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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5708_Библиотеки_им_академика_М_И_Перельмана.pdf
257
Chemical Kinetics
T
HEORY When iodine is mixed with acetone, the following reaction occurs:
CH
3
COCH
3
I
2
CH
3
COCH
2
I H
I
The reaction is quite slow in the pH range of 4–7, but it occurs quite rapidly if
the pH is below 3 or above 8 indicating an acid or base dependence. The reac-
tion rate increases with the concentration of H
in acidic solution or with the
concentration of OH
in basic solution. The rate law can be written as
Rate d CH COCH /dt d I /dt
CH COCH I H
33 2
332
−
[]
−
[]
[][]
⎡
⎣
⎢
⎤
⎦
⎥
k
ab
c
where k is the rate constant and [CH
3
COCH
3
] represents the concentration of
species CH
3
COCH
3
in mol/L. The exponents a, b and c indicate the order of the
reaction with respect to acetone, iodine and hydrogen ion, respectively.
Procedure
1. Place acetone, iodine solution, and H
2
SO
4
in thermostat for half an hour.
2. Prepare different concentrations of iodine and acetone in four different
flasks labelled 1, 2, 3 and 4, and mix them for few minutes as shown below:
Flask Acetone (mL) 0.5 N H
2
SO
4
(mL)
0.1 N Iodine
solutions (mL)
Distilled water
(mL)
1 5 25 10 70
2 7.5 22.5 12.5 66.5
310 20 15 55
4 10 25 7.5 57.5
3. Pipette out 10 mL of solution from flask 1 in a conical flask and add
10 mL of 1 N sodium acetate.
4. Titrate the sample with N/100 sodium thiosulphate using starch as indi-
cator towards end point.
5. Pipette out 10 mL of solution again after 10 min and titrate as above.
6. Pipette out 10 mL of solution after every 10 min and record the titre val-
ues as above.
7. Repeat the procedure with other three flasks. Note down the titre values
for each of the flask.
Observations
S. no. Flask 1 Flask 2 Flask 3 Flask 4
Time
(min)
Titre
value(mL)
Time
(min)
Titre
value(mL)
Time
(min)
Titre
value(mL)
Time
(min)
Titre
value(mL)
1
2
3
4
258
Practical Pharmaceutical Analytical Chemistry
Calculations
Plot a graph between time and titre value and find out slope of the each con-
centration of the solution. Calculate the velocity constant of the reaction from
the slope of the graph.
Result The reaction follows order of reaction and velocity constant
of the reaction was found to be .
Experiment 4 Study the saponification of ethyl acetate.
Requirements
Ethyl acetate, 0.5 N HCl, 0.1 N NaOH, phenolphthalein, methyl orange ther-
mostat, conical flask, pipette, thermometer, burette
Procedure
1. Prepare and standardize the 0.02 N HCl and 0.02 N sodium hydroxide
solution as described in Experiment 1.
2. Prepare 0.01 M ethyl acetate by dissolving 0.22 g of ethyl acetate in 250 mL
of distilled water.
3. Place 50 mL of 0.02 N NaOH and 0.01 M methyl acetate separately in two
conical flasks in a thermostat or in a trough of water for about 30 min.
4. Keep ready a 100-mL conical flask containing 10 mL of HCl solution.
5. Add 50 mL of 0.02 N NaOH solution to the flask containing 0.01 M
methyl acetate solution and mix the contents thoroughly by shaking the
flask.
6. Start the stop watch immediately when the two solutions are mixed and
immediately pipette out 10 mL of the mixture into the conical flask con-
taining 10 mL of HCl solution.
7. Titrate against 0.02 N NaOH solution using phenolphthalein as indicator.
8. Record the time as t
o
and note down the volume of sodium hydroxide
consumed as V
o
.
9. After 5 min, again pipette out 10 mL of the mixture into the conical flask
containing ice-cold water and titrate against 0.02 N sodium hydroxide
solution using phenolphthalein as indicator.
10. Record the time as t
5
and note down the volume of 0.02 N sodium hydrox-
ide consumed as V
5
.
11. Take out 10 mL of reaction mixture and place in thermostat or water bath
at 60°C for 1 h.
