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Index

S.No.

Name of the experiment

Page No.

1.

Reaction kinetics - Decomposition of benzene-diazonium

chloride
2.

Enzyme catalysis using UV-Vis spectrophotometer

3.

Kinetics of iodination of acetone

4.

Determination of pKa using UV-Vis spectrophotometry

5.

Heat of solution by solubility measurement

10

6.

Estimation of excess thermodynamic properties

12

7.

Estimation of thermodynamic functions from EMF data

15

8.

Determination of stability constant of silver-ammonia

17

complex
9.

Electrochemical oxidation of L-cystine to L-cysteic acid

19

10.

Verification of Beer-Lambert Law using gold nanoparticles

21

11.

Estimation of free energy of protein denaturation using


intrinsic fluorescence of protein
Fluorescence quenching of fluorescein by inorganic anions:
Determination of quenching constants and estimation of free
energy change and activation energy for the quenching process
Critical micelle concentration of CTAB

25

Acidity determination in zeolites by temperature programmed


desorption (TPD) of ammonia

31

12.
13.
14.

27

29

1. REACTION KINETICS - DECOMPOSITION OF BENZENEDIAZONIUM CHLORIDE


1.1 Aim
To determine the first order rate constant for the decomposition of benzene-diazonium chloride.
1.2 Chemicals required
Aniline, conc. HCl, sodium nitrite (NaNO2)
1.3 Apparatus required
5 mL measuring flask, 200 mL standard flask, ice water, stop watch and dropping funnels
1.4 Principle
Guggenheim method is useful to obtain the first order rate constant when the initial amount of
reactant cannot be determined by employing a physical method such as measuring the change in
pressure or volume in the course of a reaction. In this method a series of readings of the
concentration of product (reading Y) at times t that are spread over an interval which is two to
three times the half life of the reaction. A second series of readings (Y) can also be made, each
at time (t+ ), where is an exactly constant interval after the time of the corresponding reading
Y. The period must be at least two to three times the half life of the reaction.
Integrated first order rate law:

( )

We can also write

ln (

)=

When benzene diazonium chloride (BDC) is heated in acidic aqueous solution it decomposes,
following first order kinetics, to yield phenol, HCl and N2. The rate constant is determined as a
function of temperature so that the activation energy for the reaction can be calculated.
NaNO2 + HCl HNO2 + NaCl
The nitrous acid formed is reacted with aniline to give benzenediazonium chloride:
C6H5NH2 + HNO2 + HCl [C6H5N+N][Cl]
1.5 Procedure
1. Dissolve 1.3 mL of aniline in 5 mL of conc. HCl
2. Cool the solution in an ice bath.
3. Prepare a solution of sodium nitrite by dissolving 2 g of the salt in ~ 30 mL of water.
4. Add slowly with stirring the sodium nitrite solution to the aniline solution maintaining the
temperature at 0 C.
2

5. Dilute the resulting solution to 200 mL with ice cold water.


6. Transfer 50 mL of solution to the reaction vessel and put it into the constant temperature bath
maintained at 45 C.
7. After 10 minutes connect the T- tube into the burette filled with water. When water level in
the burette is zero note this initial reading as Vo. Start the stop watch.
8. Take a series of 40 readings, Vi------Vn, at times ti-------tn at fixed intervals of time (30 sec).
9. Choose a time interval (t) such that it is about 2-3 times the time of the half reaction. For
every Vt at time t note the corresponding Vt+t at t+t as in table below
S. No.

Time
(t) sec

Volume
(vt) mL

S. No.

Time
(t+ t) sec

Volume
(Vt+t) mL

Log (Vt+t Vt)

10. Plot Log (Vt+t Vt) versus t. Find the slope for the straight line. 2.303 slope = -k

Log10 (V1-V)

Table and graphs:

Time (t)

Result
The first order rate constant of the decomposition of benzene diazonium chloride is k = 0.961 x
104 sec-1 (literature value)
References
1. P.W. Atkins, Physical Chemistry, 6th ed. pp. 767-769 (1998);
2. B. Viswanathan and P.S. Ragavan, Practical physical chemistry, pp. 132-134 (2005)

2. ENZYME CATALYSIS USING UV-VIS SPECTROPHOTOMETER


2.1 Aim
To verify MichaelisMenten equation
2.2 Chemicals required
100 mL of 0.1 % Starch, 100 mL of 0.0006 N Iodine, enzyme (10 mg - amylase in 100 mL
water)
2.3 Apparatus required
UV-Visible spectrophotometer, 12 boiling test tubes, four 50 mL burettes
2.4 Principle
Michaelis Menten Mechanism
k1
k2

ES
E + S
E + product
k 1

Initial velocity, V0 =

Vmax [S]
K M +[S]

where Vmax = k 2 [E]0 ; K M

K 1
1
1
=
+ M
V0 Vmax Vmax S

k1 k2
. k 2 is called turnover number
k1

2.5 Procedure
1. Prepare stock solutions of 100 mL 0.1% starch solution, 100 mL 0.0006 N Iodine and
enzyme (10 mg - amylase in 100 mL water). Fill them in 50 mL burettes.
2. Take 12 boiling test tubes and prepare the following solutions
Set 1: Blank solutions
S.No.
0.1 % starch (mL) Iodine (mL) Water (mL) Conc of starch %
1
3
1
6
0.03
2
4
1
5
0.04
3
5
1
4
0.05
4
6
1
3
0.06
5
7
1
2
0.07
6
8
1
1
0.08
Set 2: Test solutions to which - amylase is to be added while starting the reaction
S.No.
7
8
9
10
11
12

0.1 % starch (mL)


3
4
5
6
7
8

Iodine (mL)
1
1
1
1
1
1
4

Water (mL)
5
4
3
2
1
0

Conc of starch %
0.03
0.04
0.05
0.06
0.07
0.08

3. Set the spectrophotometer at 590 nm and take absorbances of first six solutions (Set 1).
4. To the 7th solution (Set 2) add 1 mL of enzyme solution while starting the stop watch. Mix
solution well transfer into a cuvette. Monitor the reaction for about 3 min by measuring the
absorbance at every 10 sec.
5. Repeat the step 4 with solutions 8. 9. , 12. Tabulate the reading as follows
S. No.

Time
sec
Blank
10
20
30

0.03
%

0.04
%

0.05
%

0.06
%

0.07
%

0.08
%

180

1/V

6. Plot absorbance of iodine versus time. Obtain the initial rate [V]0 for every concentration of
starch, [S]
7. Plot [V]0 versus [S] and verify the Michaelis-Menten equation.
8. Plot 1/ [V]0 versus 1/[S] and obtain the MichaelisMenten constant KM, according to the
linear equation given above.

