ANALYTICAL CHEMISTRY II

QUIM 3065L
THEORY - PART I

SPRING 2017
Prepared by: Dr. Marisol Vera Coln
 Analytical Method Classification

Classic Methods Instrumental Methods

Gravimetric (mass) Electroanalytical

Mass of the Analyte is Spectroscopic
measured
Separation (Chromatographic)
Volumetric (titrations)
Mass, Volume, or
Others
concentration of the analyte is
studied relavite to a standard
 EXPERIMENTS PART I

Experiment Format
Gas Chromatography Theoretical Exercise Form
GC-TCD: Analysis of Solvents in an Unknown
Full
Industrial Lot
Simultaneous Determination of a Two Component
Short
Mixture by UV-VIS Spectroscopy
Electrogravimetric Determination of Cu in CuO Accuracy
Spectrophotometric Analysis of Iron in Mohrs Salt
Full
and an Iron Supplement

Method Types: Separation, Spectrophotometric, Electroanalytical

GAS CHROMATOGRAPHY
 GAS
CHROMATOGRAPHY
Two exercises involve gas
chromatography:
Gas Chromatography Theoretical Exercise
GC-TCD: Analysis of Solvents in an Unknown
Industrial Lot
Quantitative Analysis
Instrument: Agilent Gas Chromatography
 ANALYTICAL SEPARATIONS

Chromatography: separation is based on the

interaction between a solute, a stationary phase
and a mobile phase.
The two most common types of chromatography
(based on type of mobile phase) are:
Gas chromatography (mobile phase is gas)
Liquid chromatography (mobile phase is liquid)
HOW IT OCCURS?
CHROMATOGRAPHIC METHODS BASED
ON ANALYTE-STATIONARY PHASE
INTERACTIONS

Affinity Chromatography
Sample is applied under conditions that
favor specific binding of the target
molecule(s) to a complementary
binding substance.
 GAS CHROMATOGRAPHY
The gaseous analyte is transported through the
column by means of a mobile phase, a gas in this
case (carrier gas)
The sample (gas or volatile liquid) is injected
through a rubber septum into the hot injection
port
Most common carrier gases are: He and N2
The detector is maintained at a temperature
higher than the least volatile analyte
SCHEMATIC DIAGRAM OF A GAS
CHROMATOGRAPH (GC)

Agilent GC uses a capillary

column
 TYPES OF COLUMNS IN GC:

Packed
Consists of a glass or
stainless steel tube
packed with a solid
support coated with a
thin layer of the
stationary phase
 TYPES OF COLUMNS IN GC:

Capillary Columns
Capillary Columns (also
known as Open tubular (OT))
Column made of fused silica
(SiO2) :
typical diameters: 2mm
i.d.
typical lengths: 15-100m.
 TYPES OF COLUMNS IN GC:

Packed (Left)
Capillary (Right)
 COMMON
STATIONARY
PHASES IN GC
Principle of like dissolves
like to select wall coated
liquid phases.
Ej. Non polar columns are
better for non polar solutes.
Polar for polar solutes..
Bonded Phases
chemically modified solid
supports: more stable to high
temperatures.
STATIONARY PHASE MATERIAL USED
IN EXPERIMENT:

ZB-WAX (equivalent to Carbowax 20M):

polyethylene glycol strongly polar
with working Temps from 40 250C.
MOST COMMONLY USED GC
DETECTORS:
(Thermal Conductivity Detector TCD)

(Flame Ionization Detector FID)

(Electron Capture Detector ECD)

(Mass Spectrometry-MS)
GC/MS (hyphenated technique)
TCD THERMAL CONDUCTIVITY
DETECTOR
-Most general -responds to changes in the thermal
conductivity of the carrier gas (e.g. He) in the presence of
solute
-Least sensitive of all GC detectors - useful only with
packed columns, not for capillary columns (OT)
SCHEMATIC REPRESENTATION OF A
TCD
 DESIRABLE PROPERTIES OF A GC
DETECTOR

Inexpensive
Simple and easy to operate (user friendly)
Responds linearly over a wide range of concentrations
Uniform response to all analytes or highly predictable and selective
toward one or more type of analyte
Good signal stability and reproducibility
Adequate sensitivity (10-7-10-15 g analyte/s)
A temperature range from room temperature to at least 400 C
A short response time that is independent of the flow rate
Does not destroy the sample
Interpretation of Chromatographic
Data
Qualitative Quantitative

