Indian Institute of Science Education and Research Kolkata (IISER Kolkata)

Mohanpur, Nadia, West Bengal- 741246. www.iiserkol.ac.in

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 Contents
SI. No Experiments Page No Date of
the exp.
1 Determination of Iso-Electric Point of an 5-12
Amino acid.

2 Determination of the Degree of Hydrolysis 13-16

and the Hydrolysis Constant by
Potentiometry.

3 Determination of the pKIn Value of an Acid- 17-21

Base Indicator by Spectrophotometric
Method.

4 Determination of the Strength of a Solution 22-26

of a Strong Acid and a Strong Base by
Conductometric Titration.

5 Molecular Modelling of Organic/Inorganic 27-28

Molecules and basics of Electronic
Structure Theory
6 HOMO-LUMO Energy Optimization of a 29-30
Few Organic/Inorganic Molecules Using
Computational Calculation
Notes-Rough work 30-35

CH 1202

Semester II, April 2021 - July 2021

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GENERAL INSTRUCTIONS

1. Attendance is mandatory. In case of illness etc. the student must contact the
instructor and fix a schedule for making up the missed lab. All labs must be
completed in order to get a passing grade.
2. All data and results should be recorded directly in the lab notebook. The
recording should include, title of the experiment, date of experiment,
working formula, data in tabulated forms, results and calculations.
3. The instructor should sign the data before the student leaves the lab.
4. Graph papers and computer print-outs may be directly pasted on the lab
notebook.

Grading: (Total of 6 experiments:

The marking scheme in the lab will be as follows:

1. Lab notebook / submission of results … (8 Marks for each expt.) …..…. 48

2. Attendance …………………………………….... 12
3. Continuous assessment by teacher …………………………………......... 10
4. Final examination ……………………………………………..... 30

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Mandatory!!!

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 EXPERIMENT 1

DETERMINATION OF ISOELECTRIC POINT OF AN AMINO ACID

PRINCIPLE: Amino acids are molecules that contain both a base site (an -NH2 group) and an acid
site (a -COOH group). Individual amino acids differ only in the identity of the group, -R. On
dissolution of an amino acid in water, the proton from the -COOH group gets transferred to the -
NH2 end of the molecule as the NH2 group is a stronger base than -COO- resulting a zwitterion.
Depending on the pH of the solution the amino acids will be either in cationic form (low pH) or in
anionic form (high pH).

➢ The pH at which the presence of these two types of ions in the same concentration is called
the isoelectric point (pI). At this pH, the amino acid does not migrate in an electric field.
(gel electrophoresis)
➢ pI is the pH at which the amino acid is neutral, i.e. the zwitterion form is dominant.

INTRODUCTION:

HA(aq) + H2O = H3O+(aq) + A-(aq)

The extent of this reaction is indicated quantitatively using the equilibrium constant, Keq. The
equilibrium constant is given as

K eq = K a =
H O
3 (aq)A− (aq)
+
…………. (1)
HA(aq)
The equilibrium constant for reaction of an acid with water is usually symbolized as Ka to
remind us the type of reaction being dealt with.

➢ The concentration of water, since present in high concentration and thus essentially
a pure liquid, is not included in eq. (1).
➢ The strength of an acid in aqueous solution is defined in terms of the magnitude of
Ka. Strong acids have Ka values larger than 1 and that of weak acids is less than 1.
The equilibrium established when a weak acid reacts with water can be explored using the
following pH titration: The pH of the solution must change as the titration proceeds.

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 1. At the beginning of the process, before base is added, the pH of the solution is fairly low
because it contains acid.
2. As titration proceeds, acid is neutralized by the added base, and pH rises.
3. Addition of base after all of the acid has been neutralized produces a basic solution, with a
high pH.
4. The pH of the solution at each interval is monitored by a pH meter.
A plot of pH versus the volume of titrant added to the solution gives the so-called titration
curve.
➢ The curve is shaped like "S". All titration curves have this characteristic shapes.

