,N-diethyl-p-phenylenediamine) is oxidized by chlorine, causing a
magenta (red) color. The intensity of color is directly proportional to the chlorine concentration.
DPD reacts in much the same way with other oxidants, including bromine, chlorine dioxide,
hydrogen peroxide, iodine, ozone and permanganate.
Chemical reactions
Free available chlorine
Hypochlorous acid and the hypochlorite ion oxidize DPD causing a magenta color. The reaction is
pH dependent. DPD and appropriate buffer are packaged together in DPD Free Chlorine Reagent
Powder Pillows to handle high levels of hardness without precipitation.
Total chlorine
Potassium iodide is added to the reaction to determine combined available chlorine forms and total
chlorine. Chloramines oxidize the iodide to iodine; then the liberated iodine reacts with DPD to
form the magenta color. DPD Total Chlorine Reagent Powder Pillows from Hach contain DPD,
potassium iodide and a buffer.

Page 1565
NH3
NH3
+Cl2
I3
N+
H 5C 2
N+
C2H5
(Colorless)
H 5C 2
Wrster Dye (Red)
Figure 53 DPD chemical reactions

Page 1566
C2H5
Chromium tests explained
Chromium
Total and Hexavalent Methods
Introduction
Chromium may be present in water as the hexavalent (chromate) or the trivalent form, although
trivalent chromium rarely occurs in potable water. Hexavalent chromium enters a water supply
through industrial wastes from metal plating baths and from industrial cooling towers where
chromate is used to inhibit metal corrosion. Chromium is an objectionable contaminant in public
drinking water supplies due to its suspected carcinogenic effects. Chromium present in potable
waters above a 3-g/L level indicates the possible presence of industrial wastes. Concentrations
greater than 50 g/L are sufficient grounds to reject the water supply.
Chemical reactions
Hexavalent chromium
Hexavalent chromium is determined by the 1,5-Diphenylcarbohydrazide Method using a single dry
powder formulation called ChromaVer 3 Chromium Reagent. This reagent contains an acidic
buffer combined with 1,5-Diphenylcarbohydrazide which reacts to give a purple color when
hexavalent chromium is present. The method is applicable to fresh water and wastewater
samples. Color development is directly proportional to the amount of hexavalent chromium
present.
R
N
+ Cr6+
Cr
C
C
H
O
O
R
N
H
1,5-diphenylcarbohydrazide
Figure 54 Hexavalent chromium chemical reaction

Total chromium
In the analysis for total chromium, the sample is heated to the boiling point under strong alkaline
conditions in the presence of hypobromite. The trivalent chromium is converted to hexavalent
chromium. The proper chemical conditions for this oxidation are provided by Chromium 1 Reagent
Powder.
After the oxidation is complete, excess hypobromite is destroyed by the addition of Chromium 2
Reagent Powder. Then ChromaVer 3 Chromium Reagent, which contains an acidic buffer
combined with 1,5-Diphenylcarbohydrazide, is added. A purple color develops with an intensity
directly proportional to the total chromium concentration. The trivalent chromium can be
determined by subtracting the hexavalent chromium test results from the results obtained in the
total chromium test.
2Cr
3+
+ 3 OBr + 10 OH 2CrO 4
+ 3Br + 5H 2 O
Chromium
Page 1567
Cobalt tests explained
Cobalt
For water
1-(2-Pyridylazo)-2-Naphthol (PAN) Method
Introduction
Cobalt is valuable because of its ability to increase the strength and corrosion resistance of alloys.
It is associated with nickel, silver, lead, copper and iron ores, from which it is most frequently
obtained as a by-product. Cobalt is often found in industrial wastewaters as a corrosion product of
alloys of iron, nickel and cobalt, but it seldom occurs in natural waters.
Toxicity of cobalt to aquatic life varies depending on pH, the species or organism, and synergetic
effects. It is considered to be relatively nontoxic to humans. Methods for detection of low levels of
cobalt historically have been limited to expensive and time-consuming techniquesmainly atomic
absorption. By comparison, cobalt can be determined quantitatively by a simple colorimetric
procedure using a spectrophotometer. Accuracy and precision rivals atomic absorption
measurements. The very sensitive 1-(2-Pyridylazo)-2-Naphthol (PAN) Method is capable of
detecting 0.1 mg/L cobalt. This unique method is relatively free from interferences and provides for
simultaneous determinations of nickel and cobalt on the same sample portion without special
treatments.
Chemical reactions
PAN is suspended in water by use of surfactants to allow it to form complexes with the metals in
the sample. A complexing agent can be used to decompose all PAN chelates except those of
cobalt, nickel and iron. A pH adjustment using the Phthalate-Phosphate Reagent aids in the
masking of iron up to 10 mg/L, and also enhances the rate of development of the colored cobalt
and nickel PAN complexes.
+ Charge for Co
0 Charge for Ni
N
O
2
N
Co2+
+ 2H+
Co (or Ni)
N
OH
O
N
Figure 55 Formation of Co-PAN or Ni-PAN complex1

1
Cheng, K. L., and Bray, R. H., Journal of Analytical Chemistry, 27, 1955, page 783.
Cobalt
Page 1568
Cobalt
The absorbance of the cobalt PAN complex at 560 nm is the same as at 620 nm; however,
absorbance caused by the nickel PAN complex is zero at 620 nm. This difference in absorbance
wavelengths allows cobalt to be determined without interference from nickel at a wavelength of
620 nm. Therefore, the nickel can be determined on the same sample by measuring the
absorbance at 560 nm and subtracting the absorbance at 620 nm.
Ni
650
630
640
610
620
590
600
570
580
550
560
530
540
520
Absorbance
Co
Nm
Figure 56 Typical absorbance scan for Co and Ni
Cobalt
Page 1569
Copper tests explained
Copper
Bicinchoninate, Porphyrin and
Bathocuproine Methods
Introduction
Although copper comprises only 0.007% of the earths crust, it is a very important element. Copper
occurs in both free and combined forms throughout nature in many minerals. Copper may occur in
natural waters, wastewaters, and industrial waste streams as soluble copper salts or as copper
compounds precipitated on suspended solids. Forms of copper in water can be classified as
insoluble, dissolved (free and complexed), and total recoverable. Insoluble copper includes
precipitates such as copper sulfides and hydroxides. All copper in solution is known as dissolved
copper, including Cu1+ (cuprous) and Cu2+ (cupric) ions and copper chelates such as CuEDTA.
Copper concentrations in potable water are usually very low. Copper is not considered a health
hazard to humans although more than 1 mg/L can impart a bitter taste to water and large oral
doses can cause vomiting and may eventually cause liver damage. Copper salts, such as copper
sulfate (CuSO4), may be used to control algae; however, they may also be toxic to fish and wildlife.
Hachs simplified test procedures for copper use a variety of reagents to satisfy the desired range
of detection and the form of copper to be measured. Hach procedures use primarily the
Bicinchoninate and the Porphyrin Methods.
The Copper reagents and applications table lists proprietary reagents and applications.
Table 429 Copper reagents and applications

Form measured
Reagent
Application
Without pretreatment
CuVer 11
Free
Total recoverable
CuVer 2
Total dissolved copper
Total recoverable
Free
Total recoverable
hard water, wastewater,

seawater
Free
Total recoverable
extremely low levels in water,

wastewater and seawater
Free copper reagent

Porphyrin reagents
1
With digestion
water, wastewater
CuVer is a trademark of Hach Company.
Chemical reactions
Bicinchoninate method
Copper can be determined by the reaction of copper with 2, 2-biquinoline-4,4-dicarboxylic acid
(bicinchoninic acid). Bicinchoninate reacts with Cu1+ to produce a purple-colored complex.
Bicinchoninate does not react readily with Cu2+. Determination of Cu2+ begins by reducing it to
Cu1+. The CuVer 1 Reagent combines the bicinchoninate reagent with a buffer and reducing
agent, allowing determination of Cu1+ and Cu2+. Total recoverable copper can be determined with
this method if the sample is first digested to convert all of the copper present (including insoluble
forms and complexed forms) to free copper.
Complexed copper forms such as CuEDTA react directly with CuVer 2. Digestion is not
necessary, and high levels of hardness do not interfere. The results will be in terms of total
dissolved copper (free and complexed). When using CuVer 1, digestion is necessary and high
levels of hardness interfere.
Copper
Page 1570
Copper
HOOC
HOOC
2
HOOC
Cu+
COOH
COOH
Cu
HOOC
Figure 57 Reaction of Cu1+ and bicinchoninic acid

Use Free Copper Reagent Powder Pillows to determine free copper separately from complexed
copper. The powder pillows contain bicinchoninate, a reducing agent and an inhibitor to eliminate
calcium and magnesium interference. The results will be in terms of free copper. Complexed
copper may then be determined by adding Hydrosulfite Reagent, repeating the analysis and
subtracting the results of the two analyses.
Porphyrin method
The porphyrin method for determining copper is a very sensitive test, capable of detecting free
copper (Cu1+ and Cu2+) and total recoverable copper (with digestion) in the range of 0150 g/L.
Because of the sensitivity of the method, it is difficult to obtain water of high enough quality to
establish a blank value. The porphyrin method uses a split sample. One half of the split sample is
treated with a masking agent to complex the free copper forms; then, porphyrin reagent, a buffer,
and a reducing agent is added. This forms a zero blank without the need for special copper-free
water. Porphyrin reagent is added to the second half of the split sample, where it reacts with the
free copper.
Interference caused by the reaction of porphyrin with other metals is minimized by using the split
sample because interferences are compensated for in the blank. Porphyrin reacts slowly with
Cu2+, but a special formulation of the porphyrin and addition of a buffer allow the reaction with free
copper to be completed within seconds. A reducing agent is also added in order to destroy
unreacted porphyrin (which would otherwise interfere). An intense absorbance at 425 nm makes
this method very sensitive when using a colorimeter or spectrophotometer. However, strong visual
color development does not occur.
Copper
Page 1571
Copper
CH3
N
N
CH3
N
N
Cu
N
N
CH3
Figure 58 Final structure from the porphyrin method
Copper
Page 1572
CH3
Cyanide tests explained
Cyanide
Pyridine-Pyrazolone Method
Introduction
Cyanide is extremely toxic and occurs primarily in industrial effluents. Metal-cleaning and
electroplating baths, gas scrubbers, gas works, coke ovens and other chemical treatments are the
main sources of the cyanide found in industrial wastes. Natural waters do not contain cyanide; its
presence usually indicates contamination from an industrial source. Proper neutral or alkaline
chlorination of cyanide-containing wastewaters will reduce the level well below toxic limits.
Chemical reactions
The cyanide test involves the following 4 steps:
(1)
2CN
Cl2
2CNCl
Cyanide is reacted with chlorine to produce cyanogen chloride (CNCl); the chlorine is provided by
CyaniVer 3 Reagent.
(2)
CNCl
+ Cl
N+
CN
An intermediate nitrile is then formed by the addition of pyridine; the pyridine is provided by the
addition of CyaniVer 4 Reagent. Excess chlorine is destroyed at this point.
H
4H2O +
(3)
+ Cl
N+
CN
H
C
+ 2NH3 +
CO2
HCl
The nitrile is hydrolyzed to glutaconaldehyde; the reagent is provided in the CyaniVer 4 Reagent
from the previous step.
(4)
C
H
H
C
+2
H3C
O
H
C
N
C
H
CH3
O
N
+ 2 H2 O
H
CH3
Cyanide
Page 1573
Cyanide
Finally, CyaniVer 5 Reagent, containing an excess of pyralozone, is added. The reaction with the
glutaconaldehyde results in a blue color. The intensity of the color is directly proportional to the
amount of cyanide present in the sample.
Cyanide
Page 1574
Fluoride tests explained
Fluoride
SPADNS, SPADNS 2 and Ion-selective
Electrode Methods
Introduction
Fluoride occurs naturally in some ground waters, and a 1-mg/L level is normally maintained in
public drinking water supplies for the prevention of dental cavities. Excessive amounts of fluoride
cause an objectionable discoloration of tooth enamel called mottling. For this reason, a
permissible level in drinking water has been established by the USEPA in accordance with the
Safe Drinking Water Act.
Chemical reactions
SPADNS method
The fluoride analysis involves the reaction of fluoride with a dark red zirconium-dye complex.
Fluoride combines with part of the zirconium to form a colorless zirconium-fluoride complex with
the net effect of bleaching the color. Measurement of the decrease in color intensity provides an
accurate determination of the fluoride concentration. The SPADNS Method is the preferable
colorimetric method due to its rapid reaction with fluoride and the stability of the SPADNS reagent.
HO3S
HO3S
OH
N
N
H+
OH
Zr
O
HO3S
6F
OH
SO3H
(Red)
+
OH
ZrF62
NH2O
OH
HO3S
SO3H
(Colorless)
Figure 59 Chemical reaction for SPADNS method

SPADNS 2 method
Sodium Arsenite is used in the SPADNS method as a reducing agent to prevent interference from
chlorine and other oxidants that are typically present in drinking water. The SPADNS 2 test
eliminates arsenic from the original SPADNS formulation by using a non-toxic proprietary reducing
agent to achieve identical results and test performance. All other chemistry remains the same as
the SPADNS method.
Method of analysis
Ion-Selective electrode method
The Ion-Selective electrode method requires a Hach sension ISE Meter and an electrode
system consisting of a silver/silver chloride reference electrode and a standard fluoride ionselective electrode. Fluoride measurement is accomplished when a voltage potential is
established across the lanthanum fluoride crystal on the end of the electrode; this potential is in
direct proportion to the fluoride concentration of the sample. The meter is calibrated with fluoride
standards bracketing the expected range. The concentration may be read directly from the meter.
A total ionic strength adjustment buffer (TISAB) is used to eliminate interferences in the test, to
adjust the pH to an optimum value and to introduce sufficient sodium chloride to mask variations in
Fluoride
Page 1575
Fluoride
ionic strength. TISAB reagent uses sodium 1,2-cyclohexanediaminetetraacetic acid (CDTA) for
chelation of interfering metals, such as Al3+ and Fe3+, as well as other complexing and buffering
agents.
Fluoride
Page 1576
Formaldehyde tests explained
Formaldehyde
For water
MBTH Method
Introduction
Formaldehyde is used in the treatment of fabric in textile industries, in metal plating baths, as a
preservative for biological tissues and as a disinfectant in dialysis and reverse osmosis equipment.
The MBTH Method is a sensitive colorimetric test for low range measurement of aldehydes; it is
most sensitive for formaldehyde.
Chemical reactions
MBTH Method
MBTH (3-methyl-2-benzothiazoline hydrazone) is added in excess to a sample containing
formaldehyde, triggering multiple reactions. First, MBTH and formaldehyde react to form an azine
(1). Excess MBTH is oxidized by addition of a developing solution (2). Oxidized MBTH reacts with
the azine to form a species with an intense blue color (3). Intensity of the blue color is proportional
to the original concentration of formaldehyde.
MBTH is contained in MBTH Powder Pillows. Liquid reagent for oxidizing excess MBTH is
contained in the Developing Solution for Low Range Formaldehyde.
CH3
CH3
H
N
C
(1)
NH2
CH2 + H2O
CH3
N
(2)
NH+
S
(Oxidized MBTH)
CH3
CH3
N
C
(3)
S
CH
C
S
(Blue-colored complex)
Formaldehyde
Page 1577
Formaldehyde
CH3
CH3
H
N
C
(1)
S
NH2
C
H
N
C
O
S
Figure 60 Chemical reaction for the MBTH method
Formaldehyde
Page 1578
CH2 + H2O
Hardness tests explained
Hardness
EDTA Titration and Calmagite Colorimetric

