Course Manual

Physical Chemistry Laboratory –I (CY 457)

School of Chemistry
University of Hyderabad
Prepared by: Prof. Anunay Samanta, Prof. T. P. Radhakrishnan and Dr. Debashis Barik
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Contents

General Information Page

Preparation for the laboratory class and laboratory practices 3

Course evaluation / grading 4

List of experiments

1 Kinetics of Iodine Clock Reaction 5-7

2 Study of an Oscillating Reaction 8 - 10

3 Determination of the Change in Dipole Moment on Electronic Excitation 11 - 12

4 Adsorption of Acetic Acid on Charcoal 13 - 14

5 Fluorescence Study of Critical Micellar Concentration 15-16

6 Spectrophotometric Determination of pKa 17-19

7 Conductometric Study of Critical Micellar Concentration 20-21

8 Conductometric Titration of a Charge Transfer System 22-23

9 Determination of solubility diagram for a three-component liquid system 24-25

10 Determination of excited state acidity constant 26-27

11 Study of enzyme-substrate catalysis reaction 28-30

12 Potentiometric titration of a redox reaction 31-32

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Preparation for the laboratory class and laboratory practices

1. Read the instruction manual carefully; discuss with the instructors any point that
is not clear.
2. Read and understand the principles of the experiment before coming to the
laboratory class.
3. You must arrive for the laboratory on time; late entry into the laboratory is not
allowed.
4. Follow strictly, the instructions given in the laboratory by the instructors.
5. Safe practices in the laboratory are of utmost importance:
a. Wear shoes that cover your feet properly.
b. Wear preferably cotton clothing; clothing made of easily inflammable
materials such as silk should be avoided.
c. Do not use your mouth to draw solutions into the pipette or similar equipment.
6. Inside the laboratory, maintain absolute discipline in your behaviour and focus
full attention on the experiment you are carrying out. Do not engage in any
conversations and discussions. Do not compare your experimental observations
and results with those of others; you are supposed to report your own results.
7. Keep your laboratory bench clean and organized; careless and disorderly handling
of chemicals, glassware or equipment can lead to major laboratory accidents.
8. Maintain your laboratory notebooks properly. You must have two notebooks; (i)
the Observation Notebook for recording the various observations and data during
the experiment in the laboratory class and (ii) the Record Notebook for reporting
the details of the experiments, observations and conclusions in the following
laboratory class.
9. After you finish the experiment, ensure that you obtain the signature of the course
instructor on the data/observations recorded in your Observation Notebook. The
same observations along with details of the experiment, analysis, results and
conclusions must be recorded neatly in the Record Notebook and submitted
promptly at the beginning of the following laboratory class. If this is not followed
strictly, the grade for the specific experiment will not be awarded.
10. At the end of the laboratory class, hand over to the laboratory staff, the glassware,
chemicals, equipment etc. after proper cleaning.
11. Reagent bottles and their stoppers should not be left at the work bench. They
should be returned to their proper place upon the shelves immediately after use.
If a reagent bottle is empty, bring it to the attention of laboratory staff.
12. Use the chemicals/samples provided to you very carefully, without wasting any
material; extra samples will not be given under any circumstance.
13. Handle all the equipment carefully, following strictly the instructions for
appropriate usage.

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Course evaluation / Grading

Step 1. Continuous assessment (60 marks): All experiments will be given equal
weightage. The grading for each experiment will be based on an assessment
(for that experiment) of: (a) the performance and experimental work in the
laboratory, (b) quality and correctness of the observations and recording of
data, (c) presentation in the Record Notebook and (d) responses to questions
during the evaluation of the Record Notebook in the following week.
Step 2. End semester written examination (40 marks): This will be a 1 h examination
to assess the understanding of the basic theory and conceptual details of all the
experiments carried out during the semester.

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Experiment 1
Kinetics of Iodine Clock Reaction

Kinetics of the oxidation of iodide by hydrogen peroxide is studied; the clock refers to
the periodic cycling between iodide and iodine (visualized by the starch-iodine complex)
effected by the hydrogen peroxide in the forward direction and thiosulfate in the reverse

Basic concepts
Kinetics of the oxidation of iodide (I-, often in the form I3-) by hydrogen peroxide (H2O2)
is studied in this experiment. By an ingenious trick of converting the iodine (I2) formed,
back to I- using sodium thiosulfate, Na2S2O3, the concentration of I- is maintained constant
so that the reaction is controlled essentially by the concentration of H2O2 alone which keeps
decreasing with each run. By successive addition of small amounts of Na2S2O3, several
different runs of the experiment are mimicked in a single flask. The reactions involved
are:
2I- + H2O2 + 2H+  I2 + 2H2O; I2 + 2S2O32-  2I- + S4O62-

Experimental procedure
1. Prepare the following solutions:
(a) 100 ml 0.01 M H2O2 (the solution provided should be diluted appropriately
based on the strength indicated on the bottle)
(b) 100 ml 0.1 M KI
(c) 100 ml 0.01 M Na2S2O3.5H2O
(d) 50 ml pH 4.5 buffer solution (mix equal volumes of 0.2 M sodium acetate and
0.2 M acetic acid)
(e) Fresh starch solution (0.5 g starch made into a paste and dissolved in 50 ml
boiling water)
(f) 100 ml 0.02 M oxalic acid (H2C2O4.2H2O) (f) – (h) are required for
(g) 100 ml 0.004 M KMnO4 estimating H2O2
(h) 100 ml 2.5 M H2SO4
2. Standardise the potassium permanganate solution using the oxalic acid, and then the
hydrogen peroxide solution, using the potassium permanganate solution.