[S]

1/[S]
Results
1. Michaelis-Menten equation is verified.
2. The Michaelis-Menten constant KM is
References
1. K. J. Laidler, Chemical Kinetics, 5th edition.
2. P.W. Atkins, Physical Chemistry 6th edition (1998).

3. KINETICS OF IODINATION OF ACETONE


3.1 Aim
To study the kinetics of iodination of acetone using a UV-visible spectrophotometer
3.2 Chemicals required
Iodine (0.005 M), 0.5 M acetone and 0.5 M HCl
3.3 Apparatus required
UV-Visible spectrophotometer, 25 mL standard flasks (6), 5 mL, 10 mL, 25 mL burettes, 5 mL
pipette
3.4 Principle
The experiment is conducted with a view to study the kinetics of iodination of acetone. The
balanced equation cannot predict the rate law. The rate law can only be obtained by experimental
determination. The form of rate law would help to reach conclusions about the mechanism of the
reaction.
The chemical equation for this reaction is (CH3)2CO + I2 CH3-CO-CH2I + I +H+
The reaction rate increases with the concentration of H+ in acid solutions and with OH
concentration in basic solutions. The rate of halogenation of acetone is independent of the
concentration of the halogen except at very high acid concentration.
3.5 Procedure
1. Suitable concentrations of iodine, acetone and HCl solutions are prepared (0.5 M each)
2. Different compositions of the reaction mixtures are prepared in 25 mL standard flasks as
shown in the chart below. Iodine, water and acetone are taken in 25 mL standard flask. HCl
should be added only at the time of starting the reaction.
S. No

1
2
3
4
5

Volume of
I2
(mL)
10
5
7.5
7.5
7.5

Volume of
HCl
(mL)
1.25
1.25
2.5
2.5
0.5

Volume of
H2 O
(mL)
1.25
6.25
2.5
12.5
4.5

Volume of
Acetone
(mL)
12.5
12.5
12.5
2.5
12.5

3. When half of the HCl has been added, start the stopwatch. Mix the solution well and transfer
to the cuvette and place in the UV-Vis spectrophotometer. Measure the absorbance at 450 nm
at various intervals of time for 15 20 minutes.

3.6 Calculations
1. In each case absorbance is plotted as a function of time.
2. From the plot of optical density versus time, the initial slope is obtained which gives the
initial rate of the reaction.
3. From (1) and (2) the order with respect to iodine is determined.
4. From (3) and (4) the order with respect to acetone is determined.
5. From (3) and (5) the order with respect to acid can be found.
Table and graphs:
Time
Absorbance

Absorbance

Time

Results
1. The order with respect to iodine is
2. The order with respect to acetone is
3. The order with respect to acid is

References
1. S.W. Benson, The Foundations of Chemical Kinetics, McGraw-Hill, pp 569-573, (1960).
2. A.R. Knight, Introductory Physical Chemistry, Prentice-Hall, Chapter 6, (1970).
3. J.L. Latham, Elementary Reaction Kinetics, Butterworths (1964).
4. G.M. Harris, Chemical Kinetics, D.C.Heatch and Company (1966).

4. DETERMINATION OF pKa USING UV-Vis SPECTROPHOTOMETRY


4.1 Aim
To determine pKa of a weak organic acid or a base using UV-Vis spectrophotometry
4.2 Chemicals required
Methyl red, phosphoric acid, sodium hydroxide, distilled water and methanol.
4.3 Apparatus required
Scanning UV-Vis spectrophotometer, pH meter and 10 mL, 5 mL, 2 mL pipettes.
4.4 Principle
The protonated and deprotonated form of a weak organic acid or base often have distinctly
different electronic spectra. Since the absorbance of a prototropic species is proportional to its
concentration, the ratio of the concentration of the acid and base forms can be conveniently
monitored as a function of pH of the medium. Using Henderson-Hasselbach equation,
pH = pKa + log{[base form]/[acid form]}
A plot of log {[base form]/[acid form]} against pH gives pKa as the y-intercept.
4.5 Procedure
1. Prepare 100 mL of 0.001 N H3PO4 and 10 mL of 0.1 N NaOH.
2. Prepare a series of phosphate buffer solutions ( approximately 10 mL) using 0.1 N NaOH as
the adjusting parameter to the desired pH range of pH 3, 4, 4.5, 5.5, 6, 6.5, 7, 8, 10.
3. Make a stock solution of the test compound (10-3 M Methyl Red) in 30 % MeOH/H2O (v/v)
of 25 mL so that the longest wavelength absorption maximum has A 2.
4. Add 1 mL of this stock solution to 9 mL of each buffer solution.
5. Take the absorption spectra. Identify the isobestic point and the suitable wavelengths
corresponding to the acid form (pH 3) as well as the base form (pH 10). You will find the
spectra change significantly from one form to another over a narrow pH range.
6. Obtain the absorbance values corresponding to the two prototropic forms at the two selected
wavelengths (step 5).
7. Use Henderson-Hasselbach equation and plot log [Abase/Aacid] against pH and get pKa from
y-intercept.

Table and graph


pH

Abase

Aacid

Abase/Aacid

Log[Abase/Aacid]

Log A

base

/A

acid

S. No.

pH

Results
The pKa value of the given weak acid is 5.0 (literature value)
(i) Calculated
(ii) From graph
Reference
1. Hideo Yamazaki, R. P. Sperline, and Henry Freiser, Analytical Chemistry, Vol.64, 27202725 (1992).