Dead Time (tm) Peak Height

Retention time (tr) Peak Area

 CHROMATOGRAM: GRAPH OF THE DETECTOR
RESPONSE AS A FUNCTION OF ELUTION TIME.
 INTERPRETING A
CHROMATOGRAM

Quantitative: Area (A)

Qualitative: tr
~ (0.5*W)*H
 SUMMARY OF
CHROMATOGRAPHIC
EQUATIONS

Factor de capacidad:
Tiempo que pasa un soluto en
fase estacionaria / tiempo que
pasa en fase mvil.
 Capacity Factor

Tr

Capacity Factor (k): Describe the migration rate of the analyte in the column.
Separations will be performed under conditions where k lies between 1-5.
 Selectivity Factor

Selectivity Factor (): The partition ratio between the more retained and the more rapidly eluted

species. By definition it must be greater than unity. It is fairly independent from flow rate.
 RESOLUTION

If the resolution is greater than 1.50, total separation is assumed.

Peak resolution also depends on N or H, and k.
 Resolution Enhancement
The main objective in the improvement of column efficiency is to provide the
highest resolution in the minimum amount of time.

There are some experimental conditions that can be varied in order to enhance
peak resolution, these conditions are:
Varying column length
Changing the stationary phase
Varying the mobile phase flow rate
Changing the column temperature (more effective in GC)
Preparing a derivative of the analyte
Incorporating into the stationary phase species that selectively
interacts with one or more components of the sample
The efficiency of a column
is defined in terms of:

N: Number of theoretical plates

N(dimensionless)
H: Height Equivalent per Theoretical Plate (H)
(typical units: mm)

H=L/N
where L is the length of the column
 Number of Theoretical Plates
Number of Theoretical Plates (N): The
number of regions where the partition
process occurs.

N = 16*(tr / W)2 (Base

Line)

N = 5.55*(tr / W1/2)2 (Half

Peak)

where:

N = number of theoretical plates

 Under non-ideal conditions the eluted bands are asymmetric

Peak Asymmetry as: Asymmetric peaks indicate poor column inertness.

Because asymmetry is most severe near the baseline, we determine the
value at a point 10% of the peak height above the baseline. A high quality
capillary column provides As values between 0.9 and 1.1 for appropriate
 The best chromatographic separation is the
one that results in chromatograms with narrow
and well separated bands.

Why bands broaden?

Why bands broaden?
A . Multiple Paths
B. Longitudinal
Diffusion

C. Mass transfer rate

- Linear Velocity
 VAN DEEMETER
RELATIONSHIP
Relates how the flow rate of the mobile phase
affects the efficiency of the column (defines in
terms of H) B
H A+ + Cux
ux
Where A, B, and C are constants for a given column and
stationary phase

ux=linear velocity of the mobile phase

A=term referring to multiple paths-Eddy diffusion

B=Longitudinal diffusion. Term that is proportional to Dm

(diffusion coefficient of mobile phase)

C=Finite equilibration time between phases. Term that is

proportional to the column radius and inversely
proportional to Dm)
 Finding optimum flow rate
Plotting HETP vs. flow rate, or linear velocity of the
mobile phase, usually shows the efficiency of the
column

Plot of HETP vs. flow rate. The optimum flow rate

 Effect of temperature on chromatogram

Isothermal Analysis Constant Temperature

Programmed Temperature temperature varies during analysis

Isothermal Analysis Constant Temperature

(Ej. 150 oC) Programmed Temperature temperature varies
during analysis (50 250 oC at 8 oC/min)
EXPERIMENT: GAS CHROMATOGRAPHY (GC)-
THEORETICAL EXERCISE

Handout (in lab manual )

Answer questions, do calculations and hand in final results at the end
of lab period.

NOTE:
Bring to laboratory a calculator & ruler.
You will be expected to complete exercise in lab
Bring copy of exercise and appendices (chromatography)
experiment:
lysis of Solvents in an Unknown Industrial Lot
Purpose: Introduce the student to the use of a gas
chromatograph (GC) for quantitative analysis. Illustrates
the use of the standard addition method to determine the
amount of hexane and methanol in an Unknown (Industrial Lot
sample), using acetone as internal standard.