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3. In a 250 ml conical flask, mix 20 ml of the buffer, 10 ml of the potassium iodide

solution and 50 ml of distilled water; add 2 ml of the starch solution and 1 ml of the
sodium thiosulfate solution. Maintain the reaction mixture at a temperature (T).
4. To the mixture prepared in 3. above, add 20 ml of the hydrogen peroxide solution,
starting the clock when the solution is half down the pipette.
5. Stir the solution continuously and note the time taken for the appearance of the blue
color (indicating the formation of iodine).
6. Add 1 ml of the sodium thiosulfate solution, starting the clock simultaneously; the
blue color disappears immediately.
7. Stir the solution continuously and note the time taken for the reappearance of the
blue color.
8. Repeat steps 6 and 7 at least twelve times.

Data and analysis

1. The calculations proceed as follows:
a. Concentration of H2O2 prepared =xM
b. Volume of H2O2 taken for the reaction = v ml
c. Quantity of H2O2 taken for the experiment = xv mmol
d. Initial concentration of H2O2 (C0) = xv/V M V is the total volume in ml, of the
reaction mixture; it need not be
e. Concentration of Na2S2O3 taken =yM known explicitly as will be seen
below
f. Volume of Na2S2O3 added initially = v1 ml
g. Quantity of Na2S2O3 present initially = yv1 mmol
h. Quantity of H2O2 consumed in the first step = ½ yv1 mmol
The volume change is
i. Final concentration of H2O2 in the first step (C) = [xv - ½ yv1]/V M assumed to be negligible
j. ln (C0/C) = ln{xv/[xv - ½ yv1]}
k. Time taken for the step =ts
l. First order rate constant (k) = [{ln (C0/C)}/t] s-1
2. For step 2, C0 is equal to the C calculated in step 1; the calculations may be repeated
for further steps.
3. Tabulate the data as follows:

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Volume of Amount of H2O2 (mmol) Time

Step Na2S2O3 Beginning End ln (C0/C) taken, k (s-1)
added (ml) (xv) (xv - ½ yv1) t (s)
1
2
3
4
5
6
7
8
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10
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12

4. Estimate the average value of the rate constant, k at temperature T.

5. The experiment may be repeated at different temperatures; Arrhenius plot can be
used to determine the activation energy of the reaction, and the pre-exponential
factor.

Further reading
http://www.kbcc.cuny.edu/academicDepartments/PHYSCI/PL/chm12/Documents/CHM1
2_Experiment_5_Kinetics.pdf

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Experiment 2
Study of an Oscillating Reaction

The oscillating reaction (Belousov-Zhabotinsky) is studied using the Ce3+/Ce4+ system;

dependence of the oscillation period on the metal ion concentration is monitored.

Basic concepts
Several natural phenomena show periodic (oscillatory) behavior, for example night and
day or cell division. Oscillatory behavior in chemical reactions is associated with regular
and periodic fluctuation in the concentration of a specific chemical component. This
occurs when (i) the reaction follows a complex mechanism with at least one auto-catalytic
step (where the product catalyzes the reaction step) coupled with the other steps, (ii) the
system is far from equilibrium with some components in large excess and some are
removed from the system, for example, as gases, (iii) there are large variations in the rate
constants of different steps. The most celebrated oscillatory reaction is perhaps, the
Belousov-Zhabotinsky reaction; a typical case involves a solution of malonic acid and
bromate ions with a metal ion or metal ion complex acting as a catalyst in acidic medium.
The metal ion or complex commonly used are Ce4+/Ce3+ and Fe(phen)33+/Fe(phen)32+
(ferriin/ferroin). An explanation for the oscillation of the metal ion concentration was
provided by several models such as Field-Körös-Noyes (FKN) and the ‘Oregonator’. Even
though a large number of steps are likely to occur in this complex reaction, the essence of
the oscillatory process can be captured using the following cycle which is expanded further
in the table below.

Process A: BrO3- reduced Process B: Ce3+ is oxidized

by Br- to form Br2; MA to Ce4+ in the autocatalytic
converted to BMA step, B1

[Br-]cr
[Br-]

Process C: Ce4+ is reduced

to Ce3+ by BMA; Br- and
organic byproducts form

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Process A
1 Br− + BrO3− + 2H+ → HOBr + HBrO2 P+S→Q+R
2 Br− + HBrO2 + H+ → 2HOBr P + R → 2Q
−
3 Br + HOBr + H → Br2 + H2O
+

4 CH2(COOH)2 + Br2 → BrCH(COOH)2 + H+ +Br−

Process B
1 HBrO2 + BrO3− + 2Ce3+ + 3H+ → 2HBrO2 + 2Ce4+ + H2O R + S + 2X → 2R + 2Y
2 2HBrO2 → HOBr + BrO3− +H +
2R → Q + S
Process C
1 BrCH(COOH)2 + Ce4+ + CH2(COOH)2 + H+ → Br− + Ce3+ + organic products BMA + Y → P + X + …
-
P = Br ; Q = HOBr; R = HBrO2; S = BrO3-; 3+ 4+
X = Ce ; Y = Ce ; BMA = bromomalonic acid

Depending on the concentration of bromide ion, whether it is above or below the

critical value, [Br-]cr, the processes A and B cycle back and forth; the products of
these processes BMA and Ce4+ react accordingly. The concurrent oxidation of the
organic species in the step C1, reduces Ce4+ to Ce3+, and regenerates Br-; this is
accompanied by the color change form blue to red (in presence of the ferroin
indicator). Oxidation of Ce3+ to Ce4+ in the step B1 shows the reverse color change.
The experiment can be carried out in a number of ways; we will follow one of them.

