Civl407labmanual 20072

CIVL 407 Lab Manual
Prepared for Dept. Of Civil Engineering by Susan C.M. Harper September 2007
Table of Contents
Course Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2 First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3
L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5

Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 2: COD, DO and BOD 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory 3: Bacteriological Examination of Sewage . . . . . . . . . . . . . . . 17 Laboratory 4: Chloride Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Laboratory 5: Chlorine and Chlorine Demand . . . . . . . . . . . . . . . . . . . . . 34 Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity . . . . 39 Laboratory 7a: Coagulation and 7b: Coagulation & Softening . . . . . . . . . 47
Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54
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Course Description
(Environmental Laboratory Analysis - 1-3-0, 0-0-0) Testing procedures used in water quality studies and in the operation of water and wastewater treatment plants. Prerequisite Chem 151. Sc h e d u le Lecture CEME1204: Laboratory CEME1301: Wednesday 1:00 pm 1) Tuesday (time to be arranged by consensus) 2) Wednesday 2:00 to 5:00 pm 3) Thursday (time to be arranged by consensus) 4) Friday (time to be arranged by consensus) (Note: Lab Session must have at least 4 students to run)
Subject No Lab No Lab Laboratory Safety & Orientation (~1 hr) #1 Solids determination #2 Dissolved oxygen, COD, BOD No lab - Thanksgiving week - catch up #3 Bacteriological Examination of waste water #4 Chloride #5 Chlorine demand #6 Acidity, alkalinity, hardness, colour, turbidity No lab - Remembrance day - catch up #7a Coagulation #7b Coagulation and Softening
Date 2007 Sep. 4 - 7 Sep. 11 - 14 Sep. 19 Sep. 25 - 28 Oct 2 - 5 Oct 9 - 12 Oct. 16 - 19 Oct. 23 - 26 Oct. 30 - Nov.2 Nov. 6 - 9 Nov. 13 - 16 Nov. 20- 23 Nov. 27 - 30
Lecturer: Lab instructor: T.A.s: Lab: Marker:
Victor Lo, email: Susan Harper, email: Margaret Parsotan, email: Kazi Parvez Fattah, email:
kvlo@interchange.ubc.ca scharper@civil.ubc.ca barathird@yahoo.co.uk parvez@interchange.ubc.ca
(604) 822-4880 (604) 822-4397
September 2007 CIVL 407 Lab M anual
Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)

The following items will be maintained in each stations supply drawer or at station on bench when required: 2 pair safety glasses 2 permanent marker pens 2 stir bars and a bar retriever 1 stir plate 2 buret funnels 4 30 ml beakers 4 50 ml beakers 2 100 ml beakers 2 150 ml beakers 2 250 ml beakers 2 1 L plastic beakers 2 600 ml plastic beakers 2 400 ml plastic beakers 2 10 ml grad cylinders 2 25 ml grad cylinders 2 50 ml grad cylinders 2 100 ml grad cylinders 1 buret stand 1 double buret clamp 2 50 ml burets 2 25 ml burets 2 10 ml burets 0-14 pH strips Thermometer 2 Stir stick or spatula Filter funnel (Nalgene magnetic) Tweezers (Millipore or Gelman for membrane filters)) On benches each row: Latex gloves- small, medium and large Non-latex gloves (keep on reserve for latex sensitive persons only) Filter racks with funnels #4 Whatman filter paper or equivalent Vacuum flasks and hoses 8 1 L grad cylinders 8 3 L plastic beakers
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First Session: Laboratory Safety and Orientation
1) 2) 3) 4) NO Food or drink. No sandals or open-toe or heel shoes. Personal Protective Equipment: Lab coat (long), eye protection, gloves as required. Sanitation: antibiotic soap for hand washing, disinfectant for bench surfaces. Hygiene: At times you will be working with sewage. Be aware that handling your pens, calculators, packs, etc. with contaminated gloves will contaminate those items. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. Wearing lab coats out of the lab is forbidden. Please take your labcoat away in a plastic bag. Safety Equipment: Eye Wash Station, Safety Shower. WHMIS: Workplace Hazardous Materials Information System is a government regulation. You must be informed of the hazards of a substance before using it. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. Spills: Wipe up all spills immediately, wash down and dry the bench. Please ask for assistance if chemicals are spilled. Broken Glass: Be careful not to break glass, but if you do please ask for assistance. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. If you cut yourself, no matter how minor, please get attendant help. Clean-up: You must leave your work station as you found it - clean and dry. All glassware must be rinsed well (at least 5 times with tap water) and put on paper towels at your station or on drying racks, if there is room. Do not put lids and small items onto racks that may slip through to the filthy drip tray. Remember to empty and rinse burets as well. Pipettes must be placed with tips pointing up into the pipet basket supplied. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6)
7) 8)
9)
10)
Volumetric pipettes are the most accurate way to measure volume. They are available in 0.5, 1.0, 2.0...up to 10.0 ml, 15, 20, 25, 50 and 100 ml. These should be used whenever standard solutions are measured or when upmost accuracy is required. Most pipettes are calibrated to deliver or TD and should never be blown out. They are calibrated by weighing the volume of distilled water that will flow from them by gravity, with the tip against the side of the receiving vessel. A small amount of liquid always remains in the tip and must not be blown out. Some pipettes may be to
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deliver with Blow-Out. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. They are usually identified by a double etched or coloured band at the top of the pipette. Graduated or measuring pipettes are not as accurate as volumetric (bulb type) pipettes and the one having the maximum capacity closest to the volume required is the most accurate one to select. Some are blow-out, some are not meant to drain completely - pay attention to the markings. NEVER mouth pipette. Pipette bulbs or pumps must be used and great care must be taken not to contaminate bulbs. A demonstration will be given. If liquid should enter a bulb or pump, please rinse out immediately and set aside to dry. Do not squish bulbs in the direction of another person. Graduated cylinders: Not as accurate as pipettes, especially at smaller volumes. They are useful for measuring samples 20 mls - 1000 mls. Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. Ie If you want to measure 20 mls, do not use a 1 L graduated cylinder. Cylinders are available: 5 mls, 10 mls, 25 mls, 50 mls, 100 mls, 250 mls, 500 mls, 1, 2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms; markings are not accurate at all - merely approximations. Used to hold/transfer approximate volumes of samples, chemicals or for titrations.. Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). Erle n m e y e r or conical flask markings are not accurate. They are containers best suited for swirling liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6). 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. Undetected gas leaks can be deadly. Even the force of air from the air taps can be dangerous because it can contain grit or water. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. They can be unpredictable in force and are close to eye level. Chemical residues can be splashed in you face. 13) Stir bars: Please remove from containers before pouring contents down the drain. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. Additional supplies are available in the supply room (around the corner). Pipettes are in drawers indicated. Chemicals will be put out as required for each lab and should be used sparingly, not wastefully as quantities are limited. Make sure that you label beakers and flasks to avoid confusion and waste. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS.
L ABORATORY R EPORT W RITE U P G UIDELINES

Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes and summarized here. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate. The mark allocation for some sections for both reporting format is indicated in parenthesis in the table below. Please note that the marking outline is intended only as a guideline to assist in report write-up and that marks would also be allocated for overall presentation and clarity of the report.
R EPORT E LEMENTS Title page Objective Materials and Method
S HORT R EPORT L ABS 1 TO 6 Required Required Details are not required. State that the procedures were in accordance with CIVL 407 Lab Manual and note any deviations to the manuals procedure. Required [1] Required [2] Brief discussion required [2] Required Required [4] Required
Observations and Results Sample Calculations Discussion Including sources of error Conclusions Questions and Answers Other Students Data and Results References Presented in an acceptable format. Total Mark
L ONG R EPORT L AB 7 Required Required Required Include a brief description of the procedure. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported. Required [2] Required [2] Required [5]
Required [2] Required [6] Required [1] Required
[10]
[20]
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Laboratory Exercises
La b o ra to ry 1: So lid s D e te rm in a tio n
WARNING
You will be handling sewage samples which may contain pathogenic bacteria. Use pipette bulbs, wipe up all spills and wash with disinfectant, keep hands and pencils away from your mouth and wash hands frequently. Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. Alternatively a place will be provided in the lab to store your coat.
OBJECT:
-to become familiar with a number of the most common sewage treatment plant tests and to illustrate some of the difficulties in performing these tests; to measure sewage treatment plant efficiency in removing residue. MATERIALS: -Imhoff cones, samples of raw sewage, sludge, final effluent, oven muffle furnace, desiccators, balance, evaporating dishes, tin dishes, graduated cylinders, distilled water bottles.
A. SETTLEABLE SOLIDS 1. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark.. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample, if necessary. 2. Settle for 45 minutes, gently dislodge any solids that have clung to the sides using a stirring rod, settle for 15 minutes longer, and record the volume of settleable solids. If large pockets of liquid form between the particles of settled matter, subtract an estimated volume from the measured volume of matter. Do not include any surface floating material as settled solids.
[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. This is a different test to the one we are doing using Imhoff cones and should not be confused. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. 2. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed, dried, pre-fired and desiccated - done for you, prior to session). Mix the sample in the carboy thoroughly, pour about 500 mls (for Part B & Part C) into a beaker and then, immediately after stirring the beaker, rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. Quickly record the exact volume and pour the sample into the dish.[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish - so be quick.] Using very small amounts of distilled water, rinse the cylinder, adding the rinsings to the dish. Make sure that you have recorded all the pertinent details: Dish #, tare weight, sample source and sample volume. Put the dish in the 103-105 C oven for drying. Do not write on crucibles as high temperatures during the next steps will burn writing off. After drying overnight, the dish will be transferred to a desiccator. Obtain the gross weight of the dish [Note: it should be room temperature before weighing.]. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. The dish containing the dried total solids can now be placed in a muffle furnace or, if instructed, on a cart in preparation for staff to do so when all are ready. The samples will be fired at 550 C for one hour. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator. Once dishes are at room temperature they can be re-weighed. The loss in weight represents the volatile solids lost from the originally measured sample volume. Conversely, the solids remaining represent the fixed solids. Calculate the mg/L volatile and fixed solids.
3.
4. 5. 6.
C. SUSPENDED SOLIDS - TOTAL AND VOLATILE 1. 2. 3. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed, dried and fired). Assemble filtering apparatus, position the filter and begin suction (vacuum tap). Wet filter with a small volume of distilled water to seat it. Using the sample poured out for Part B (for consistency), stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder. [Hint: pour out small portions (say 25 mls at a time for Effl, 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion; when filtering slows significantly do not add more. Record the total volume filtered.] Rinse the graduated cylinder with small amounts of distilled water and add to filter. Suggested portions: 100-200 ml of final effluent, 25-50 mls of raw sewage and 5-10 ml of sludge u s in g g ra d u a te d c y lin d e rs to measure - not beakers. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. Pinch sides of dish in a bit to protect the filter from oven drafts. Place the aluminum dish into the 103C oven to dry for at least one hour (or leave drying
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4.
5. 6.
7. 8.
overnight). Transfer dish to a desiccator, cool and weigh. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. Calculate as the mg/L total suspended solids. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550C for at least 30 minutes (or until a constant weight is achieved). For the time being, place the dishes on a cart designated by the instructor. Lab personnel will perform the timed-firing after the class. After firing the dishes will be moved to a desiccator. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. Please return tomorrow to obtain final weight. Calculate the mg/L total volatile solids and total fixed suspended solids. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample. Enter all data in the tables on following page.
Calculate the percent solids reduction through the treatment plant. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. What major types of solids are removed in primary treatment? in secondary treatment? 2. If sludge is used in Part A, compute the sludge volume index (SVI). Discuss the meaning and significance of this index. 3. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. Your hands may have moisture and natural and/or applied oil/lotion on them. What, if any, affect would there be to the TSS and TVSS results if your bare hands were used to handle a tin dish a) prior to obtaining the initial tare weight and b) after obtaining the initial tare weight? 5. A raw sewage sludge goes through an anaerobic digestion process, where the volatile solids are reduced from 65% to 40%. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1.3 and fixed solids 2.50, what is the percentage reduction in solids. 6. Classify each of the following into its proper solids category(s) i.e. dissolved, suspended, volatile, fixed (use more than one adjective to describe each substance.) a) a bacterium b) sodium chloride c) sugar d) clay
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DATA AND RESULTS Raw Sewage Aerobic Sludge

A. Settleable Matter After 1 hour (mL/L) B. Total Solids Dish marking (do not use ink - use permanent mark on dish) Sample volume (mL) Gross weight - oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight - fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C. Suspended Solids Tin dish marking (do not use ink - use embossed mark) Sample volume (ml) Gross weight - oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight - fired (G) Loss on ignition (G) Volatile suspended solids (mg/L) Fixed suspended solids (mg/L) Dissolved solids (mg/L)
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Final Effluent
La b o ra to ry 2: CO D , D O a n d B O D 5
WARNING You will be using concentrated acid and other corrosive and toxic chemicals, as well as handling samples probably containing pathogenic bacteria. Wipe up all spills immediately, wash/rinse bench tops with water/disinfectant. NEVER pour water into acid. Keep fingers, pencils, etc out of your mouth. Wash hands frequently with soap. Always use and store pipettes with the top higher than the tip, otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely.
OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters; to illustrate the shortcomings of these tests; and to demonstrate the use of dissolved oxygen probes.
I.
COD - Closed Reflux, Colourimetric Method NOTE: Open reflux method is the most accurate means of determining COD because a large sample size is used (20 mls). The closed reflux method is more economical but because a small sample volume (2 mls) is used, homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. Digests from either method can be titrated or read colourimetrically.
Materials:
Raw sewage and final effluent samples, vials with pre-measured reagents, Hach block digester, spectrophotometer, potassium hydrogen phthalate standards: 800, 400, 200, 100 and 50 mg/L as COD, pipettes, etc.
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Procedure: 1. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods, except without mercuric sulfate). Put identifying labels on each tube ie your initials, Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. 2. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present, in which case, use wide mouth graduated pipettes) in duplicate, into the appropriate tubes and screw cap on snugly. If sample is known to be outside the standard range (above 800 mg/L), a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. Make sure sample is well mixed with the acid contents of the vial. [Note: it should look homogenous and be careful it will be very hot!] 3. Take vials to fumehood and place in block digester - noting the location of your well-labelled, well-mixed samples in the 25-position digester. 4. Standards have been digested for you, before the lab, and are at the spectrophotometer. Allow samples to digest for 30 minutes (normally this would be 2 hours). Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary). 5. The instructor will demonstrate the use of the spectrophotometer, which is set at 600 nm to read absorbance of light by the sample. The vials should be clean and dry on the outside. Read your samples and the set of COD standards: 800, 400, 200, 100, 50 and 0 COD as mg O 2 /L. 6. Prepare calibration curve, absorbance versus concentration and calculate the COD in samples. If less than two mls of sample or diluted sample was used, a volumetric correction must be made to the value obtained from the curve. II. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples, dilution water, alkaline-iodide-azide reagent, manganous sulfate, concentrated sulphuric acid, starch indicator, 0.025 N sodium thiosulphate, thermometers, BOD bottles, 10-ml graduated pipettes, burette stand with burettes, 250-ml graduated cylinder, 500-ml Erlenmeyer flasks.
Note: You are going to measure DO on four different liquids using two different methods. The four liquids are cold tap water, aerated dilution water, raw sewage and final effluent. You will use a chemical titration (Winkler - the azide modification of the iodometric method) and a dissolved oxygen probe and meter. The probe has been previously calibrated. DO NOT ADJUST THE SETTINGS ON THE METER.
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A. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. 1. Fill four BOD bottles with the four samples (cold tap water, aerated dilution water, raw sewage or influent and final effluent), right to the top and place stopper on bottle. (Fill carefully, taking care not to entrain additional air. For the cold tap water sample, submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). Determine DO, using the DO probe and meter, for each of the four samples. Instructions on the use of the probe will be given in the lab. Record the temperature of each sample using the DO meter or a thermometer. (Note: DO meter temperature is usually not accurate.) You can save these bottles for B.
2.
B. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. Do not mix droppers and do not allow tap water into the reagent bottles. 1. 2. Fill a BOD bottle with each of the four samples (or use ones saved from A ). Stopper the bottles without trapping any air bubbles. Place the bottles in the sink and one at a time remove the stopper, add 1 (one) ml of manganous sulfate reagent using plastic dropper provided (keeping the dropper tip just below the surface) and quickly replace the lid. To the same bottles, add 1 ml of alkaline-iodide-azide solution the same way, again quickly replacing the lid. Take each stoppered bottle, tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times, as demonstrated, to thoroughly mix contents. Set back in sink. Allow the floc to settle to about 1/3 the bottle volume, invert several times again and allow to settle again. When the floc has settled the second time to about 1/3 bottle volume, remove the stopper, add 1 ml of concentrated H 2 SO 4 and quickly restopper. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. The chemical floc should completely dissolve. (Note: biological solids originally present in the sample will not dissolve.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. Use the specially marked volumetric flask to dispense the 201 ml. Titrate this volume with 0.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains. Note: if solution already seems pale yellow add starch right away. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate, dropwise, until the first complete disappearance of the blue colour. Neglect any reappearance of the blue colour. Record the volume of titre. (Hint: if you measured very low DO level with the meter, then the same sample will require little or no titre and may not turn blue when starch is added - think about it!) Complete all four titrations. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated. Determine the percent saturation for each sample.
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3. 4.
5.
6. 7.
8. 9.
Dissolved Oxygen Data

Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc. (mg/L) DO conc. (Probe) Temp (oC) (thermometer) Saturation concentration Percent saturation
DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD For a quick and easy determination of the percent saturation value for dissolved oxygen at a given temperature, use the saturation chart above. Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. Draw a straight line between the water temperature and the mg/l of dissolved oxygen. The percent saturation is the value where the line intercepts the saturation scale. Note that this nomogram assumes that the water is at sea level The saturation value can also vary slightly depending on barometric pressure with lower values expected when a storm front moves through as compared to bright and sunny "high pressure" days.
Raw Sewage
Final Effluent
Tap Water
Diln Water
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III. BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. Be sure to note this in your lab write-up. Materials: Raw sewage and final effluent samples, dilution water, BOD bottles, 10-ml graduated pipettes, graduated cylinders. Dissolved oxygen meter and probe.
1.
Using the volumes and samples provided by the instructor, carefully measure the waste water samples into BOD bottles. Two dilutions will be made for each sample and each dilution will be in duplicate for a total of eight bottles (plus one bottle for the blank mentioned in Step 4). Be sure to record the bottle numbers and sample volumes on the following table - do not write on the bottles and do not use the numbers on the lids. Carefully top up the BOD bottles with DILUTION WATER, to bring the levels above the ground glass neck of the bottle. Try not to aerate the contents by keeping the filling tube below the surface of the liquid in the bottle or carefully running the water down the side of the bottle. Measure the Initial DO using a calibrated probe and meter. In addition, fill one BOD bottle with DILUTION WATER alone. Because you want to be sure that the dilution water has no BOD, be careful not to contaminate it with sample. ie Rinse the filling tube if it has been in the sample. The blank is used to check that the dilution water has negligible BOD. Measure the Initial DO of the blank using the calibrated probe and meter. There should be negligible BOD in the Dilution water, but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation. If the dilution water BOD is greater than 1 ppm, it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. Stopper the BOD bottles, being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water. All bottles should have liquid in the well around the stopper - if not add a some dilution water (or distilled water) to the well. Place plastic caps over all bottles. These caps are designed to prevent the evaporation of the liquid around the stopper, thereby maintaining a water seal for each bottle. Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues. groups). After five (or six)days, remove bottles and measure the DO, preferably using the same meter and probe used to get IDO reading. Calculate the 5-day (or 6-day if unavoidable) BOD, showing sample calculations. Note: Each group should have 9 BOD bottles in the incubator.
3.
4.
5.
6.
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BIOCHEMICAL OXYGEN DEMAND (BOD) DATA Sample source: ________________________________ Raw Sewage Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Final Effluent Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Dilution water Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L)
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
_____ _____ _____ _____
BOD Calculation When dilution water is not seeded (our water is not seeded for this exercise):
When seeded water is used:
where: D 1
= D2 P B1 B2 f mg/L.
DO of diluted sample immediately after preparation, mg/L = DO of diluted sample after 5 d incubation at 20 oC, = = = = decimal volumetric fraction of sample used DO of seed control before incubation, mg/L DO of seed control after incubation, mg/L, and ratio of seed water in diluted sample to seed in seed control= (%seed in diluted sample) / (%seed in seed control).
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Summary Raw Sewage, Diln. 1 Raw Sewage, Diln. 2 Final Effluent, Diln. 1 Final Effluent, Diln. 2 ________ ________ ________ ________ ________ ________ ________ ________ Average ________
Average ___________
________
Percentage BOD reduction through treatment plant Lab 2 Questions 1)
At what relative times should influent and effluent samples be taken from a sewage treatment plant in order to determine true % removal efficiency? Give two reasons why samples for dissolved oxygen should be fixed in the field, if at all possible. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol.
2)
3)
4)
Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes. Both wastes have equal BOD 5 temperature and volume. One waste has a K value of 0.17, the other 0.25. If an industrial waste has a COD of 450 mg/L, what would you expect its I) BOD 5 and ii) BOD L to be. What interference does the Azide Modification of the Winkler Dissolved Oxygen method overcome and why is it the most common method used for domestic sewage? Why is a blank titrated in the COD test? (Refer to standard methods, open reflux, titrimetric method.) Why is your COD result different from the BOD result?
5.
6)
7)
8)
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La b o ra to ry 3: B a c te rio lo g ic a l Exa m in a tio n o f Se w a g e
WARNING As you are handling and pipetting samples probably containing pathogenic organisms please observe the following rules: Wear gloves, lab coats, eye protection; DO NOT MOUTH PIPETTE; wipe up all spills and disinfect the area of the spill. Keep hands, pencils, etc., away from your mouth. Wipe lab benches with disinfectant before and after each lab. Wash hands with soap before leaving. Do not wear lab coat out of lab - leave it here or take away in a plastic bag to launder.
OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes, lauryl tryptose broth tubes, Brilliant Green Lactose Broth tubes, EC tubes and/or Hach A1 tubes. Demonstration only. Membrane filter method: dilution tubes, sterile pipettes, sterile dilution water, filtering apparatus, membrane filters, LES Endo agar plates, 35 oC incubator. Heterotrophic plate count: dilution tubes, sterile pipets, agar tubes, plates, 35oC incubator, Quebec colony counter. HACH P/A Broth with MUG. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample, not both. Get raw data from another group and include in your report.
F iv e d if f e re n t p ro c e d u re s w ill b e s tu d ie d - b e c a u s e o f lo g is tic s it is n o t p o s s ib le f o r y o u to p e rf o rm th e la b a s p re v io u s ly w ritte n . T h e re f o re , y o u w ill d o th o s e m a rke d b y ? only
17
A. Multiple-tube Fermentation or Most Probable Number: Serial dilutions of sample are incubated in lauryl tryptose broth at 35oC for 48 hours (swirl after 24 hrs). Gas formation indicates the possible presence of coliform organisms. Another series of tubes are inoculated from these positive lauryl tryptose tubes. Brilliant green lactose bile (BGLB) tubes are inoculated and incubated at 35oC for 48 hours (confirmed test for total coliforms) and EC tubes are inoculated and incubated at 44.7oC for 24 hours (confirmed test for faecal coliforms). Bacterial numbers are calculated from the numbers of positive tubes. Hachs Most Probable Number Method 8368, A-1 Medium, is USEPA-accepted for testing non-potable waters. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. Note: these methods will be demonstrated but data must be recorded. ? B. Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC.
? C. Heterotrophic Plate Count - Pour Plate Method: Serial dilutions of samples mixed
with agar and incubated for 48 hrs at 35oC. The number of colonies counted directly. D. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E.coli, using a ready-to-use P/A Broth with MUG. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E.coli. The broth will turn yellow indicating total coliform and fluoresce in the presence of E.COLI in 4 to 24 hours. Demonstration. E. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination. (Single colonies are obtained by streaking LES Endo agar plates with an inoculum from positive BGLB or EC broth tubes and incubating for 24 hours.)
Methods:
? Recording BGLB, EC or A-1 tubes positives:

1.
All tubes have been examined for gas production. Formation of gas in any amount in the inverted vial of the BGLB broth fermentation tube at any time within 48 hr 3 hr constitutes a positive confirmed phase. Calculate the MPN value from the number of positives using the Table 1. Express as Total coliforms. Results will be provided for the demonstration set. Consider positive EC broths incubated at the elevated temperature (44.5 oC) as a positive completed test response for both Total and faecal coliform. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. Use Table 2 on page 27.
2.
3.
Completed Phase LES Endo agar Plates: A p la te w ill b e a v a ila b le to lo o k a t. 1. Using aseptic technique, as demonstrated, streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. Streak plates in a manner to insure presence of some discrete colonies.
18
2.
Flame loop between second and third quadrants to improve colony isolation. Incubate plates (inverted) at 35 oC for 24 2 hr. Prepare Gram Stain slides for examination under a microscope..
?B. Membrane Filter Technique -Total Coliform 1. Dilution tubes. If you havent already, prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. 2. Filtering. The filtration apparatus has been sterilized by autoclaving. Mount apparatus on suction flask with valve sideways to block suction. Using sterile forceps, place a sterile membrane filter (grid side up) onto the filtration apparatus. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. Pour the full volume of dilution tube # 6 onto the filter, open the valve slowly and allow all the liquid to be pulled through. Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. 3. When filtering is complete and the valve is closed, remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter). Place the filter on a small LES Endo agar plate carefully, from one edge, with a motion that excludes air as it comes in contact with the agar. Do not push down on the filter except on the outside edge and use only sterile forceps - applying gentle pressure. 4. Repeat for the rest of the dilution tubes to # 2. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. Be sure dishes are labelled with your name and dilutions. 5. Counting. After 20-22 hr, choose appropriate dilutions for counting. Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. Atypical coliform colonies can be dark red or nucleated without sheen. Colonies that lack sheen may be pink, red, white, or colourless and are considered to be non-coliforms. The total count of colonies (coliform and noncoliform) on Endo-type medium has no consistent relationship to the total number of bacteria present in the original sample. A high count of non-coliform colonies may interfere with the maximum development of coliforms. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. Verification is usually done by a test for lactose fermentation. Gram staining technique can be also be used. In general, coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). -Note: typical colonies count as coliforms in this procedure, therefore the results table should specify #coliform colonies/100 ml. ?C. Heterotrophic Plate Count - Pour Plate Method 1. Dilution tubes. Prepare one set of six dilution tubes as described in Section A.1. 2. Inoculating. Using a sterile pipette, transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish, removing the cover just enough to insert pipette tip and dispense sample, then immediately replace the cover.
19
3. 4.
5. 6.
Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). Take 5 tubes of melted agar from the incubator. Let them cool down from the 60C that they are kept at but not enough to solidify. Test temperature on your wrist - too hot for a baby - too hot for a bacterium. Slide the cover of a Petrie dish back just far enough to pour the agar, pour and replace cover. Gently swirl the dish, on the bench, to mix the sample with the agar. Repeat for the rest of the dilutions. Work quickly or the agar will solidify in the tube before you get a chance to pour it. Allow the agar to solidify in the dish before inverting, marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. Counting. After 48 hr incubation, select the appropriate dilution to count. Ideally, use the plate(s) that will give from 30 to 300 colonies/plate. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. A hand counter is also available. Compute bacterial density and report as colony-forming units (CFU) per ml. Do not report as too numerous to count if all plates exceed 300 colonies. Instead, if fewer than 10/cm 2, count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. Preferably select seven consecutive squares horizontally and six consecutive squares vertically. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). If more than 10/cm 2, count four representative squares, average and multiply by 57 (for plastic Petrie dish). If necessary, refer to Standard Methods for more instruction.
D. Presence/Absence Testing - d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. Add 100 mls of sample directly to pre-measured medium provided. 2. Incubate at 35 oC for 24-48 hours. ?3. Examine for yellow colour (total coliforms) and fluorescence (E.coli). ?4. Record results of previously prepared test. E. Microscope Slides: 1. Slide preparation: With a flame sterilized inoculating loop, place 1 loopful of sterile dilution water in the centre of a slide. Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Do this for a typical and an atypical colony. Label the slide using a grease pencil (T or A). 2. Slide staining. a. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. b. Flood slide with crystal violet solution and allow to act for 30 seconds. c. Wash off stain with water and then with iodine solution (Lugols). d. Allow iodine to act for 30 seconds. e. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. Flood the slide with fresh alcohol-acetone solution to act for 30 seconds. f. Wash slide in tap water.
20
g. h.
Apply fuchsin or safranin (counter)stain for 30 seconds. Wash in water, blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts).
?3. Microscopic examination. a. Examine the prepared slides - do not turn knobs except the positioning ones. b. In general, coliform are Gram-negative (pink), nonsporing, usually motile, short, plump rods, sometimes coccus-like and 0.5 - 3 m in size. Cells occur singly, in pairs, or in short chains. Note: in the staining process: before decolourisation all organisms appear Gram positive (blue), after decolourisation Gram negative organisms are no longer visible. After application of the counterstain, Gram negative organisms are visualized (pink). c. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny, dull, metallic, rough, smooth, concave), transparency, colour, odour; and on the slide: typical or atypical, gram positive or negative, cocci or rods, length to width ratio, cell groupings (single, pairs chains).
Schedule Summary Day 1. - Record positives in all Multiple Tube Fermentation inoculations: LTB, BGLB and EC and AI tubes - counts will be provided on the board. - Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. - Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. - Membrane filter prepared dilutions, place on LES Endo agar dishes and incubate. - Record Presence/absence testing results of Hach Prepared Media bottles. - Examine prepared slides. Day 2. - Come in and count colonies on LES Endo Membrane Filter dishes. Day 3. - Come in and count Heterotrophic Plate colonies after 48 hours. Plates ready on a weekend will be put in cold room until Monday.
21
Bacteriological Examination Data