5. HEAT OF SOLUTION BY SOLUBILITY MEASUREMENT


5.1 Aim
To determine H, G, S of solution for potassium dichromate by solubility measurements
5.2 Chemicals required
Starch, K2Cr2O7, KI, H2SO4 (2 N), sodium thiosulphate
5.3 Glassware Required
Pipette, 20 mL measuring cylinder, conical flask, weighing bottle, beaker and thermometer
5.4 Reactions involved
K2Cr2O7 + 6KI + 7H2SO4 Cr2(SO4)3 + 4K2SO4 + 7H2O + 3I2
I2 + 2Na2S2O3 2NaI + Na2S4O6
5.5 Principle
Solubility of substances, in general, increases with temperature. There are a few exceptions to
this observation. In all cases, the effect of temperature on the solubility of a substance can be
predicted from its heat of solution if no chemical reaction occurs during dissolution.
Le Chateliers principle indicates that the substances which absorb heat during dissolution
process (H is positive), dissolve to a greater extent at higher temperature. In the same
way it can be shown that the substances, which evolve heat (H is negative) during dissolution,
dissolve to a greater extent at low temperatures.
From thermodynamics, the following relationship can be derived

where S1 and S2 are the solubilities of the substances in 100 g of water at temperatures T1 and T2
in K and R is the gas constant in JK-1mol-1. T2 is greater than T1.
5.6 Procedure
Prepare saturated solutions of potassium dichromate around 20 C, 30 C (room
temperature) and 40 C by keeping in the ice cold water bath and hot water bath as the case may
be. Keep them immersed in bath for around 5 minutes for equilibrium. Note down the room
temperature.
Quickly pipette out 1 mL of the saturated solution at 20 oC and deliver into a weighing
bottle previously weighed. Allow sometime for the solution to attain the room temperature.
Wipe out the outside by means of filter paper gently to remove the condensed moisture and
weigh again. From these two weights, calculate the density of the saturated solution at 20 C.
By the same procedure, find out the density of the saturated solution of K2Cr2O7 at 40 C and at
the room temperature.
10

Solubility determinations
1. Pipette out 1 mL of saturated solution of K2Cr2O7 at 20 C into a conical flask.
2. Add about 15 mL of 2 N H2SO4 and a little solid KI.
3. Titrate the liberated I2 against standard 0.2 N thiosulphate solution using starch as indicator.
4. From the normality of potassium dichromate solution calculate the weight of K2Cr2O7 in 1
mL of the solution.
5. Let it be X. If X1 is the weight of 1 mL of the saturated solution at 20 C the solubility S1
at 20 C is given by
(
)
6. Calculate the solubility S2 of K2Cr2O7 at 30 C and S3 40 C in a similar fashion.
7. From the equations described earlier, calculate the H, G, S of solution, using the
solubilitys at 20 C, 30 C and 40 C.

Results
Solubility (in gm/ 100 gm water) of potassium dichromate at 20C, 30C and 40C respectively
are 12.3, 18.1, 26.3
The H, G, S of solution are

Reference
1. F. Daniels and R.A. Alberty, Physical Chemistry, Wiley pub., 3rd Ed. (1996)

11

6. ESTIMATION OF EXCESS THERMODYNAMIC PROPERTIES


6.1 Aim
Estimation of excess thermodynamic properties of binary liquid mixture from density and
ultrasonic velocity measurements
6.2 Chemicals required
Benzene and cyclohexane
6.3 Glassware required
Beaker, Pipette (5 mL, 10 mL) and pycnometer
6.4 Principle
Raoult's Law for the mixture of two volatile liquids states that the total vapour pressure of
the mixture is equal to the sum of the individual partial pressures for ideal solution.
Total Vapour Pressure = PA + PB
Many pairs of liquids are present in which there is no uniformity of attractive forces i.e.
the adhesive & cohesive forces of attraction are not uniform between the two liquids, so that they
show deviation from the Raoult's law.
Deviation from ideal behaviour: When adhesive forces between molecules of A & B are greater
than the cohesive forces between A & A or B & B, then the vapor pressure of the solution is less
than the expected vapor pressure from Raoult's law. This is called as negative deviation from
Raoult's law. e.g. chloroform and acetone show such an attraction by formation of a hydrogen
bond.
When the cohesive forces between like molecules are greater than the adhesive forces,
the dissimilarities of polarity or internal pressure will lead both components to escape solution
more easily. Therefore, the vapor pressure will be greater than the expected from the Raoult's
law, showing positive deviation. If the deviation is large, then the vapor pressure curve will show
a maximum at a particular composition, e.g. benzene & ethyl alcohol, carbon disulfide &
acetone, chloroform & ethanol.
Excess thermodynamic properties: Knowledge of excess thermodynamic properties of the multicomponent systems is essential for the design calculations involving separations, heat transfer,
mass transfer, and fluid flow. Further, these properties have relevance in the theoretical research
to get the insight into intermolecular forces and microscopic structure of liquids.
The excess molar volumes and excess isentropic compressibility are the properties very
sensitive to different kinds of association in the pure components and in the mixtures and often
they are related to local order. They have been used to investigate the molecular packing,
molecular motion and various types of intermolecular interactions and their strength, influenced
by the size, shape and the chemical nature of component molecules.
6.5 Procedure
1. Find the weight of the dry pycnometer.
2. Prepare 10 mL binary mixture of benzene and cyclohexane with 0.2, 0.4, 0.6 and 0.8 mole
fractions by narrow-mouth specific gravity bottles / pycnometer taking due precautions to
minimize the evaporation loses.

12

3. For preparation of binary, measure the volume as specified in table below. As exact volume
is difficult to measure so after transferring the required volume (v1, v2) measure the
corresponding weight (w1, w2)
4. The table given below is of standard value and is merely to guide you the amount of volume
you have to take. Make your own table as below only excluding v1, v2 column.

Benzene + cyclohexane mixture


S.No.

x1

x2

v1

v2

w1

w2

0.0

1.0

0.0

10.0

0.000

7.790

0.2

0.8

1.7

8.3

1.498

6.458

0.4

0.6

3.5

6.5

3.110

5.026

0.6

0.4

5.5

4.5

4.847

3.482

0.8

0.2

7.7

2.3

6.726

1.812

1.0

0.0

10.0

0.0

8.765

0.000

28.4

31.6

24.946

24.568

5. Calculate X1 and X2 by the given formula


X1 =

X2 = 1-X1

Where wi, Mi are weight and molar weight respectively


6. Calculate density by weighing the pycnometer with sample for each solution.
7. Measure u for each binary solution and pure component with the help of Ultrasonic
interferometer.
8. Measure ultrasonic velocity of two pure liquids and four binary mixtures by using the formula u
= where is wavelength and is frequency of ultrasonic wave (both and are
obtained from interferometer)
9. Repeat the procedure number 6, 7 and 8 at 50 oC using water bath
10. From the density and ultrasonic velocity data of two pure liquids and four mixtures calculate the
excess thermodynamic properties using following equations:

Excess molar volume:

Where xi, Mi and i represents mole fraction, molar weight and density of ith component in
mixtures respectively
Isentropic Compressibility: or S= 1/ (.u 2)
Excess Isentropic Compressibility (E or SE):

13

Vi, i and Cp,i are the molar volume, isobaric thermal expansion coefficient and molar isobaric
heat capacity respectively of pure component i and i = xiVi/xjVj is the volume fraction of i in
the mixture, stated in terms of the unmixed components.
7. Draw the graph between and T for each solution and find out the slope. Calculate i by
dividing the slope with density as given by the equation

= Results
Plot of excess molar volume and excess isentropic compressibility of binary mixture versus
mole fraction of component A are shown
References
1. S.L. Oswal, M.M. Maisuria1, R.L. Gardas , Journal of Molecular Liquids, vol.109, 155166
(2004).
2. Rowlinson, and F.L. Swinton, Liquid and Liquid Mixtures, 3rd Ed. Butterworth, London,
(1981).