Experimental procedure
1. Prepare (or procure) the following solutions:
(a) 100 ml 0.006 M ceric ammonium sulphate [Ce(NH4)4(SO4)4.2H2O] in 0.5 M
sulfuric acid
(b) 100 ml 0.09 M potassium bromate (KBrO3) in 0.5 M sulfuric acid
(c) 100 ml 0.30 M malonic acid (CH2(COOH)2) in 0.5 M sulfuric acid
(d) Ferroin indicator solution
(e) 20 ml each of 0.0045, 0.003, 0.0025 and 0.0015 M ceric ammonium sulphate in
0.5 M sulfuric acid [these may be prepared by appropriate dilution of (a); use 0.5
M sulfuric acid for the dilutions]
2. Prepare reaction mixtures containing equal volumes of the solutions (a), (b) and (c);
note down the effective concentration of each in the mixture. Maintain the mixture
at a constant temperature. Add a few drops of ferroin; wait for a few minutes for the

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color oscillation to start. Note down the oscillation period at least 10 times, and
estimate the average value and standard deviation.
3. Repeat step 2. using each of the ceric ammonium sulphate solutions prepared in (e).
4. Prepare a reaction mixture containing equal volumes of the solutions (a), (b) and (c).
Record the electronic absorption spectrum when the solution shows a clearly visible
color. Note down the peak position (max).
5. Record the time variation of the absorbance at max.

Data and analysis

1. Plot the oscillation period (with standard deviation) determined from steps 2 and 3,
as a function of the concentration of ceric ammonium sulphate solution.
2. Plot the time variation of the absorbance at max determined in step 5.
3. Compare the period of the oscillation determined from the visual and the spectral
observations.
4. Write down the rate equations for the concentrations, [P], [R] and [Y] using the 5
symbolic equations in the last column of the Table {note that solution of these
differential equations provide insight into the oscillatory nature of the reaction}.

Further reading
1. O. Benini, C. Rinaldo, P. Fetto, J. Chem. Ed. 1996, 73, 865.
2. https://www.rose-hulman.edu/mathjournal/archives/2002/vol3-n1/paper1/v3n1-
1pd.pdf

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Experiment 3
Determination of the Change in Dipole Moment
on Electronic Excitation

Change in the dipole moment on electronic excitation of a push-pull chromophore is

determined by studying the solvatochromism of its fluorescence emission Stokes shift.

Basic concepts
UV or visible light excitation of molecules can lead to significant electron density
redistribution and hence change in the dipole moment, between the ground and excited
electronic states. Energy level of polar ground or excited electronic states can be shifted
by solvation effects. For example, the electronic excitation energy can increase markedly,
if the molecule has an appreciable ground state dipole moment, and is placed in a solvent
with a high dielectric constant. In a similar solvent, the fluorescence emission energy
would decrease if the molecule has an excited state with a high dipole moment. These
solvatochromic effects therefore provide insight into the dipole moment of the molecule in
the relevant electronic states. In this experiment, the change in dipole moment on
excitation (Δµge) of a ‘push-pull’ molecule (N,N-dimethylamino
benzonitrile, DMABN) will be determined by measuring the
solvatochromism of its Stokes shift (shift in energy between the
excitation and fluorescence emission). Solvent mixtures with
varying polarity will be used to observe the solvatochromic effect,
and a standard betaine dye (known as the Reichardt’s dye) will be
used as a reference. Incidentally, DMABN has a complex excited
state profile, which leads to interesting consequences in the
fluorescence emission spectrum.

Some data required for the experiment: solvent polarity value, 𝐸𝑇𝑁 for toluene (T) –
acetonitrile (A) mixtures:
Vol. % of A in T-A mixture 20 30 40 50 60 70 80 90
𝐸𝑇𝑁 0.286 0.312 0.349 0.360 0.379 0.397 0.410 0.420

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Experimental procedure
1. Prepare mixtures of toluene (T) and acetonitrile (A) with 20, 30, 40, 50, 60, 70, 80 and
90% by volume of acetonitrile. The solvent polarity parameter of each of these
mixtures denoted by the 𝐸𝑇𝑁 value is provided above. What is 𝐸𝑇𝑁 ?
2. Dissolve equal weight of DMABN in each of the solvent mixtures prepared in step 1,
to make approximately 1 µM solutions.
3. Record the electronic absorption and fluorescence emission spectra of all the solutions
prepared in step 2; the wavelength corresponding to the maximum of the absorption
spectrum (max) may be used as the excitation wavelength for recording the What is the
basis for
fluorescence spectrum.
choosing exc ?
4. Record on paper, the absorption and fluorescence spectrum of only one solution; note
down the max for both spectra of all the solutions.

Data and analysis

1. Calculate the Stokes shift (Δ𝜐̅ in cm-1) for the different DMABN solutions, What is the
origin of the
corresponding to each of the peaks (if more than one is observed) in the fluorescence peaks in the
spectrum. fluorescence
spectrum ?
2. Plot the Stokes shifts against the 𝐸𝑇𝑁 values. Carry out a least square fit analysis and
determine the least square fit straight line; note down the slope.
3. Δµge for DMABN is given by following equation:
Δ𝜇𝑔𝑒 2 𝑎 3
Δ𝜐̅ (𝑐𝑚−1 ) = 11307.6 {( Δ𝜇 ) ( 𝑎𝐷 ) } 𝐸𝑇𝑁 + 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡
𝐷

where ΔµD (9 D) and aD (6.2 Å) are the dipole moment change on excitation and the
Onsager radius respectively, of the betaine dye used for the 𝐸𝑇𝑁 determination. Δµge
and a are the corresponding quantities for DMABN; a of the molecule may be
approximated by half the length of the molecular dipole.
4. Compare the value of Δµge you have determined with what is reported in the literature.
5. Discuss the reason for the solvatochromism observed.
6. Find out the structure of the betaine dye; why is it uniquely suited for solvent polarity
measurements ?

