PART

2. & 3.

II
In the world of
science, nothing
CHROMATOGRAPHY

invigorates the
a universally
mind so much as to

s o m e small
seed of discovery to
watch a concept from decades, chromatography,
technology. In the last four
applicable crude technique,
has grown into a
and very
the once mysterious reliable separation
technology.
and
very sophisticated Chromatography, 1998.
R.P. W. Scott, Chiral
-T.E. Beesly and

the Greek) is
INTRODUCTIONN

colour-writing from
Chromatography
(meaning, isolation and
for the separation,
an important
technique employed e v e n when they
are
of a mixture,
i d e n t i f i c a t i o n of the
components
method is particularly
This
amounts.
small
have almost the
same
present only in very
the mixture
components in be separated
useful when the hence cannot
chemical properties and
physical and
methods of separation.
by the other usual introduced by the
was first
technique the separation
The chromatographic who used it for
Tswett (1906),
Russian botanist Mikahil
Since the scope
of this technique
coloured pigments
from plants. idea of colour
of the
colourless mixtures,
extended to
has been widely
connected with the concept. for
1S no longer versatile tool
and
is now apowerful be
Chromatography addition, it can
In
related chemical species. determination
Separating closely and quantitative
identification
used for the qualitative
of separated species.
MIGRATION
DIFFERENTIAL
PRINCIPLE OF in which

is a physical
method of separation
Chromatography distributed between
two phases,
to be separated
are other (the
n e components while the
(stationary phase) Definition,
One of which is stationary direction (IUPAC
in a definite
mobile phase) moves

21
22 ADVANCED PRACTICAL ORGANIC
CHEMISTRY
1993). The stationary phase (either solid or liquid) is
usuallu in
the form of a column or a thin film of adsorbent
mobile phase (either liquid or gas) moves on.
through which
During the chromatography
technique, the components
mixture are
separated by allowing the sample (analyte) toofa
he
transported through a packed bed of material (the stationary
by a fluid mobile phase. If the individual phasel
mixture mové through the components in the
stationary phase at different rates then
separation will occur, the degree of separation depending
difference in the rates of on the
different components takes migration. Thus the
separation of the
place due to the
differential
depends on the relative affinitymigration
of components, iwhich in turn
the stationary
and mobile towards
phases. The component which is less
strongly held by the stationary phase would tend to move
the mobile
phase and vice versa. faster in

CLASSIFICATION OF CHROMATOGRAPHIC
METHODS
Depending upon the various combinations
and the
stationary phase, there are different of the mobile phase
chromatography.
is known as
If the fluid mobile
phase
techniques of
is a liquid, the
liquid chromatography; if it is a technique
8as), then the gas (called the carrier
technique is called
technique may be further
subdivided
gaschromatography. Each
Each
stationary phase (the mobile phase is according to the nature or tne
indicated first).
Table 1. Different
Nature of
Techniques of Chromatography
mobile phase Nature of
Name
stationary phase
Liquid Solid
Liquid-solid
chromatography (LSC)
Liquid Liquid-liquid
Gas chromatography (LLC)
Solid Gas-solid
chromatography (GSC)
Liquid Gas-liquid
chromatography (GLC)
CHROMATOGRAPHY 23
The above approach of classification is rather too general and
further systems have been suggested.

Depending upon the mode of separation, chromatographic methods

can be broadly divided into adsorption chromatography and partition
chromatography. In adsorption, the binding of a compound to the
surface of the solid phase takes place; whereas in partition, the
relative solubility of a compound in two phases results in the partition
of the compound between two phases.
In adsorption chromatography, the stationary phase is a solid
and the mobile phase may be liquid or gas. When the solid stationary
phase is taken as a column, it is known as column chromatography.
When the solid adsorbent is used as a thin layer coated on a glass
plate, it is called thin layer chromatography (TLC).
In partition chromatography, the stationary phase is a liquid
held on an insert solid support and the mobile phase may be a
liquid or a gas. Here also, a column of insert solid can be used to
retain the liquid film and then it is called partition column
chromatography. Liquid-liquid chromatography can be carried out
not only on columns but also on a strip of paper or a thin layer of
glass plate. Here, a thin liquid
supporting material coated on a

plate will act as the

film of the solvent held on the paper or

stationary phase. These methods are called paper chromatography

(PC) and thin layer chromatography (TLC) respectively.
26 ADVANCED PRACTICAL ORGANIC CHEMISTRY
THIN LAYER CHROMATOGRAPHY (TLC)
Thin layer chromatography (TLC) is described as a method
for chromatographic analysis on thin layers of adsorbents. TLC is
a liquid-solid chromatographic technique, in which the moving liquid
phase is allowed to ascend a thin layer of adsorbent coated on a
backing support. The technique is rapid and helps in the separation
of micrograms of the substances. Though TLC is very much
similar to paper and column chromatography, it is considered to
be far superior to both of them because in TLC the
isS separation
sharpest, and it requires less time and less amount of the substances.
TLC is special of
a case
adsorption chromatography and uses
a
chromatoplate i.e., a glass strip coated with a thin uniform
layer of the adsorbent like alumina or silica gel which constitute
the stationary
phase. To ensure that the absorbent adheres to the
glass, a binder such starch or Plaster of Paris is added while
as