Methods
Introduction
Hardness in water is caused by dissolved minerals, primarily divalent cations, including calcium
(Ca2+), iron (Fe2+), strontium (Sr2+), zinc (Zn2+) and manganese (Mn2+). Calcium and magnesium
ions are usually the only ions present in significant concentrations; therefore, hardness is generally
considered to be a measure of the calcium and magnesium content of water. Considerations
should be given when other cations contributing to hardness are present in significant amounts.
Titration methods
Hardness in water can be determined quickly by titration and the use of color indicators. By proper
choice of pH, total hardness (Ca2+ and Mg2+) or the portion contributed by calcium and
magnesium individually can be measured. The traditional test for hardness involves pH
adjustment to 10.1 with an ammonium buffer, addition of Eriochrome Black T indicator [1-(1hydroxy-2-naphthylazo)-6-nitro-2-naphthol-4-sulfonic acid] and then titration with Na2EDTA
(ethylenediaminetetraacetic acid, disodium salt) solution.
HOOCH2C
CH2COOH
NCH2CH2N
HOOCH2C
CH2COOH
Figure 61 Chemical structure of EDTA-Ethylenediaminetetraacetic acid

Some other indicators are more stable, giving a faster reaction and a more distinct end point than
Eriochrome Black T. One of the best is calmagite, 1-(1-hydroxyl-4-methyl-2-phenylazo)-2naphthol-4 sulfonic acid, which is used in Hach total hardness tests.
Colorimetric method
The Colorimetric Method is for low level measurement of hardness. The interference of some
metals with the Titration Methods will be rendered inconsequential after diluting the sample to
bring it into the range of this test. Calmagite indicator and two chelating agents, EGTA and EDTA,
are used in the test.
Chemical reactions
Total hardness
Several solutions including digital titrator cartridges are described in the following section for
titrating prepared water samples containing calmagite indicator. TitraVer Hardness Titrant
(0.020 N EDTA) is the most widely used. Other strengths of TitraVer Hardness Titrant are available
for titrating high hardness samples. HexaVer Hardness Titrant also is available. HexaVer is
CDTA (cyclohexanediaminetetraacetic acid, disodium salt). It gives slightly sharper end points and
can tolerate higher levels of iron interference than TitraVer.
Hardness
Page 1579
Hardness
CH2COONa
N
CH2COOH
CH2COOH
N
CH2COONa
Figure 62 Chemical structure of CDTA, disodium salt
Calmagite indicator is available in special formulations as ManVer and UniVer. The ManVer
formulations of calmagite have been specially prepared to enhance stability and to be free from
most interferences. Interferences caused by metal ions, such as copper or iron, can be removed or
masked by the use of the magnesium salt of CDTA. It is effective, yet safe to use. Cyanide
compounds also may be used to overcome interferences. Their use is avoided where possible
because of potential environmental and health hazards.
CH3
CH3
O
N
N
+
OH
SO3
Calmagite (blue)
Mg
2+
Mg
N
H+
SO3
Calmagite-Mg complex (wine red)
Figure 63 Reaction between magnesium and calmagite indicator

The reaction of calmagite is pH-dependent; it has been determined that a pH of 10.1 is ideal.
Traditionally, ammonia buffers have been used; however, they have a strong odor. Hach methods
use Hardness 1 Buffer (2-amino-2-methyl-1-propanol), which is stable, safe to use and has a less
objectionable odor.
The sequence of analysis in the hardness tests begins with pH adjustment and addition of
inhibitors followed by formation of the Mg2+ and Ca2+ complexes with calmagite. The calcium
forms a weak complex with calmagite at this pH. The solution is titrated with TitraVer (EDTA) or
HexaVer (CDTA). The titrant first complexes any calcium, then magnesium. Color change from
wine red to blue is an indication that all calcium and magnesium have been removed from the
calmagite and complexed with the titrant.
Hardness
Page 1580
Hardness
H2
OOC
C
N
O
C
O
C
Mg
C
O
N
OOC
C
O
H2
Figure 64 Magnesium complexed with TitraVer
Expression of results of the hardness titration is mg/L as CaCO3. The reaction of TitraVer with
Ca2+ and Mg2+ is a 1:1 ratio.
Calcium hardness
The test for calcium hardness is very similar to the total hardness test. Traditionally, either
murexide indicator (ammonium purpurate) or Eriochrome Blue-Black R indicator is followed by
titration with EDTA. CalVer 2 Calcium Indicator has been developed by Hach to replace these
indicators. CalVer 2 (hydroxy naphthol blue) is more sensitive and has a sharper end point color
change.
CalVer 2 Calcium Indicator forms a red-violet complex with calcium and changes to pure blue after
TitraVer removes calcium from the complex. The pH is elevated to at least 13 to precipitate
magnesium. A few drops of Magnesium Standard Solution may be added to the reaction to
sharpen the end point color change. This may seem inconsistent because magnesium is
precipitated by elevating the pH. However, the added magnesium is chelated preferentially by the
dye and the quantity of chelated magnesium is very small; thus any error caused by addition of
magnesium is negligible.
The pH adjustment is accomplished by addition of potassium hydroxide prior to addition of CalVer
2. Potassium cyanide may also be added to complex interfering metals prior to the addition of
CalVer 2.
Calcium hardness and total hardness may be determined sequentially using the same sample.
After the calcium hardness is determined the sample pH can be adjusted downward, using sulfuric
acid. Then Hardness Buffer 1 and ManVer 2 are added and titration with TitraVer is resumed.
Hardness
Page 1581
Hardness
DO NOT use this procedure if potassium cyanide has been used in determining
calcium hardness! The addition of sulfuric acid will cause deadly hydrogen cyanide gas
to evolve.
Colorimetric method
Calmagite, contained in Calcium and Magnesium Indicator Solution, is added to a sample and the
pH is elevated to about 12.5 by using a buffer. Adding calmagite prior to pH adjustment prevents
the calcium and magnesium precipitation that ordinarily would occur at this elevated pH. The
sample is then split into three equal portions.
EDTA is added to the first portion to sequester calcium and magnesium, thereby breaking the Caand Mg-calmagite complexes. This solution is used as a zero reference blank to standardize the
spectrophotometer.
EGTA or ethyleneglycol-bis (2-aminoethylether)-N,N,N,N-tetraacetic acid, is added to the second
sample portion. EGTA selectively chelates calcium under conditions of the test; only absorbance
due to the Mg-calmagite complex remains to be measured. The result is expressed as mg/L Mg as
CaCO3. After measurement, the spectrophotometer is adjusted to read zero on this portion.
Absorbance of the third sample portion (containing no chelant) is measured to determine mg/L Ca
as CaCO3. Adjust the spectrophotometer to a reading of zero after measurement of the second
sample portion to compensate for absorbance due to magnesium in the sample.
OOCH2C
CH2COOH
N -CH2-CH2-O-CH2-CH2-O-CH2-CH2-N
HOOCH2C
CH2COO
Figure 65 Chemical structure of EGTA
Hardness
Page 1582
Hydrazine tests explained
Hydrazine
For water and boiler water
p-Dimethylaminobenzaldehyde Method
Introduction
Hydrazine is used as an oxygen scavenger for high pressure boilers in power plants and other
industries to reduce corrosion of metal pipes and fittings. The test for hydrazine is a modification of
the p-Dimethylamino-benzaldehyde Method, in which several solutions have been formulated into
a single, stable reagent called HydraVer 2 Hydrazine Reagent. The method is both sensitive and
easy to perform. It is used mostly for the determination of small amounts of hydrazine in boiler
feedwater. There are no common interferences.
Chemical reactions
Under acid conditions, hydrazine combines with p-Dimethylaminobenzaldehyde to form a yellowcolored azine complex. Color development follows Beers law and is stable after maximum color is
developed in 10 to 15 minutes.
+ :N
N:
CH3
2
CH3
Hydrazine
p-Dimethylaminobenzaldehyde
CH3
CH3
N
CH3
C
H
C
H
+2H2O
N
CH3
Azine complex (yellow in dilute solution)

Figure 66 Chemical reaction for p-Dimethylaminobenzaldehyde method
Hydrazine
Page 1583
Iron tests explained
Iron
1,10-Phenanthroline, FerroZine, TPTZ and

Titration Methods
Introduction
Natural waters contain variable, but minor, amounts of iron, despite its universal distribution and
abundance. Iron in ground waters is normally present in the ferrous (Fe2+), or soluble state, which
oxidizes easily to ferric (Fe3+) iron on exposure to air. Iron can enter a water system from leaching
of natural deposits, iron-bearing industrial wastes, effluents of pickling operations, or from acidic
mine drainage.
Iron in domestic water supply systems stains laundry and porcelain, causing more of a nuisance
than a potential health hazard. Taste thresholds of iron in water, 0.1 mg/L for Fe2+ and 0.2 mg/L for
Fe3+, result in a bitter or astringent taste. Water used in industrial processes must contain less
than 0.2 mg/L of total iron.
Three methods of colorimetric iron analysis are used in Hach procedures. The 1,10Phenanthroline Method is the best-known test for iron. The Fe2+ procedure uses Ferrous Iron
Reagent Powder containing 1,10-Phenanthroline as an indicator. Total iron determination or
analysis uses FerroVer Iron Reagent. FerroVer Iron Reagent contains 1,10-Phenanthroline,
combined with a reducing agent, to convert all but the most resistant forms of iron present in the
sample to Fe2+.
The FerroZine Method for total iron is more than twice as sensitive as the 1,10-Phenanthroline
Method. Researchers at Hach have patented a process to manufacture high purity FerroZine Iron
Reagent, ideal for iron measurement, in economical quantities. FerroZine is highly specific for iron,
forms an intensely-colored stable complex and performs in the pH range of 37.5. The FerroZine
Method requires boiling to dissolve rust.
The TPTZ Method for total iron has the advantages of simplicity, sensitivity and freedom from
common interferences. Iron in the sample, including precipitated or suspended iron such as rust, is
converted to Fe2+ by a reducing agent. A highly colored Fe2+-TPTZ complex is formed.
Hach Methods also include a high-range titration procedure utilizing sulfosalicylic acid as the
indicator and EDTA as the titrant.
Chemical reactions
1,10-Phenanthroline method
1,10-Phenanthroline, contained in Ferrous Iron Reagent Powder, reacts with Fe2+ to form a
characteristic orange-colored complex. The intensity of color development is directly proportional
to the amount of Fe2+ in the sample. Total iron also can be determined with FerroVer Iron Reagent.
(When Environmental Protection Agency reporting is necessary, digestion of the sample is
also required)
Iron
Page 1584
Iron
N
N
+ Fe
3
N
2+
Fe
Figure 67 Chemical reaction for 1,10-Phenanthroline method

FerroZine method
Very low concentrations of iron can be determined using an ultra-sensitive iron indicator, FerroZine
Iron Reagent, 3-(2-pyridyl)-5, 6-bis (4-phensylsulfonic acid)-1, 2, 4-triazine, monosodium salt.
FerroZine Iron Reagent also can be used to analyze samples containing magnetite (black iron
oxide) or ferrites. The test is performed by adding a solution of FerroZine Iron Reagent to the water
sample. The sample is thereby buffered to a pH of 3.5 and a purple-colored complex directly
proportional to the iron concentration is formed. A reducing agent is included to convert any Fe3+
to Fe2+(which forms the colored complex).
SO3H
Where:
SO3 Na+
N
SO3H
FerroZine
N
N
N
N
SO3 Na++ Fe2+
3
N
Fe
N
Figure 68 Chemical reaction for FerroZine method
Iron
Page 1585
Iron
Titration method
The Titration Method is intended for high iron concentrations, such as oil-field water
determinations. In this method the iron present in the sample is oxidized to Fe3+ by an oxidizing
agent. The Fe3+ is then detected with sulfosalicylic acid, which forms a wine red complex with
Fe3+. The solution is titrated with TitraVer (EDTA) to a colorless to yellow end point. A buffer is
added to stabilize the Fe3+
Sulfosaliclylic acid
SO3
OH
OH
C
O
CO2 H
HO3 S
+ Fe
3+
HO
C
O
Fe
O
O
O
C
SO3
Figure 69 Titration method
Iron
Page 1586
OH
SO3
+ 6H+
Iron
TPTZ method
TPTZ, 2,4,6-tripyridyl-s-triazine, reacts with Fe2+ to form a deep blue-purple color. Reducing
agents are added to convert iron in the sample to the Fe2+ form. TPTZ, reducing agents and pH
buffers are combined in one simple reagent TPTZ Iron Reagent Powder Pillows.
TPTZ
N
N
+ Fe2+
Fe
N
N
N
N
N
N
N
N
N
Figure 70 Chemical reaction for TPTZ method
Iron
Page 1587
Langelier and Aggressive indices
Langelier and Aggressive