Further Reading
M. Ravi, A. Samanta, T. P. Radhakrishnan, J. Phys. Chem. 1994, 98, 9133.

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Experiment 4
Adsorption of Acetic Acid on Charcoal

Adsorption of acetic acid on charcoal is studied, and the adsorption isotherm determined.

Basic concepts
Adsorption is the process of adhesion of atoms, molecules or ions from a gaseous or liquid
phase on to a surface, usually of a solid material; the interaction is generally weak and
reversible, if it is a case of physical adsorption (chemical adsorption involves a bond
formation and is stronger). Materials with a large surface area per unit weight (for example,
charcoal) is capable of adsorbing significant amount of molecules (like acetic acid or oxalic
acid) from an aqueous solution. Adsorption from solution often follows the Freundlich
isotherm:
𝑋
( ) = 𝑘𝐶𝑒𝑛
𝑚
where X is the number of moles of the adsorbate (acetic acid) adsorbed by m g of the
adsorbent (charcoal); 𝐶𝑒 is the equilibrium concentration of the adsorbate and k and n are
constants for a particular adsorbate-adsorbent combination. The equation can be rewritten
as :
𝑋
𝑙𝑜𝑔 ( ) = 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 + (𝑛 × 𝑙𝑜𝑔𝐶𝑒 )
𝑚
𝑋
implying that a plot of 𝑙𝑜𝑔 (𝑚) versus log(𝐶𝑒 ) is linear.

Experimental procedure
1. Prepare (or procure) the following:
(a) 250 ml 0.5 N acetic acid solution
(b) 100 ml 0.5 N sodium hydroxide (solution)
(c) Phenolphthalein indicator
(d) Powdered charcoal
2. Weigh 2.00 ± 0.01 g charcoal each into 6 stoppered conical flasks or bottles.
3. Add into the flasks, acetic acid solution + water as follows:
Flask No. 1 2 3 4 5 6

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0.5 N acetic acid (ml) 50 40 30 20 15 10

Water (ml) 0 10 20 30 35 40
4. Shake thoroughly each sample regularly for a period of ~ 20 min. Allow to stand for
~ 5 min. Justify the
precautions
5. Filter through a filter paper; take care to use a small filter paper. Collect the filtrate in taken during
appropriately labeled flasks; discard the first 5 ml while collecting the filtrate. filtration.
6. Titrate 10 ml of each filtrate and titrate against 0.5 N NaOH solution; use
phenolphthalein as the indicator.
7. Titrate also 10 ml of the stock 0.5 N acetic acid solution.

Data and analysis

1. Titration value for the 10 ml of stock 0.5 N acetic acid = x ml (NaOH).
2. From x, the concentration of acetic acid in each flask before adsorption can be
estimated (in terms of the volume equivalent of 0.5 N NaOH), considering the
dilution involved (use the Table in step 3 of the experimental procedure);
titrations in step 6 provides the concentrations after adsorption ie. at equilibrium.
3. Amount of acetic acid adsorbed (X) on charcoal can be estimated using the initial
(before adsorption) and final (equilibrium) amount of acetic acid, in terms of the
volume equivalent of 0.5 N NaOH; see the following table:
Flask Weight of Concentration of acetic acid 𝑿 𝑿
log ( ) log Ce
No. charcoal, (volume equivalent in ml of NaOH) 𝒎
𝒎
m (g) Initial, C Final, Ce X = C - Ce
1
2
3
4
5
6

𝑋
4. Plot log (𝑚) versus log Ce; verify if the Freundlich isotherm is followed.

5. Plot the data appropriately to see if it follows the Langmuir adsorption isotherm.

Further Reading
P. Atkins, J. de Paula, Atkin’s Physical Chemistry, Oxford University Press, 2010.

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Experiment 5
Fluorescence Study of Critical Micellar Concentration

The critical micellar concentration of a surfactant is determined by measuring the steady

state fluorescence intensity of an environment-sensitive probe as a function of the
concentration of surfactant

Basic concepts
Surfactant molecules aggregate to form a micellar structure only when the concentration
exceeds a particular value, which is commonly termed as critical micellar concentration
(CMC). In this experiment, the CMC of an anionic surfactant, sodium dodecyl sulfate
(SDS), is determined by following the variation of fluorescence intensity of 8-anilino-1-
naphthalene sulfonate (ANS), an environment sensitive fluorescence probe, for various
concentrations of SDS. As ANS molecules exist in aqueous environment below the CMC,
but above it they get incorporated into the micelle, wherein the polarity is very different
from that in aqueous medium, the formation of micelle is associated with a sharp change
in the plot of fluorescence intensity (If) versus [SDS].

Experimental procedure
1. Prepare 100 ml of ~10-4 M aqueous solution of ANS.
2. Divide this solution to 10 x10 ml volumetric flasks.
3. Label these solutions from 1 to 10
4. Add appropriate amount of solid SDS to these solutions (except for the solution
labelled 1) such that [SDS] in flasks labelled 2-10 are 0.5, 0.8, 1.0, 2.0, 5.0, 8.0,
10.0, 15.0 and 20.0 mM, respectively.
5. Measure the fluorescence spectra of the solutions under identical instrumental
settings.
6. Plot the fluorescence intensity (If) of the system versus [SDS].
7. Determine CMC of SDS from the plot and compare it with the literature value.

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Suggested reading
1. Photochemistry in Microheterogeneous Systems, K. Kalyanasundaram
2. Any other book on fluorescence spectroscopy or photochemistry

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Experiment 6
Spectrophotometric Determination of pKa

The pKa of a weak acid is estimated by measuring the concentrations of its neutral
(acidic) and deprotonated (basic) forms spectrophotometrically at various pH.