Experimental: Known amounts (spikes) of hexane (HEX)

and methanol (MetOH) will be added to the unknown
sample solutions, which are injected in the GC. The peak
areas for HEX, MetOH and the internal standard
(Acetone) will be determined by electronic integration. 1-
propanol will be used as solvent.
 Experimental conditions
Column: ZB-WAX capillary Agilent GC 6890N
column (polar); length: 30m
Gas: Nitrogen
Hexane 50% v/v
Methanol 50% v/v
Acetone 50% (internal
standard)
1-propanol (solvent)
Unknown
10mL volumetric flasks
100 L syringes
Operating conditions:
Oven temperature: 80C
isothermal
Injector temperature: 100C
Detector temperature: 150C
Flow rate: 10mL/min
Method: Q3065L.M
Sequence: Q3065L.S
Column 1; Front detector
(TCD)
Sample preparation
 STANDARD ADDITIONS METHOD:
The composition of standard solutions should closely
approximate that of the unknown. All of the
components making up a sample, except for the
analyte, are called the matrix.

The method of standard additions is an analytical

procedure in which known quantities of analyte are
added to the unknown; the accompanying increase in
the signal allows the operator to determine how much
analyte is present in the original solution.

The unknown solution itself is used to make the

standard solutions by adding known amounts of the
standard to known volumes of the unknown.
STANDARD ADDITIONS METHOD GRAPHIC REPRESENTATION
 STANDARD ADDITIONS CURVE
Small variations in the sample
injection volume can be
corrected by normalizing the
peak areas to that of an internal
standard.

A calibration curve is plotted

where the y-axis is the ratio of
the area under the peak for the
standard to that of the peak for
the internal standard and the x-
axis is the concentration of
added standard.
-b
Given Y=mX+b; set Y=0 and X=
solve for X m
 MANUAL INJECTION VS.
AUTOSAMPLER
Programmed
Injection with GC
Autosampler

Manual injection with 1

L syringe
 INTRODUCTION TO
ULTRAVIOLET-VISIBLE
SPECTROSCOPY
 SPECTROSCOPY
The use of the absorption, emission, or scattering of
electromagnetic radiation by matter to qualitatively or
quantitatively study the matter or to study physical processes.
The Electromagnetic
Spectrum
 Properties of
Electromagnetic Radiation
Made up of packets of energy called photons
Ranges between the high frequency gamma
to the low frequency radio waves
Largely invisible to the human eye
Humans can only see radiation in the range of
750-450 nm
Relation between energy and
frequency

E = h
h = 6.62x10 J .s -34

Remember Energy increase if:

Frequency (Hz) increases (Units: Hz)
Wavelength (nm) decreases (nm)
Wavenumber increases (cm-1)
 Types of interaction between light
and matter
Absorption: A transition from a lower level to a higher level with
transfer of energy from the radiation field to an absorber, atom,
molecule, or solid.

Emission: A transition from a higher level to a lower level with

transfer of energy from the emitter to the radiation field. If no
radiation is emitted, the transition from higher to lower energy levels
is called nonradiative decay.

Scattering: Redirection of light due to its interaction with matter.

Scattering might or might not occur with a transfer of energy, i.e.,
the scattered radiation might or might not have a slightly different
wavelength compared to the light incident on the sample.
Types of interaction between light and
matter
Absorption of Electromagnetic
Radiation (EMR)
Occurs when the energy of the radiation emitted by
the light source matches the energy required to
promote a transition of an electron from its ground
state to an excited state.
 Electronic Transition
Example of some of the possible electronic transitions in
the ultraviolet-visible region

n*

n*
Energy

What kind of information we get from the
interaction of matter and EMR?

Quantitative Information:
Using Beers Law (A=bC)
UV-Vis Spectroscopy (molecular spectroscopy)
Atomic Absorption Spectroscopy

Qualitative- identification of compounds

Infrared Spectroscopy
 Definitions
Chromophore is a functional group thats promotes the absorption of
UV-VIS radiation at higher wavelengths
Conjugated groups

Auxochrome- A functional group that does not absorb radiation by itself

but induces the absorption of UV-VIS radiation
Common Chromophores
 Definition
Beers Law:
A=bc
Where is the molar
absortivity coefficient

It describes the ability of the P

compound to absorb energy.