14

7. ESTIMATION OF THERMODYNAMIC FUNCTIONS FROM EMF


DATA
7.1 Aim
I.
To determine the standard electrode potential of Cu2+/Cu half cell.
II.
To calculate the thermodynamic functions G, S, H.
7.2 Chemicals required
Copper sulphate, distilled water
7.3 Apparatus required
Copper electrode, 100 mL standard flask, 25 mL standard flask (4), 50 mL beaker, salt bridge,
calomel electrode, potentiometer, water bath and thermometer
7.4 Principle
The electrode potential is defined as the tendency of the electrode to loose or gain electrons.
Here the potential of the electrode is determined using a reference electrode (calomel electrode).
When a chemical reaction is carried out reversibly and isothermally in a galvanic cell, the
decrease in free energy is the net electrical work done by the system ie G = nFE
(joules/mole) where n is the number of electrons involved in the redox reaction, F is the faraday
constant (96497 coulombs/equivalent) and E is the reversible EMF of the cell.
7.5 Procedure
1. Prepare 25 mL of each 0.1 M, 0.05 M 0.01 M 0.005 M of copper sulphate solution.
2. For determining the standard electrode potential, set up a galvanic cell by dipping a copper
electrode into 0.005 M solution of copper sulphate solution taken in a beaker and combine
this with the reference electrode (calomel electrode) through salt bridge.
3. Connect the positive end of the potentiometer to the copper electrode and negative end of the
potentiometer to the calomel electrode.
4. Switch on the potentiometer and note the reading.
5. The cell is placed in the water bath and the EMF is measured at different temperatures (20
C, 25 C (RT) and 30 oC)
6. Repeat the experiment with each of the above indicated concentration of copper sulphate.
7.6 Calculations
Write the half cell representations of the both the half cells.
Calculate the standard electrode potential of copper electrode from the measured EMF values,
molarity of copper sulphate.
Eobs = E1 E2
E2= EMF of the standard calomel electrode (0.2422 V vs SHE) and E0 can be calculated from

is the activity of copper ions (use concentration in place of activity for calculation
purpose).

15

From the potential data calculate the G, S, H from the below equations.
G = nFE1
(

H = G +T S
Results
1.
Concentration of copper
sulphate (M)
0.1
0.05
0.01
0.005

EMF of the concentration cell


taken at (temp)

EMF of the concentration cell


taken at (temp)

2. Values of G, S, H (for any two concentrations)


Concentration of
H (kJ mol-1)
S (J K-1 mol-1)
copper sulphate (M)
0.1
0.01

References
1. D. E. Smith, J. Chemical Education, vol.60, 1983, 299.
2. P. A. Rock, J. Chemical Education, vol.52, 1975, 787.
3. C. A. Vincent J. Chemical Education, vol.47, 1970, 365.

16

G (kJ mol-1)

8. DETERMINATION OF STABILITY CONSTANT OF SILVER AMMONIA COMPLEX


8.1 Aim
To determine the stability constant of silver- ammonia complex according to the reaction
Ag+ + 2 NH3 [Ag(NH3)2]+
8.2 Chemicals required

0.1 N AgNO3 solution, 4 N ammonia solution


8.3 Apparatus required
50 mL beaker, silver electrode, salt bridge, 100 mL standard flask, 50 mL standard flasks (5),
potentiometer.
8.4 Principle
The determination of stability constant of a complex is based upon the determination of the
activity of the metal ion in a dilute solution containing the complex. The determination of the
metal ion activity is as usual done by constructing a suitable galvanic or concentration cell. The
stability constant of the equilibrium Ag+ + 2 NH3 [Ag(NH3)2]+ is given by
Where x = conc. of complex at equilibrium
a = initial conc. of AgNO3
b = initial conc. of ammonia
Using the Nernst equation, from the conc. cell we will obtain conc. of free ions in the complex
solution i.e., C1. Therefore
Substituting x in eqn1 we get
(

8.5 Procedure
1. The following concentration cell is set up
Ag| 0.025 N AgNO3 in 1 N NH3 || KNO3 (sat) || 0.01 N AgNO3 | Ag
Where in the pure 0.01 N AgNO3 is taken as reference.
2. Two silver electrodes are dipped in the respective half cells and the silver electrode placed in
the solution of the complex is connected to the negative end of the potentiometer while the
silver electrode is placed in silver nitrate solution is connected to the positive end of the
potentiometer. The voltage reading is noted.
3. The experiment is repeated with different concentrations of AgNO3. (i) 0.025 N in 1 N NH3
(ii) 0.0125 N AgNO3 in 1 N NH3 (iii) 0.006 N AgNO3 in 1 N NH3 (iv) 0.0125 N AgNO3 in 2
N NH3 (v) 0.0125 N AgNO3 in 1.5 N NH3.
8.6 Calculations
The observed EMF of the cell is given by the equation

17

}=

From eqn3, C1 i.e., the conc. of free Ag+ ions in the complex equilibrium, is calculated.
Substituting a, b, C1 in eqn2 we obtain the equilibrium constant K.
For Example in (i) 0.025 N AgNO3 in 1 N NH3; a = 0.025, b = 1, and C1 is given from the eqn3
substituting these in eqn2 we get the equilibrium constant K.
Result
The stability constant of the silver ammonia complex is 1.67 x107 (literature value)
References
1. V. D. Athawale, Experimental Physical Chemistry, New Age International Ltd, page 208.
2. R. W. Ramette, J. Chemical Education vol.49, 1972, 423.

18

9. ELECTROCHEMICAL OXIDATION OF L-CYSTINE TO LCYSTEIC ACID


9.1 Aim
To determine the mechanism of electrochemical oxidation of L-cystine to L-cysteic acid and
hence to find the diffusion coefficient of Br- ion by using Randels-Sevcik equation
9.2 Chemicals required
10 mM L-cystine, 10 mM HBr, 0.5 M H2SO4
9.3 Apparatus required
Electrochemical cell, glassy carbon electrode, platinum electrode and Calomel electrode
9.4 Principle
The oxidation of L-Cystine to L-Cysteic acid is performed by electrochemically generated
bromine using HBr solution.