preparing the adsorbent. The glass plate is then dried in an air

oven, as a result of which a thin
uniform layer of firmly bound
adsorbent is left. The mixture to be
end of the plate and this end is analysed is applied near the
dipped
solvent in a closed tank so that the vertically
into the suitable
above the surface of the solvent. applied spot is about l cm

SLURRY
KNOB-
LID
PLATE IN THE
GLASS PLATE COURSE OF
SLURRY SPREAD DEVELOPMENT
OVER INATHIN
LAYER SOLVENT-
(CHROMATOPLATE)
PAPER ROUND SIDES OF TANK-

Fig. 7.
Apparatus for thin layer chromatography
Development of the chromatogram occurs by
of the solvent up the adsorbent capillary movement
layer. After the
some distance, the plate is withdrawn and dried. solvent has moved
If the
of the mixture are coloured, the components
spots are readily located. If the
components are colourless, the dried plate is with sprayed iodine
vapour or a solution of a suitable
compound to make the spots
CHROMATOGRAPHY
27
the poSitions ot the components are revealed,
coloured. In this way, the Rf values of unknown
identification is possible by comparing
Now different solvents.
samples and known compounds using
is largely used in qualitative analysis.
Thin layer chromatography
chromatography in several ways, one
TLC is better than paper
minute quantities of numerous
that it can be used to separate
being
classes of compounds. In addition,
separation is rapid in this process.
benefited from TLC, where it is
Forensic chemistry has greatly
of a wide variety of
extensively employed for initial screening
drugs.
The Technique of Thin Layer Chromatography
In its simplest form, the TLC technique employs the following
basic operations
)Preparation of thin layer plates
(i) Application of sample
(ii) Development of chromatogram
(iv) Detection and identification of separated substances
Preparation of TLC plates
Solid layer TLC plates are prepared on microscopic slides or
large glass plates. Two such glass plates are held back to back
and dipped in a slurry of the adsorbent (alumina, silica gel etc)
prepared in solvents like chloroform, methanol or a mixture or
both. After dipping the glass plates in the slurry. they are withdrawn
SiOwly to let the solvent drain off. The plates are then separated
and activated by drying and heating them in an air oven.
LOOse layer TLC plates may be prepared by spreading a suspensiOn
Or adsorbent on a glass plate with the help of an applicator.
wole: TLC plates should never be handled or touched on the
Surface, but carefully held only by the edges. This will avoid
possible contamination due to
perspiration).
(i) Application of sample (spotting)
The sample is dissolved in a suitable solvent and a concentrated
Winis prepared. A drop of this solution is applied on the plate
with the The spot is
help of a
microsyringe or a capillary tube.
28 ADVANCED PRACTICAL ORGANIC CHEMISTRY
kept as small as possible (~ 2 mm in diameter) and on the middle
of a line drawn by pencil, 1.0 - 1.5 cm above the bottom edge.

(iii) Development of chromatogram

After drying the spot, the plate is placed in the developing
chamber, which is a glass jar or beaker containing a suitable
solvent. The plate is placed in such a way that the lower edge of
the adsorbent layer is dipped in the solvent but the spot applied is
above the solvent level. The developing chamber is closed from
the top and the plate kept undisturbed for 2 to 3 minutes. Now the
solvent gradually rises in the plate. When the solvent front has
reached 1 to 2 cm below the upper edge, the plate is taken out, the
solvent front is immediately marked and dried.

- TLC plate

Developing9
chamber

Mixture of
components A &B
**
Solvent

Fig. 8. Development of chromatogram in TLC

-Solvent front

-
Separated
components
-
b

Fig. 9. Developed chromatogram

CHROMATOGRAPHY 29

(iv) Detection and identification of substances

If the substances to be separated are coloured, then coloured
directly visible on the plate. For
spots of separated substances
are

colourless substances one of the following methods is used:

when normally colourless

(a) Irradiation with UV light
even

substances become coloured under UV light and can be seen.

iodine
(b) Placing the dried plate in a desiccator containing
crystals which produce coloured spots.
(c) Spraying with reagent which reacts with substances
some
to form coloured spots e.g. ninhydrin for amino acids or p-anisidine
phosphate for sugars.
The position of the spots should be marked in pencil, for the
colours fade after some time.