Multiple Tube Fermentation : indicate the number of positives for each dilution Tube # Mls of sample/diln tube LTB 24 hr (+ or -) BGLB 48 hr (+ or -) EC 24 hr (+ or -) AI 24 hr (+ or -) Infl Effl Infl Effl Infl Effl Infl Effl Infl Effl
From MPN Index table find MPN Index per 100 ml. Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1, 1/ 10 and 1/100 are used..
Heterotrophic Plate Count - pour plate method, 35 oC/48 hr Tube # 1 2 3 Mls of inoculum Infl Effl Plate count Infl Effl *CFU/ml Infl Effl *CFU= colony-forming units Membrane Filter Technique Tube # Dilution (mls) Infl Effl Coliform Colonies Infl Effl #Colonies/100 mls Infl Effl 1 2 3
22
Discussion: -sources of error? -compare MPN, MF results. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count. Do they agree? Why/why not? What is expected? What is a heterotroph? -were the reductions through the sewage treatment plant as expected? Additional notes Presence/Absence (P/A) Testing: Media chosen may be Presence/Absence broth (P/A) or Lauryl Tryptose broth with Bromcresol Purple broth (LT/BCP) to indicate acid formation. Both media meet USEPA guidelines for testing of total coliforms in drinking water. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation. If zero total coliform is the maximum contamination goal, it is not necessary to enumerate. With MUG reagent added to P/A Broth, you can simultaneously detect total coliforms and E.coli. A long-wave UV light is required to detect fluorescence. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E.coli because it produces a fluorogenic product when hydrolyzed by glucuronidase, an enzyme specific to E.coli. E.coli will fluoresce as early as 4-24 hours. Estimation of Bacterial Density - Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions, use the results from only three of these in computing the MPN. To select the three dilutions to be used in determining the MPN index, choose the highest dilution that gives positive results in all five portions tested (no lower dilution giving any negative results) and the two next succeeding higher dilutions. Use the results at these three volumes in computing the MPN index. In the examples given below, the significant dilution results are in boldface. The number in the numerator represents positive tubes; that in the denominator, the total tubes planted; the combination of positives simply represents the total number of positive tubes per dilution: Example a b c 1 ml 5/5 5/5 0/5 0.1 ml 5/5 4/5 1/5 0.01 ml 2/5 2/5 0/5 0.001 ml 0/5 0/5 0/5 Combination of positives 5-2-0 5-4-2 0-1-0 MPN Index /100 ml 5000 2200 20
23
Table 1 - MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL, 1.0 mL, 0.1 mL)
Comb. Of Positiv es
0-0-0 0-0-1 0-1-0 0-2-0 1-0-0 1-0-1 1-1-0 1-1-1 1-2-0 2-0-0 2-0-1 2-1-1 2-2-0 2-3-0 3-0-0 3-0-1 3-1-0 3-1-1 3-2-0 3-2-1 4-0-0 4-0-1 4-1-0 4-1-1 4-1-2
MPN Index/ 100 ml
95% Confidence Limits
Lower
1 1 1 1 1 1 2 2 1 2 3 3 5 3 4 4 6 6 7 5 7 7 9 12
Upper
Comb. Of Positives
MPN Index/ 100 ml 22 26 27 33 34 23 30 40 30 50 60 50 70 90 80 110 140 170 130 170 220 280 350 240 300 500 900 1600
95% Confidence Limits Upper Lower
<2 2 2 4 2 4 4 6 6 4 7 9 9 12 8 11 11 14 14 17 13 17 17 21 26
1 10 13 11 15 15 18 18 17 20 24 25 29 24 29 29 35 35 40 38 45 46 55 63
4-2-0 4-2-1 4-3-0 4-3-1 4-4-0 5-0-0 5-0-1 5-0-2 5-1-0 5-1-1 5-1-2 5-2-0 5-2-1 5-2-2 5-3-0 5-3-1 5-3-2 5-3-3 5-4-0 5-4-1 5-4-2 5-4-3 5-4-4 5-5-0 5-5-1 5-5-2 5-5-3 5-5-4 5-5-5
9 12 12 15 16 9 10 20 10 20 30 20 30 40 30 40 60 80 50 70 100 120 160 100 100 200 300 600 -
56 65 67 77 80 86 110 140 120 150 180 170 210 250 250 300 360 410 390 480 580 690 820 940 1300 2000 2900 5300 ----
$1600
24
Hach - MPN Table 2
25
Further information can be found in the laboratory library (do not remove without permission): Standard Methods: -Part 9000 Microbiological Examination
HACH publications:-Catalogue listing testing methods with descriptions. -The Use of Indicator Organisms to Assess Public Water Safety - http://www.hach.com/ Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D.D. Mara: -Bacterial Indicators/ Health Hazards Associated with Water
-Bacteriology for Sanitary Engineers
Microbiology 321: -Manual of Microbiological Techniques Website: http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.htm Lab 5 Questions: 1) Compare the advantages and disadvantages of the multiple tube fermentation and membrane filter techniques. What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis.
2) 3)
26
La b o ra to ry 4: Ch lo rid e D e te rm in a tio n
WARNING
You will be using concentrated acid and other very dangerous chemicals. Mercuric thiocyanate is very toxic. Use personal protective equipment: gloves, goggles or glasses and lab coats. If chemicals are spilled alert instructors, immediately.
OBJECT: -to acquaint you with the use of specific ion electrodes, known addition and calibration techniques and colourimetric and potentiometric titrations; to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. MATERIALS: -millivolt meter, chloride specific ion electrode, reference electrode (double junction), magnetic stirrer, beakers, buffer, standards, semi-log graph paper; spectrophotometer, 125 ml Erlenmeyer flasks, graduated cylinders; pH meter with double-junction electrode and silver billet electrode, burettes, conc. HNO 3, standard chloride solution (0.5 mg/L), standard AgNO 3 solution, volumetric pipettes; standard Hg(NO 3)2, indicator-acidifier reagent, indicator, NaHCO 3, beakers; sample.
Note and consider carefully: Different methods of analysis have different optimum ranges. You will be told the approximate concentration in the sample and it is up to you to make sure that the sample will fit within the specified ranges. That means you will probably have to dilute the sample by the most accurate means available. Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section.
27
I. CHLORIDE - SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. Standards of 1000, 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. Unless already done by a previous group, pour out 100 mls of each standard into separate, labelled beakers. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). Unless already done by a previous group (values written on the board), place the lowest standard on the stirrer and lower the electrodes into it. Turn on stirrer and while stirring record the millivolt (mv) reading when the reading appears to be stable. As probe tends to drift for some time it may be expedient to select a standard time to read (i.e. 2 minutes) and use it for all standards and samples. Repeat for rest of standards, rinsing and blotting dry the electrodes between each. Measure 100 mls of sample, added 2 mls of ISA, stir and obtain a reading for it. Using semi-log paper or software equivalent, plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve. Determine the concentration of chloride in the sample. Method of Standard Addition :Add 5 mls of the stock (4 mg/ml) chloride solution (V s) to the sample measured in Step 3 (E 1) and take a new reading after value is stable (E 2 ). Calculate the chloride concentration, where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 - E 1 volume of sample volume of stock solution added mg Cl- added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2).
2.
3. 4.
5.
6.
mg Cl- in sample =
mg/L Cl- =
____________________
28
II. CHLORIDE - BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl- + Hg(SCN)2 HgCl2 + 2SCN SCN - + Fe+++ Fe(SCN) ++ (Reddish brown) 1. Using a 25 ml graduated cylinder, measure 25 mls of zero standard or blank (halide-free distilled water), the three standards (2.0, 4.0 and 8.0 ppm), and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total). Marking the time (T=0), add 5 ml of the combined reagent (containing ferric alum and mercuric thiocyanate solutions) to the blank first, using the dispenser provided. (Use extreme caution as this is a corrosive & toxic chemical.) Mix thoroughly by gently swirling the flask. Wait two or three minutes and then add the reagent to the first (lowest) standard; after a few more minutes, add the reagent to the second standard and so on for the rest of the standards and sample (which may need to have been diluted). [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required - they cant all be at 10 minutes at once.] After 10 minutes has passed (since the combined reagent was added to the blank), use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ), discard and refill at least twice to rinse cuvette. Insert cuvette into the spectrophotometer (wavelength must be set at 463 nm) and press zero or ref set (depending on model) to make the meter read zero. (Alternatively, zero instrument on distilled water and obtain an absorbance value for the blank, which then must be subtracted from the standards and sample.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample, fill, insert in the spectrometer and record the absorbance displayed on the meter. (No further adjustment is required after the blank or distilled water has been set to zero.) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards. Determine the chloride concentration in the sample from the calibration curve. If sample is above the highest standard, it should be rerun from the start after diluting.
2.
3.
4.
5.
6.
III. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl- ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. After all Cl- ions have been bound, the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point). Hg ++ + 2Cl- HgCl2 1. 2. and Hg ++ (excess) + DPC violet colour
3.
Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker.. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. The colour of the solution should be green-blue at this point. (Light green indicates a pH of less than 2.0; pure blue, a pH of greater than 3.8. For most samples, the 1 ml of the indicator-acidifier reagent will adjust the pH to 2.5 0.1. Titrate with 0.014N Hg(NO 3) 2 to a definite purple end-point. Near the end-point, the solution should be green-blue and will turn to pure blue (no green) within a few
29
4.
drops of the purple end-point. Determine the blank by titrating 100 ml of distilled water containing 10 mg of NaHCO 3 which serves to provide some alkalinity to the blank (to make it similar to the sample) and provides a correction for alkalinity and reagents - if one is necessary. Dont forget to add the indicator. The titre from this step must be subtracted from the titre for the sample. Calculate the chloride concentration:
5.
where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. The first group will do the standard (part one) in duplicate or triplicate depending on the size of the group and the other will do the sample (part two) in duplicate (or triplicate). This way each station will have data for each part. A reference set of data will be provided for comparison. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. Part one: Standard 1. Place 10.0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker, dilute to about 100 ml with distilled water, and add 2.0 ml of concentrated HNO 3 (use plastic dropper). Nitric acid is in the sink and is very corrosive.. 2. Immerse stir bar, place beaker on stir plate and lower electrodes into the solution. Take care that stirrer is not hitting the electrodes. 3. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. 4. Begin titrating, in increments, with standard AgNO 3, recording the volume and millivolt reading for each increment. At the start, large increments (1-2 mls) may be added; then, as the end-point of the reaction is approached ( mV/ml increases), smaller increments (0.1 to 0.2 ml or drop-wise) should be added, until past the endpoint when larger increments can be used again ( mV/ml decreases). Continue the titration about 5 ml past the end-point. The end-point occurs at the greatest mV change per unit addition of AgNO 3. 5. Plot a differential titration curve to determine the exact end-point.. 6. Calculate the Normality of the AgNO 3 using the following equation:
30
Part Two: Sample 1. 2. Measure 100 ml of sample or portion made up to 100 ml, if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). Add 2 ml of HNO 3 and proceed as described in Part One. (If pH of the sample were quite high it would be necessary to first adjust the pH to about 4 and then add 2 ml HNO 3. It is not, in this case.) Plot three curves for this titration: a. Millivolt reading versus ml of titrant. b. Millivolts per ml versus ml of titrant. c. Millivolts per ml per ml versus ml of titrant. In plotting b and c, which are merely first and second derivatives, be careful to use the average value of ml of titrant on the abscissa. Calculate the chloride concentration in the sample using the following equation:
3.
4.
Where:
A = ml AgNO 3 B = ml Blank N = normality of titrant (AgNO 3) D = ml of sample
ml From graph a b c
mg/L Cl-
S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q
31
Chloride - Potentiometric Titration