The effect of peak current on scan rate and concentration in cyclic voltammetry is given by
Randels-Sevcik equation which is given by

Where ip= peak current (A), n= no of electrons, A= area of the electrode (cm2), D- diffusion
coefficient (cm2s-1), C- concentration of L-Cystine (mol cm-3), -scan rate (Vs-1) .
9.5 Procedure
1. The electrochemical cell and the three electrodes are rinsed with distilled water.
2. 10 mL of 0.5 M sulphuric acid is taken in the cell. The electrode is immersed in the solution
and connected to the potentiostat.
3. The Cyclic Voltammetry is run with the following parameters
a. Initial potential 0.0 V and final potential 1.8 V vs SCE.
b. The current range is assigned in the milliampere range.
c. Scan rate is chosen as 0.05 V/s.
4. The CV is started by giving the run command. The data is then stored.
5. To the 10 mL of 0.5 M sulphuric acid add 10 mM in 10 mL of L-cystine. Record CV with the
above given parameters.
6. In another cell again 10 mL of 0.5 M sulphuric acid is taken and 10 mM in 10 mL of HBr is
added. Record the CV with the same parameters. Save the recorded CV
7. To the solution of step 6 add 10 mM in 10 mL L-cystine and run the experiment.
8. Repeat the experiment with different scan rates.
19

9. Repeat the steps 6 to 8.


10. Record the CV with different concentrations of L-cystine from 10 mM to 50 mM.
11. Save all the recorded CVs.
9.6 Calculations
1. A graph is drawn by taking concentrations of L-Cystine on the x-axis and peak current of
different concentration of L-cystine.
2. A plot of peak current of L-Cystine and square root of scan rates is made.
3. From the slopes calculate the diffusion coefficient using Randels-Sevcik equation

The typical cyclic voltammogram recorded looks like the following

Figure1. cyclic Voltammograms of L-Cystine oxidation at glassy carbon electrode surface


(a) 0.5 M H2SO4, (b) 10 mM of L-Cystine in 0.5 M H 2SO4, (c) 10 mM of HBr in 0.5 M
H2SO4 and (d) 10mM of HBr in 0.5 M H 2SO4 with different concentrations of L-Cystine
from 10 mM to 50 mM.
Results
1. The electrochemical oxidation of L-cystine is performed and the mechanism is elucidated.
2. The linearity of the obtained from the two graphs verifies the Randels-sevcik equation.
3. Diffusion coefficient obtained from Randels-Sevcik equation is 2.08 cm-2sec-1 (literature)
References
1. Wang X, Zhao, Chemistry Letters, vol.33, 2004, 332.
2. G. Sanchez-cano, V. Montiel, A. Aldaz, Tetrahedron, vol.47,1991, 877.
20

10. VERIFICATION OF BEER-LAMBERT LAW USING GOLD


NANOPARTICLES
10.1 Aim
I.
Preparation of gold nano particles using the citrate method and verification of BeerLambert law.
II.
Preparation of gold nanorods of different aspect ratios and to determine the effect of
concentration of NaBH4 on the position of LSP.
10.2 Chemicals required
Tetrachloroauric acid trihydrate (HAuCl43H2O), sodium borohydride (NaBH4), silver nitrate,
cetyltrimethylammonium bromide (CTAB), ascorbic acid, trisodium citrate (Na3C6H5O7)
10.3 Apparatus required
50 mL flat bottomed flask, 5 mL standard flasks (10), 50 mL, 100 mL, 250 mL standard flasks, 1
mL, 100 L pipettes, 25 mL measuring jar, heating mantle and cuvettes
10.4 Principle
The Beer-Lambert law, also known as the Beer's law or the Beer-Lambert-Bouguer, is the
linear relation between the absorbance and concentration of an absorbing species. According to
this law, when light passes though a material, there exists a linear dependence between the
absorbance (A) of light and the product of absorption coefficient () with the path length (l) of
the light (distance though which light travels in the material). Absorption coefficient of a
material in turn depends on the molar absorptivity or molar extinction coefficient or molar
absorption coefficient () and the concentration (c) of the absorbing species in the material. Thus,
according to Beer-Lambert law, absorbance is given by,
where is constant, for a given wavelength of light, and can be obtained from the absorbance
values of solutions with different concentrations. Absorbance is unitless, while the units of , l, c
are L.mol-1.cm-1, mol.L-1 and cm-1, respectively.

parent AuNPs
4 mL AuNPs + 1 mL water
3 mL AuNPs + 2 mL water
2 mL AuNPs + 3 mL water
1 mL AuNPs + 4 mL water

Absorbance

1.2
0.9

0.6
0.3

500

600

Absorbance (a.u.)

Appearance of wine-red color in step 6 indicates the formation of colloidal gold or gold
nanoparticles. The recorded UV-Vis spectrum of the solution (typically 400-1100 nm window),
in step 10, shows the absorbance maximum at a wavelength (max) of ~520 nm, as shown in Fig.
1a. These features are attributed to the plasmon resonance of gold nanoparticles. The intensity of
the peak is reduced with increase in dilution.
1.2

0.9

B
B

0.6
0.3
500

700
-1

600

700
-1

Wavelength (cm )

Wavelength (cm )