12. After every 10 min, repeat the steps 8-10, till t
50
is recorded.
13. After 1 h of placing reaction mixture in thermostat or water bath at 60°C,
pipette out 5 mL of the mixture and titrate with 0.02 N sodium hydroxide
259
Chemical Kinetics
solution using phenolphthalein as indicator. This will be t
f
and the volume
of sodium hydroxide consumed is V
f
.
14. Record all the readings in table, as mentioned below, and calculate the
velocity constant and t
½
of the reaction.
Observations and calculations
Room temperature °C
Observations
S. no Time
(min)
Volume of sample
withdrawn (mL)
Burette reading
(mL) V
t
(a x)
(V
f
V
t
)
(b x)
(V
f
V
t
)
1 t
0
10
V
0
2 t
10
10
V
10
3 t
20
10
V
20
4 t
30
10
V
30
5 t
40
10
V
40
6 t
50
10
V
50
7
t
f
10
V
f
In a second-order rate reaction, the constant is calculated by the following equation:
k
ta b
ba x
ab x
=
−
()
−
()
−
()
2 303
10
.
log
where a and b are initial concentrations of alkali and ester
a V
- V
0
(a x) amount of NaOH present at time t, or amount of HCl
consumed
V
V
t
x a (a x)
V
t
V
0
(a b) excess of NaOH over ester
unreacted amount of NaOH at the end of the reaction
(V V
f
)
b can also be as taken as a (a x)
V
V
0
(b x) (V
V
0
) (V
t
V
0
)
(V
V
t
)
Calculate the value of k by using the above-mentioned equation.
Result The saponification of ethyl acetate was studied at room temperature
and it was observed that the saponification of ethyl acetate follows order
rate equation.
260
Practical Pharmaceutical Analytical Chemistry
Judge yourself
1. What do you understand by kinetics of a reaction?
2. What is temperature coefficient?
3. What are various methods of determining the order of reaction?
4. What is half-life of a reaction and how is it determined?
5. Write down the units of first- and second-order rate constants.
6. How will you determine the first-order rate constant?
7. What is molecularity?
8. What is the difference between molecularity and the order of reaction?
9. What are the factors that affect rate of reaction?
10. How does the size affect in heterogeneous reactions?
11. Define collision and activation energy?
12. Name the theories of chemical kinetics.
13. Write down the characteristics of zero- and first-order rates.
Chapter
10
Chapter
Chromatography
10.1 Introduction
Chromatography (colour-writing) is a technique used to separate and purify a
mixture of compounds. The separation in chromatography is based on differential
affinities of the solute between two immiscible phases. The two phases are
classified as mobile phase that moves through or over the surface of the fixed
phase and stationary phase, which is a fixed bed of large surface area.
Definitions
Chromatography may be defined as the technique or method used to separate
a mixture of components into individual components through equilibrium
distribution between two phases. The bands of colour obtained from
chromatography is known as chromatogram.
Components of chromatography
1. Solute: The mixture to be separated must
a. be of molecular dimension;
b. be in the solution form or in the vapour state;
c. have reversible affinity for each of the phase.
2. Stationary phase: The fixed bed used for separation may
a. be porous or finely divided solid;
b. be liquid coated as thin layer over an inert supporting material;
c. have particles as small and homogenous as possible to give large surface
area helping in sorption and desorption process.
3. Mobile phase: The solvent system used for the development of
chromatogram
a. should be nonreactive;
b. have to be selected on the basis of nature of stationary phase.
c. can be a single solvent or mixture of two or more solvents;
d. can be either liquid or gas.
262
Practical Pharmaceutical Analytical Chemistry
Classification of Chromatography
According to nature of stationary and mobile phases chromatographies are
classified. The following two chromatographies are based on the nature of
stationary phase
1. Adsorption chromatography: In this stationary phase is solid.
2. Partition chromatography: In this stationary phase is liquid.
These two categories are further classified into two more subcategories depending
on the nature of the mobile phase
1. Adsorption chromatography
a. Liquid–solid chromatography: In this mobile phase is liquid with solid
stationary phase.
b. Gas–solid chromatography: In this mobile phase is gas with solid
stationary phase.