This provides the method for determining the equivalence point:

We successively add small volumes of base, measure pH after each addition, and plot the titration
curve, from which we may find Vbase at the inflection point (the equivalence point).

Moles of acid in the original aliquot is calculated as : Moles of acid = Vbase at inflection point x M base

A derivative plot needs to be created to determine the pKa values accurately. The steps are as
follows:

1. Calculate pH/V from the collected pH data for each addition of the titrant.
2. Plot pH/V against V (volume of titrant added).
3. The plot gives sharp peaks at the equivalence points corresponding to the sharp jumps in
the titration plot.

In the case of amino acids, titration of the zwitterion with standard NaOH would provide the
Ka value for the -NH3+ acid, which is expected to be similar to that of NH4+ (pKa = 9.25). However, Ka

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value for the -COOH group could also be determined. It is possible to generate the acid form in
solution by adding a strong acid to the zwitterion. The strong acid transfers a proton to the -COO-
group of the zwitterion, resulting into a cation. Titration of a solution of this cation with standard
NaOH should then yield two equivalence points, one for each acid. It should thus be possible to
measure both the Ka values.

APPARATUS: pH meter, beaker, burette, pipette, glass rod, spatula.

REAGENT AND MATERIALS: Potassium hydrogen phthalate, glycine, alanine, HCl, NaOH,
phenolphthalein.

EXPERIMENTAL PROCEDURE:

i) Standardisation of NaOH solution

a) Prepare 100 mL 0.1 M KHP (Potassium hydrogen phthalate) solution.
b) Standardize the supplied ~0.1M NaOH solution against KHP solution using phenolphthalein
indicator (three results).
ii) Amino acid titration and estimation of equivalence point
a) Transfer exactly 10 mL of the supplied protonated amino acid solution to a clean 100 mL
beaker.
b) Add 15 mL of distilled water to the beaker so that the total volume of the amino acid solution is
25 mL.
c) To homogenize the solution, place the beaker on the top plate of a magnetic stirrer and place a
1-inch stir bar in the beaker. Rinse the pH electrode and submerge it in the solution containing
protonated amino acid. Make sure that the tip of the electrode is clear of the magnetic stir bar
in the beaker before starting the stirrer. The rotation rate should be reasonably fast, but not so
vigorous that splashing of the solution occurs.
d) Record the initial pH of the solution. Initiate the pH titration by adding 0.5 mL of NaOH solution
from burette.
e) On each addition of base solution, note the pH of the solution. Continue this addition until you
find larger gaps between two subsequent pH values. This indicates approach of the equivalence
point. Reduce the volume of addition of the alkali solution to 0.1 mL until you comfortably cross
the sudden jump in pH, indicating the equivalence point. After the equivalence point is passed,
increase each volume of addition to 0.5 mL. Repeat this process if you expect more than one
equivalence points.
f) Discard the solution on completion. Rinse the pH electrode with distilled water till pH meter
reading is approximately equal to that of distilled water. Leave the pH electrode in beaker of
distilled water and turn the meter off.
RESULTS:

Table 1. Preparation of 100 mL standard 0.1 N KHP solution

Weight taken (g) Weight to be taken (g) Strength of KHP solution

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Table 2. Standardization of NaOH solution using standard KHP solution

Sl. Volume of Burette reading (mL) Average Strength of NaOH

No. KHP (mL) volume (mL) solution
Initial Final Difference

Table 3. Titration of amino acid solution using standard NaOH solution

Volume of amino acid (mL) =

Sl. No. Volume of pH V (mL) pH pH/V

NaOH (mL)

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DISCUSSION:

Amino acids are more complicated than simple weak acids since amino acids have at least 2
ionizing groups. Glycine, for example, has both a carboxylic acid and an amino group that can
ionize: If we dissolve the free base of glycine in pure water (ie neutral pH), it will ionize by
protonating itself. The equilibrium is far to the right so most of the glycine is in the charged form
called the zwitterion and glycine is still neutral because the +ve charge is neutralized by the -ve
charge. Glycine is always in the zwitterion form at neutral pH.