21

Fig.1a. UV-Vis spectra of gold nanoparticles of different dilutions. Fig.1b. Plot of absorbance vs.
concentration of gold nanoparticles
Fig. 1b shows the plot of absorbance vs. concentration for the gold nanoparticles of different
dilutions. It can be observed that all the data points fit to a straight line, thus conforming to the
Beer-Lambert law. Slope of the above plot gives the absorption coefficient of the gold
nanoparticles. For the calculation of absorption coefficient, path length is being taken as equal to
the width of the cuvette used for recording the UV-Vis spectrum (we have taken it as 1 cm).
From the above data, the value of absorption coefficient is calculated as ~3.36 L.mol-1.cm-1. By
making use of the above plot, we can calculate the concentration of gold nanoparticle of
unknown dilution.
Nanorods belong to the class of non-spherical or anisotropic nanoparticles. Such particles
require more than one parameter to describe them, such as length and width in the case of rod
shaped particles. Most of the synthesized nanorods are cylindrical in shape. They are
characterized by their aspect ratio which is the ratio of the major dimension to the minor
dimension. Nanorods are often classified as 1-D nanostructures and have less aspect ratio than
other class of 1-D nanostructures (e.g., nanowires or nanotubes). Size of a nanorod ranges within
1-100 nm and aspect ratio of a nanorod is greater than 1 but less than 20, with typical values
between 3 and 5. Gold nanorods belong to an important class of anisotropic nanomaterials.
Chemical methods to synthesize gold nanorods are very recent.
Absorption spectrum of gold nanorod is characterized by two surface plasmons. They are
transverse surface plasmon (TSP) resonance and longitudinal surface plasmon (LSP) resonance.
TSP of a nanorod is observed in the range of ~505-520 nm and LSP occurs at higher wavelength
(600-1300 nm). There is negligible blue shift in TSP with increase in aspect ratio, whereas LSP
is very sensitive and increases with increase the aspect ratio of nanorods. In this experiment, we
will find a linear dependence between the position of LSP and aspect ratio.
Chemistry of gold nanorods is considerably different from colloidal gold due to its surface
structure and the presence of the {110} facet on nanorod surface owing to the use of
cetyltrimethylammonium bromide (CTAB) as the capping agent. CTAB stabilizes the {110}
facet, although it is of higher energy. In a nanocrystal, different planes have different surface
energies, which can be related to the stability (hence reactivity) of the surfaces.
Fig.2c. shows the TEM images of gold nanorods prepared this way at different magnifications.
Aspect ratio of gold nanorods can be obtained from such a TEM image. There are also
theoretical ways of obtained this by calculating the absorption spectrum for a particle of specific
shape.
Fig.2a. shows the absorption spectrum of the gold nanorods having two surface plasmons at 511
and 796 nm corresponding TSP and LSP, respectively, suggesting an aspect ratio as 3.4. This is
obtained from an optimized synthesis. Aspect ratio can be tuned by varying the volume of
NaBH4 added to the growth solution, as shown in Fig. 12.4. It is observed that the aspect ratio
decreased with increase in the volume of NaBH4 used, finally resulting in spherical nanoparticles
(aspect ratio, 1) at 0.5 mL (keeping all other parameters the same). Under optimum conditions,
gold nanorods are formed have an aspect ratio of 3.4, by the addition of 50 L of 1.67 mM
NaBH4 (trace a). With further increase in the amount of NaBH4 (500 L of 1.67 mM NaBH4),
the LSP merged with the TSP and a single absorption peak was obtained, characteristic of
spherical particles (trace e).
22

A plot of the position of LSP and the concentration of NaBH4 is shown in Fig.2b. An
approximate estimate of residual NaBH4 available in the volume of seed solution added to the
growth solution is calculated from the fact that one mole of BH4- can reduce eight moles of Au1+
existing in the growth solution.
Absorbance (a. u.)

1.2

0.8
d
0.4
0.0

e
450

600 750 900


Wavelength (nm)

1050

Position of LSP (nm)

850

800
750
700
650
0.0

50 nm

5 nm

0.2
0.4
0.6
Effective NaBH4 (mole)

Fig.2A. Absorption spectrum of the gold nanorods having two surface plasmons. Fig.2B. UVVis spectra of gold nanorods formed with increasing amount of NaBH4: (a) 50 L, (b) 100 L,
(c) 200 L, (d) 300 L, and (e) 500 L. Fig.2C. Position of LSP versus effective NaBH4
concentration in growth the solution

10.5 Procedure -I
1. Prepare 10 mM HAuCl4 solution in a 5 mL standard flask by dissolving 17 mg of
HAuCl43H2O in water. HAuCl43H2O is hygroscopic and a standard solution will be given
to you which may be diluted to make the required solution.
2. Prepare ~0.5% solution of trisodium citrate in a 10 mL sample bottle flask by dissolving 25
mg of Na3C6H5O7 in 5 mL of water.
3. Take 0.5 mL of 10 mM HAuCl4 solution in a 50 mL flat bottomed flask using a 1 mL pipette
and add 13 mL of distilled water to it using a 25 mL measuring jar.
4. Heat the solution over a heating mantle and bring the solution just to a boil. (marked by the
appearance of bubbles from the colourless solution).
5. To the boiling solution, add 1 mL of ~0.5 % trisodium citrate solution using a 1 mL pipette.
6. Continue heating (with boiling) till the colour turns to wine-red (this may take about 1-2
minutes).
7. Remove the flask from the mantle and keep it for cooling in air, for about 15-20 minutes.
8. To account for loss of water during boiling, make the solution to 14.5 mL (i.e. to the original
volume) in a 25 mL measuring jar.
9. Calculate the concentration of gold nanoparticles in the solution in terms of gold.
10. Prepare the following solutions of different concentrations.

5 mL prepared solution

4 mL prepared solution + 1 mL distilled water

3 mL prepared solution + 2 mL distilled water


23

2 mL prepared solution + 3 mL distilled water

1 mL prepared solution + 4 mL distilled water

11. Collect the UV-Vis spectrum of undiluted solution along with those of above diluted ones,
with distilled water as the reference, using UV-Vis absorption spectroscopy.
12. Note the value of absorbance maximum and the corresponding wavelength (max) of the each
spectrum.
13. Plot a graph of absorbance vs. concentration using the values obtained. Slope of the above
plot gives the absorption coefficient of the gold nanoparticles.
10.6 Procedure - II
1. Prepare the growth solution containing 5 mL of 100 mM CTAB, 250 L of 10 mM
HAuCl43H2O, 32.5 L of 10 mM AgNO3, and 35 L of 100 mM AA.
2. Prepare 10 mL of 1.67 mM of NaBH4 in ice-cold water.
3. Prepare various samples of the following proportions:
a. 50 L of freshly prepared ice-cold NaBH4 + growth solution of (~5 mL)
b. 100 L of freshly prepared ice-cold NaBH4 + growth solution of (~5 mL)
c. 200 L of freshly prepared ice-cold NaBH4 + growth solution of (~5 mL)
d. 300 L of freshly prepared ice-cold NaBH4 + growth solution of (~5 mL)
e. 400 L of freshly prepared ice-cold NaBH4 + growth solution of (~5 mL)
f. 500 L of freshly prepared ice-cold NaBH4 + growth solution of (~5 mL)
2. Collect the absorption spectrum of each sample.
3. Plot position of LSP vs conc. of NaBH4
Results
1. Beer lamberts law is verified.
2. The absorption coefficient of the gold nanoparticles is ~3.36 L.mol-1.cm-1.
3. The effect of NaBH4 is plotted.
Reference
1. J. P. Juste, I. P. Santos, L. M. Liz-Marzan, P. Mulvaney. Coord. Chem. Rev., vol. 249,
2005, 18701901.
2. C. J. Murphy, T. K. Sau, A. M. Gole, C. J. Orendorff, J. Gao, L. Gou, S. E. Hunyadi, T.
Li, J. Physical Chemistry B, vol.109, 2005, 13857-13870.
3. T. S. Sreeprasad, A. K. Samal, T. Pradeep, Langmuir, Vol. 23, 2007, 9463-9471.
4. A. K. Samal, T. S. Sreeprasad, T. Pradeep, J. Nanopart. Reserarch, vol.12, 2010, 17771786.