2. Partition chromatography
a. Liquid–liquid chromatography: In this both stationary and mobile
phase is liquid in nature.
b. Gas–liquid chromatography: In this mobile phase is gas with liquid
stationary phase.
There are two other categories in addition to these:
3. Ion-exchange chromatography
4. Size-exclusion chromatography
Retention factor (R
f
): It can be defined as the ratio of the distance travelled by
the solute from the origin to the distance travelled by the solvent in a particular
time.
R
f
value=
Distance from the origin to the centre of spot
Distance of th
ee solvent front from the origin line
Retention time (T
R
): The time which elapsed from the start of the
chromatogram to the elution maximum of the solute is called the retention time.
10.2 Paper Chromatography
This technique was first introduced by Schonbein in 1961 under the name
capillary analysis. It may be defined as the technique of separation of mixture of
substances by the flow of solvent on specially designed filtre paper. On the basis
of the nature of filter paper it may be categorized into two:
1. Paper-partition chromatography: The technique in which paper containing
water or moisture in the pores of cellulose fibre is used as stationary phase.
2. Paper-adsorption chromatography: In this technique a modified paper, i.e.
paper impregnated with silica or alumina act as stationary phase.
263
Chromatography
Principle:
It is mainly based on the partition rather than adsorption. In this
technique substances are partitioned between two liquids, i.e. one is the stationary
liquid which is held in the fibres of the paper and called the stationary phase and
the other is the moving liquid or developing solvent and called the mobile phase.
Experimental Procedure
1. Selection of filter paper: The 98-99% of filter paper is made of is
a-cellulose. Rest of the paper is mineral content. Different types of paper
are available for the separation depending on R
f
values, requirement speed,
etc. of the substance to be separated like
For wide apart R
f
values : Whatmann 31ET
For close R
f
values : Whatmann 20, Schleicher and
Schull 2045
Macherey Nogel 261, Edrol 208
For preparative purpose : Whatmann 3MM
For good resolution : Whatmann 20
For fast separation : Whatmann 4
Some times paper is modified to achieve the desired properties like
a. impregnated with diatomaceous earth, alumina, silica gel and
ion exchange resins to get the different techniques of separation;
b. increase of carboxyl content by partial oxidation to increase the
exchange capacity during separation of polar substances;
c. acetylated or treated with silicone or impregnated with inert
nonpolar type organic polymers which gives hydrophobic property
to paper which helps in retaining of hydrophobic type solvent rather
hydrophilic type (a basis of reverse phase chromatography).
2. Mobile phase: Pure solvents, buffer solutions or mixture of solvents are
used. Some of the examples are as follows:
a. For hydrophilic substances
Isopropanol : Ammonia : Water (9 : 1 : 2)
n-Butanol : Acetic acid : Water (4 : 1 : 5)
Methanol: Water (3:1)
t-Butanol : Water : Formic acid (40 : 20 : 5)
b. For intermediate hydrophilic substances
Formamide : Chloroform
c. For hydrophobic substances
DMF : Cyclohexane
3. Development
a. Cut a piece of filter paper to fit into development chamber.
b. Mark a line on the bottom edge of paper approx. 2 cm from the rim.
c. Put a spot of sample on origin line.
264
Practical Pharmaceutical Analytical Chemistry
d. Keep a distance of 2 cm between two spots.
e. Select an air tight development chamber.
f. Add mobile phase to the chamber so that it is about 2 cm deep.
g. Seal the chamber and wait for saturation of atmosphere of development
chamber.
h. Suspend the piece of spotted paper using thread in the air for
10 min.
i. Finally immerse the paper’s bottom edge into the developing
chamber.
j. Remove paper when solvent has travelled 3/4th length of paper.
On the basis of the development of chromatogram, chromatography can be
categorized into the following:
1. Ascending chromatography
a. When the development of the paper is done by allowing the solvent
to travel up the paper, it is known as ascending chromatography.
b. In it mobile phase or solvent moves against the gravitational force.
c. The rate of movement of solvent becomes slower with increasing
height and this slowness in the rate provides enough time for
partition equilibrium to take place between two phases
2. Descending chromatography
a. When the development of the paper is done by allowing the solvent
to travel down the paper, it is known as descending chromatography.
b. This is carried in a special chamber where the solvent holder is at the top
c. The movement of the liquid is caused by the capillary action as well
as by the pull of the gravity.
d. In this technique the rate of movement of solvent is more rapid.
e. This technique allows a separation over a longer distance and allows
an increase in resolution.