Glycine

Now if we put Glycine at an acidic pH where it is fully protonated, we can titrate it to reveal its
both the pK values for the alpha-carboxylic acid group and the alpha-amino group.

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From the pK values, the pI (called the isoelectric point or the place where Glycine has no net
charge) can be calculated:
pI = (2.4 + 9.6)/2  6 ; (pK1 = 2.4, pK2 = 9.6)

Glycine is neutral at pH 6; it has no net charge here.

Some amino acids are classified as triprotic. This is because, in addition to the ionizable
protons of the -COOH and -NH3 groups, they also have a dissociable proton in their R group.
Although triprotic amino acids can exist as zwitterions, under physiological conditions these amino
acids will be charged. If the net charge under physiological conditions is negative, the amino acid is
classified as an acidic amino acid because the R group has a proton that dissociates at a pH
significantly below pH 7. The remaining triprotic amino acids are classified as basic amino acids due
to a) their having a net positive charge under physiological conditions and b) an R group dissociable
proton with a pKa near or greater than pH 7. Titration curves for triprotic amino acids generate the
same information as those for the diprotic amino acids. The pI for a triprotic amino acid can be
determined graphically, although this is somewhat more challenging.

Glutamic Acid Lysine

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 EXPERIMENT 2

Determination of the Degree of Hydrolysis and the

Hydrolysis Constant by Potentiometry

Working
Electrode Reference
Electrode

PRINCIPLE: A potentiometer is used to determine the difference between the potential

of two electrodes. The potential of one electrode—the working electrode—responds to the
analyte’s activity, and the other electrode—the reference electrode—has a known, fixed
potential.

INTRODUCTION:

Anilinium hydrochloride, C6H5NH3+Cl- when dissolved in water, ionizes to form C6H5NH3+

and Cl- ions, and the cation establishes the following hydrolytic equilibrium.
C6H5NH3+ + H2O C6H5NH2 + H3O+
The equilibrium constant for this hydrolytic process is called the hydrolysis constant for the
salt and is given by,

Kh = H
(
a +  aB )
a BH +
where, aH + is the activity of the free acid (H3O+); a B is the activity of the free base
(C6HsNH2)· and aBH + is the activity of the unhydrolysed salt (C6H5NH3+Cl-). However, for
dilute solutions, we may replace activities by concentration terms;
Hence,
Kh = [H+] [B ] / [BH+] (1)
Hydrolysis constant can also be related to the dissociation constant, Kb, of the base
through the ionic product of water, Kw as
Kh = Kw / Kb (2)
If c equivalents of the salt is dissolved in a litre of water, c equivalents each of free base
and free acid will be formed due to hydrolysis ( is the degree of hydrolysis). Thus, the pH
of the solution may be related to the degree of hydrolysis as,
pH = - log [H+] = - log (c)
Hence, by measuring the pH of the solution, c can be calculated from which the degree
of dissociation  can be obtained at a given concentration.

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 Also, expressing K, in terms , using Kh = c2/( 1 – ), the hydrolysis constant can be
calculated. Substituting for Kh in equation (2) and taking Kw = 1.0 x 10-14 at 25°C the
dissociation constant of the base, Kb can be evaluated.

Apparatus : Potentiometer, Platinum electrode and calomel electrode.

Chemicals : Anilinium hydrochloride, quinhydrone,

PROCEDURE:

➢ Prepare an N/10 aniline hydrochloride solution by dissolving appropriate quantity of

the substance in distilled water (100 mL).
➢ From this stock solution, dilute appropriately and get 25 mL of N/20, N/50 and N/100
solutions. Then construct the following cell:
➢ Transfer the 25 mL solution to 100 mL beaker; add a pinch of Quinhydrone, stir
properly to dissolve it, dip the electrodes (Pt and Calomel electrodes) in to the
solution.
Pt|0.1 M Aniline Hydrochloride, Quinydrone // Calomel
➢ Determine the potential of the cell. Repeat the experiment with each of the other
solutions.