Indices
Method 8073
Langelier saturation index

The Langelier Saturation Index (LI), a measure of a solutions ability to dissolve or deposit calcium
carbonate, is often used as an indicator of the corrosivity of water. The index is not related directly
to corrosion, but is related to the deposition of a calcium carbonate film or scale; this covering can
insulate pipes, boilers and other components of a system from contact with water. When no
protective scale is formed, water is considered to be aggressive and corrosion can occur. Highly
corrosive water can cause system failures or result in health problems because of dissolved lead
and other heavy metals. An excess of scale can also damage water systems, necessitating repair
or replacement.
In developing the LI, Langelier derived an equation for the pH at which water is saturated with
calcium carbonate (pHs). This equation is based on the equilibrium expressions for calcium
carbonate solubility and bicarbonate dissociation. To approximate actual conditions more closely,
pHs calculations were modified to include the effects of temperature and ionic strength.
The Langelier Index is defined as the difference between actual pH (measured) and calculated
pHs. The magnitude and sign of the LI value show waters tendency to form or dissolve scale and
thus to inhibit or encourage corrosion.
Although information obtained from the LI is not quantitative, it can be useful in estimating water
treatment requirements for low pressure boilers, cooling towers and water treatment plants, as
well as serving as a general indicator of the corrosivity of water.
Parameter measurement
The Langelier Saturation Index can be calculated easily by using Hach products to determine the
pH of calcium carbonate saturation (pH) and the actual pH of a solution. Using a simple formula,
the pH is derived from the values for calcium hardness, total alkalinity at pH 4.5, temperature and
total filterable residue (total dissolved solids). All of these procedures are included in this manual.
Temperature:
Temperature can be measured in degrees celsius with a laboratory thermometer
(Catalog. Number. 566-01). If only a Fahrenheit thermometer is available, conversion to degrees
Celsius will be necessary.
[C = 5/9 x (F 32)].
Calculation
After the preceding parameters have been determined, calculate the pH from the following
formula:
pH s = A + B C D
Where:
Constant A takes into account the effect of temperature. It is found by selecting the value from the
Water temperature table that corresponds to the measured temperature in degrees Celsius.
Constant B is a correction for the ionic strength of the sample. It is determined using the TDS table
by taking the value that corresponds to the measured total filterable residue or the estimated total
dissolved solids (TDS).
Langelier and Aggressive Indices

Page 1588

Value C is obtained from the Hardness or alkalinity table by reading the value corresponding to the
calcium hardness (in mg/L CaCO3) of the sample.
Value D is obtained from the Hardness or alkalinity table by reading the measured value for total
alkalinity (in mg/L CaCO3) of the sample.
The Langelier Saturation Index is the difference between the actual pH of the solution and pHs
calculated above.
LI = pH
actual
pH
Interpretation
The LI is a gauge of whether a water will precipitate or dissolve calcium carbonate. If the pHs is
equal to the actual pH, the water is considered balanced. This means that calcium carbonate will
not be dissolved or precipitated. If the pHs is less than the actual pH (the LI is a positive number),
the water will tend to deposit calcium carbonate and is scale-forming (nonaggressive). If the pHs is
greater than the actual pH (the LI is a negative number), the water is not saturated and will
dissolve calcium carbonate (aggressive). In summary:
pHS = pHactual, water is balanced
pHS < pHactual, LI = positive number, water is scale forming (nonaggressive)
pHS > pHactual, LI = negative number, water is not scale forming (aggressive)
It is important to remember that the LI value is not a quantitative measure of calcium carbonate
saturation or corrosion.
Because the protective scale formation is dependent on pH, bicarbonate ion, calcium carbonate,
dissolved solids and temperature; each may affect the waters corrosive tendencies independently.
Soft, low-alkalinity waters with either low or excessively high pH are corrosive, even though this
may not be predicted by the LI. This is because insufficient amounts of calcium carbonate and
alkalinity are available to form a protective scale.
Waters with high pH values and sufficient hardness and alkalinity may also be corrosive, even if
the LI predicts the opposite. This is the result of calcium and magnesium complexes that cannot
actively participate in the scale forming process. Analytical procedures do not distinguish between
these complexes and available calcium and magnesium; therefore, the LI value is not accurate in
such situations.
Corrosive tendencies may also be exhibited by water containing high concentrations of sulfate,
chloride and other ions which interfere with uniform carbonate film formation.
As a result of these and other problems, the LI is useful only for determining the corrosivity of
waters containing more than 40 mg/L of alkalinity, sufficient calcium ion concentration and ranging
between pH 6.5 and 9.5.
Aggressive index
The Aggressive Index (AI), originally developed for monitoring water in asbestos pipe, is
sometimes substituted for the Langelier Index as an indicator of the corrosivity of water. The AI is
derived from the actual pH, calcium hardness and total alkalinity. (Use procedures contained in
this handbook). Where it is applicable, it is simpler and more convenient than the LI. Because the
AI does not include the effects of temperature or dissolved solids, it is less accurate as an
analytical tool than the LI.
Calculation
After obtaining the pH, total alkalinity and calcium hardness, use the following formula to calculate
the AI:
Al = pHactual + C + D

Page 1589

Where:
Value C is obtained from the Hardness or alkalinity table by reading the value corresponding to the
calcium hardness (in mg/L CaCO3) of the sample.
Value D is obtained from the Hardness or alkalinity table by reading the measured value for total
alkalinity (in mg/L CaCO3) of the sample.
Interpretation
As with LI, the AI is not a quantitative measure of corrosion, but is a general indicator of the
tendency for corrosion to occur and as such, should be used with proper reservation. An AI of 12
or above indicates nonaggressive (not corrosive) water. AI values below 10 indicate extremely
aggressive (corrosive) conditions. Values of 1011.9 suggest that the water is moderately
aggressive. Corrosivity characteristics of water as indicated by the LI and AI are compared in the
Corrosion characteristics table.
Table 430 Water temperature

Water temperature, C
2.60
2.50
2.40
12
2.30
16
2.20
20
2.10
25
2.00
30
1.90
40
1.70
50
1.55
60
1.40
70
1.25
80
1.15

Page 1590

Table 431 TDS
TDS, mg/L
9.70
100
9.77
200
9.83
400
9.86
600
9.89
1000
9.90
Table 432 Hardness or alkalinity

Calcium hardness or total alkalinity in mg/L CaCO3
C1 or D2
10
1.00
20
1.30
30
1.48
40
1.60
50
1.70
60
1.78
70
1.84
80
1.90
100
2.00
200
2.30
300
2.48
400
2.60
500
2.70
600
2.78
700
2.84
800
2.90
900
2.95
1000
3.00
Factor C is the logarithm (base 10) of the calcium hardness expressed in mg/L
Factor D is the logarithm (base 10) of the total alkalinity expressed in mg/L
Table 433 Corrosion characteristics

Corrosive characteristics
Langelier index
Highly aggressive
< 2.0
Aggressive index
< 10.0
Moderately aggressive
2.0 to 0.0
10.00 to 12.0
Nonaggressive
>0.0
>12.0

Page 1591

References
1. Langelier, W. F., The Analytical Control of Anticorrosion Water Treatment Journal of
American Water Works Association 1936, 28, 1500.
2. Larson, T.E.; Buswell A. M. Calcium Carbonate Saturation Index and Alkalinity
Interpretations Journal of American Water Works Association 1942, 34, 1667.
3. Langelier, W. F., Chemical Equilibria in Water Treatment Journal of American Water Works
Association 1946, 38, 169.
4. Maguire, J. J.; Polsky, J. W. Simplified Plant Control Test for Boiler Water Dissolved Solids
Combustion 1947, May, 35.
5. Betz Handbook of Industrial Water Conditioning 1962, 6th ed., Betz Laboratories: Trevose,
PA.
6. Robinson, R. A.; Stokes, R. H. Electrolyte Solutions 1965, Butterworth & Co. LTD: London.
7. Federal Register 1980, 45 (168), August 27, 1980, p. 57338.

Page 1592
Lead tests explained
Lead
LeadTrak Method
Introduction
Lead is seldom found in ground water in more than trace quantities; it averages around 10 g/L.
Surface waters contain very low levels of lead because it is precipitated by a variety of substances.
Lead may be found in potable water systems as a result of the corrosion of lead service lines,
lead-based solder joints, or lead-based plumbing fixtures.
Lead and its compounds are poisonous and accumulate in the bone structure when ingested in
amounts exceeding the natural elimination rate of about 300 micrograms per day. Accumulation of
significant amounts of lead in the body may cause severe and permanent brain damage,
convulsions and death. Environmental concern with lead poisoning has resulted in a national
program to reduce the concentration of lead in consumer products.
Chemical reactions
LeadTrak method
The LeadTrak Method for determining soluble lead as Pb2+ in potable water first involves the
acidification of the sample to keep all lead ions soluble and to prevent the lead from being lost by
precipitation or absorption on the sample container walls. Complexing and buffering agents are
then added to the sample to modify the lead ions into a form which allows them to be retained by
the cellulose medium in the concentrator column. Other competing ions, such as iron, copper and
zinc, pass through the column and are eliminated.
The lead ions are then eluted from the concentrator column with a nitric acid solution. The nitric
acid eluant is neutralized and reacted with meso-tetra (4-N-methylpyridyl) porphine tetratosylate to
form a faintly colored complex. The absorbance of the complex is measured at 477 nm. EDTA is
then added to the complex. The EDTA complexes with the lead and removes the lead from the
porphine complex. The absorbance of the sample is read again and the lead concentration is
determined by the difference between the two readings.
Lead
Page 1593
Manganese tests explained
Manganese
Periodate Oxidation and PAN Methods
Introduction
Manganese is present in ground waters primarily as the divalent ion (Mn2+), due to the lack of
subsurface oxygen. Surface waters may contain combinations of manganese in various oxidation
states as soluble complexes, or as suspended particles.
The occurrence of manganese in public water supplies presents more of an economic problem
than a potential health hazard. Manganese causes dark stains in laundry and on plumbing fixtures,
tends to deposit in water lines, and imparts an objectionable taste to beverages such as coffee and
tea. Manganese levels in natural waters rarely exceed 1 mg/L, but levels of 0.1 mg/L are sufficient
to cause the taste and staining problems. The recommended allowable manganese level in public
water supplies is 0.05 mg/L.
Two methods for manganese determination are used in test procedures. The Periodate Oxidation
Method gives a simple, rapid test for high levels of manganese. The 1-(2-Pyridylazo)-2-Napthol
(PAN) Method is a sensitive, rapid procedure for low levels of manganese.
Chemical reactions
Periodate oxidation method
Manganese is oxidized to permanganate using periodate in a slightly acidified water sample. No
indicator is necessary. Intensity of the purple color of the permanganate ion is a direct indication of
the amount of manganese present in the sample.
3H2 O + 2Mn
2+
+ 5IO 4 2MnO 4 + 5IO 3 + 6H
PAN method
The PAN method employs an Alkaline-Cyanide Reagent. PAN Indicator is added and forms an
orange-red colored complex with the manganese ion.
N
N
2
N
Mn2+
Mn
2H+
N
O
OH
N
Figure 71 Chemical reaction for PAN method

Manganese
Page 1594

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
Edition 7
Mercury cold vapor tests explained
Mercury, Cold Vapor

Introduction
Mercury is sometimes dumped into rivers and lakes by industry. Farmers have also used mercury
compounds to prevent fungi attack on seeds. In nature, bacteria change elemental mercury to
organic mercury compounds that are very toxic to all living organisms. These compounds
bioaccumulate in the food chain and can eventually reach humans. At high enough levels, mercury
causes nerve damage, mutations and death. Because mercury is so toxic, environmental
monitoring is necessary especially at sources of industrial discharge.
Chemical reactions
The sample is digested to convert all forms of mercury to mercuric ions (Hg2+) and to destroy
interfering substances that may be present. The sample is then treated with a reducing agent to
convert the mercuric ions to elemental mercury (Hg). The elemental mercury is volatilized from the
sample in a semi-closed system by bubbling air through the sample.
The airstream containing the mercury vapor is swept through an absorber column that contains
hypochlorous acid and hypochlorite ions. The vaporized mercury ions react in the column and
form mercuric chloride (HgCl2). The mercuric chloride is then eluted off the column with an acid
solution.
The eluate is treated with HgEx Reagent 3 to destroy the excess hypochlorous acid and
hypochlorite, and to make the solution alkaline. A sensitive indicator (HgEx 4) forms a complex
with the mercuric ions, and HgEx 5 maximizes the mercury-indicator reaction, thus increasing the
sensitivity of the test. The spectrophotometer is zeroed on this solution at the absorbance peak
(412 nm) of the unreacted indicator. HgEx 6 is added to break the mercury-indicator bond. The
analyst then measures the absorbance increase of unreacted indicator. This increase is
proportional to the amount of mercury in the sample.
Mercury, Cold Vapor