Higher , the better

chromophore is the compound. A=-log T=- log (P/P0)
 Parameters that affect
Absorbance

Note that the Law is not

A = bc tells us that absorbance
obeyed at high
depends on the total quantity of
concentrations, and linearity
the absorbing compound in the
is lost.
light path through the cuvette
 Relation between Concentration
and Absorbance

A=(b)C+0
If the relation is linear
between A and C. Y=(m)X+b
m = *b
If b= 1cm, m=
 Function of Main components in a
UV-VIS spectrophotometer

Source- provide the energy to promote an electronic transition

Deuterium and Tungsten lamps

Wavelength selector to select the desired wavelength

eg. Monochromator- Have a grating

Sample holder:
Cuvette: made of quartz (for UV-VIs), glass or plastic (for Vis
only)

Detector- detects signal (converts photons to electrical signal)

photomultiplier tube (PMT)
 BASIC COMPONENTS OF A UV-VIS
SPECTROPHOTOMETER
(SINGLE BEAM INSTRUMENT)

Quartz Cell Detector

Monochromator

Slit
Source
Readout Device
Signal Processor
 UV-VIS ABSORPTION PHENOMENA (SCAN MODE)
Pi
A = log
Po

Slit

Tungsten: VIS
Deuterium: UV
1.2

Absorbance 0.6

0.2
0
220 320 420 520 620
Wavelength
 UV-VIS ABSORPTION PHENOMENA (FIXED )

P0 Pi

Slit
Tungsten: for VIS
Deuterium: for UV
Pi
A = log 0.2548
Po
Inside Look of the Monochromator
Range of the Deuterium
and Visible Lamps
Advantages and Disadvantages
of UV-Vis
Advantages
High sensitivity
Moderately high selectivity (requires
chromophore)
Lots of applications
Moderately low detection limits

Disadvantages
Requires low analyte concentrations (Not
useful for conc. more than 0.01M due to
solute-solute interactions)
fluorescence or phosphorescence of the
sample
Simultaneous Determination of
Caffeine
and Benzoic Acid
The UV-Vis Spectroscopy is a good method when is
desired to quantify one compound in a unknown.

What happens when a sample have a mixture of

compounds that absorbs radiation?

Can it be analyzed by UV-Vis technique?

 Did you Know?
Caffeine is a common organic molecule found in
many beverages such as coffee, tea, and cola.

It is a stimulant to the central nervous system.

Like many conjugated organic molecules, caffeine

absorbs radiation.

Sodium Benzoate is added to the beverages as a

preservative.
 General Information

The addition of HCl to all the solutions is to

transform Sodium Benzoate to Benzoic Acid.

For the determination of Sodium Benzoate you

need to use the Stoichiometric factor.
Caffeine UV-Spectrum
0.9
275
0.8

Absorbance 0.7

0.6

0.5

0.3

0.1

0
210 220 250 280 290
-0.1

Wavelength
 Standard Solutions of
Caffeine

For a series of standards of Caffeine can you measure ?

How many can you measure?
Benzoic Acid UV-Spectrum
0.9 230

0.8
Absorbance
0.7

0.6

0.5

0.3

0.1

0
210 220 250 280 290
-0.1

Wavelength
Standard Solutions of Benzoic
Acid

Which is the value of at 230 and 275 nm?

At which wavelength Benzoic acid is a better
chromophore?
 Mountain Dew Sample:
Multi-Component System

0.9
230
0.8 275
Absorbance

0.7

0.6

0.5

0.3

0.1

0
210 220 250 280 290
-0.1

Wavelength
 Beers Law for a
Multi-Component System

A = Ai

A'T 1 = A1 + A2 = 1bC1 + 2bC2 (1)

A"T 1 = A1 + A2 = '1 bC1 + '2 bC2 (2)

If b= 1cm
 Beers Law for a Multi-
Component System, and
b=1 cm

1. Absorb total 230 = Absor Benzoic Acid + Absor Caffeine

Absorb total 230 = Benzoic Acid 230 * [conc Benzoic Acid] + cafeine 230 * [Caffeine]