24

11. ESTIMATION OF FREE ENERGY OF PROTEIN DENATURATION


USING THE INTRINSIC FLUORESCENCE OF THE PROTIEN
11.1 Aim
To determination of free energy of protein denaturation using the intrinsic fluorescence of the
protein (bovine serum albumin / human serum albumin / lysozyme).
11.2 Chemicals Required
Protein (bovine serum albumin), urea, guanidine hydrochloride, citrate buffer and HCl tris buffer
11.3 Apparatus required
5 mL, 10 mL, 25 mL, 50 mL, 100 mL standard flasks, 0.1 mL, 1 mL, 5 mL, 10 mL pipettes, 25
mL, 50 mL beakers, fluorimeter and pH meter.
11.4 Principle
Denaturation is a process in which proteins lose their secondary structure and/or tertiary structure
by application of external stress like heat or chemicals such as urea, guanidine hydrochloride.
Proteins have an intrinsic fluorescence due to tryptophan, tyrosine, and/or phenylalanine. Most
of the emissions are due to excitation of tryptophan residues, with a few emissions due to
tyrosine and phenylalanine. The table summarizes the fluorescence characteristics of the three
aromatic residues:
Lifetime Absorption
Fluorescence
-1
-1
(nm) (M cm ) (nm) Quantum
Tryptophan
2.6
280
5,600
348
0.20
Tyrosine
3.6
274
1,400
303
0.14
Phenylalanine 6.4
257
200
282 .
0.04
The fluorescence of the aromatic residues varies in somewhat unpredictable manner in various
proteins. Compared to the unfolded state, the quantum yield may be either increased or decreased
by the folding. Accordingly, a folded protein can have either greater or lesser fluorescence than
the unfolded form. Fluorescence intensity, wavelength of the emitted light or a combination of
both can serve as a probe for studying perturbations of the folded state or denaturation.
Tryptophan residues that are exposed to water, have maximal fluorescence at a wavelength of
about 340-350 nm, whereas totally buried residues fluoresce at about 330 nm.
An assumption made in this experiment is that at any concentration of the denaturant, lysozyme
will exist in only two states, the native (N) and completely unfolded or denatured (D) states.
These two states are assumed to be in equilibrium, as shown in Scheme I.

From the measured parameters and their calculated baseline and maximum values, the fraction of
protein in the denatured state at any denaturant concentration may be calculated, assuming a two
state model, shown in the following equation
Y is the measured parameter (fluorescence intensity, I), Yo is the baseline value of the parameter
at low denaturant concentration, Ymax the maximum value of the parameter when the protein is

25

completely unfolded. The apparent equilibrium constant, KD, for the denaturation process may
then be calculated as shown in following equation
From KD, the free energy change may be calculated from

GD = -RT lnKD.

From these G values, the G value at zero denaturant concentration may be obtained using the
linear extrapolation method.
11.5 Procedure
1.
2.
3.
4.

Buffer 1: Prepare 0.050 M citrate buffer, pH = 3.


Solution 1: Prepare 25 mL solution of 10 M urea in buffer 1.
Stock 1: Prepare 5 mL, 0.12 mM solution of the protein in buffer 1.
Sample set 1: Prepare a series of solutions containing 3M protein each and varying
concentrations of urea (3.0 9.0 M) from stock 1. (Nt: The final volume of each sample
should be 4.0 mL).
5. The solutions were allowed to sit for at least 1 h
6. Measure the fluorescence intensity. (ex = 280 nm, em = 300-460 nm).
7. Calculate the GD from the equations given above.
8.
S. No
[Urea]
Vol. of Urea sol.
Vol. Protein
Buffer
in M
in mL
in mL
in mL
1
0
2
3
3
4
4
5
5
6
6
7
7
8
8
9
Result
The value of free energy of protein denaturation ( GD) is 17-21kJM-1(literature value)
Reference
1. R. F. Greene, Jr. and C. N. Pace, Journal of Biological Chemistry, vol. 249, 1974, 5388
5393.

26

12. FLUORESCENCE QUENCHING OF FLUORESCEIN BY


INORGANIC ANIONS: DETERMINATION OF QUENCHING
CONSTANTS, ESTIMATION OF FREE ENERGY CHANGE AND
ACTIVATION ENERGY FOR THE QUENCHING PROCESS
12.1 Aim
To determine the fluorescence quenching constant and calculate the activation energy as well as
free energy change of the quenching process in fluorescein by selected anions.
12.2 Chemicals required
Fluorescein, Quenchers: NaBr, NaI and Na2SO4
12.3 Appartus required
5 mL, 10 mL, 25 mL standars flasks, 5 mL, 10 mL pipettes, 1 mL graduated pipette, 25 mL, 50
mL beakers
12.4 Principle
The fluorescence quenching constant from fluorescein was analyzed using Stern-Volmer
[ ]
kinetics.
According to equation 1, a plot of I0/I vs [Q] will be a straight line with KSV as the slope, where
I0 and I are the intensity of emission in the absence and presence of Q, respectively.
KSV=kq, where kq is the quenching constant and is the excited state lifetime of the fluorophore.
The free energy change in the electron transfer process can be calculated from Treinin and
Hayon equation 2.