Page 1596
Molybdenum, molybdate tests explained
Molybdenum, Molybdate
Mercaptoacetic Acid and Ternary Complex
Methods
For water
Introduction
Molybdenum (molybdate) salts are commonly used as corrosion inhibitors in cooling water
systems. There are numerous procedures for determining molybdenum as molybdate (MoO42) in
water. The Mercaptoacetic Acid Method is one of the most frequently referenced methods for
determining molybdenum. The Hach procedure improves and simplifies this time-proven
procedure.
Chemical reactions
Mercaptoacetic acid
The MolyVer (Mercaptoacetic Acid) Method utilizes three reagent powders. First, a MolyVer 1
Reagent Powder Pillow is added. MolyVer 1 contains a buffer to control the pH, in addition to a
chelating agent to mask interferences.
Low test results can be caused from reduction of Mo6+ to Mo5+, because the test is specific for
Mo6+. MolyVer 2 Reagent Powder is added to prevent reduction of the Mo6+ ion. Finally,
mercaptoacetic acid, contained in MolyVer 3 Reagent Powder, is added. The reaction of MolyVer 3
with Mo6+ results in formation of a characteristic yellow color. Development of the yellow color
follows Beers Law over the range of the test.
MoO42
2HSCH2 COOH
2H+
Mercaptoacetic acid
Molybdate
CH2
HO
Mo
CH2
2H2O
OH
Figure 72 Chemical reaction for the Mercaptoacetic Acid method
Page 1597
Ternary complex method
The ternary complex method is a two-reagent method for molybdenum in the
03 mg/L range. First, the molybdenum-containing sample reacts with Molybdenum 1 Reagent,
which contains colorimetric indicator, pH buffer, and reducing agent. The reducing agent
counteracts interference by iron, a common contaminant in boiler and cooling water samples. The
indicator forms a colored, binary complex with molybdenum. Depending on molybdenum levels,
binary-complex color will range from pale yellow to rusty orange.
Second, Molybdenum 2 Reagent will combine with the binary complex to form an intensely
colored, blue, ternary (three-part) complex directly proportional to sample molybdenum
concentration. The eye perceives the color as ranging from yellow to green because the blue color
is superimposed over a yellow background. These colors correspond to 0 mg/L Mo (yellow) up to 3
mg/L Mo (dark green).
MoO4
Sample:
Contains
molybdatemolybdenum
Molybdenum 1 reagent
Indicator-Molybdate
binary complex
Powder Pillow:
ph Buffer
Reducing agent
Colorimetric indicator
Weak color:
Yellow to orange
Molybdenum 2
reagent solution
Figure 73 Chemical reaction pathway for the Ternary Complex method
Page 1598
Indicator-MolybdateMolybdenum 2
reagent Ternary
complex
Yellow to green
color
Nickel tests explained
Nickel
For water
Heptoxime and PAN Methods
Introduction
Nickel is seldom found in natural waters, but often present in industrial wastewater as a direct
product of metal plating baths, and as a corrosion product of stainless steel, nickel or cobalt alloys.
Nickel is considered relatively nontoxic to humans. The toxicity of nickel to aquatic life varies
widely and is influenced by species, pH, synergetic effects, and other factors. Nickel salts at
concentrations between 0.5 and 1.0 mg/L have been shown to be toxic to some plant species.
Two methods for determining nickel, the Heptoxime Method and the PAN Method, are used in
Hach procedures. The well-known heptoxime indicator has been formulated into a dry, stable
powder packaged in pillows for the nickel determination. A second powder pillow is used to
overcome interference from other metals present. The heptoxime forms a yellow complex, which is
extracted into chloroform for measurement.
The PAN procedure is a sensitive method for detecting nickel and cobalt in concentrations less
than 1 mg/L. The method is unique because simultaneous determinations of nickel and cobalt
concentrations can be made on the same sample portion without the need for solvent extraction or
sample preconcentration steps.
Chemical reactions
Heptoxime method
Nickel is analyzed quantitatively through its reaction with Heptoxime to form a yellow-colored
complex, which is then extracted into a chloroform layer to concentrate the color and to enable a
more sensitive colorimetric determination. Chelating agents are added to the sample to overcome
the interferences caused by cobalt, copper and iron.
NOH
2
NOH
2+
Ni
Ni
H
O
H
N
2H+
Figure 74 Chemical reactions for Heptoxime method

PAN method
The PAN method for nickel is discussed in detail in the PAN method for cobalt, because both
cobalt and nickel can be determined on the same sample.
Nickel
Page 1599
Nitrogen, Ammonia tests explained
Nitrogen, Ammonia
Nessler Method and Salicylate Method
Introduction
Ammonia is a product of the microbiological decay of animal and plant protein. It can be directly
reused by plants to produce protein. Ammonia and ammonia compounds are applied directly as
fertilizers.
The presence of ammonia nitrogen in surface water usually indicates domestic pollution. Ammonia
in ground water is normal and is due to microbiological processes. Two methods for determining
ammonia, the Nessler method and the Salicylate method, are used in Hach products and
procedures.
Chemical reactions
Nessler method
In the ammonia test, Nessler Reagent (K2HgI4) reacts with the ammonia present in the sample
(under strongly alkaline conditions) to produce a yellow-colored species. The intensity of the color
is in direct proportion to the ammonia concentration.
2K 2 HgI 4 + NH3 + 3KOH Hg2 OINH 2 + 7KI + 2H 2 O
Salicylate method
The Salicylate method is a variation of the well-known Phenate Method, but it has an advantage of
being free from mercury salts and phenol. This method is most useful for low range ammonia
nitrogen determinations. Although the procedure involves multiple reactions before a final green
color is developed, all reagents are contained in convenient powder pillows (Salicylate Reagent
Powder Pillows and Alkaline Cyanurate Powder Pillows) or a combination of powder pillows and
TNT vials.
Ammonia compounds are initially combined with hypochlorite to form monochloramine (1), which
then reacts with salicylate to form 5-aminosalicylate (2).
(2)
NH2Cl +
OH
H 2N
COO
OH
Cl
COO
Oxidation of 5-aminosalicylate is carried out in the presence of a catalyst, nitroprusside or

Fe(CN)5NO2 (also called nitroferricyanide), which results in the formation of indosalicylate, a
blue-colored compound. The blue color is masked by the yellow color (from excess nitroprusside)
causing a green-colored solution. The intensity of the color is directly proportional to the ammonia
concentration in the sample.
COO
COO
Figure 75 Chemical structure of Indosalicylate
Nitrogen, Ammonia
Page 1600
Nitrogen, Kjeldahl tests explained
Nitrogen, Kjeldahl
Peroxide Digestion Method
Introduction
The test for Kjeldahl nitrogen, also referred to as crude protein, is used to determine ammonia and
organic nitrogen present in a sample. Only small fractions of nitrite and nitrate nitrogen are
included in the test. A preliminary digestion is used to oxidize carbon compounds to carbon
dioxide, and to convert organic forms of any nitrogen present (amino acids, proteins, peptides) to
ammonia. The traditional digestion uses sulfuric acid and various combinations of metallic
catalysts and salts. Digestion of at least 2 hours is followed by addition of sodium hydroxide to the
digest and then distillation of the ammonia into a boric acid or buffer solution. Ammonia in the
distillate is measured with back titration or nesslerization. This procedure requires several hours
for reagent preparation, digestion, distillation and final measurement.
The Hach Digesdahl Digestion Apparatus and the Peroxide Digestion Method allow completion of
the Kjeldahl test within 15 minutes or less, depending on the nature of the sample. First, the
sample is charred in concentrated sulfuric acid. Fifty percent hydrogen peroxide is fed into the
reaction mixture, where it oxidizes organic carbonaceous matter and converts organic nitrogen
into ammonium bisulfate. For example, the reaction with glycine, a simple amino acid is:
NH2CH2COOH + 2H2O2 + H2SO4 NH4HSO4 + CO2 + 2H2O
Glycine
Digestion apparatus
The Digesdahl digestion apparatus includes a fractionating column. Hydrogen peroxide, added
slowly, trickles into the reaction mixture in the flask below. The temperature of the reaction is
maintained near the boiling point of sulfuric acid (300 C, 572 F). Vapors from the reaction rise to
the column where SO2 and water vapors are drawn off by an aspirator. Hydrogen peroxide vapors
condense in the column and return to the reaction mixture.
It should be noted that no metal catalysts or salts are used in digestion. The digest is suitable for
nesslerization for final measurement without an intermediate distillation step. The digest is also
suitable for mineral analysis of Ca, Mg, Mn, K, P and Zn. See Nitrogen, Ammonia for more
information about the Nessler method.
Nitrogen, Kjeldahl
Page 1601
Nitrogen, Nitrate tests explained
Nitrogen, Nitrate
Introduction
Nitrate represents the most completely oxidized state of nitrogen, and is commonly found in water.
Nitrate-forming bacteria convert nitrites into nitrates under aerobic conditions; lightning converts
large amounts of atmospheric nitrogen (N2) directly to nitrates. Many granular commercial
fertilizers contain nitrogen in the form of nitrates.
High levels of nitrate in water may indicate biological wastes in the final stages of stabilization, or
run-off from heavily fertilized fields. Nitrate-rich effluents discharged into receiving waters can
degrade water quality by encouraging excessive growth of algae. Drinking waters containing
excessive amounts of nitrates can cause infant methemoglobinemia (blue babies). For this
reason, a maximum concentration level in drinking water has been established by the USEPA in
accordance with the Safe Drinking Water Act.
Two methods of analysis are used in the high range tests. The NitraVer5 high range method is a
modification of the Cadmium Reduction Method, using gentisic acid in place of 1-naphthylamine.
All the necessary reagents have been combined into a single stable powder.
The Chromotropic Acid high range nitrate method involves the reaction of nitrate in a strong acid
medium with chromotropic acid. The final reaction mixture is contained in the screw-capped
Test N Tube vial.
The low range nitrate test is also a modification of the Cadmium Reduction Method, and uses a
very sensitive chromotropic acid indicator. Both methods register nitrate and nitrite nitrogen.
Chemical reactions
High rangeNitraVer 5
In the NitraVer 5 high range test, cadmium metal is used to reduce nitrates (NO3) to nitrites
(NO2) (reaction 1). Next, the nitrite ions react in an acidic medium with sulfanilic acid to form an
intermediate diazonium salt (reaction 2) which, when coupled with gentisic acid (reaction 3), forms
an amber-colored compound. Color intensity of the compound is directly proportional to the nitrate
concentration of the water sample.
(2)
NO2 + H2N
SO3H + 2H+
Sufanilic Acid
HO3S
+
N
+ 2H2O
Diazonium Salt
Nitrogen, Nitrate
Page 1602
Nitrogen, Nitrate
High rangeChromotropic acid method
In the Chromotropic Acid test, sample is added to a Test N Tube vial containing sulfuric acid.
This sample/sulfuric acid mixture is used to zero the spectrophotometer. Chromotropic acid is then
added as NitraVer X Reagent B. Two moles of nitrate react with one mole of chromotropic acid to
form a yellow reaction product, which exhibits maximum absorbance at 410 nm.
OH
(2)
OH
OH
H+
NO3
SO3
SO3
OH
2ON
NO2
+
SO3
2H+
SO3
Chromotropic acid
Nitrate
Yellow color
Low range
In the low range nitrate test, cadmium metal is used to reduce the nitrates to nitrites. The cadmium
is provided in NitraVer 6 Reagent Powder Pillows. Nitrite ions react with sulfanilic acid to produce
an intermediate diazonium salt, as in the high range test. The diazonium salt then forms a redorange complex with chromotropic acid. The color intensity is in direct proportion to the nitrate
concentration in the sample (reaction 4). In the low range test the sulfanilic acid and chromotropic
acid are contained in NitriVer 3 Reagent Powder Pillows.
OH
(3)
+
N
HO3S
OH
COOH
HO3S
OH
Amber colored species
OH
Diazonium salt
Gentisic acid
OH
(4)
HO3S
+
N
Diazonium Salt
OH
OH
HO3S
COOH + H+
HO3S
SO3H
Chromotropic Acid
OH
HO3S
H+
SO3H
Red-Orange color
Nitrogen, Nitrate
Page 1603
Nitrogen, Nitrite tests explained
Nitrogen, Nitrite
Ferrous Sulfate and Diazotization Methods
Introduction
Nitrite nitrogen occurs as an intermediate stage in the biological decomposition of compounds
containing organic nitrogen. Nitrite-forming bacteria convert ammonia under aerobic conditions to
nitrites. The bacterial reduction of nitrates can also produce nitrites under anaerobic conditions.
Nitrites are often used as corrosion inhibitors in industrial process water and cooling towers; the
food industry uses nitrite compounds as preservatives.
Because nitrites readily oxidize to nitrates, they are not often found in surface waters. The
presence of large quantities of nitrites indicates partially decomposed organic wastes in the water
being tested. Drinking water concentrations seldom exceed 0.1 mg/L of nitrite.
The high range nitrite test is a modification of the classical brown ring test for nitrate using ferrous
sulfate. By controlling the sample pH, the nitrite present is reduced to nitrous oxide, which reacts
with the indicator to form a greenish-brown color. Nitrates are not registered in the test. All
necessary reagents have been combined in a single powder pillow form called NitriVer 2 Nitrite
Reagent Powder. A special agent helps prevent color formation or precipitation of common
interfering ions.
The low range nitrite test uses chromotropic acid and sulfanilic acid as the indicator. The indicator
and a buffer are combined in a single powder NitriVer 3 Nitrite Reagent. The test is sensitive to low
nitrite concentrations.
Chemical reactions
High range, ferrous sulfate method
In an acidic medium ferrous sulfate reduces nitrogen in nitrite (NO2) to form nitrous oxide (NO).
Ferrous ions combine with the nitrous oxide to form a brown-colored complex ion, the color
intensity of which is in direct proportion to the nitrite present in the water sample. Color
development follows Beers Law.
2Fe2+ + 4H+ + 2NO2 2Fe3+ + 2NO + 2H2O
NO + FeSO4 FeSO4 NO
Low range, diazotization method

In the low range nitrite test, nitrite ions react with sulfanilic acid to form an intermediate diazonium
salt. This reacts with chromotropic acid to produce a red-orange complex directly proportional to
the amount of nitrite present. A measurement of the color intensity will provide an accurate
determination of the nitrite concentration in the water sample.
Nitrogen, Nitrite
Page 1604
Nitrogen, Nitrite
+
NH2 + 2H
NO2 + HO3S
HO3S
+ 2H2O
Sulfanilic acid
OH
HO3S
OH
OH
HO3S
OH
SO3H HO3S
SO3H + H+
SO3H
Chromotropic acid
Figure 76 Chemical reaction for Low Range Diazotization method
Nitrogen, Nitrite
Page 1605
Nitrogen, Total tests explained
Nitrogen, Total
Titanium Chloride and Persulfate Digestion
Methods
Introduction
Total nitrogen methods measure nitrogen loads on influent streams, at intermediate stages of
water treatment for sludge, and on effluent to gauge overall treatment plant efficiency. Assessing
nitrogen levels allows process monitoring, adjustment and nitrogen reduction efficiency throughout
the treatment.
Titanium chloride reduction method (Total Inorganic Nitrogen)
Titanium (III) ions reduce nitrate and nitrite to ammonia in a basic environment. After centrifugation
to remove solids, the ammonia is combined with chlorine to form monochloramine.
Monochloramine reacts with salicylate to form 5-aminosalicylate, a green solution, as in the
salicylate method in Ammonia Nitrogen (see Nitrogen, Ammonia).
Persulfate digestion method (Total Nitrogen)
An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfate is
added after the digestion to eliminate interferences from halogen oxides. Under strongly acidic
conditions, nitrate reacts with chromotropic acid to nitrate the biphenyl rings at several locations,
forming several nitrated products (Figure 77). The nitrated products that form are measured
at 410 nm.
SO3H
HO3S
OH
OH
Figure 77 Chromatropic acid structure, including available reaction sites for nitrate
Nitrogen, Total
Page 1606
Total Organic Carbon tests explained