2. Absorb total 275 = Absor Benzoic Acid + Absor Caffeine

Absorb total 275 = Benzoic Acid 275 * [conc Benzoic Acid] + cafeine 275 * [Caffeine]
 Mountain Dew
Absorbances
Mountain Dew Soln 275 nm 230 nm
ABS 0.1214 0.3919

3. Substitute the slopes values in their repectives wavelength

Absorb total 230 = 0.0907 * [conc Benzoic Acid] + 0.0255* [Caffeine]
Absor total 275 = 0.0077 * [conc Benzoic Acid] + 0.0479 * [Caffeine]

4. The values for the Total Absorbances are the readings for the Mountain Dew Sample

0.3919 = 0.0907 * [conc Benzoic Acid] + 0.0255* [Caffeine]

0.1214 = 0.0077 * [conc Benzoic Acid] + 0.0479 * [Caffeine]
 Answer
The two equations contain two unknowns: [X] and [Y].

The equations can be solved using simultaneous equations or

using determinants. If using determinants, the solutions are
obtained using:

Where the determinant D is solved as :

a b
= (a * d) - (b * c)
c d
ELECTROGRAVIMETRIC
DETERMINATION OF
COPPER
 ELECTROGRAVIMETRIC DETERMINATION
OF COPPER

PURPOSE:
Determine the percent of copper in an impure sample of
copper oxide by means of an electrogravimetric analysis.

THEORY:
An electrogravimetric analysis consists on depositing,
quantitatively, the product of an electrolytic reaction on a
previously weighed inert electrode. The determination of
copper is one of the classic examples of this technique.
The process does not require a 100% current efficiency.
 Electrogravimetric Methods
The potential of the working electrode is adjusted to a value were only the reaction
of interest can occur
The sensitivity of the method is limited by the difficulty in determining the
differences in mass between the free (clean) electrode and the electrode plus the
deposit
It requires the washing and drying of the electrode
It is limited only to reactions that forms insoluble substances
The technique needs smooth and adherent metal platings and deposits
The physical characteristics of the deposit depends on the:
form of the metal ion in solution
presence of adsorbable surface active agents in solution
other factors not completely understood
 Electrode array for Electrogravimetric
analysis

B C
A
A typical electrode array used in electro-gravimetric techniques:
(A) A schematic representation of an electrogravimetric analysis
(B) The Pt gauze electrode to serve as cathode
(C) The Pt gauze electrode to serve as anode. The solution can be stirred by
means of a motor, instead of using a magnetic stirrer.
 Electrochemical reaction

The process consists of reducing Cu2+ ions to Cu0, in an acidic

solution, on the surface of the platinum electrode that acts as
the cathode.

At the cathode, Cu0 is deposited as a thin film of metallic

copper.
Cu 2+(ac) + 2e- Cu0(s) (1)

At the anode, the oxidation of water occurs:

2H2O(l) O2(g) + 4H+(ac) + 4e- (2)

The overall reaction is: Eq(1)+ Eq(2)

2Cu 2+(ac) + 2H2O(l) 2Cu0(s) + O2(g) 4H+(ac)
 Experimental Considerations
A mixture of nitric and sulfuric acids is used in the
analysis. The presence of nitric acid leads to more
satisfactory deposits since it prevents the evolution
of hydrogen gas.
If the concentration of the acid is too high,
however, there is a tendency for the deposition to
be incomplete.
The presence of Cl ions should be avoided since
platinum anodes are attacked by the Cl2 formed by
electrolysis of those ions.

Metals such as nickel, cobalt, cadmium, and zinc

will not deposit provided the acidity is kept
sufficiently high.
 Platinum electrodes immersed
in the copper oxide (blue)
solution.

The cathode (left) is where

the Cu0 deposits (notice
color).

Bubbles are observed near

the anode.

The stirring rod Is spun inside

the cathode electrode.
 The
electrodeposition of
the copper present
in the sample takes
about an hour,
after drying,
weighing and Figure: Pt gauze electrode after Cu0
dissolving the electrodeposition. The color of copper coated
region is similar to that of the 1 cent coin.
samples.