Paper
suspended
in trough
Anti-siphon
rod
Solvent
Solvent
Gas-jar cover
Cotton
thread
Clips
Rising
Solvent
front
Paper strip
Original spot
mixture
Serrated edge
to allow solvent
to flow uniformly
off the paper
A
B
C
D
Fig. 10.1
Ascending and descending
paper chromatography
265
Chromatography
3. Ascending–descending chromatography
a. This is a hybrid of above two techniques.
b. In this technique upper part of the ascending chromatography can
be folded over a glass rod allowing the ascending development to
change over into the descending after crossing the rod.
4. Radial paper chromatography
a. It is also known as circular paper chromatography.
b. Circular paper is selected and cut parallel to the radium from edge
to centre.
c. Material to be separated is placed at the centre of circular paper
having a wick.
d. The components get separated in the form of concentric circular
zones.
e. This technique allows spotting of more samples.
k. Open the lid of development chamber and remove the chromatogram.
l. Mark the solvent front with a pencil and allow the chromatogram
to dry.
4. Visualization: After the chromatogram has dried, one can identify the spots.
This can be achieved by either physical means (e.g. UV chamber) or chemical
means. The chemicals used for visualization is known as chromogenic reagents
or visualizing reagents. Some of the common visualizing reagents are:
Iodine chamber method: Brown or amber spots are observed when the paper is
kept in a jar containing few iodine crystals.
Some of the chemicals can be used for spraying for identification of specific
compounds:
Ninhydrin reagent: for amino acids,
Dragendorff reagent: for alkaloids,
2,4-dinitrophenyl hydrazine: for aldehydes, ketones, etc.
Cir ar i ter
paper
ri ina p t
itre
i paper t
t dip in ent
Cir ar
i ter paper
etri di h
er
etri di h
ent
Fig. 10.2
Radial or circular paper
chromatography
266
Practical Pharmaceutical Analytical Chemistry
5. Calculation: The R
f
value for every spot should be calculated. It is specific
for any given compound for the same set of stationary and mobile phase.
R
f
value=
Distance from the origin to the centre of spot
Distance of th
ee solvent front from the origin line
10.3 Thin Layer Chromatography (TLC)
This technique was first introduced by Izmailov and Shraiber in 1938. In
1958, Stahl introduced TLC as a means of separation based on adsorption
chromatography. It may be defined as the solid–liquid form of chromatography
where a thin layer of some inert material such as alumina, silica, etc. is used as a
substrate, as stationary phase, in the place of a filter paper. It is also known as drop,
strip, spread layer, surface and open column chromatography.
Principle: It is mainly based on the adsorption rather than partition. In this
technique substances get adsorbed on stationary phase such as silica or alumina.
This method can be used for partition or ion exchange chromatography
depending on the nature of the stationary phase.
Experimental procedure
1. Stationary phase: Silica gel, alumina, kiesleguhr and cellulose powder are
used as inert coating material for preparation of thin layer over glass plate. The
adsorbent do not adhere to the glass plate so binders like gypsum, starch, hydrated
Gypsum is most widely used binder. In many cases, a fluorescent indicator like
zinc silicate is mixed to simplify the visualization later on. Uniform thin layer of
adsorbent is placed on the support by various techniques like
a. Pouring
b. Dipping
c. Spraying
d. Spreading
Sometimes, a precoated plates which have thickness usually varying from 0.1 to
0.2 mm are used. After preparation of thin plates, the liquid or moisture associated
with the thin layer has to be removed. This is done by drying the plates for
30 min in air and then in an oven at 110
o
C for another 30 min. This drying
procedure makes adsorbent layer active and the process is known as activation of
plates. For very active layer, plates can be heated to about 150
o
C for about 4 h.
2. Mobile phase: If we know the chemical nature of components to be separated
the solvent or mobile phase can be selected by using Stahl’s triangle. This inter
relates adsorbent activity, nature of the solute and nature of the solvent.