Direct Method
Introduction
Total Organic Carbon (TOC) testing is important in drinking water treatment as an indicator of
potential disinfection by-product formation. In wastewater, TOC is valuable as a surrogate for COD
testing and has applications in domestic wastewater pre-treatment standards, effluent discharge
limitations, and industrial process waters.
The colorimetric TOC test measures the total amount of non-volatile organic carbon in a sample.
The method is based on controlled digestion/diffusion in a sealed glass assembly*. Sample carbon
is oxidized to carbon dioxide by persulfate oxidation. The carbon dioxide diffuses into a colored pH
indicator solution where it is converted into carbonic acid. The resulting color change is
proportional to the concentration of carbon present in the sample.
Chemical reactions
Inorganic carbon is removed from the sample by adjusting the sample to pH 2 with a buffer, and
stirring vigorously for 10 minutes:
TOC = Total Carbon Inorganic Carbon
A suitable volume of treated sample and potassium persulfate is added to a 16-mm screw top
digestion vial containing Acid Digestion Solution Reagent. A 9-mm sealed glass ampule containing
the TOC Indicator Solution is opened and placed inside the digestion vial. The whole assembly is
then sealed with a screw cap and digested at 103105 C (217221 F) for 2 hours.
In the presence of acidic persulfate and with increased pressure and elevated temperature, the
samples organic carbon is oxidized to carbon dioxide. For example, in the persulfate digestion of
a sample that contains formate, the chemical reaction is:
S2O82 + HCOO HSO4 + SO42 + CO2
The evolved CO2 then diffuses and is trapped in an aqueous solution containing a pH indicator.
The absorbed CO2 forms carbonic acid according to:
CO2 + H2O 2H+ + CO32
The pH indicator (prior to CO2 absorption) is in its deprotonated, or basic, form (D). As the
absorbed CO2 level increases, the hydrogen ion level will also increase, resulting in an increase of
the protonated form of the indicator:
D (Color A) + H+ DH (Color B)
The concentration of the carbon in the sample is proportional to the color change, either the
change in Color A (D), or the change Color B (DH) or the sum (D + DH).
* U.S. Patent 6,368,870

Page 1607
Oxygen Demand, Mn III tests explained
Oxygen Demand, Chemical, Mn III

Introduction
Chemical oxygen demand (COD) is defined as a measure of the oxygen equivalent of the organic
matter content of a sample that is susceptible to oxidation by a strong chemical oxidant.* Trivalent
manganese (Mn III) is a strong, non-carcinogenic chemical oxidant that changes quantitatively
from purple to faint pink when it reacts with organic matter. Manganese III COD results are
measured colorimetrically, and the color intensity is inversely proportional to the amount of COD in
the sample.
The digestion time is 60 minutes, but can be extended when samples are difficult to completely
oxidize. The reagent typically has an oxidation efficiency of about 80% for standards prepared
from potassium acid phthalate and domestic wastewater samples. No oxygen demand test will
oxidize all organic compounds with 100% efficiency. With non-typical samples, standards can be
prepared from other reference materials. Studies have shown that the Mn III COD procedure
correlates very well to biochemical oxygen demand (BOD) and dichromate COD tests.
Many COD reagents contain mercury, chromium and silver. The absence of these materials in the
Mn III COD Reagent significantly minimizes the disposal cost and reduces exposure of the analyst
to hazardous compounds.
Inorganic materials may interfere with the Mn III COD Reagent. Chloride is the most common
interference and is removed by sample pretreatment with the Chloride Removal Cartridge.
Ammonia interferes with the test when present with chloride. The interference is severe at high
ammonia and chloride concentrations.
Chemical reactions
Trivalent manganese oxidizes organic materials in the sample to CO2 and H2O. In the process,
manganese III is reduced to manganese II. The reaction occurring in the Mn III COD Reagent Vial
is represented by the equation below:
2 KC8H5O4 + 30 Mn2(SO4)3 + 24 H2O 16 CO2 + 60 MnSO4 + 28 H2SO4 + 2 KHSO4
Chemical oxygen demand results are usually expressed by the amount of oxygen consumed
during the oxidation of organic matter.
When oxygen is used as the primary oxidant in the oxidation of potassium acid phthalate, the
equation below describes the reaction:
KC8H5O4 + 7.5 O2 8 CO2 + 2 H2O + KOH
Seven and one-half molecules of oxygen (O2) consume one molecule of potassium acid phthalate
(KHP). On a weight basis, the theoretical oxygen demand for KHP is 1.175 mg O2 per mg KHP.
The interference from chloride is minimized by sample pre-treatment with the Chloride Removal
Cartridge (CRC). The CRC contains a proprietary reagent to remove chloride from the sample
solution. The flow rate through the CRC has been optimized for chloride removal, while at the
same time minimizing any effect the CRC might have on other sample components.
* APHA Standard Methods for the Examination of Water and Wastewater, 19th ed., 1995

Page 1608

Suspended solids, which may contain oxidizable organic compounds, are filtered out of the
sample with a glass fiber filter located in the upper part of the Chloride Removal Cartridge. After
the sample has been filtered into a Mn III COD Reagent Vial, the glass fiber filter, along with any
suspended solids present, is transferred into the COD Reagent Vial. The glass fiber filter is binder
free and has no oxygen demand.
Comparability of the Mn III COD to other tests
For samples from a specific source, the Mn III COD results can be related empirically to BOD,
dichromate COD, organic carbon, or organic matter. The test is useful for monitoring and control
after correlation has been established. The test can also be used to estimate dilutions for the five
day BOD test. This will ensure reliable BOD data for pre-treatment and compliance monitoring. For
samples with constant chloride concentrations, reliable correlations can be developed without the
chloride removal pre-treatment.
Test oxidation efficiency
Different test methods may oxidize sample components with different efficiencies. The Mn III COD
Reagent will oxidize KHP standards with about 80% efficiency. Many wastewater samples are also
oxidized with 80% efficiency.
For example, an ASTM Wastewater Reference Sample was analyzed for COD using both the
dichromate and Mn III COD. The dichromate COD result was 1018 mg/L and Mn III COD result
was 1008 mg/L. These test results are comparable despite different oxidation efficiencies because
the instrument calibration is based on KHP.
Correlation of test results
To demonstrate how to correlate the results between the two different tests, a glutamic acid
standard was prepared to contain a theoretical chemical oxygen demand of 500 mg/L. The
dichromate COD result was 506 mg/L and the Mn III COD result was 459 mg/L. To correlate the
Mn III COD results with the dichromate COD, a correlation factor is determined.
Cr COD
506 mg/L
------------------------------ = ------------------------- = 1.102 (Correlation Factor)
Mn III COD
459 mg/L
Mn III COD, mg/L 1.102 = Dichromate COD, mg/L
Calibrations based on reference materials other than KHP

It may be desirable for COD results to closely match the theoretical demand of the sample.
Occasionally, samples contain a major component that is incompletely oxidized. In this situation,
calibration standards can be prepared from known values of that sample component. In the
example above, calibration standards would be prepared from glutamic acid.

Page 1609
COD explained

Reactor Digestion Using Potassium
Dichromate Method
For wastewater
Introduction
The Dichromate Chemical Oxygen Demand (COD) test measures the oxygen equivalent of the
amount of organic matter oxidizable by potassium dichromate in a 50% sulfuric acid solution.
Generally, a silver compound is added as a catalyst to promote the oxidation of certain classes of
organics, and a mercuric compound may be added to reduce interference from the oxidation of
chloride ions by the dichromate. End products are carbon dioxide, water, and various states of the
chromium ion.
After the oxidation step is completed, the amount of dichromate consumed is determined
titrimetrically or colorimetrically. Either the amount of reduced chromium (chromic ion), or the
amount of unreacted dichromate, can be measured. If the latter method is chosen, the analyst
must know the precise amount of dichromate added.
Chemical reactions
In the oxidation of organic materials by dichromate in sulfuric acid, most of the carbon is converted
to carbon dioxide while any hydrogen present in the organic compound is converted to water.
Other elements also may be oxidized.
Chemical oxygen demand results are usually expressed by the amount of oxygen consumed
during the oxidation of organic matter. When oxygen is used as the primary oxidant in the
oxidation of potassium acid pthalate, the equation below describes the reaction.
KC 8 H 5 O 4 + 7.5O 2 8CO 2 + 2H 2 O + KOH
Seven and one-half molecules of oxygen (O2) consume one molecule of potassium acid pthalate
(KHP). On a weight basis, the theoretical oxygen demand for KHP is 1.175 mg O2 per mg KHP.
There are two basic methods, titrimetric and colorimetric, for determining the amount of chromium
in a particular valence state. There are several variations of each method.
When titration is used in the measurement process, the amount of Cr6+ left is determined. It is
done in one of two ways; in both cases, the precise initial amount of Cr6+ ion must be known. This
is necessary because one must be able to subtract the final Cr6+ level from the initial level to yield
the amount that was reduced to Cr3+. This difference is used to calculate the COD. The initial
amount is known either through calculation, because primary standard grade potassium
dichromate is readily available, or by testing the bulk solution before running the individual tests.
The final amount of dichromate is most commonly determined by direct titration using ferrous
ammonium sulfate as the titrant and ferroin (1,10-phenanthroline ferrous sulfate) as the indicator.
The Fe2+ in the titrant reacts with the chromic ions:
3Fe
2+
+ Cr
6+
3Fe
3+
+ Cr
3+
1,10-phenanthroline forms an intense color with Fe2+ but no color with Fe3+. When reduction of
Cr6+ to Cr3+ is complete, Fe2+ reacts to form the ferroin complex and the solution color changes
sharply from greenish-blue to orange-brown, signaling the end point. The end point also can be
detected potentiometrically.

Page 1610

The colorimetric determination has several advantages over titration. Colorimetric determination is
quicker and easier to run, and does not require additional reagents. In the Reactor Digestion
Method, the digestion vials can be checked during the digestion process while they are hot to
determine when no further oxidation is taking place, resulting in a shorter digestion time. When
using the Reactor Digestion Method the spectrophotometer is set at 420 nm or 365 nm for the low
ranges, and 620 nm for the high ranges. Low range measurement determines the remaining
yellow Cr6+. High range measurement determines the amount of green Cr3+ produced.

Page 1611
Dissolved oxygen tests explained
Oxygen, Dissolved
Azide Modification of Winkler Method and
Luminescence Measurement (LDO) Method
For water, wastewater, and seawater
Introduction
The dissolved oxygen test is one of the most important analyses in determining the quality of
natural waters. The effect of oxidation of wastes on streams, the suitability of water for fish and
other organisms, and the progress of self-purification can all be measured or estimated from the
dissolved oxygen content. In aerobic sewage treatment units, the minimum objectionable odor
potential, maximum treatment efficiency and stabilization of wastewater are dependent on
maintenance of adequate dissolved oxygen. Frequent dissolved oxygen measurement is essential
for adequate process control.
Dissolved oxygen is essential for the survival of aquatic plant and animal life. Generally, 45 mg/L
of dissolved oxygen content is a borderline concentration for an extended time period. For
adequate game fish population, the dissolved oxygen content should be in the 815 mg/L range.
Dissolved oxygen concentration varies with water depth, sludge deposits, temperature, clarity and
flow rate. Thus a single water sample is rarely representative of the over-all condition of a body of
water.
Chemical reactions
Azide modification of Winkler method
In the analysis, Mn2+ (manganous ion) reacts with the dissolved oxygen present in the alkaline
solution to form a Mn4+ oxide hydroxide floc (1). Azide is added at this time to suppress
interference from any nitrate present (which would react with the iodide). The solution is then
acidified, and the manganese floc is reduced by iodide to produce Mn2+ and free iodine as I3(I2 +
I in solution, see equation 2). The iodine gives the clear supernate a brown color. Phenylarsine
oxide (PAO) or thiosulfate is then used to titrate the iodine to a colorless end point (3). (Starch
indicator can be added to enhance the determination of the end point by producing a color change
from dark blue to colorless.) The dissolved oxygen of the sample is then calculated from the
quantity of titrant used.
(2)
MnO(OH)2
6I
6H+
Mn2+
2I3
3H2O
OH
(3)
2H2O
I3
As = O
2HI + I
As = O
OH
Oxygen, Dissolved
Page 1612
Oxygen, Dissolved
Luminescence measurement of dissolved oxygen (LDO)
The luminescence-based sensor procedure measures the light emission characteristics from a
luminescence-based reaction at the sensor-water interface. A light emitting diode (LED) provides
incident light required to excite the luminophore substrate. In the presence of dissolved oxygen the
reaction is suppressed. The resulting dynamic lifetime of the excited luminophore is evaluated and
equated to dissolved oxygen concentration.
Figure 78 LDO probe

Dissolved oxygen
The Dissolved oxygen saturation in water (mg/L) table lists the mg/L dissolved oxygen in water at
saturation for various temperatures and atmospheric pressures. The table was formulated in a
laboratory using pure water. The values given are only approximations for estimating the oxygen
content of a particular body of surface water
Table 434 Dissolved oxygen saturation in water (mg/L)