Once the process is completed, the Cu-coated electrode

is rinsed, dried and weighed to obtain the weight
of the metallic copper by the difference relative to
the initial weight of the electrode prior to deposition.
 Apparatus and materials
Eberbach Electrogravimetric Analyzer
Copper Oxide sample (CuO) (MW: 79.545)
50% sulfuric acid
50% nitric acid
Acetone
4 Pt gauze electrodes
Electroanalyzer:
Instrument which uses inert
platinum electrodes, is used
for the determination.
 Sample Preparation
Weigh two 2.0000 to 3.0000g samples of the unknown.

Add to each sample

22 mL of dH2O
4 mL of 50% H2SO4
2 mL of 50% HNO3

Cover each beaker with a watch glass and heat the solutions on
a hot plate, without boiling, until the samples are completely
dissolved.

NOTE: The solution should be perfectly clear and blue

colored. Do this inside the fume hood. This process will also
remove HNO2, which prevents complete deposition of copper.
Cool down the samples and add 100 mL of dH2O to each. Cover
again with the watch glass.
 Experimental
Clean the Pt gauze electrodes:
Immersion in hot 50% HNO3 for 5 minutes (in the fume
hood).
Tap water
Distilled water
Acetone

Dry in an oven at 1100 C for 5 minutes.

Cool in a desiccator

Weigh carefully on an analytical balance.

Attach the cathode (the large electrode) to the negative

terminal and the anode to the positive one.
 Experimental cont.
Elevate the beaker containing the solution so that all but a few millimeters
of the cathode are covered and the lower edge of the electrode almost
touches the bottom of the beaker.
The solution can be diluted, if necessary, with distilled water.
Turn Power ON and start stirring.

Adjust the applied potential so that a current of 0.5 Amp passes through
the cell for about 30 minutes.
Increase current to 1.0 Amp and run for another 30 minutes.

The blue color of the copper ion should disappear. Add sufficient water to
raise the level on the cathode solution by a detectable amount and
continue the electrolysis for another 5 minutes. If no copper deposit
appears on the newly covered portion of the cathode, the electrolysis is
complete.
If additional copper deposits, continue test for completeness
 Experimental cont.
When no more copper is deposited:
Slowly lower the beaker.
Rinse the electrodes thoroughly with a fine stream of water.

NOTE: Do not turn off the applied potential until rinsing is complete or the copper
may be redissolved by the acid.
Disconnect the cathode carefully rinse with distilled water and then with a stream of
acetone.
Dry the electrode in an oven for 2 to 3 minutes at 90 C, cool in a desiccator and
weight. (REACHING CONSTANT WEIGHT STEP)
(Two consecutive weights not differing by more than 0.0004 g).
Repeat this step until constant weight.
(REACHING CONSTANT WEIGHT STEP)
Do not heat the electrode any longer than this since the copper surface oxidizes easily.

To Finish
Clean the Pt gauze electrodes:
Immersion in hot 50% HNO3 (in the fume hood).
Tap water
Distilled water
Return to your laboratory instructor
 Calculations
Mass of copper recovered in each sample.
mass of Cu = 0

m after electrolysis - mass before electrolysis

Percentage of copper (%Cu)

0
for each sample.
mass of Cu
%Cu = 100
mass of sample

Average percentage of Cu. (For 2 samples)

Relative standard deviation (ppt) of your

results.
SPECTROPHOTOMETRIC
ANALYSIS OF IRON IN
MOHRS SALT AND IRON
SUPPLEMENT
 EXPERIMENTAL
In the experiment, the dissolved iron is reduced to Fe (II) with
hydroquinone (as reducing agent).

The pH of the solution is adjusted between 3 and 6 (e.g. 3.5)

with sodium citrate, and an excess of o-phenanthroline is
added.
After the color of the complex has developed, the
absorbances of a series of standards (A std ) and sample
solutions (Asample) are measured.
A calibration curve (Astd vs. Cstd) is constructed and used to
determine the concentration of iron in the samples.
 SAMPLES ANALYZED:
a Mohrs salt unknown (for which your instructor knows its exact iron
content),
Mohrs salt is the common name for the ferrous ammonium sulfate
hexahydrate (FeSO4 (NH4)2SO4.6H2O, MW 392.14). Ferrous ammonium
sulfate hexahydrate is soluble in water.
An iron supplement tablet (for which the label provides an estimate value
of its iron content).
The iron supplement tablet must be dissolved in hot acid solution until
all the iron is dissolved; and the solution filtered to remove all insoluble
particles before selecting an aliquot for analysis.
The efficiency of your sampling process (how well you handle your real
life sample) will relate to the accuracy of your analysis.
 The iron must be in the +2 oxidation state to form the complex with o-
Phenanthroline
Fe+3 present is reduced after the reaction with Hydroquinone:

Hydroquinone is the reducing agent

pH of the solutions must be between 3-6 to form the complex
 After the Redox reaction, the complex formation is
described as follows:

Fe(II) forms a very stable, orange complex with o-phenanthroline and

this complex is a good chromophore.
Fe(II) complex has an absorption maximum around 512 nm and a
molar absorptivity coefficient at 1.1x103 cm-1 M-1.
 REACTANTS AND MATERIALS:
Hydroquinone Solution (Freshly Prepared 10g/L). Stored in Amber Bottle.

O- Phenantroline (2.5g in 100 mL Ethanol, add 900 mL water). Stored in

Amber Bottle.
Fe+3 present is reduced after the reaction with Hydroquinone.

Hydroquinone is the reducing agent.

Iron must be in the +2 oxidation state to form a stable complex with O-

phenanthroline.

Since the complex shows color, it can be analyzed by Visible spectroscopy.

 PROCEDURE:
Prepare 1L Std Fe stock solution ( 0.04gFe/L): Weight 0.28g of the Mohrs salt.
Transfer into 1L volumetric flask. Add 2mL of 50% sulfuric acid and 200mL distilled
water. Stir until the salt is dissolved. Fill the flask to the mark with distilled water and
mix.
Prepare Mohrs salt unknown solution: Weight 0.28g of the Mohrs salt. Transfer
into 1L volumetric flask. Add 2mL of 50% sulfuric acid and 200mL distilled water. Stir
until the salt is dissolved. Fill the flask to the mark with distilled water and mix.
Prepare Iron Supplement solution: Place one tablet in 100mL beaker. Add 25mL of
6M HCl and boil gently (in the fume hood) for 15 min. Filter into 100mL volumetric
flask. Wash the beaker, filter into the same volumetric flask (repeat several times). Fill
the flask with distilled water. Transfer 5.00mL into a second 100 mL volumetric flask.
 PROCEDURE:
How to estimate the amount of sodium citrate buffer needed to maintain the
pH solution at 3.5?
Pipet 10mL aliquot of the Fe solution (Std, Mohrs salt or Fe tablet solution) into
a beaker.
Counting the drops, add sodium citrate buffer drop by drop until a pH of 3.5 is
reached.

Example: If 30 drops of buffer were needed for the 10 mL aliquot to reach a pH

of 3.5, an aliquot of 5mL will need 15 drops of the buffer.
 PROCEDURE:
Std solutions preparation
Estimate the amount of buffer (sodium citrate) needed to maintain the iron
stock solution at a pH of 3.5
Pipet 10mL of Fe stock solution into 100mL volumetric flask. Add the sodium
citrate buffer (amount was previously determinate). Add 2mL of hydroquinone
solution and 3mL of o-phenanthroline solution. Dilute to the mark. LABEL as S5.
Repeat previous step with 5mL, 2mL, 1mL and 0mL of the Fe stock solution.
Label as S4, S3, S2, and S1, respectively.
 PROCEDURE:

Prepare The Iron tablet sample

Estimate the amount of buffer (sodium citrate) needed to maintain the iron
tablet at pH of 3.5
Transfer 10 mL of the tablet solution into 100mL volumetric flask. Add the
sodium citrate buffer (amount was previously determinate). Add 2mL of
hydroquinone solution. Add 3mL of o-phenanthroline solution. Dilute to the
mark.
 PROCEDURE:

Prepare Mohrs sample

Estimate the amount of buffer (sodium citrate) needed to maintain the iron
tablet at pH of 3.5.
Transfer 10 mL of the Mohrs solution into 100mL volumetric flask. Add the
sodium citrate buffer (amount was previously determinate). Add 2mL of
hydroquinone solution. Add 3mL of o-phenanthroline solution. Dilute to the
mark.
 PROCEDURE:
Determine the optimum wavelength (should be aprox. 508nm)
Measure the absorbance of all solutions at the optimum wavelength.