RESULTS:

1. pH is given by pH = (-Eobs + EQH + Ecal) / 0.0591 where EQH = 0.6996 V and Ecal = -
0.242 V (Oxidation Potential). From this relation, pH of the solution can be
calculated.
2. As pH = - log [H+] = - log (c), pH = - log c - log , the degree of hydrolysis  can be
calculated at every given concentration.
3. From , calculate the hydrolysis constant using the relation, Kh = c2/(1 - )
4. The dissociation constant, Kb can be calculated from the relation Kb = Kw/Kh

C6H5NH3+Cl- Eobs(V) pH  Kh Kb
N/10
N/20
N/50
N/100

Mean Kh = - ... , Mean Kb = ...

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 EXPERIMENT 4

DETERMINATION OF THE pKIn VALUE OF AN ACID-BASE

INDICATOR BY SPECTROPHOTOMETRIC METHOD

PRINCIPLE: Spectrophotometric methods will be used to determine the acid dissociation

constant of an acid-base indicator (Bromocresol green), the light absorption characteristics of its
acid and base form. This experiment will provide you with opportunities to refine your
understanding of absorption process while providing an opportunity to apply many aspects of acid-
base chemistry.

INTRODUCTION: Acid–base indictaors are weak acids or bases having distinctly different
colours in acidic and alkaline solution, and by virtue of change of colour they indicate the end points
of acid-base titrations. To illustrate this point, consider the case for Bromocresol green (an organic
acid):

As shown above, this proton can be donated/or received to water to obtain a hydronium ion. If we
represent the acidic form of the bromocresol green as (HIn) and the conjugate base as (In-) then the
dissociation reaction looks like:

HIn ↔ H+ + In- H3O+ + In-

The acid dissociation constant (Equilibrium constant) can be represented as:

K In =
H In 
+ −

HIn
The strategy of this experiment is to adjust [H3O+] to known values using a buffer and then to
measure the ratio [In-]/[HIn] spectrophotometrically. Knowing this ratio and the value of [H3O+/pH]
will allow us to calculate Ka using the above equation.

➢ The trick then is knowing how to determine the ratio [In-]/[HIn] using light absorption
measurements .

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ABSORPTION SPECTRAL CHARACTERISTICS OF BROMOCRESOL GREEN:

The acid form of bromocresol green (HIn) absorbs light in a different region of the spectrum
than the basic form of bromocresol green (In-). From the figure below, you may see that the two
species have distinctly different values for λmax. Acid-base indicators are useful for determining pH
and indicating end-points because they change color as the pH of the solution changes. It is
important to note that the two solutions used to measure these absorption spectra have the same
concentration of the bromocresol green indicator. We want to select a wavelength that will allow us
to determine the relative concentration of each species present. One very poor choice occurs at
about 507 nm, because at this wavelength both the species absorbs equally well. The best choice is
the wavelength which has the largest difference in absorbance for the two species. This may be at >
565 nm where the acid form hardly absorbs light.

507 nm

THEORY: The ionization equilibria of a weak acid indicator (HIn) may be represented according
to,
HIn ↔ H+ + In- …………… (1)

Acidic form Alkaline form

for which the ionization constant (KIn) in dilute solution may be defined as the concentration
quotient (2)

K In =
H In 
+ −
………………… (2)
HIn
where, [ ]’s represent the molar concentrations of the respective species. Transforming the
equation (2) in logarithmic form one obtains,
[In - ]
pH = pKIn + log ……………… (3)
[HIn]
(where, pKIn = -log10 KIn and pH = -log10 [H+] in dilute solution).
Thus, if a fixed amount of the indicator is placed in the same volume of a series of buffer solutions of
different known pH values, the ratio, [In-]/[HIn], will increase with increase of pH. If the values of the
ratio at different pH are determined by measuring the colour intensity of the indicator solutions
then the pKIn value of the indicator can be found out if the pH of the buffer solutions is known.
If the alkaline form of the indicator (In-) absorbs at a selected wavelength and Beer’s law is
obeyed in the range of concentration of the indicator used, then the absorbance (A) of the indicator