RAW DATA A Volume AgNO 3 Added (Vol) (ml) B Meter Reading (E) (mV) C FIRST DERIVATIVE D SECOND DERIVATIVE F G H
(Vol)
(ml)
(E)
(mV)
E ave (AgNO 3 ) Volume (ml)
(E)/ (Vol)
(mV/ml)
( E/ Vol)
(mV/ml)
[Ave(Vol)]
(ml)
I Ave of Ave(Vol) (ml)
( E/ Vol)
/ml (mV/ml/ml )
Extra pages are at the back of this lab manual in appendix.
32
INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide, fluoride and bromide titrate as chloride. # Other interferences: ferricyanide, chromate, dichromate, ferric ion. # Pretreatment of environmental samples usually required. MERCURIC NITRATE TITRATION METHOD # Iodide, fluoride and bromide titrate as chloride. # Chromate, ferric, and sulfite ions interfere when concentration greater than 10 mg/L. # Colour may obscure or interfere with end-point determination. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. # Environmental samples usually require complex pre-treatment. COLOURIMETRIC METHOD # Bromide, iodide, fluoride, cyanide, thiosulphate, hydrazine, and nitrite interfere. # Colour in the sample may interfere in the absorbance measurement. # Environmental samples usually require pretreatment. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane, causing electrode malfunction. Also, strongly reducing solutions may form a surface layer of silver. # Mercury must be absent from samples. # In practice, specific ion electrodes are rarely usable in environmental samples. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. In the above tests, known addition cannot correct for any ions that test as chloride ion. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. Each sample type requires development of an appropriate method which, in itself, requires knowledge of the compounds in the sample which are likely to interfere. This is a complex, timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. Lab 3 Questions 1. In the Mohr method for chlorides (described in Sawyer and McCarty) what would the equilibrium concentration of silver ions be (in mg/L) when the chloride concentration has been reduced to 0.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration. From your chloride results, can you see any advantage in suing the first or second derivative curve? Discuss.
2. 3.
33
La b o ra to ry 5: Ch lo rin e a n d Ch lo rin e D e m a n d
WARNING Glacial acetic acid is very corrosive. Bleach/chlorine solutions are strong oxidants. Personal protective equipment required at all times when working in the lab are: eye protection, gloves and lab coats. When weighing chemicals at the balance, clean up all spills with a brush into a container and wash crystals down the drain. Wipe up all liquid spills immediately.
OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals; to illustrate the concepts of free and combined chlorine residuals, chlorine demand and break-point chlorination. Materials: -600 ml beakers, 250 ml Erlenmeyer flasks, 250 ml volumetric flasks, pipettes, burettes and stands, 100 ml graduated cylinder, bleach or hypochlorite solution, 0.025 N sodium thiosulphate, chlorine free water, (concentrated) glacial acetic acid, potassium iodide (KI) crystals, starch indicator, phosphate buffer solution, N,N-diethyl-p-phenylenediamine (DPD) indicator solution, standard ferrous ammonium sulphate (FAS) solution.
REFERENCES: Standard Methods 19th ed. 1995. pages 4-56 to 4-57 or older/newer editions. I. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method - total chlorine residual) 1. Prepare a stock solution of strong chlorine water by adding 5 mls of bleach solution (sodium hypochlorite) to about 200 mls of water in a 250 ml volumetric flask. Make up to the 250 ml and invert flask several times to mix. Rinse a burette and fill it with this stock chlorine solution.. 2. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide, about 50 ml of chlorine-free water (distilled water will do), about 5 ml of glacial acetic acid (use a graduated cylinder), and exactly 10 ml of 0.025 N sodium thiosulphate solution (use a volumetric pipette). 3. Mix well (on stir plate with stir bar), add 1 ml of starch solution, allow to mix again. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant
34
purplish-blue colour. 4. From the amount of chlorine solution used, calculate the chlorine concentration in the stock solution (See Data and Results section for formula). Work in groups of three or four for this part, as there is lots to do - COORDINATE! IIa.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. Set up a numbered row of six 600 ml beakers each containing 500 mls of the sample of water provided (it has been prepared using Ammonium chloride and Glutamic acid). 2. Allow at least 5 minutes of time to elapse between applications of chlorine doses (use the buret containing diluted chlorine solution from Part I above) to successive beakers in order to allow enough time for titrating the free and combined chlorine residuals, refilling burette, etc. Sequentially add the necessary volumes of chlorine stock solution to each beaker at the appropriate time spacing to provide the applied dosages given by the instructor (see board). 3. After 15 minutes of contact time, and again after 1 hour of contact time determine the free and combined chlorine (see below) in 100 ml samples taken from each of the beakers at the appropriate time spacing (15 minutes and 60 minutes after the addition of the chlorine dose to each beaker). IIb. Determination of Free and Combined Chlorine by the DPD Method 1. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. These 6 flasks can be prepared at once to make them ready for the next step. 2. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. (*NOTE: If total chlorine exceeds 5 mg/L use a smaller sample and dilute to a total volume of 100 mls with chlorine-free water. Mix usual volumes of buffer reagent and DPD indicator solution with distilled water b e fo re adding sufficient sample to bring total volume to 100 ml or the test will not work. It is likely that the highest dose will require dilution.). a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. Record the burette reading (mls) = Reading A. b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. Continue titrating until red colour (if any) is discharged once again (Reading B). Go to c. c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). For dichloramine concentrations greater than 1 mg/L, let stand 2 min more if colour drifts back indicating incomplete reaction. d) Alternatively: (Not to be done in this lab.) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading, add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample, mix to dissolve, let stand for two minutes and titrate until the red colour (if present) is discharged. Let stand 2 minutes more; if colour drifts back (indicating an incomplete reaction), titrate to colourless again. Record the burette reading (Reading C). Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C - A Monochloramine= B-A Dichloramine= C-B For interferences, please refer to Standard Methods, 18th edition, Method 4500-Cl F. pp 4-43.
35
DATA AND RESULTS I. Standardization of chlorine stock solution: Volume of sodium thiosulphate used Normality of sodium thiosulphate used Volume of stock chlorine solution Cl concentration of stock solution =
= = =
______ mls (A) ______ (N) ______ mls (B) = _________mg/L Cl as Cl2*
*To determine bleach concentration as percent you must multiply first by your dilution (i.e. 250) then convert mg/L to g/100 mls = %. Guaranteed analysis is normally printed on the container and is usually 5-6%. II. FAS titrant concentration is 1 ml = 100 g Cl as Cl2 a. Determine the concentration of total, free and combined residual chlorine for each applied chlorine dose. b. Make a plot of the three residual chlorine components against the applied chlorine dosage. Make a separate graph for the 15 minute and 1 hour contact time. c. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. d. How would you expect the forms of residual chlorine and their speed of formation to change as a function of 1) temperature and 2) pH?
Lab 5 Questions
1.
Why is it important to determine chlorine residuals in domestic water supplies?
2. Given: HOCl W H + + OClK eqv = 2.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3. Under what conditions is the practice of break point chlorination necessary? 4. a. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. If this is true, what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0.5 mg/L if 50% are killed in 1 minutes at this concentration? b. If a sewage sample contains 10 x 10 6 coliforms/100 ml, how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes?
36
15 MINUTE CONTACT TIME BEAKER NUMBER

Vol. of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl dosage (mg/L) Volume of FAS to endpoint A Volume of FAS to endpoint B (includes A) Volume of FAS to endpoint C (includes A & B) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) Time of Cl addition Time of Residual measurement
37
60 MINUTE CONTACT TIME