Pressure in millimeters and inches Hg
mm
Temp
775
760
750
725
700
675
650
625
inches
F
30.51
29.92
29.53
28.45
27.56
26.57
25.59
24.61
32.0
14.9
14.6
14.4
13.9
13.5
12.9
12.5
12.0
33.8
14.5
14.2
14.1
13.6
13.1
12.6
12.2
11.7
35.6
14.1
13.8
13.7
13.2
12.9
12.3
11.8
11.4
37.4
13.8
13.5
13.3
12.9
12.4
12.0
11.5
11.1
39.2
13.4
13.1
13.0
12.5
12.1
11.7
11.2
10.8
41.0
13.2
12.8
12.6
12.2
11.8
11.4
10.9
10.5
42.8
12.7
12.4
12.3
11.9
11.5
11.1
10.7
10.3
44.6
12.4
12.1
12.0
11.6
11.2
10.8
10.4
10.0
46.4
12.1
11.8
11.7
11.3
10.9
10.5
10.1
9.8
48.2
11.8
11.6
11.5
11.1
10.7
10.3
9.9
9.5
50.0
10
11.6
11.3
11.2
10.8
10.4
10.1
9.7
9.3
51.8
11
11.3
11.0
10.9
10.6
10.2
9.8
9.5
9.1
53.6
12
11.1
10.8
10.7
10.3
10.0
9.6
9.2
8.9
55.4
13
10.8
10.5
10.5
10.1
9.8
9.4
9.1
8.7
57.2
14
10.6
10.3
10.2
9.9
9.5
9.2
8.9
8.5
Oxygen, Dissolved
Page 1613
Oxygen, Dissolved
Table 434 Dissolved oxygen saturation in water (mg/L) (continued)
59.0
15
10.4
10.1
10.0
9.7
9.3
9.0
8.7
8.3
60.8
16
10.1
9.9
9.8
9.5
9.1
8.8
8.5
8.1
62.6
17
9.9
9.7
9.6
9.3
9.0
8.6
8.3
8.0
64.4
18
9.7
9.5
9.4
9.1
8.8
8.4
8.1
7.8
66.2
19
9.5
9.3
9.2
8.9
8.6
8.3
8.0
7.6
68.0
20
9.3
9.1
9.1
8.7
8.4
8.1
7.8
7.5
69.8
21
9.2
8.9
8.9
8.6
8.3
8.0
7.7
7.4
71.6
22
9.0
8.7
8.7
8.4
8.1
7.8
7.5
7.2
73.4
23
8.8
8.6
8.5
8.2
8.0
7.7
7.4
7.1
75.2
24
8.7
8.4
8.4
8.1
7.8
7.5
7.2
7.0
77.0
25
8.5
8.3
8.3
8.0
7.7
7.4
7.1
6.8
78.8
26
8.4
8.1
8.1
7.8
7.6
7.3
7.0
6.7
80.6
27
8.2
8.0
8.0
7.7
7.4
7.1
6.9
6.6
82.4
28
8.1
7.8
7.8
7.6
7.3
7.0
6.7
6.5
84.2
29
7.9
7.7
7.7
7.4
7.2
6.9
6.6
6.4
86.0
30
7.8
7.6
7.6
7.3
7.0
6.8
6.5
6.2
87.8
31
7.7
7.4
7.4
7.2
6.9
6.7
6.4
6.1
89.6
32
7.6
7.3
7.3
7.0
6.8
6.6
6.3
6.0
91.4
33
7.4
7.2
7.2
6.9
6.7
6.4
6.2
5.9
93.2
34
7.3
7.1
7.1
6.8
6.6
6.3
6.1
5.8
95.0
35
7.2
7.0
7.0
6.7
6.5
6.2
6.0
5.7
96.8
36
7.1
6.8
6.9
6.6
6.4
6.1
5.9
5.6
98.6
37
7.0
6.7
6.7
6.5
6.3
6.0
5.8
5.6
100.4
38
6.9
6.6
6.6
6.4
6.2
5.9
5.7
5.5
102.2
39
6.8
6.5
6.5
6.3
6.1
5.8
5.6
5.4
104.0
40
6.7
6.4
6.4
6.2
6.0
5.7
5.5
5.3
105.8
41
6.6
6.3
6.3
6.1
5.9
5.6
5.4
5.2
107.6
42
6.5
6.2
6.2
6.0
5.8
5.6
5.3
5.1
109.4
43
6.4
6.1
6.1
5.9
5.7
5.5
5.2
5.0
111.2
44
6.3
6.0
6.0
5.8
5.6
5.4
5.2
4.9
113.0
45
6.2
5.9
5.9
5.7
5.5
5.3
5.1
4.8
114.8
46
6.1
5.8
5.9
5.6
5.4
5.2
5.4
4.8
116.6
47
6.0
5.7
5.8
5.6
5.3
5.1
4.8
4.7
118.4
48
5.9
5.7
5.7
5.5
5.3
5.0
4.8
4.6
120.2
49
5.8
5.6
5.6
5.4
5.2
5.0
4.7
4.5
122.0
50
5.7
5.5
5.5
5.3
5.1
4.9
4.7
4.4
Oxygen, Dissolved
Page 1614
Oxygen scavenger tests explained
Oxygen Scavengers
For water
(DEHA) Iron Reduction Method
Introduction
Diethylhydroxylamin (DEHA) is used as an oxygen scavenger to reduce corrosion in boilers. It is
also used in photographic processes and in the manufacture of certain silicon compounds.
Analytical methods to monitor DEHA concentrations have been limited to complex techniques not
suitable for on-site use. Hach has developed a relatively simple test, available in both laboratory
and field test kit formats.
OH
N
H5C2
C2H5
Figure 79 Chemical structure of N,N-Diethylhydroxylamine (DEHA)
Chemical reactions
DEHA reacts quantitatively with Fe3+ (ferric iron) and reduces it to Fe2+ (ferrous iron). The Fe2+
can then be determined by use of FerroZine, a sensitive ferrous iron indicator.
A buffer at an optimum pH of 2.93.0 enhances color development. Best results are obtained if the
sample temperature is 25 C (77 F), and with a reaction time of 10 minutes in the dark. All
chemicals for this procedure are packaged in two reagents. DEHA Reagent 1 Powder Pillows
combine buffer and FerroZine indicator. DEHA Reagent 2 is a liquid formulation containing a
source of Fe3+ for the reaction. An explanation of the reaction between FerroZine and Fe2+ can be
found in the FerroZine Method for Iron explanation.
This method is also applicable to other oxygen scavengers, such as hydroquinone, erythorbic acid
(iso-ascorbic acid) methyl ethyl ketoxime and carbohydrazide, by use of the appropriate
conversion factor.
Oxygen Scavengers
Page 1615
Ozone tests explained
Ozone
Indigo Method1
For water
1
Adapted from: Analytical Aspects of Ozone Treatment of Water and Wastewater; Lewis Publishers: Chelsea, Michigan, 1986; pages 153
156.
Introduction
Ozone (O3), a powerful oxidant, is being increasingly used for water disinfection. It was first used
in the Netherlands in the late 1800s to disinfect drinking water, and is now used worldwide in
drinking water and wastewater facilities, swimming pools, spas, and in the bottled water and
beverage industries. Ozone quickly provides microbial sterilization and disinfection, organic
compound destruction and conversion of iron or manganese salts to insoluble oxides which can be
precipitated or filtered from the water. The major reaction by-products are oxygen, water and
carbon dioxide. For environmental safety, unreacted or residual ozone should be monitored.
Chemical reactions
As ozone reacts quantitatively with indigo trisulfonate (blue indigo dye), the color of the solution
fades. Color intensity, inversely proportional to the amount of ozone present, is then measured at
600 nm with a photometer (colorimeter or spectrophotometer). The reagent is formulated to
prevent interference from any chlorine residual which may be present.
Traditionally, ozone loss during sampling is a major cause of analytical error. Ozone is liberated
when the sample is transferred from container to container, the loss causing erroneously low
determinations. The evacuated AccuVac Ampuls draw the sample directly from the water stream
or source in seconds. Ozone liberated while rushing into the Ampul is trapped there and reacts
immediately with the indigo reagents. The reagent buffers the sample solution to pH 2.5. The
Ampul is then placed directly in a photometer and measurements are taken in the reaction vial,
eliminating cross contamination between samples.
AccuVac Ampuls for analysis cover three ranges: low range (00.25 mg/L), medium range (00.75
mg/L), and high range (01.50 mg/L). The lower ranges are necessary because small amounts of
ozone will bleach the indigo dye only slightly. This slight decrease in color is difficult to detect if the
original blue color is very intense, as it is with the high-range Ampuls.
The low and medium-range tests are designed with less intense color so that a slight bleaching
can be more easily detected, thereby producing results accurate to as little as 0.01 mg/L.
Conceptually the reaction may be described as:
O
KO3S
2O3
SO3K
O2
Ozone
O
KO3S
N
Potassium Indigotrisulfonate
O
H
SO3K
4
N
Potassium Isatin sulfonate
H
SO3K
Figure 80 Chemical reaction for ozone determination, indigo method
Ozone
Page 1616
pH tests explained
pH
Introduction
pH is a measure of the hydrogen ion activity in a solution and is defined as:
log10 a H+ where a H+ is the activity of the hydrogen ion. Practically, this means that at pH 0, the
hydrogen ion concentration is 1 x 1014 times greater than at pH 14. This also means the hydroxyl
ion concentration at pH 14 is 1 x 1014 times greater than at pH 0. When the hydrogen and hydroxyl
ions are present in equal numbers (the neutral point), the pH is 7. Values from pH 0 to pH 7 are
termed Acidic and those from pH 7 to pH 14 are termed Basic. It is important to note that a pH
change of one unit (for instance, from pH 6 to pH 7) actually is a factor-of-10 change (decade
difference) in the hydrogen ion concentration.
The pH electrode used in pH measurement consists of a glass sensing half-cell and a reference
half-cell. Together the two half-cells form an electrode system. The sensing half-cell is a thin pHsensitive glass membrane separating two solutions. The outer solution is the sample to be tested.
The internal solution is enclosed within the glass membrane and has a known pH. An electrical
potential is developed on the inside surface of the glass membrane with the internal solution and
remains constant. Another electrical potential is developed on the outside surface of the glass
membrane with the sample solution. This potential varies with the pH of the sample solution. The
amount of variation is linear when the temperature is constant. The change in potential per pH unit
is termed the Electrode Slope.
A second half-cell, or reference half-cell, in contact with the sample solution has a constant
potential. Therefore, any changes in the potential of the electrode system at a given temperature
will be due to changes in the pH of the sample solution. Sensing and reference half-cells may be
contained in two separate electrodes, or they may be combined and called a combination
electrode; see the Platinum series combination electrode figure.
Temperature effects on pH measurements depend on the reference electrode used, pH of the
solution within the pH electrode and pH of the test solution. At a certain pH, temperature will have
no effect on the potential of the electrode system. This is known as the isopotential point. Also, at
some pH level, the system will exhibit 0 millivolts. This is known as the zero potential point. Both
the isopotential and zero potential points are features designed into electrodes. Hach electrodes
are designed so the isopotential and zero potential points are at pH 7. This minimizes temperature
effects in the majority of typical samples.
Conventional electrode design

The porous junction of a conventional reference half-cell is made of ceramic or fiber. With time, the
junction will become clogged with silver chloride or contaminants, causing large variation in the
reference potential. In addition, reference solution can be contaminated or diluted by back
diffusion of sample into the junction. Particular contaminants may be introduced into the junction.
Clogged or fouled junctions can cause drift along with inaccurate, noisy, erratic and sluggish pH
measurements. The performance of conventional porous junctions deteriorates as they age
because of clogging; see Figure 81.
pH
Page 1617
pH
New
When new,
conventional porous
reference junction
allows electrolyte
solution to flow freely.
Used
Even after just a few days,
conventional reference
junctions can become
clogged, causing slow,
unstable response and
inaccurate results.
Figure 81 Conventional combination electrode
Platinum series electrode design

Platinum Series Electrodes solve this clogging problem because they use a continually renewed
liquid junction, also known as a free diffusion junction (FDJ); see Figure 82. There is no ceramic or
fiber plug to become clogged and therefore the electrode lasts longer. The free diffusion junction
has been shown to have a faster and more stable response than conventional electrodes
(Figure 81). Figure 84 shows the accuracy and stability of the Platinum Series Electrode.
Figure 85 shows the variation of the Platinum Series Electrode and a conventional electrode. The
Platinum Series Electrode clearly provides repeatable results.
Free flowing reference

junction with reference
element
pH Sensing
half-cell
Figure 82 Platinum series combination electrode

Note: The free-flowing reference junction design prevents clogging. Every measurement is rapid, accurate
and stable, regardless of electrode age.
pH
Page 1618
pH
The Platinum Series Electrode provides stable results in one minute in this deionized water
sample. The conventional ceramic frit junction electrode shows a slow, noisy response.
Platinum series electrode

Actual pH
Conventional ceramic frit electrode
10
20
30
Time in minutes
Figure 83 Response times and electrode drift for Platinum Series and conventional electrodes
The initial pH reading obtained with a conventional reference junction for a deionized water
sample was incorrect, and shifted by 0.36 pH units when the sample ionic strength was increased
by adding 50 mg of ultrapure KCl. The same electrode with a Platinum Series reference electrode
showed significantly improved stability and accuracy.
Conventional ceramic frit reference junction
5.0
Platinum series
reference junction
Actual pH
+KCI
6.0
5
10
20
30
Time in minutes
Figure 84 Accuracy of Platinum Series and conventional electrodes
pH
Page 1619
pH
Platinum series pH systems (white) provide repeatable results every time. The conventional
electrode (gray) varies greatly from one measurement to the next.
+0.1
Expected pH
-0.1
Conventional ceramic
frit electrode
Platinum series electrode
Conventional Ceramic Frit

Electrode
Figure 85 Repeatability comparison between Platinum Series and conventional electrodes
pH
Page 1620
pH Indicators explained
pH Indicators
Introduction
Acid-base indicators behave as weak acids or bases in water or solvents. An equilibrium
established between the hydronium ion (H3O)+ and the indicator ion (In ) can be represented as
follows for an aqueous medium:
H2O + HIn H3O+ + In
(In as a weak acid)
(acid color)
(base color)
In + H2O InH+ + OH
(In as a weak base)
(base color)
(acid color)
The ratio of HIn to In or In to InH+ varies as a function of pH. However, the human eye cannot
detect all the color changes as result of the varying concentrations of In in the solution. The color
change between the predominately acid form of the indicator and the predominately base form is
clearly visible, and therefore suitable for visual acid-base titration.
Most of the Hach Acid-Base Indicators can be divided into three classes based on the structure.
The phthalein indicators are sparingly soluble in water but are quite soluble in alcohols. Alcohol is
the preferred solvent for indicator solution preparation. Equilibrium of the phthalein-type indicators
is exemplified by phenolphthalein represented as follows:
HO
OH
+ H2O
O
HO
OH
+ H3O+
OH
O
C
O
O
+ H3O+
C
O
C
O
pH Indicators
Page 1621
pH Indicators
Many of the sulfonphthalein indicators exhibit two useful visual transition ranges. These indicators
exhibit good stability toward strong alkali solution. Indicator solutions are usually prepared in 20%
alcohol solution. The equilibria of this type of indicator are exemplified by cresol red, which is
represented as follows:
Acidic transition range
OH+
HO
H3C
CH3
SO3
H2O
(orange)
HO
H3C
CH3
SO3
H3O+
(yellow)
Basic transition range
H3C
CH3
SO3
+ H3O
pH Indicators
Page 1622
pH Indicators
Most of the azo indicators exhibit a color change from red to yellow. The equilibrium for azo-type
indicators is exemplified by methyl orange, as shown below:
H
SO3
N(CH3)2
SO3
N(CH3)2 + H+
SO3
N(CH3)2
Indicator choice
The visual transition range is the main factor in selection of pH indicator. Indicator solubility also is
important. When using the indicator for an acid-base titration, ease of identifying the color change
and slope of the titration curve must be considered.
Figure 86 illustrates titration of a strong acid with a strong base. Any of the three indicators would
be used with satisfactory results in the appropriate pH ranges. However, as the slope of the curve
decreases, the selection of an indicator with the proper transition range is more important.
In Figure 87, titration of a weak acid with a strong base; only phenolphthalein would be
satisfactory. Use of methyl red would give an incorrect result because the transition range of the
indicator does not correspond with the actual titration equivalence point.
Phenolphthalein range
Bromthymol Blue range