Where, Ectts is the charge transfer to solvent transition energy of the quenchers, E1/2 is the redox
potential and ES is the singlet transition state energy for fluorescein. All the units are in electron
volt.
A plot of logKq and G will show a linear plot, which suggests that electron transfer is the rate
determining step in the process. The equation for the linear plot will be given by equation 3.
Since kET is the rate determining step, equation 3 can be re-written as

From Arrhenius equation,


Where A is the collisional frequency for which a value of 1010 can be assumed. Substituting the
value of kET from equation 5 in equation 4, and assuming that kdiff ~ k-diff,
From equations 4 and 6, the following expression can be obtained for room temperature
experiments.
12.5 Procedure
1. The quenchers are NaBr, NaI and Na2SO4. A stock solution 25 mL of fluorescein (2 x 10-6
M) in water was prepared.
27

2. Six separate solutions of the fluorescein ex = 484, (10-6 M) at identical concentrations were
prepared containing increasing amounts of quencher concentrations (0, 0.5 M).(Nt: The final
volume of each sample should be 10 mL).
3. The emissions intensity at 520 nm from each solution is measured and each I0/I values was
plotted against [Q] to determine the fluorescence quenching constants with all the quenchers
given above.
4. Using the calculated G values, the corresponding activation energy is calculated using
equation7.
5. Plot IO/I= Ratio of fluorescence intensity without or with quencher vs [Q]= Quencher
concentration

S.No

Vol. of NaI
(mL)

Vol. fluorescein
(mL)

Water
(mL)

Io/I

1
2
3
4
5
6

[Q]
in M
0
0.1
0.2
0.3
0.4
0.5

Conc. Of [KI] (M)

Results
1. The fluorescence quenching constant in fluoroscein derivatives by selected anions is
2. Free Energy Change (G) for the Quenching Process is -50 eV
3. Activation Energy (E a) for the Quenching Process is 1.125 eVmol -1
References
1. P.K. Behera, T. Mukherjee and A.K. Mishra, Indian Journal of Chemistry:A, vol.34(A),
1995, 419-422.
2. Lakowiz J. R. Principles of fluorescence Spectroscopy, 1999.

28

13. CRITICAL MICELLE CONCENTRATION OF CTAB


13.1 Aim
To study the aggregation behaviour and determination of the critical micelle formation
concentration of cationic surfactant in water by surface tension measurement.
13.2 Chemical required
Hexadecyltrimethylammonium bromide(CTAB)
13.3 Glassware required
Beaker (50 mL), Tubing with a balloon, Weighing bottle
13.4 Principle
The surfactant concentration at which micelle formation begins is known as the critical micelle
formation concentration (CMC). Micelles are spherical or ellipsoid structures on whose surface
the hydrophilic heads of the surfactant molecules are gathered together whereas the hydrophobic
tails project inwards.
The critical micelle formation concentration (CMC) can be determined by carrying out surface
tension measurements on a series of different surfactant concentrations. If the liquid with known
surface tension is used for one of them (water), the surface tension of the other liquid can be

calculated from the equation:

Surfactants exhibit a specific surface tension curve as a function of the concentration. Initially
the surfactant molecules increasingly enrich themselves at the water surface. During this phase
the surface tension decreases linearly with the logarithm of the surfactant concentration. When
the CMC is reached, i.e. when the surface is saturated with surfactant molecules, a further
increase in surfactant concentration no longer has any appreciable influence on the surface
tension.
Determination of the critical micelle formation concentration

This means that in order to determine the CMC the two linear sections formed by the measuring
points obtained from the series of different concentrations must be determined. The CMC is
obtained from the intersection of the straight lines for the linear concentration-dependent section
29

and the concentration-independent section. The surface excess concentration of surfactant ions
s and the area per molecule was calculated from the slope of the straight line in the surface
tension plot (d/d ln C) below CMC, using appropriate form of Gibbs adsorption equation:
(
)

13.5 Procedure
1. Mount the clean and dry stalagmometer on the vertical stand.
2. Weigh the mass of the weighing bottle m0.
3. Fill the beaker with distilled water. Mount the tubing with balloon on the top end of
stalagmometer. Immerse the bottom end of stalagmometer into water and fill it up, such that
the water level is above the wide part of stalagmometer.
4. Remove the balloon and collect 20 water drops into the weighing bottle.
5. Weigh the mass of the weighing bottle with water and determine the mass of 20 drops.
6. Empty the weighing bottle and stalagmometer, dry them and prepare for the next
measurement.
7. Repeat steps 2-6 for liquids with the unknown surface tension.
8. Knowing the temperature in laboratory, determine the water surface tension using values
from the table 1, and calculate the surface tensions of studied liquids according to the
equation.
Table: The temperature dependence of the surface tension of distilled water
Temp
o
C

20

21

22

23

Surface tension

0.07275

0.07259

0.07244 0.07228

24

25

30

0.07213

0.07197

0.07118

N/m

Result
1. The critical micelle concentration of CTAB is
References
1. A. Domnguez, A. Fernndez, N. Gonzlez, E. Iglesias and L. Montenegro, Journal of
Chemical Education. Vol. 74, 1997, 1227-1231.
2. Peter Atkins, Physical Chemistry, 9th Ed., W. H. Freeman and Co., (2009)

30

14. ACIDITY DETERMINATION IN ZEOLITES BY AMMONIA


TEMPERATURE PROGRAMMED DESORPTION
14.1 Aim
To analyse various zeolite samples for its qualitative and quantitative determination of acidic
sites by Temperature Programmed Desorption of ammonia
14.2 Chemicals required: ZSM-5 Zeolite
14.3 Apparatus required: TPD
14.4 Principle
Zeolites are microporous aluminosilicate structures, important for a variety of applications
including selective reactions and adsorptive separations. It is important to estimate the acidic
(both Brnsted or Lewis) sites present in zeolites. Temperature-programmed desorption (also
called thermal desorption spectroscopy, TDS) of ammonia is the most widely used technique for
characterizing acidity in zeolites. Using this method the number of acid sites can be determined
from the amount of desorbed ammonia molecules adsorbed directly on the acid sites. TPD
experiment typically involves saturation of the surface with ammonia under a set of adsorption
conditions, followed by linear ramping of the temperature of the sample in a flowing inert gas
stream. Ammonia concentration in the effluent gas can be measured by absorption/titration or
mass spectroscopy. The experiment can also be carried out in a microbalance and changes in
sample mass can be recorded continuously as a function of temperature. The amount of ammonia
desorbing above some characteristic temperature is taken as the acid-site concentration, and the
peak desorption temperatures can be used to calculate heats of adsorption. This method provides
information on the number of acid sites and their strength.
14.5 Procedure
1. 100 mg of dry zeolite sample pre-treated at 500 C in 25 mL/min Pure Helium gas flow for
30 min.
2. Temperature cooled to 100 C and then 10% NH3 in Helium gas mixture (25 mL/min) is
passed through the sample for 30 min.
3. Physisorbed Ammonia gas is removed by flushing with Helium gas for 15 minutes.
4. Temperature programmed heating started from 100 C to 500 C @ 10 C per min and NH3
evolution recorded.
5. The peak area will give the volume of NH3 desorbed which is directly proportional to the
acidity at that peak temperature.
Temp.
C

TCD Signal
(a.u.)

TCD signal (a.u.)

0.140

S. No.

0.136

0.132

0.128

0.124
0

31

100

200

300

400

Temperature ( oC)

500

600