Basic concepts
The extent of dissociation of a weak acid can be determined spectrophometrically when
the characteristic absorption of its acidic and basic forms are measured. In this experiment,
the dissociation constant and pKa of a weak acid, methyl red, is estimated by determining
the concentrations of its acidic (HMR) and basic (MRˉ) forms at different pH by
spectrophotometric measurements. In the case of methyl red, its two forms have strong
characteristic absorption in the visible region with HMR (red) having an absorption peak
at ~520 nm and MRˉ (yellow) at 430 nm. The transformation between the two forms are
observable in the pH interval of 4 – 6 and this pH range can be conveniently obtained by
CH3COONa - CH3COOH buffer. The determination of pKa involves three steps: (i)
determination of peak wavelengths, A and B, for HMR and MRˉ, respectively, (ii)
verification of Beer’s law for both forms at A and B and (iii) determination of relative
amounts of HMR and MRˉ as a function of pH of the medium.
The relative amount of HMR and MRˉ present in solution can be calculated by relating the
measured absorbance, AA (at A) and AB (at B) as:
AA = dA,HMR [HMR] + dA,MR [MRˉ] (1)
AB = dB,HMR [HMR] + dB,MR [MRˉ] (2)
By estimating dA,HMR, dB,HMR, dA,MR, and dB,MR from the plots (discussed later), the ratio,
[MRˉ]/[HMR] in solution can be calculated by solving simultaneous equations (1) and (2),
and pKa can be determined using
pKa = pH – log[MRˉ]/[HMR] (3)

Experimental procedure
1. Prepare a stock solution (in a 50 ml volumetric flask) of methyl red by dissolving
0.1 g in 30 ml ethanol and then diluting it by water to 50 ml.

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2. Take 5 ml of this stock solution in a 100 ml volumetric flask. Add 50 ml ethanol

and then make up the volume to 100 ml by water. This is your standard solution.
3. Then prepare solution A by diluting a mixture of 10 ml of standard solution and
10 ml of 0.1 M HCl solution to 100 ml. (the pH of this solution is expected to be
~2 and methyl red will be present exclusively as HMR).
4. Prepare solution B by diluting a mixture of 10 ml standard solution and 25 ml
0.04 M CH3COONa solution to 100 ml. (the pH of this solution is expected to be
~8 and methyl red will be present entirely as MRˉ)
5. Measure the absorption spectra of solution A and solution B over the range of 350
– 600 nm and determine A and B.
6. Measure out 40, 25 and 10 ml of solution A into three separate 50 ml volumetric
flasks and make up the volume to 50 ml by 0.1 M HCl solution. The solutions will
contain 0.8, 0.5 and 0.2 times the initial [HMR].
7. Measure out 40, 25 and 10 ml of solution B into three separate 50 ml volumetric
flasks and make up the volume to 50 ml by 0.01 M CH3COONa solution. The
solutions will contain 0.8, 0.5 and 0.2 times the initial [MRˉ].
8. Measure the absorbance of this six solutions at A and B.
9. Plot absorbance versus relative concentration of methyl red (Plot 1). In each case,
a linear plot is obtained.
10. Prepare the following solutions in four 100 ml volumetric flasks by mixing 10 ml
standard solution, 25 ml 0.4 M CH3COONa solution and
(i) 50 ml 0.02 M CH3COOH solution and 15 ml water
(ii) 25 ml 0.02 M CH3COOH solution and 40 ml water
(iii) 10 ml 0.02 M CH3COOH solution and 55 ml water
(iv) 5 ml 0.02 M CH3COOH solution and 60 ml water
11. Measure the pH of each solution.
12. Measure the absorbance of each solution at A and B.
13. For each solution, obtain dA,HMR, dB,HMR, dA,MR, and dB,MR at relative
concentration of 1.0 from Plot 1.
14. Solve simultaneous equations (1) and (2) to determine [MRˉ]/[HMR] in each
solution.

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15. Now, calculate pKa using equation (3).

Suggested reading
1. Physical Chemistry, by P.W. Atkins and J. de Paula

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Experiment 7
Conductometric Study of Critical Micellar Concentration

The critical micellar concentration of a surfactant is determined by monitoring the

change in specific conductivity of the solution as a function of the concentration of
surfactant

Basic concepts
Surfactant molecules aggregate to form a micellar structure only when the concentration
exceeds a particular value, which is commonly known as its critical micellar concentration
(CMC). In this experiment, the CMC of an anionic surfactant, sodium dodecyl sulfate
(SDS), is measured by following the variation of specific or/and molar conductivity of the
solution as a function of the concentration of SDS. Below the CMC, addition of SDS to an
aqueous solution increases the number of change carriers and hence, the conductivity.
Above CMC, addition of surfactants increases the micelle concentration maintaining the
monomer concentration approximately same. As micelles are much larger than the
monomers and diffuse more slowly, the conductivity increases much slowly beyond the
CMC. Thus CMC can be determined from the break point in the plot of specific/molar
conductivity versus [SDS].

Experimental procedure
1. Use redistilled water for all measurements.
2. Prepare 25 ml of ~0.04 M aqueous stock solution of SDS.
3. Pipette 25 ml of water into the conductance cell. Add 0.5 ml SDS stock solution
using a pipette. Stir the solution slowly for a minute or two (without creating too
many bubbles) and then read the conductance.
4. Add additional 0.5 ml stock solution and measure the conductance. Continue the
process of adding stock solution and measuring conductance until 40-45 aliquots
have been added.