Methyl Red range
Volume Base (mL)
Figure 86 Titration of a strong acid with a strong base
pH Indicators
Page 1623
pH Indicators
Volume Base (mL)
Figure 87 Titration of weak acid with a strong base
pH Indicators
Page 1624
pH Indicators
The chart below shows the ranges of some pH indicators available from Hach Company.
10
11
12
13
14
Crystal Violet (yellow to blue-violet)

(yellow to purple)
Cresol Red (orange to yellow)
(yellow to blue)
Thymol Red (red to yellow)

Erythrosin B (orange to red)
2, 4-Dinitrophenol (colorless to yellow)
Bromphenol Blue (yellow to blue)
Methyl Orange (pink to yellow)
Bromcresol Green (yellow to blue)

Methyl Red (pink to yellow)
Eriochrome* Black T (red to blue)
Bromcresol Purple (yellow to purple)
Alizarin (yellow to red)
(red to purple)
Bromthymol Blue (yellow to blue)

Phenol Red (yellow to red)
m-Nitrophenol (colorless to yellow)
The pH ranges shown are approximate.
Specific transition ranges depend on the indicator
solvent chosen.
* Trademark CIBA GEIGY CORP.
o-Cresolphthalein (colorless to red)

Phenolphthalein (colorless to pink)
Thymolphthalein
(colorless to blue)
Alizarin Yellow GG
(yellow to orange)
pH Indicators
Page 1625
Phenols test explained
Phenols
4-Aminoantipyrine Method
Introduction
Phenols are produced as waste in oil refineries, coke plants, and in some chemical manufacturing
plants. Natural waters normally contain less than 1 g/L, but concentrations up to 20 g/L occur in
some areas. Levels of 10 to 100 g/L phenol are detectable by taste and odor. A 1-g/L phenol
concentration can impart an objectionable taste to water following slight chlorination.
Chemical reaction
Phenols and all substituted phenols (except those with para substitution), are determined by
buffering the sample to a pH of 10.0 and adding 4-aminoantipyrine to produce a yellow or ambercolored complex in the presence of ferricyanide ion. The color is intensified through extraction of
the complex into chloroform. Measurement of this color quantitatively determines the phenol
concentration of the sample.
OH
H3 C
H3C
H3 C
4-Aminoantipyrine
H3C
NH2
Phenol
Figure 88 Chemical reaction for 4-Aminoantipyrine method
Phenols
Page 1626
Phosphonate tests explained
Phosphonates
For water
Ultraviolet Photochemical Oxidation Method
Introduction
Phosphonates are employed as chemical additives to function as threshold antiscalants, corrosion
inhibitors, chelants, sludge conditioners, deflocculants, dispersants and crystal growth modifiers in
various industrial water treatment processes. They are used predominantly as scale and corrosion
preventatives for boiler and cooling tower waters. Phosphonates exist in various formulations as
acids or salts and are marketed in the form of concentrated solutions.
Until recently, analytical methods for phosphonates have been difficult, time consuming and
subject to many interferences. The Ultraviolet (UV) Photochemical Oxidation Method involves a
photochemical oxidation of phosphonate followed by conventional colorimetric determination of
the liberated orthophosphate by the Ascorbic Acid Method. The UV Photochemical Oxidation
Method is rapid, easy to use, relatively free from interferences, and applicable to both field and
laboratory situations.
Chemical reactions
Phosphonic acids are organic compounds of the form R-PO3H2. Structures of two commonly used
treatment chemicals are shown below; the phosphonic acid group is shown in parentheses.
Phosphonates are the corresponding anions formed by ionization of one or more of the acidic
hydrogens.
Hydroxyethylene diphosphonic
acid
Aminotri
(methylene phosphonic acid)
CH2
(PO3H2)
(PO3H2 )
CH2
CH3
(PO3H2 )
OH
(PO3H2)
(PO3H2 )
CH2
Figure 89 Chemical structures of two common phosphonic acids

Decomposition of these compounds by oxidation will liberate the organically bound phosphate as
orthophosphate. The combined action of the UV radiation and oxygen will liberate orthophosphate
rapidly without the necessity of heat or corrosive agents. When the photo-oxidation is carried out in
the absence of acid, no significant degree of depolymerization or hydrolysis of condensed (pyro,
meta or other poly) phosphates occurs, making the method a true test for organic phosphate.
Presence of excess oxygen is ensured by the addition of a small amount of potassium persulfate.
In this oxygen-rich environment UV light will rapidly catalyze the oxidation of the phosphonate C-P
bond.
UV
PO3H2
+ H3PO4
The orthophosphate formed can then be determined colorimetrically using the Ascorbic Acid
method. Reagents for the Ascorbic Acid Method for orthophosphate have been combined into a
single reagent powder, PhosVer3. Determination of orthophosphate using PhosVer 3 is
described in the Phosphorus methods.
Phosphonates
Page 1627
Phosphorous tests explained
Phosphorous
Amino Acid, Ascorbic Acid and
Molybdovanadate Methods
Introduction
Phosphorus occurs in natural water and wastewaters almost solely as phosphates. Phosphates
may enter water from agricultural run-off and as biological and industrial wastes. They may be
added to water in municipal and industrial water treatment processes to control corrosion. A
certain amount of phosphate is essential for most plants and animals, but too much phosphate in
water can contribute to eutrophication, especially when large amounts of nitrogen are also
present.
Phosphorus can be classified as orthophosphate, condensed phosphate or organically bound
phosphate. Condensed phosphates are formed by dehydrating the orthophosphate radical; they
include metaphosphate, pyrophosphate and polyphosphate. The only form of phosphate
determined directly is orthophosphate; other forms require pretreatment for conversion to
orthophosphate for analysis. When no pretreatment is used, phosphate analyses determine
Reactive Phosphorus. Reactive phosphorus is a measure of orthophosphate, plus a small fraction
of condensed phosphate that may have been hydrolyzed during the test.
Hach offers high and low range tests for reactive phosphorus. High range tests can be completed
with the Amino Acid Method or the Molybdovanadate Method. The Molybdovanadate Method uses
a single reagent and has a faster reaction than the Amino Acid Method. Both methods have a
broad range and are free from most interferences. Low range tests use the Ascorbic Acid Method.
Condensed phosphates plus orthophosphate can be determined by acid hydrolysis using sulfuric
acid, followed by the reactive phosphorus test for the appropriate range. A small amount of
organically bound phosphorus will be included in this measurement. The results of the test are
reported as acid-hydrolyzable phosphorus. Total phosphorus (orthophosphate, condensed and
organically bound) can be determined by acid oxidation with persulfate, followed by the reactive
phosphorus test. Organically bound phosphate can then be determined by subtracting the acidhydrolyzable phosphorus.
Chemical reactions
Pretreatment steps
Reactions for pretreatment to determine acid-hydrolyzable and total phosphorus are illustrated
below:
O
R
R'
K2S2O8
H2SO4
OH
H3PO4 + 2K+ + 3SO42
Various organic fragments
Figure 90 Example of potassium persulfate oxidation of organically bound phosphorus1

1
R and R represent various organic groups
Phosphorous
Page 1628
Phosphorous
Amino acid and ascorbic acid methods
Reactive phosphorus is determined in essentially two steps for either the Ascorbic Acid Method
(low range) or the Amino Acid Method (high range). The first step involves reaction of
orthophosphate with molybdate in acid solution, which forms a yellow-colored phosphomolybdate
complex:
12MoO3 + H2PO4 (H2PMo12O40)
The phosphomolybdate complex is then reduced by either an amino acid or ascorbic acid, causing
a characteristic molybdenum blue species. Various structures for the molybdenum blue species
have been suggested in the literature. For example, see Killeffer, D. H., Molybdenum CompoundsTheir Chemistry and Technology, Interscience Publishers, 1952.
All reagents for the Ascorbic Acid Method are contained in PhosVer3 Reagent Powder Pillows.
Reagents for the Amino Acid Method are contained in Amino Acid Reagent Solution and
Molybdate Reagent Solution.
Molybdovanadate method
Reactive phosphorus combines with molybdate in an acid medium to form a phosphomolybdate
complex. Vanadium, contained in Molybdovanadate Reagent, reacts with the complex to form
vanadomolybdophosphoric acid. Intensity of the resulting yellow color is proportional to the
concentration of reactive phosphorus. One possible formula for the complex is suggested below.
The exact structure is not known.
[ PO 4 VO 3 16MoO 3 ]
Phosphorous
Page 1629
Potassium tests explained
Potassium
Tetraphenylborate Method
Introduction
Potassium, one of the most abundant elements, is found in many minerals. Soils contain
approximately 1 to 4% potassium. Concentrations of potassium in most drinking water is usually
less than 20 mg/L; occasionally brines may contain more than 100 mg/L. The greatest areas of
interest in measurement of potassium levels probably are medicine and agriculture, due to the
importance of potassium as a mineral for plants and animals. Potassium salts, particularly potash,
are common in fertilizers.
The Tetraphenylborate Method for determination of potassium in water is accurate, rapid, and
inexpensive. In the reaction, a precipitate is formed and the resulting increase in turbidity is
measured. All necessary reagents are packaged in three powder pillows to provide reagent
stability, convenience and accuracy.
Chemical reactions
Potassium combines with sodium tetraphenylborate to form potassium tetraphenylborate, a white
precipitate. The precipitate remains in suspension in samples with low concentrations of
potassium, causing an increase in turbidity.
NaB(C6H5)4 + K+
KB(C6H5)4
Na+
Figure 91 Chemical reaction between potassium and tetraphenylborate