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solution at a particular pH will be proportional to its concentration, provided the acid form (HIn) does
not absorb at this wavelength.
A =  [In-] l (4)
In a strongly alkaline solution, HIn is practically absent, and the absorbance (A) will correspond to the
total concentration (TIn) of the indicator.
A =  [TIn] l (5)
Where,  = molar extinction coefficient of In- and l = optical path length in cm.
Mass balance equation of the indicator is,
TIn = [HIn] + [In-] (6)

 [HIn] = TIn - [In-] (7)

( A - A )
From (5) – (4) one obtains, = [HIn] (8)
l
(A)
= [In-] (9)
l
Substituting these values of HIn and In- in equation (3) one obtains,

 A 
pH = pKIn + log10   (10)
 A - A 
A and A may be measured colourimetrically. Therefore, by plotting log10 [A/(A - A) ] against pH of
the buffer solutions a straight line of slope =1 will be obtained, of which the intercept on the pH axis
will give pKIn .

PROCEDURE:
You will be provided with ~0.5 N NaOH and~0.5 N acetic acid. Prepare 0.5 N oxalic acid (50
mL) and follow the procedure.
1. Standardisation of NaOH (~ 0.5 N) using oxalic acid. Then standardise the acetic acid.
2. Prepare 50 mL of exact 0.4 N acetic acid (pKH= 4.74 at 25oC) and 50 mL of exact 0.4 N NaOH
solutions separately by usual procedure.
3. Take 6 hard glass test tubes of uniform dimensions and label them from 1 to 6. Prepare the
following series of solutions by proper mixing (experimental pH values may be obtained from
chart below, or, may be determined using a pH meter).
Test Vol. of 0.4 N Volume of 0.4 N Volume of pH (Expt.) A A/(A’-A)
tube acetic acid (mL) NaOH (mL) Water (mL)
1 5.0 0.5 4.5 3.72
2 5.0 1.5 3.5 4.27
3 5.0 2.5 2.5 4.63
4 5.0 3.5 1.5 4.99
5 5.0 4.5 0.5 5.57
6 0 2.5 7.5 A’ =

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 4. Add a few drops of bromocresol green indicator to test tube number 6 using a dropper.
5. Set spectrophotometer at 570 nm, adjust the transmittance of water to 100%.
6. Measure the transmittance of the solution in test tube 6. If the transmittance is below 15%
(i.e. Absorbance is above 0.82), take test tube 7 and add fewer number drops of the
indicator to it and measure the transmittance. In this way by adjusting the number of drops
of the indicator, adjust the transmittance of the alkaline form between 25 to 15 %
(absorbance is above 0.60 but below 0.82) using test tube numbers 6 to 8 as required.
7. Add the same number of drops of the indicator as adjusted in step 5 to each of test tubes 1 –
5 and measure their transmittance.
8. Calculate the absorbance (A) values of solutions 1 - 5 and the absorbance (A) of the alkaline
solution of the indicator (6, 7 or 8) using the relation:
A = log (100/T %) = 2 – log T
9. Plot pH against log10 [A/(A - A)] and draw the best straight line of unit slope passing
through the experimental points, using the same scale for pH and log10 [A/(A - A)] axis. Find
pKIn from the intercept on the pH axis.

Table 1: Standardisation of NaOH

SI. Vol. of Oxalic Burette reading Avg. Strength

No acid (mL) Initial Final Diff. Vol(mL) of NaOH

Table 2: Standardisation of Acetic Acid

SI. Vol. of Acetic Burette reading Avg. Strength

No acid (mL) Initial Final Diff. Vol(mL) of Acetic
Acid

CONCLUSION: pKIn of bromocresol green is .............