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5. In order to study the effect of electrolyte, prepare 100 ml of ~0.02 M NaCl

solution. Use this as solvent for making up 25 ml of ~0.04 M aqueous stock
solution of SDS. Pipette 25 ml of salt solution into the conductance cell and
measure the conductance. Add 0.5 ml of the SDS stock solution and record the
conductance. Repeat the same for about 30-40 additions.
6. Prepare appropriate Table to record the conductance and [SDS] data.
7. Find out the cell constant of the conductivity cell using standard KCl solution and
convert the conductance values into specific conductance (κ) and molar
conductance (Λm). Then plot κ vs [SDS] and Λm vs [SDS] and determine CMC.
8. Compare the estimated CMC of SDS with the literature value.

Suggested reading

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Experiment 8
Conductometric Titration of a Charge Transfer System

The formation of charge transfer complex between an electron donor and acceptor is
studied and the stoichiometry of the complex is determined by following the variation of
conductance of the solution with concentration of the donor and acceptor.

Basic concepts
A charge transfer complex is formed between an electron donor with low oxidation
potential and an acceptor having high electron affinity. The complex dissociates into ions
in medium of high dielectric constant and contribute to the conductivity. Thus formation
of charge transfer complex can be studied by monitoring the conductance of a solution
containing electron donor and acceptor. In this experiment, the formation of charge transfer
complex between p-phenylenediamine (donor) and phthalic acid (acceptor) is investigated
by measuring the conductance of the solution for several concentrations of the donor and
acceptor.

Experimental procedure
1. Prepare 250 ml of 3.4 mM solution of p-phenylenediamine in acetone.
2. Prepare 250 ml of 3.4 mM solution of phthalic acid in acetone.
3. 50 ml solution of phthalic acid is taken in a beaker and its conductivity is measured.
4. The phenylenediamine solution is taken in a burette and 1 ml of it is added to the
phthalic acid solution. The solution is then stirred and conductivity measured.
5. Repeat the process of addition and measurement of conductivity until the latter
reaches its maximum value and then start showing a decreasing trend (140-150 ml
may be required).
6. Similarly, 50 ml phenylenediamine is taken in another beaker, phthalic acid
solution is then added in small quantities from a burette and the conductivity is
measured.

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Data and analysis

1. Estimate molar concentrations of the electron donor (D) and acceptor (A) using
MA = MA0.VA/ (VA+VD) and MD = MD0.VD/ (VA+VD), where, MA0 and MD0 are the
initial concentrations and VA and VD refer to the volume of the stock solution of A
and D, respectively.
2. Plot a graph of conductance versus MD (1st titration of addition of donor) and
another one versus MA (2nd titration of addition of acceptor) on the same graph
sheet.
3. Determine the molar concentrations of phenylenediamine and phthalic acid for
which maximum conductance is observed in each case and determine the
stoichiometry of the complex.

Suggested reading

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Experiment 9
Determination of solubility diagram for a three-component liquid
system
The triangular phase diagram of a three-component liquid system involving acetic
acid, chloroform and water is determined.
Basic Concepts:
According to the phase rule (F = C – P + 2; F: degrees of freedom, C: component and P:
no of phases) there are at most four degrees of freedom, for a system containing three liquid
components. If the pressure and temperature be kept constant, it follows that the
concentrations of only two of the three components can be independently varied. When
only two of the three parameters are independently variable, all three parameters can be
represented in one plane by means of the triangular diagram shown below.\

Fig. 1: Three component solubility diagram or phase diagram

In this diagram each side of the equilateral triangle represents 0-100 percent of one of the
components. Let us fix the point that represents a mixture containing 25 percent of A, 25
percent of B and 50 percent of C. Let us mark the point a on side AB, which represents 25
percent of A. Now draw a dotted line from a to the opposite side (AC) parallel to the side
BC. All mixtures containing 25 percent of A will fall on this. Mark a point b on BC, which
represents 25 percent of B. Draw a dotted line from b to the opposite side (AC) parallel to

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AB. All mixture containing 25 percent of B will fall on this. Draw dotted line from c on
AC to BC parallel to AB. The three dotted lines intersects at some d which represents the
composition of the mixture.
It may be noted that only two of the three dotted lines are required to find out d. Any
mixture of only two components can be represented on one side of the triangle. For
instance, the point x represents the composition of a mixture containing 30 percent of C
and 70 percent of B.
Experimental procedure:
1. Procure the following liquids: glacial acetic acid, chloroform and water
2. Set up three clean and dry burettes and fill them with acetic acid, chloroform and
water separately. (caution: acetic acid is corrosive)
3. Make up 7 mixtures of chloroform and acetic acid in clean and dry glass-stoppered
bottles (or conical flasks) as shown in the Table 1. The total volume of each mixture
should be 20 ml.
4. Add water into each mixture, shaking the mixture well after each addition until the
clear solution becomes permanently turbid. Note the volume of water for each
experimental set up.

Table 1: Data table for the experiment.

Setup No. Vol. of Vol. of Vol. of Wt % of Wt % of Wt % of
CHCl3 CH3COOH added CHCl3 CH3COOH added
(ml) (ml) H2O (ml) (ml) H2O
(ml) (ml)
1 1.5 18.5
2 2.0 18.0
3 3.5 16.5
4 6.5 13.5
5 10.0 10
6 12.5 7.5
7 16.0 4.0
8 99.0 1.0
9 0.8 99.2

Data and analysis:

1. Calculate the wt % of the homogeneous mixture from the data table.
2. Plot the three-component phase diagram using the data

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Experiment 10
Determination of excited state acidity constant

The acidity constant in the excited state is studied using UV-Vis absorption and
fluorescence spectral measurements.
Basic concepts:
The acidity constant of a molecule in the excited state can be quite different from that in
the ground state because of the difference in the electron distribution in the two states. This
experiment provides a simple method of determination of the change in pKa of 2-naphthol
on electronic excitation from UV-Vis absorption and fluorescence spectral measurements.
2-Naphthol is a weak acid in the ground state with pKa value of 9.5. Therefore, the
compound exists almost exclusively in the neutral form (AH) at pH in between 4-5 in the
ground state. However, on excitation of AH at this pH, the compound exhibits dual
fluorescence that can be assigned to emission from AH* (maximum at around 360 nm) and
A−* (maximum at around 420 nm). The fluorescence excitation spectrum obtained on
monitoring the long wavelength band is identical with the absorption spectrum of AH
indicating that A-* is formed as a result of the excited state deprotonation reaction, not due
to direct excitation of A-.