The sodium tetraphenylborate is contained in Potassium 3 Reagent Powder Pillows. Ammonium
salts, magnesium and calcium interfere with the precipitation. Potassium 1 Reagent Powder
Pillows and Potassium 2 Reagent Powder Pillows prevent these interferences.
Potassium
Page 1630
Selenium tests explained
Selenium
Diaminobenzidine Method
Introduction
Selenium levels in natural waters seldom exceed 0.01 mg/L and concentrations greater than
0.50 mg/L are rare. The appearance of selenium in natural waters may be due to seepage from
soils containing selenium or intrusion of industrial wastes.
Selenium, very toxic to man and animals, exhibits properties similar to those of arsenic. Selenium
is suspected of causing dental caries and of being carcinogenic, but trace amounts also have been
found essential to maintain normal body metabolism. For this reason, a maximum concentration
level in drinking water has been established by the USEPA in accordance with the Safe Drinking
Water Act.
The Diaminobenzidine Extraction Method uses diaminobenzidine indicator, which develops a
yellow color with selenium. This method measures only the tetravalent selenium (Se4+). Selenium
present as Se2+ and Se6+ is not detected unless the sample is first distilled.
Chemical reactions
In the test for selenium, an EDTA masking agent is first added to the sample to remove
interferences such as iron. The addition of formic acid adjusts the sample to an optimum pH range
of 12. Under these conditions, diaminobenzidine reacts with all selenium present in the Se4+ form
to give a yellow-colored piazselenol complex. (Selenium existing in the selenate form is not
determined by this method.) The sample is then buffered to a pH of 1011, where the piazselenol
complex can be extracted into toluene. Measurement of the color intensity directly indicates the
amount of Se4+ in the sample.
H2 N
NH2
H2N
NH2
H2SeO3
Diaminobenzidine
H2N
H2N
Se
N
3H2O
Selenium
Page 1631
Silica tests explained
Silica
Silicomolybdate/Heteropoly Blue Method
Introduction
Silicon is the second most abundant element in nature. Accordingly, it is not surprising that most
waters contain compounds of silicon, usually as silica (SiO2) or silicates (SiO4 and SiO32). Silica
concentration in water is commonly less than 30 mg/L, although concentrations greater than
100 mg/L are not unusual; concentrations exceeding 1000 mg/L are possible in brines and
brackish water.
Silica and silicates are added to water for a number of uses, such as water conditioners,
detergents, and corrosion inhibitors. However, silica in water can cause significant problems for
industries, primarily in boiler and turbine applications. High pressures and high temperatures
cause silica deposits on tubes of boilers and heat exchangers. These glassy deposits lower the
efficiency of heat transfer and lead to premature failure. Silica deposits on steam turbine blades
decrease efficiency and necessitate costly downtime for cleaning. Silica levels must be kept below
0.005 mg/L in very high pressure applications.
Measuring silica in water is useful when efficiency of demineralizers is monitored. Testing for silica
(one of the first impurities detected when the exchange capacity of a demineralizer is exhausted)
provides a sensitive check of demineralizer performance.
Analytical procedures for silica include the Silicomolybdate Method for high range measurement
and the Heteropoly Blue Method for low range measurement. The Heteropoly Blue method is an
extension of the Silicomolybdate method to increase sensitivity.
Chemical reactions
High and low ranges
The Silicomolybdate Method involves the reaction of molybdate ion with silica and phosphate
under acid conditions to form a yellow color. Citric acid is added to destroy the phosphomolybdic
acid complex (the yellow color formed due to phosphate), but not the silicomolybdic acid complex.
For large amounts of silica, the remaining yellow color is intense enough to be read directly. For
low concentrations, an amino-naphthol sulfonic acid reducing agent is used to convert the faint
yellow color to a dark heteropoly blue species. The color formed is directly proportional to the
amount of silica present in the original sample; a colorimetric measurement of this intensity
provides an accurate means of determining the silica concentration. Some forms of silica (usually
polymeric) will not react with ammonium molybdate and must be digested with sodium bicarbonate
to be converted to a reactive form.
Silicic acid reacts with water and hydrates as follows:
H2SiO3 + 3H2O H8SiO6
This hydrated silicic acid reacts with molybdate in the presence of acids to form
silicomolybdic acid.
H8SiO6 + 12(NH4)2MoO4 + 12H2SO4 H8[Si(Mo2O7)6] + 12(NH4)2SO4 + 12H2O
This silicomolybdic acid is then reduced to a blue color (heteropoly species) by an amino naphthol
sulfonic acid for low concentrations.
Silica
Page 1632
Sulfate tests explained
Sulfate
For water, seawater and oil-field water
Introduction
Sulfate occurs in natural waters in a wide range of concentrations. Mine waters and industrial
effluents frequently contain large amounts of sulfate from pyrite oxidation and the use of sulfuric
acid.
Because of sulfates cathartic action, a secondary maximum contaminant level has been
established by the USEPA, in accordance with the Safe Drinking Water Act. The taste threshold of
magnesium sulfate is 400 to 600 mg/L; calcium sulfate is 250 to 800 mg/L. Sulfate may be either
beneficial or detrimental in water used for manufacturing and domestic supply. The presence of
sulfate is advantageous in producing desired flavors in the brewing industry. In domestic water
systems, sulfates do not appear to cause any increased corrosion on brass fittings, but
concentrations above 200 mg/L do increase the amount of lead dissolved from lead pipes.
Sulfate determination is important in oil field applications where two or more waters are mixed.
High concentrations of sulfate, along with barium, calcium, and strontium, can form insoluble
scales.
The procedure for determining sulfate is a modification of the Barium Sulfate Turbidimetric
Method. A single dry powder reagent, SulfaVer4 Sulfate Reagent, will cause a milky precipitate
to form if sulfate is present. The amount of turbidity formed is proportional to the amount of sulfate
present.
Chemical reactions
Sulfate is determined by its quantitative precipitation with barium chloride. Because the finely
divided barium sulfate turbidity formed is proportional to the amount of sulfate in the sample, a
photometric reading enables the sulfate concentration to be accurately determined.
Ba
2+
+ SO 4
BaSO 4
Sulfate
Page 1633
Sulfide tests explained
Sulfide
Methylene Blue Method
Introduction
Sulfide is a poisonous by-product of the anaerobic decomposition of organic matter and commonly
is found in sewage and industrial wastewaters. Sulfide can be present as the free sulfide ion (S2)
or as dissolved hydrogen sulfide (H2S and HS). The toxicity of hydrogen sulfide is equivalent to
that of hydrogen cyanide, but its offensive odor is detectable long before toxic levels are reached.
However, at high concentrations hydrogen sulfide quickly deadens the sense of smell; thus toxic
levels may be present but undetected.
Chemical reactions
The sulfide test is based on the ability of hydrogen sulfide and acid-soluble metallic sulfides to
convert N,N-dimethyl-p-phenylenediamine directly to methylene blue in the presence of a mild
oxidizing agent (potassium dichromate). Intensity of the methylene blue color development is
directly proportional to the amount of sulfide present in the original sample. A colorimetric
measurement of this intensity provides an accurate means to determine the sulfide concentration.
All necessary reagents are contained in Sulfide 1 Reagent and Sulfide 2 Reagent.
NH2
+ 2H2S +
Cr2O72
+ 10H+
(CH3)2N
N
+ 2Cr3+ +
2
(CH3)2N
2NH4+
7H2O
N(CH3)2
Figure 92 Chemical reaction for hydrogen sulfide using the Methylene Blue method
Sulfide
Page 1634
Sulfite tests explained
Sulfite
Titration Method
Introduction
Sulfite is most commonly found in boilers and boiler feedwater, where it is used to inhibit corrosion
by reducing dissolved oxygen. It may also be found in industrial wastes such as paper mill
effluents. Sulfite normally is not present in natural waters because it readily oxidizes to sulfate.
Chemical reactions
The water sample is acidified by the addition of Dissolved Oxygen 3 Reagent Powder Pillow,
starch indicator is then added and the solution is titrated with Potassium Iodide-Iodate Solution.
The acidified solution releases free iodine, which oxidizes sulfite to form sulfate. The iodine is
reduced to colorless iodide:
KIO3 + 5KI + 6HCI 6KCI + 3I2 + 3H2O
SO32 + I2 + H2O SO42 + 2HI
When all sulfite has been converted to sulfate, excess iodine is indicated by a blue color from the
starch-iodine reaction.
Sulfite
Page 1635
Turbidity tests explained
Turbidity
Introduction and definition
Turbidity, a qualitative characteristic which is imparted by solids obstructing the transmittance of
light through a water sample, is an important water quality indicator. Turbidity can be interpreted as
a measure of the relative clarity of water and often indicates the presence of dispersed, suspended
solids; particles not in true solution such as silt, clay, algae and other microorganisms; organic
matter and other minute particles. Turbidity is not a direct measure of suspended particles in water,
but a measure of the scattering effect such particles have on light.
The extent to which suspended solids can be tolerated varies widely, as do the levels at which they
exist. Industrial cooling water, for example, can tolerate relatively high levels of suspended solids
without significant problems. In modern high pressure boilers, however, water must be virtually
free of impurities. Solids in drinking water can support growth of harmful microorganisms and
reduce effectiveness of chlorination, resulting in health hazards. In almost all water supplies, high
levels of suspended matter are unacceptable for aesthetic reasons and can interfere with chemical
and biological tests.
Theory of light scattering

Very simply, the optical property expressed as turbidity is the interaction between light and
suspended particles in water. A directed beam of light remains relatively undisturbed when
transmitted through absolutely pure water, but even the molecules in a pure fluid will scatter light to
a certain degree. Therefore, no solution will have a zero turbidity. In samples containing
suspended solids, the manner in which the sample interferes with light transmittance is related to
the size, shape and composition of the particles in the solution and to the wavelength (color) of the
incident light.
A minute particle interacts with incident light by absorbing the light energy and then, as if a point
light source itself, reradiating the light energy in all directions. This omnidirectional reradiation
constitutes the "scattering" of the incident light. The spatial distribution of scattered light depends
on the ratio of particle size to wavelength of incident light. Particles much smaller than the
wavelength of incident light exhibit a fairly symmetrical scattering distribution with approximately
equal amounts of light scattered both forward and backward (see Figure 93).
As particle sizes increase in relation to wavelength, light scattered from different points of the
sample particle create interference patterns that are additive in the forward direction. This
constructive interference results in forward-scattered light of a higher intensity than light scattered
in other directions (see Figure 94 and Figure 95). In addition, smaller particles scatter shorter
(blue) wavelengths more intensely while having little effect on longer (red) wavelengths.
Conversely, larger particles scatter long wavelengths more readily than they scatter short
wavelengths of light.
Particle shape and refractive index also affect scatter distribution and intensity. Spherical particles
exhibit a larger forward-to-back scatter ratio than coiled or rod-shaped particles. The refractive
index of a particle is a measure of how it redirects light passing through it from another medium
such as the suspending fluid. The particle's refractive index must be different than the refractive
index of the sample fluid in order for scattering to occur. As the difference between the refractive
indices of suspended particle and suspending fluid increases, scattering become more intense.
The color of suspended solids and sample fluid are significant in scattered-light detection. A
colored substance absorbs light energy in certain bands of the visible spectrum, changing the
character of both transmitted light and scattered light and preventing a certain portion of the
scattered light from reaching the detection system.
Light scattering intensifies as particle concentration increases. But as scattered light strikes more
and more particles, multiple scattering occurs and absorption of light increases. When particulate
concentration exceeds a certain point, detectable levels of both scattered and transmitted light
Turbidity
Page 1636
Turbidity
drop rapidly, marking the upper limit of measurable turbidity. Decreasing the path length of light
through the sample reduces the number of particles between the light source and the light detector
and extends the upper limit of turbidity measurement.
Incident beam
Figure 93 Effects of particle size and light wavelength (small particles)1

1
Size: Smaller Than 1/10 the wavelength of light

Description: Scattering symmetric
Incident beam
Figure 94 Effects of particle size and light wavelength (large particles)1

1
Size: Approximately 1/4 the wavelength of light

Description: Scattering concentrated in forward direction
Incident beam
Figure 95 Effects of particle size and light wavelength (larger particles)1

1
Size: Larger Than the wavelength of light

Description: Extreme concentration of scattering in forward direction;
Development of Maxima and Minima of scattering Intensity at wider angles
General instrument description

The instrument optical system typically includes a lamp, lenses and apertures to focus the light, a
90-degree detector to monitor scattered light and optionally, a forward-scatter light detector, a
transmitted-light detector and a back-scatter light detector. The optional detectors may be added to
minimize the impact of color, stray light and lamp and optical variabilities (see Figure 96).
Turbidity
Page 1637
Turbidity
90
Detector
Transmitted
Light
Detector
Lamp
Lens
Sample
Cell
Figure 96 General turbidimeter optical system

Formazin primary standard
The chemically accepted definition of a primary standard is a standard that is prepared by the end
user, on the bench, from traceable raw materials.
Formazin meets that criteria when prepared by accurately weighing and dissolving 5.000 g of
hydrazine sulfate and 50.0 g of hexamethylenetetramine in one liter of distilled water. The solution
develops a white turbidity after standing at 25 C (77 F) for 48 hours and can be prepared
repeatably with an accuracy of 1%. This solution is equal to 4000 NTU. All other turbidity
standards are traced to formazin.
Due to the statistical reproducibility of the nephelometric scatter of white light by the formazin
polymer, instruments with the traditional tungsten filament white light optical designs can be
calibrated with a high degree of accuracy and reproducibility. The randomness of particle shapes
and sizes within formazin standards yield statistically reproducible scatter on all makes and
models of turbidimeters.
Turbidity
Page 1638
Zinc test explained
Zinc
Zincon Method
Introduction
Zinc concentrations in most water supplies average about 1 mg/L, but may range as high as
50 mg/L in some areas. Although zinc is commonly found in many natural waters, the deterioration
of galvanized iron and leaching of brass can add substantial amounts. Industrial effluents may
contribute large amounts of zinc; high concentrations suggest the presence of lead and cadmium,
both common impurities from the galvanizing process.
Zinc is essential to human metabolism and has been found to be necessary for proper growth.
High concentrations of zinc in water act as stomach irritants but the effects are temporary.
Concentrations above 5 mg/L show no harmful physiological effects but can cause a bitter taste
and/or an opalescence in alkaline drinking water.
A dry powder form of 2-carboxy-2hydroxy-5sulfoformazyl benzene indicator, commonly called
zincon, is used in the ZincoVer Method of determining zinc concentrations. This test has been
approved by the Environmental Protection Agency for National Pollutant Discharge Elimination
System-reporting purposes based on comparability studies if the sample is first digested. Testing
done for non-reporting purposes generally does not require sample digestion.
Chemical reaction
In the analysis of zinc, cyanide is added to a buffered water sample of pH 9 to form a complex with
all heavy metals present in the sample. (1) The addition of cyclohexanone then frees the zinc from
the cyanide complex (2) and enables it to react with the indicator, zincon (3) a blue-colored
complex forms in direct proportion to the amount of zinc in the sample. Measurement of the color
intensity determines the zinc concentration.
(2)
Zn(CN)42 + 4
Zn2++ 4
4H2
+ 4OH
C OH
O
C
(3)
OH
Zn2+
+
O3S
O
N
N
C
Zincon (orange)
O3S
O
C
Zn
H
N
+ H2 O
N
C
(Blue)
Zinc
Page 1639
Zinc
Zinc
Page 1640
Technical Support
Page 1641
Contact Hach Company

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Monday through Friday
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By Mail:
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P.O. Box 389
Loveland, Colorado 80539-0389 U.S.A.
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nearest you, send an e-mail to: intl@hach.com or contact:
Hach Company World Headquarters; Loveland, Colorado, U.S.A.
Telephone: (970) 669-3050; Fax: (970) 669-2932
Technical and Customer Service (U.S.A. only)

Hach Technical and Customer Service Department personnel are eager to answer questions
about our products and their use. Specialists in analytical methods, they are happy to put their
talents to work for you.
Call 1-800-227-4224 or e-mail techhelp@hach.com
Page 1642

Water Analysis Handbook - Hach

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Water Analysis Handbook - Hach

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WATER ANALYSIS

Hach Company, 2008, 2012.

Section 2 Laboratory Practices ...................................................................................................... 19

Section 3 Chemical Analysis........................................................................................................... 31

Section 4 Sample Pretreatment by Digestion ............................................................................. 47

Section 5 Water Management and Safety .................................................................................... 55

Section 6 International Guideline Comparison ........................................................................... 63

Procedures for Analysis................................................................................................................... 69

Microbiology .................................................................................................................................. 1241

Electrochemistry ........................................................................................................................... 1431

Chemical Procedures Explained ................................................................................................ 1545

Technical Support ......................................................................................................................... 1641

Pulp & Paper Mills

Power Plant Utilities

Pools & Spas

Metals/Mining, Mfg & Finishing

Pulp & Paper Mills

Power Plant Utilities

Pools & Spas

Metals/Mining, Mfg & Finishing

Pulp & Paper Mills

Pools & Spas

Power Plant Utilities

Metals/Mining, Mfg & Finishing

Abbreviations and Conversions

1.1 Procedure abbreviations

degree(s) Celsius (Centigrade)

litervolume equal to one cubic decimeter

American Chemical Society reagent grade

method detection limit

marked dropping bottle

milligrams per liter (ppm)

micrograms per liter (ppb)

Standard Methods for the Examination of

milliliter1/1000 of a liter. It is approximately

National Interim Primary Drinking Water

National Pollutant Discharge Elimination

poly chlorinated biphenyl

parts per billion

parts per million

Code of Federal Regulations

Estimated detection limit

self-contained dropping bottle

Environmental Protection Agency

free and total

total organic carbon

total petroleum hydrocarbons

United States Environmental

grains per gallon (1 gr/gal = 17.12 mg/L)

ultra low range

Abbreviations and Conversions

Abbreviations and Conversions

Abbreviations and Conversions

Figure 1 Rotate a sample cell

Figure 2 Swirl a cylinder and invert a sample cell

Arsenic Distillation Apparatus Set (Catalog No. 2265400)

Cyanide Distillation Apparatus Set (Catalog No. 2265800)

Figure 3 General purpose distillation apparatus

Figure 4 Vacuum filtration

2.5.2 Required apparatus for vacuum filtration

Filter Holder, membrane, 47-mm

Flask, filtering, 500-mL

Select one of the following:

Pump, vacuum, portable, 115 VAC