BEAKER NUMBER Vol. of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol. of FAS to endpoint A (mls) Vol. of FAS to endpoint B (mls) (includes A) Vol. of FAS to endpoint C (mls) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) Time of Cl addition Time of residual measurement 1 2 3 4 5 6
(includes A+B)
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Lab o rato ry 6: Alkalin ity , Ac id ity , p H, Hard n e s s , Co lo u r, T u rb id ity
WARNING Please use caution when handling all chemicals, particularly those labelled with specific hazards. Use care when using lab equipment. Probes, metres and calibrated glassware are available in limited quantities and are very expensive to replace. Always wear personal protective equipment.
OBJECT: -to familiarize you with the most common water treatment tests; to acquaint you with the use of pH metres, pH test paper and other water quality assessment equipment. MATERIALS: -pH meter, pH test paper, magnetic stirrer, bromphenol blue, phenolphthalein, bromcresol green, metacresol purple, standard acid, standard base, burettes and stand, graduated cylinders, beakers, flasks, colour comparator, turbidimeters, EDTA, buffer solution, hardness indicators, 1 N NaOH, water samples. I. DETERMINATION OF pH 1. Check the pH of a small aliquot of the sample by wetting a pH test paper and comparing the colour combinations to those shown on the case. (Take care not to contaminate paper in case.) 2. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. Be sure that buffers are being stirred when you perform the calibration and that when you change buffers you thoroughly rinse the probe into a waste beaker and blot dry so that you do not contaminate other buffers or your samples. Normally, the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7, then use buffers 4 and 7 to calibrate; if above 7, then use buffers 7 and 10. 3. After calibration is complete, rinse & dry probe, place a beaker containing the sample and stir-bar on the stir-plate, lower probe, turn on stirrer and obtain a stable reading.(Do not allow the stir bar to hit the probe.) Record value in following table. 4. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage.
39
II. DETERMINATION OF ALKALINITY 1. Rinse and fill a labelled burette with standard acid solution. 2. Measure 100 ml of water sample into a clean, sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. In order to remove any free residual chlorine (which interferes with the indicator colour response), add 1 drop of 0.1 N sodium thiosulphate to the sample. Add a stir-bar. 3. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red, titrate until just colourless. Record burette reading (P). (If solution is clear after addition of P...think what that means!) 4. Add a dropper full of bromcresol green (BG= MO) to this same water sample and continue the titration until the colour changes from blue to yellow (at pH 4.5). 5. Record the burette reading in the following table. (BG or MO) 6. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. Apply dilution factor to obtain final result. III. DETERMINATION OF ACIDITY 1. Rinse and fill another burette with standard base solution and label it. 2. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask. Add 1 drop of sodium thiosulphate solution. 3. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity). (If the solution is already blue after indicator....?) 4. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein. Titrate to the first uniform pink colour (colour stays). Record the volume of titrant required in the following table. IV. TOTAL HARDNESS DETERMINATION - EDTA TITRIMETRIC METHOD 1. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. 2. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. Pour into a 250 ml Erlenmeyer flask. Add stir-bar. 3. Add 1-2 ml of buffer solution using dropper bottle (3 full droppers) and one aliquot of Total Hardness Indicator using the special dispenser on the chemical bottle. The buffer elevates the pH to 10. A pH much above 10 promotes CaCO 3 precipitate. 4. Titrate slowly with EDTA, while stirring, until the reddish tinge disappears from the solution and a pure blue remains. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. White paper placed under the flask will facilitate this determination. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. If a ppt does form within the 5 minutes, it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water. Alternatively, if the approximate hardness is known (by unsuccessful attempts), add 90% of the titrant to the sample before adjusting the pH with buffer.
40
5. Record the final burette reading in the following table. V. CALCIUM HARDNESS DETERMINATION - EDTA TITRIMETRIC METHOD 1. Refill the burette with EDTA titrant and note the initial reading. 2. Measure out 50 ml of sample into an Erlenmeyer flask and add 2.0 ml of 1 N NaOH solution (use 3 droppers full), or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). Add a stir-bar and stir to mix. 3. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly, with continuous stirring to a pure blue end-point. 4. Record the final burette reading in the following table. VI. COLOUR - TRUE AND APPARENT PART A: TRUE COLOUR - HACH Spectrophotometer. - Enter the stored program number for true colour=120 1. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. 3. Place the cell containing water into the spectrophotometer and press Zero. 4. Place the cell containing sample in it and press Read. The instrument may suggest diluting if over range.. Values are reported as APHA Colour Units (eqv. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. Use the technique outlined in Part A, except on an unfiltered sample, to determine the apparent colour (measures the influence of turbidity on the reading). The reading might be higher so if you had to dilute in Part A dilute for Part B. 2. Record all colour data in the following table. VII. TURBIDITY - BY HACH 2100P Turbidity Meter. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1. Fill a sample cell to the line (about 15 mL), taking care to handle the sample cell by the top. Cap the cell.. 2. Wipe the cell with a soft, lint-free cloth to remove water spots and fingerprints. 3. Press: I/O. The instrument will turn on but will turn off automatically after a short time so you should be ready. 4. Insert the sample cell in the instrument cell compartment so the diamond or orientation mark aligns with the raised orientation mark in front of the cell compartment. Close the lid. Select automatic range by pressing the RANGE key. The display will show AUTO RNG when the instrument is in automatic range.. 5. Select signal averaging mode by pressing the SIGNAL AVERAGE key. The display will show SIG AVG when the instrument is using signal averaging. Use signal average mode if
41
the sample causes a noisy signal (display changes constantly). 6. Press: READ The display will show - - - - NTU, then the turbidity in NTU. Record the turbidity after the lamp symbol turns off.. PART B: Hellige Turbidimeter - Demonstration only 1. Carefully fill the 20 mm viewing depth tube to the mark with sample. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). Note that the rectangular door mirror is closed, that the filter selector shows "none" and that bulb B is in use. Close the door and switch on the light. 2. Immediately balance the light intensity of the central spot with the surrounding observation field by turning the dial on the right-hand side of the instrument. Take the reading where the dark central part first merges with the surrounding field. If you take too long, the turbidity may start to settle and condense on the bottom of the tube. 3. Read the scale on the dial, record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2. PART C: Jackson Candle Turbidity - demonstration only..
1. Be sure that the calibrated tubes are clean before adding samples to the tubes. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT - adding liquid to a hot tube will cause it to shatter. Try to be quick and not burn the candles more than a few minutes at a time. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes, obscuring vision. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. Remove about 10 mls of sample by pipette and slowly dispense this aliquot back into the tube until the shape of the flame is no longer distinguishable. (This allows you to make a more gradual and accurate approach to the end-point.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. Note that there are several different scales on the side of the tube. Record value in following table as J.T.U.s.
2.
3.
Result of Titration
Hydroxide Alkalinity as CaCO 3
Carbonate Alkalinity as CaCO 3
Bicarbonate Concentration as CaCO 3 T T-2P 0 0 0
P=0 0 0 P<1/2T 0 2P P=1/2T 0 2P P>1/2T 2P-T 2(T-P) P=T T 0 Where: P = Phenolphthalein alkalinity; T = Total alkalinity. See Standard Methods, 16th edition, page 272-273.
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DATA Sample 1 pH - Initial pH by test paper Initial pH by pH meter ALKALINITY- Sample volume "M.O." Alkalinity end-point (mls) Initial burette reading Net P. Alkalinity titre (mls) Net "M.O." Alkalinity titre (mls) ACIDITY- Sample volume Initial burette reading P. Acidity end-point reading (mls) Initial burette reading Net "M.O." titre (mls) Net Total acidity titre (mls) Net CO 2 Acidity titre (mls) Final burette reading (mls) Initial burette reading (mls) Net titre (mls) Total Hardness as mg/L CaCO 3 CALCIUM Sample volume (mls) Final burette reading (mls) Initial burette reading (mls) Net titre (mls) Calcium as mg Ca ++ /L __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ Sample 2 ___________ ___________
P. Alkalinity end-point reading (mls) __________
"M.O." Acidity end-point reading (mls)__________
TOTAL Hardness Sample volume (mls)__________
Calcium hardness as mg CaCO 3/L __________
43
CALCULATIONS Calcium:
Calcium
Hardness:
where:
A= B=
ml titrant for sample mg CaCO 3 equivalent to 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.0
Total Hardness (EDTA):
where:
A= B=
ml titrant for sample mg CaCO 3 equivalent to 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.0
Alkalinity: where: A= N= ml standard acid used normality of standard acid
Acidity: where:A = N=
ml standard base used normality of standard base
44
RESULTS (sample) P. Alkalinity as CaCO 3 (mg/L) "M.O." Alkalinity as CaCO 3 (mg/L) OH - Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3- Alkalinity as CaCO 3 (mg/L) OH - Alkalinity as OH - ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3- Alkalinity as HCO 3 - ion (mg/L) "M.O." Acidity as CaCO 3 (mg/L) Total Acidity as CaCO 3 (mg/L) Free carbon dioxide as CO 2 (mg/L) Total Acidity as CO 2 (mg/L) Ca Hardness as mg Ca +2 (mg/L) Ca Hardness as CaCO 3 (mg/L) Mg Hardness as CaCO 3 (mg/L) Mg Hardness as Mg+2 (mg/L) Carbonate Hardness as CaCO 3 (mg/L) Non-carbonate Hardness as CaCO 3 (mg/L) Excess alkalinity as CaCO 3 (mg/L) Colour - APHA Colour Units: True Apparent Turbidity - Hach - Units= N.T.U. ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________
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Lab 6 Questions 1. Is this water suitable for a domestic water supply? Why? 2. What errors can occur in the calcium hardness determination? Why?
3. What sanitary significance does colour have? 4. A water has the following analysis: Na + K+ Ca +2 15 mg/L 33 mg/L 12 mg/L ClSO 4-2 HCO 3 70 mg/L Mg+2 18 mg/L 42 mg/L 8 mg/L
What is the carbonate, non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why?
6. From equations (17-12) and (17-13) in Sawyer and McCarty, calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab, given K 2 = 4.7 x 10 -11 and K w = 10 -14. Does this agree with the results from Part II of this lab? Explain.
Note: You should bring a copy of this lab to the next two sessions as you will need the same instructions to perform the same tests in the Coagulation and Softening Labs.
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Lab o rato ry 7a: Co ag u latio n an d 7b : Co ag u latio n & So fte n in g
ATTENTION
This lab will require patience and cooperation from all of you as we have only two jar testers with 6 paddle sites each. Please try to coordinate your work with the rest of the class. You should have with you the part of Exercise 7 that gives instructions for the various tests.
OBJECT: -to illustrate the principles of coagulation and water softening. This is a two-step experiment, to be conducted over two lab periods. The first step will be to analyse the raw water sample and the supernate from a number of alum dosages (to be announced in the class) using the techniques learned in Lab. No.6. The optimum alum dosage for coagulation of the sample will then be chosen.for the next session (b). Several lime and soda ash dosages will also be selected for determining the efficiency of water softening. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. MATERIALS: -Jar Test Apparatus, 1000 ml beakers, alum solution (Al2 (SO 4 )3 @18H 2 O), soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH), and all of the materials from Lab. No. 7 (except Jackson Candle and Hellige Turbidimeter).
47
Part A:. COAGULATION USING ALUM - work in 2 or 3 teams to assess a total of 6 dosages for each team. 1. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure, not a beaker ) and transfer to 1000 ml beakers. 2. Place beakers on the jar tester apparatus. You may need to use blocks to position the paddle 0.5 to 1 cm off the bottom of the beaker. Make sure it is centred. 3. Add the assigned alum dosages as quickly as possible and then start the stirrer. Stir at 100 rpm for 1 minute, then reduce stirring rate to about 20 rpm for 10 minutes (just fast enough to keep the floc suspended but not so fast that the floc is sheared). Observe and make notes describing floc formation and note the time of appearance. 4. Stop stirring. Lift paddles out of beakers and secure. Record relative floc sizes (pin-point, small, medium, large); clarity of supernatant liquid (very clear, clear, hazy, cloudy); and settling characteristics of the floc (rapid, moderate, slow). 5. After the flocs have settled (allow 15 min), decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled). 6. Analyze these supernates plus a sample of raw water for pH, alkalinity, acidity, hardness (total and calcium), true colour and turbidity (Hach). A pH meter could be used for the alkalinity and acidity determinations, if available, but because of equipment limitations, use indicators as specified in Lab 6. 7. Complete the coagulation summary sheet during the lab period, if time permits, and fill in the table on the board. In the lecture, you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week. You will also determine the optimum lime and soda ash dosages to soften this water and select a range of dosages surrounding this optimum for investigation next week. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. 1. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. Add the appropriate alum dosage and immediately start stirring. Stir at 100 rpm for 1 minute. Reduce the stirring rate to about 20 rpm for 10 minutes. Stop stirrer and allow floc to settle. 2. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers, position them under stirrers. Discard the floc and clean beakers in preparation for a second decanting in Step 4. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. (NB dosage now based on 800 ml sample size). Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. Make relative estimates of floc sizes, times of appearance and descriptions and record. 3. Stop stirrer. Record observations on settling rate, floc volume, and clarity of supernatant liquid. 4. After the floc has settled (allow 15 min), decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc.. 5. Analyze the supernates for pH, alkalinity, acidity, hardness (total and calcium), turbidity and colour. 6. Determine which dosages were most appropriate for this sample. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample, drop of 0.1 N sodium thiosulphate
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@ titrate with 0.02 N acid with phenolphthalein to colourless - pH 8.3 and with bromcresol green (continuing with the same sample) to yellow - pH 4.5. @ calculation - alkalinity (mg/L CaCO 3)= mls titre x N acid x 50,000/mls sample Acidity: @ 100 ml of sample, drop of 0.1 N sodium thiosulphate @ titrate with 0.02 N base with bromphenol blue to blue - pH 3.7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink - pH 8.3 ("phenolphthalein" or total acidity) @ calculation - acidity (mg/L CaCO 3)= mls titre x N base x 50,000/mls of sample Total Hardness: @ 25 ml sample diluted to 50 ml @ add 1 ml buffer and Total Hardness Indicator (Calmagite) @ titrate quickly with EDTA to blue end-point (no pink/purple tinge - pure blue). @ calculation: hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Calcium Hardness: @ 50 ml sample @ add 1 ml 1 N NaOH (or to pH 13) and Ca Hardness Indicator (Hydroxy Naphthol Blue) @ titrate quickly with EDTA to blue end-point (no pink/purple tinge - pure blue). @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A. Coagulation 1. Analyze the response of colour and turbidity removal as a function of alum dose. 2. How does the consumption of alkalinity by alum compare with theoretical calculation based on balanced equations? How much extra soda ash should you add to compensate for the alkalinity consumption by 70 mg/L alum? B. Softening 1. Based on your chemical analysis of the raw water, show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening. 2. Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations. 3. Compare removal of hardness components under the different doses - use graphical presentation. 4. What treatment would you recommend to prepare this water for human consumption? Why?
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Coagulation Raw Data Sheet