The determination of the change in pKa of a compound on electronic excitation is based

on a method suggested by Forster which can be understood from the following diagram:

It can be seen from the above diagram, Δ𝐻 + ΔEA− = Δ𝐻 ∗ + Δ𝐸AH . Assuming the entropy
changes associated with the acid dissociation in the ground and excited states are the same,
one can write the free energy change as Δ𝐺 − Δ𝐺 ∗ = Δ𝐸AH − Δ𝐸A− and which ultimately

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can be transformed as 𝑝𝐾𝑎 − 𝑝𝐾𝑎 ∗ = (Δ𝐸AH − Δ𝐸A− )/2.303𝑅𝑇 or 𝑝𝐾𝑎 − 𝑝𝐾𝑎 ∗ =

0.625(ν̅AH − ν̅A− )/𝑇, where ν̅ is the wavenumber of the associated electronic transition
abtained from the absorption and fluorescence spectra.
Experimental procedure:
1) Prepare a stock solution of 100 ml 10-3 M 2-naphthol in 20% methanol. Using the stock
solution prepare the following solutions:
a) 25 ml 5×10-4 M solution at pH 0
b) 25 ml 5×10-4 M solution at pH 13
c) 25 ml 5×10-4 M solution at pH in between 4-5
2) Record the absorption spectra of all solutions in the range of 300–425 nm.
3) Record fluorescence emission spectra of all solutions. Note that for the emission spectra
the excitation wavelength must be at the absorption peak plus 10 nm. Scan till 550 nm
4) Record fluorescence excitation spectrum (300 – 400 nm) of the third solution
monitoring the longer wavelength emission.
5) Determine the mean 0-0 transition energies of the neutral and the anionic forms from
the spectral data and estimate Δ𝑝𝐾𝑎 (= 𝑝𝐾𝑎 − 𝑝𝐾𝑎 ∗).

Reference:
J. F. Ireland and P. A. H. Wyatt, Adv. Phys. Org. Chem. 12 (1976) 131 and references
therein

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Experiment 11
Study of enzyme-substrate catalysis reaction

The catalytic efficiency of a non-specific enzyme is studied by measuring the rate of the
enzyme-catalysed reaction.
Basic concepts:
Phosphatases are a class of enzymes that can catalyse the hydrolysis of phosphate
monoesters to give inorganic phosphate and an alcohol. An enzyme alkaline phosphatase
is a non-specific phosphatase, recognizing a wide variety of molecules as substrates, that
is found in bacteria, fungi, and higher animals. In this experiment the catalytic efficiency
of an alkaline phosphatase is investigated by measuring the rate of the enzyme-catalysed
reaction under different conditions. To measure the alkaline phosphatase activity a non-
biological substrate p-nitrophenyl phosphate is chosen as substrate (Scheme 1) that is
hyldrolyzed to p-nitrophenol by the enzyme.

Scheme 1. The hydrolysis of p-nitrophenyl phosphate as catalysed by alkaline

phosphatase.
Activity assays of alkaline phosphatase with p-nitrophenyl phosphate are terminated by the
addition of NaOH. This serves two functions; first, it stops the enzyme-catalysed reaction
by changing pH which is unsuitable for the enzyme (the enzyme is denatured at high pH
and so unable to function). Second, it deprotonates the p-nitrophenol to give the yellow
coloured p-nitrophenylate. The progress of the reaction is monitored by absorption
spectrum of p-nitrophenylate (obtained upon deprotonation of p-nitrophenol) that has an
intense yellow colour.
Experimental procedure:
1. Prepapre or procure the following solutions:
a) 100 ml of phosphate buffer of pH 6.8
b) 5 ml 7.5 mM p-nitrophenyl phosphate and 5 ml alkaline phosphatse
solutions
c) 20 mL of 1.0 M NaOH

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2. Turn on the spectrometer and set the wavelength to the λmax of p-nitrophenylate,
which you need to determine first.
3. Dispense 6 mL of phosphate buffer (pH 6.8) into a test tube.
4. Uncatalyzed reaction: Add 0.2 mL of p-nitrophenyl phosphate (7.5 mM). Place a
piece of parafilm over the tubes and gently invert 3 times (DO NOT shake the
solution!!!). Write down the time at which you added the p-nitrophenyl phosphate.
At the end of the lab you will measure the absorption of this solution. This will be
your uncatalyzed reaction, and will give you a measure of the rate of the non-
catalysed reaction.
5. Control reaction: Dispense 6 mL of pH 6.8 phosphate buffer into another tube,
0.2 mL of p-nitrophenyl phosphate, and 1.0 mL of 1.0 M NaOH. Note the time, and
place a piece of parafilm over the tube and gently invert 3 times. Transfer the
solution to the absorption cell, insert the cell into the spectrometer and record the
absorbance. This is the time 0 measurement, and will provide the absorbance of the
solution in the absence of any reaction. This value will need to be subtracted from
all other measurements to provide “net” absorption values. Transfer the contents
back to the tube and set the tube aside until the end of the lab. This will allow you
to measure the rate of hydrolysis in the presence of strong base. This is important
because the enzymatic reactions are terminated with NaOH, and the analysis is
predicated upon the assumptions that no significant reaction occurs once the NaOH
has been added.
6. Now place 6 mL of the buffer into another tube and add 0.2 mL of p-nitrophenyl
phosphate. Add 0.2 mL of the solution containing alkaline phosphatase to the tube,
note the time, place a piece of parafilm over the top, and gently invert 3 times (DO
NOT shake the solution!!!). This should all be done in 30-45 seconds.
7. After a total of 2 mins has elapsed add 1.0 mL of NaOH to terminate the reaction.
Note: the amount of time that you allow the reaction to progress is not critical, but
it is critical to know exactly what the reaction time is (i.e. whether the reaction was
run for 1 min 50 sec or 2 min 5 sec doesn’t matter, only that you know the actual
elapsed time).
8. Take the solution into spectrophotometer cell and record the absorbance.