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 EXPERIMENT 5

Determination of the Strength of a Solution of a Strong

Acid by Conductometric Titration.
PRINCIPLE:

The strength of a solution of a strong acid, namely, hydrochloric acid (HCl) will be
determined using a solution of a strong base, namely, sodium hydroxide (NaOH) from the change in
the conductance of the solution mixture.

INTRODUCTION:

Suppose an acid is taken in a beaker and NaOH solution is gradually added to it from a
burette. The reaction occurring during neutralisation is given by

HCl + NaOH → NaCl + H2O

Or, H+ + Cl- + Na+ + OH- → Na+ + Cl- + H2O

It is evident from the above equations that as NaOH solution is gradually added, the H+ ions,
having high ionic conductance, are replaced by Na+ having lower (ionic) conductance and hence the
conductivity of the solution in the beaker gradually decreases. At the equivalence point the
conductivity would be the minimum. After the equivalence point the Na+ and OH- ions will be
accumulated in the solution that increases the conductance of the solution. If the conductances
corresponding to the volume of the NaOH solution added be plotted, two straight lines having
opposite slopes will be obtained. The point of intersection of the two straight lines will give the
equivalence point.

The strength of the NaOH solution should be at least 5 times greater than that of the HCl
solution so that the effect of the volume change on the conductance be negligible.

APPARATUS:

Conductivity bridge, conductivity cell, beaker, burette, pipette.

CHEMICALS: Hydrochloric acid solution (0.05 N); Sodium hydrochloride solution (0.2 N); Oxalic
acid (0.1 N); Phenolphthalein indicator; Water.

EXPERIMENTAL PROCEDURE:

• Prepare 250 ml of approximately 0.2 N NaOH solution and standardise it by a standard

solution of 0.1 N oxalic acid using phenolphthalein indicator.
• Take NaOH in a burette.
• Take 25 ml of the supplied acid by a pipette into a 250 ml beaker and add 125 ml water to it.
• Place the conductivity cell in the beaker so that the electrodes are completely immersed in
the acid solution.
• Connect the cell to the conductivity bridge and measure the conductance of the solution.

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 • Add NaOH solution from the burette 0.5 ml at a time in the beginning, 0.2 ml at a time near
the end point point and again 0.1 ml at a time after the end point.
• Measure the conductance of the solution after each addition of the NaOH solution.
• Plot the conductance values against the corresponding titre values, draw the straight lines
and obtain the point of intersection.

RESULTS:

Strength of NaOH solution S1 = N

Volume of the supplied acid solution taken V2 = 25 ml

Table 1. Titration of the supplied acid with the standardised NaOH solution.

Volume of NaOH Observed Volume of NaOH Observed

solution added (ml) conductance (ohm-1) solution added (ml) conductance (ohm-1)

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GRAPH AND CALCULATIONS:

Let the point of intersection of the straight lines obtained by plotting conductances against
the titre values correspond to V1 ml. This gives the volume of NaOH solution required to neutralise
25 ml (V2) of the acid. Let S2 be the strength of the supplied acid solution.

V1S1 = 25 x S2

S2 = V1S1/25 (N)

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 EXPERIMENT 9

Molecular Modelling of a Few Organic/Inorganic Molecules

Using Computational Calculation

This experiment will give students an idea of visualizing molecules and show how to obtain
optimized ground state structures of these molecules. A very basic theoretical knowledge will be
provided before the hands on session.

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 EXPERIMENT 10

HOMO-LUMO Energy Optimization of a Few Organic/

Inorganic Molecules Using Computational Calculation

This experiment will discuss very brief what are the levels of theory available in Modern
Quantum Chemistry Package. As such discussion needs knowledge of advanced quantum
chemistry mostly we will discuss very elementary quantum chemistry. We will show how
Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital
(LUMO) energy levels can be calculated for some molecules.

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