DATA Raw Sample Alum mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
pH Alkalinity-sample vol ml Net titre to pH 8.5 P Net titre to pH 4.5 TOT Acidity - sample vol ml Net titre to pH 3.7 MO Net titre to pH 8.5 TOT Total Hardness sample vol Net titre Ca Hardness - sample vol Net titre
Notes:
50 September 2007 CIVL 407 Lab M anual
Coagulation Summary Sheet

DATA
Raw Sample Alum mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
pH Floc size Clarity Floc settling rate Alkalinity (mg/L CaCO 3 ) P Alkalinity (mg/L CaCO 3 ) TOT Acidity (mg/L CaCO 3 ) MO Acidity (mg/L CaCO 3 ) TOT Total Hardness (mg/L CaCO 3 ) Ca Hardness (mg/L CaCO 3 ) Apparent Colour (A.P.H.A.) True Colour (A.P.H.A.) (filtered) Turbidity (N.T.U.) unfiltered
Notes:
Coagulation and Softening Raw Data

D ATA
Lime/NaOH mg/L Soda Ash mg/L Alum mg/L
pH Alkalinity-sample vol ml Net titre to pH 8.5 Net (Total) titre pH 4.5 Acidity - sample vol ml Net titre to pH 3.7 Net (Total) titre pH 8.5 Total Hardness sample vol Net titre Ca Hardness - sample vol Net titre
Notes:
Coagulation and Softening Summary Sheet

Data
Lime/NaOH mg/L Soda Ash mg/L Alum mg/L
pH P- Alkalinity -mg/L CaCO 3 *Total Alkalinity-mg/L CaCO 3 MO Acidity -mg/L CaCO 3 *Total Acidity -mg/L CaCO 3 Total Hardness -mg/L CaCO 3 Ca Hardness -mg/L CaCO 3 Mg Hardness -mg/L CaCO 3 **Carbonate Hard. -mg/L CaCO 3
**Non-Carb Hard -mg/L CaCO 3
**Excess Alk. -mg/L CaCO 3 Apparent Colour (A.P.H.A.) True Colour (A.P.H.A.) (Filtered) Turbidity (N.T.U.)
Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include MO Acidity. Calculation
** By
4
Appendices see: CIVL407Manual_CourseNotes.pdf
1. Instructions for Laboratory Report Writing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2. Reading and Reference Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4. Normal Solutions and Equivalent Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5. Standard Solutions, Titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 6. Typical Problems for Acid-Base and General Chemistry . . . . . . . . . . . . . . . . . . . . . 68 7. Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 8. Solids Removal in Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 9. DO, BOD, COD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 10. Balancing an Oxidation Reduction Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 11. Hach Absorption Method - COD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 12. Chloride Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 13. Known Addition Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 14. Bacteriology and Water-related diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 15. Faecal Coliforms - Levels and Water Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 16. Bacteriological Examination - Additional Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 17. Chlorine - Chlorine Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 18. Equilibrium Relationships of Chlorine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 19. Acidity, Alkalinity, pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 20. Concepts and Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 21. Alkalinity Species as a Function of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 22. Colour, Turbidity, Hardness, Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 23. Hardness Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 24. Milliequivalent and mg/l as CaCO 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 25. Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 26. Chemical Requirements for Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 27. Bar Diagram Method for Water Softening Problems . . . . . . . . . . . . . . . . . . . . . . 112 28. Periodic table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 29. Extra tables for Chlorine Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
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CIVL 407 Lab Manual

L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5

Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54

Lecturer: Lab instructor: T.A.s: Lab: Marker:

kvlo@interchange.ubc.ca scharper@civil.ubc.ca barathird@yahoo.co.uk parvez@interchange.ubc.ca

(604) 822-4880 (604) 822-4397

September 2007 CIVL 407 Lab M anual

Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

L ABORATORY R EPORT W RITE U P G UIDELINES

R EPORT E LEMENTS Title page Objective Materials and Method

Required [2] Required [6] Required [1] Required

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

DATA AND RESULTS Raw Sewage Aerobic Sludge

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

Dissolved Oxygen Data

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

_____ _____ _____ _____

When seeded water is used:

September 2007 CIVL 407 Lab M anual

Percentage BOD reduction through treatment plant Lab 2 Questions 1)

September 2007 CIVL 407 Lab M anual

La b o ra to ry 3: B a c te rio lo g ic a l Exa m in a tio n o f Se w a g e

F iv e d if f e re n t p ro c e d u re s w ill b e s tu d ie d - b e c a u s e o f lo g is tic s it is n o t p o s s ib le f o r y o u to p e rf o rm th e la b a s p re v io u s ly w ritte n . T h e re f o re , y o u w ill d o th o s e m a rke d b y ? only

September 2007 CIVL 407 Lab M anual

? Recording BGLB, EC or A-1 tubes positives:

September 2007 CIVL 407 Lab M anual

Bacteriological Examination Data

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

MPN Index/ 100 ml

95% Confidence Limits

95% Confidence Limits Upper Lower

9 12 12 15 16 9 10 20 10 20 30 20 30 40 30 40 60 80 50 70 100 120 160 100 100 200 300 600 -

September 2007 CIVL 407 Lab M anual

Hach - MPN Table 2

September 2007 CIVL 407 Lab M anual

-Bacteriology for Sanitary Engineers

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

September 2007 CIVL 407 Lab M anual

A = ml AgNO 3 B = ml Blank N = normality of titrant (AgNO 3) D = ml of sample

S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q

September 2007 CIVL 407 Lab M anual

Chloride - Potentiometric Titration

E ave (AgNO 3 ) Volume (ml)

I Ave of Ave(Vol) (ml)

Extra pages are at the back of this lab manual in appendix.

September 2007 CIVL 407 Lab M anual

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