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9. Repeat the rate measurement in duplicate.

10. Repeat Steps 5-8 for 0.4, 0.6, 0.8, 1.2 mL of p-nitrophenyl phosphate (7.5 mM).
11. After the enzyme rate measurements have been made, add 1.0 mL NaOH to your
control reaction prepared in Step 3 and note the time.
12. Measure the absorption of the control reaction and the NaOH reaction prepared in
Step 4.

Data and Analysis:

1. Use the calibration curve of p-nitrophenylate to convert all of the absorbance data
to concentration using Beer’s law.
2. Now divide the concentrations by the various reaction times (in minutes) to get
reaction rates in M min-1 (mol/L min).
3. Use the Linewaver-Burk plot to find out the maximum rate of the reaction and
Michaelis constant.

Further Reading
P. Atkins, J. de Paula, Atkin’s Physical Chemistry, Oxford University Press, 2010.

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Experiment 12
Potentiometric titration of a redox reaction

Potentiometric redox titration of K3Fe(CN)6 with Co(II) to find out the concentration
of the latter in a given solution.

Basic concepts:
Cobalt (II) can be estimated by K3Fe(CN)6 in strongly alkaline medium in presence of
ammonium citrate. CoII(NH3)6 formed in alkaline medium is oxidised to CoIII(NH3)6 by
ferricyanide.
The oxidation potential of any redox system is given by Nenrst equation
𝑅𝑇 [Oxidised from]
𝐸 = 𝐸 0 + 𝑛𝐹 ln [Reduced form] (1)

Therefore, for Fe(CN)3− 4−

6 +𝑒 ⇌ Fe(CN)6 reaction
𝑅𝑇 [Fe(CN)3− ]
𝐸 = 𝐸0 + ln [Fe(CN)64−] (2)
𝐹 6

The cell used for the experiment can be represented as

Std. Calomel ∥ Fe(CN)3− 4−
6 (𝑎𝑞), Fe(CN)6 (𝑎𝑞) ∥ 𝑃𝑡(𝑠)

The observed emf of such a cell is given by

𝑅𝑇 [Fe(CN)3−
6 ]
𝐸𝑜𝑏𝑠 = 𝐸 − 𝐸Calomel = (𝐸 0 − 𝐸Calomel ) + ln (3)
𝐹 [Fe(CN)4−
6 ]

When Cobalt (II) is added to the system, Fe(CN)3−

6 is removed and the concentration of

Fe(CN)4−
6 increases, leading to gradual decrease in observed emf, 𝐸𝑜𝑏𝑠 . At the end point

there will be a sharp decrease in 𝐸𝑜𝑏𝑠 due to the sudden removal of all ferricyanide ions.
Experimental procedure:
13. Prepapre the following solutions:
d) 250 ml 0.01 M K3Fe(CN)6
e) 250 ml 0.01 M Co(NO3)2
f) Ammonium citrate solution: 25 g citric acid + 100 ml water + 67.5 ml liq.
ammonia
g) 250 ml 5N ammonia
h) Sodium thiosulphate and potassium dichromate solutions
i) Prepare or procure saturate KCl solution for salt bridge

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14. Standardize Na2S2O3 solution by potassium dichromate solution. Titrate

K3Fe(CN)6 with standardised Na2S2O3 and find out the strength of ferricyanide
solution.
15. In a 250 ml beaker take 20 ml of ferricyanide, 20 ml of ammonium citrate and 100
ml of 5 N ammonia. The mixture is covered with sufficient petroleum ether whose
function is to prevent aerial oxidation of Co(NH3 )2−
6 and to prevent evaporation of

NH3.
16. Setup the cell for the emf measurement.
17. Add cobaltous nitrate solution from a burette. Stir the solution and note down the
observed emf. Note: initially larger amounts (say 1 ml each time) may be added but
as the end-point is approached, readings are taken more frequently (0.1 or 0.05 ml)
and after the end point amounts in larger steps can be added.
18. Dilute the original cobaltous nitrate solution by de-ionized water so that its
concentration is exactly half of that of the original one. Repeat the above procedure
with this dilute solution.

Data and analysis

1. Prepare data table as given below

Experiment 1 Experiment 2
Volume of K3Fe(CN)6 = 20 ml Volume of K3Fe(CN)6 = 20 ml
Vol. of Co(II) Eobs (volt) Vol. of Co(II) Eobs (volt)
solution (ml) solution (mL)

2. Plot Eobs vs Vol. of Co(II) solution. Find out the inflection point to obtain the end
point of the titration. You may also calculate the first and second derivatives of
your data to find the inflection point.
3. Calculate the strength of the Co(II) solutions from the emf experiments and
compare the values with the indicator titration values.

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