R Value
The ratio between the distance travelled by a particular component
the mixture the distance travelled by the solvent front is
of to
known as the Rf value for that component (Rf stands for retention
factor or ratio of fronts).

Distance travelled by (the centre of) the component

i.e., R
Rf =
Distance travelled by the solvent front

For example, if 'a' and 'b' are the distances travelled by

two

'1' is the distance

components A and B present in the mixture and
travelled by the solvent front, then

Rf value for component A =

R value for component B =

distance travelicu
The Rf value is determined by measuring the
30 ADVANCED PRACTICAL ORGANIC CHEMISTRY
by a component from the SOLVENTT
FRONT
starting 1line to the middle of
FILTER
the spot, and dividing this PAPER
distance by the distance the
solvent front has travelled as Rr(A)-4 MIXTURE
(AB)
measured from the same B(B)-4
starting line. BASE PENCIL
LINE
The Re value depends on LINE

the conditions of
chromatography such as nature --. sOLVENT
of adsorbent, solvent,
Fig. 10. Determination of R
temperature etc. The Rf value
values.
is a constant for a given
component under a given set of conditions. Thus by comparing the
R values with those known samples it is possible to deduce the
identity of the component.

Experiment No. 1
TLC Separation of Organic Binary Mixture No. 1
Aim
To carry out the TLC separatiom of a mixture of p-chloroaniline
and p-nitroaniline and to calculate the Rf values of the components.
Procedure
The given mixture of p-chloroanilne and p-nitroaniline was
dissolved in CHCl3 and a concentrated solution was prepared. A
drop of this solution was applied on the TLC plate with the help of
a capillary tube. The plate was then placed in
a beaker containing
5 ml of 75% CHCl3 and 25 % of n-hexane. The plate was kept
undisturbed for 2 to 3 minutes when the solvent front reached 1 to
2 cm below the upper edge. The plate was then taken out; solvent
front was marked and dried. It was kept in an iodine chamber for
5 minutes. The positions of the spots and the solvent front were
noted and distances were measured. From this the Rf values
were calculated.
Calculation
Distance travelled by the component
N
Distance travelled by the solvent front
CHROMATOGRAPHY 31
Distance moved by solvent front =

Distance moved by component I =

Distance moved by component II =

Distance travelled by the component I

Rf value of component I = Distance travelled by the solvent front

Distance travelled by the component II

R value of component II = Distance travelled by the solvent front

Result

R value of component I =

R value of component II =

Note: Some mixtures and developing solvents that can be used

for TLC separation

Mixture Developing solvents

1. p-Chloroaniline + p-Nitroaniline - 7 5 % Chloroform +
25% n-Hexane
2. p-Chlorophenol + p-Nitrophenol - 75% Chloroform +
25% n-Hexane
3. o-Nitroaniline + p-Nitroaniline 50% Chloroform +
50% n-Hexane
4. Aniline + p-Nitroaniline 50%Chloroform+
50% n-Hexane
5. Benzil +
Benzophenone 15% Chloroform +
85% n-Hexane
6. Benzil 15% Chloroform +
+ Acetophenone
85% n-Hexane
7. Naphthalene 60% Dichloromethane +
+ Benzophenone
40% n-Hexane
8. Amino acid mixture 70% Ethanol +
30% Water
(or n-Butanol +
Acetic acid + Water in
volume)
the ratio 4:1:1 by
32 ADVANCED PRACTICAL ORGANIC CHEMISTRY
of the developed
For mixtures from 1 to 7, the spots
visualized by keeping in an iodine
chromatogram may be located or
chamber. In the case of mixture 8, a 0.3% solution of ninhydrin
a little acetic acid may
be used to make the
in n-butanol containing
spots visible.

COLUMN CHROMATOGRAPHY

Column chromatography is defined as the uniform percolation

substance. The selected
of a liquid through column of finely divided
substance selectively retards certain comnponents of the liquid.
When the column of solid used is the adsorbent, it is called adsorption
solid column
column chromatography. On the other hand, when the
then it is called
is acting only as a support to the 1liquid adsorbent,
partition column chromatography.
are silica,
The usual adsorbents used in column chromatography
sucrose etc. Selection
alumina, calcium carbonate, magnesia, starch,
in the
of the solvent is based on the nature of the components
solvents such as
mixture. The solvents commonly used are organic
carbon tetrachloride
ether, hexane, benzene, chloroform, acetone,
alcohol. The rate at which the components of a mixture
are
or

separated depends on the activity of the adsorbent and polarity of

the solvent. If the activity of the adsorbent is very high and polarity
of the solvent is too low, then separation is very slow but gives
comparatively pure components. On the other hand, if the activity
the
of the adsorbent is low and polarity of the solvents is high,
separation is rapid but the components obtained are not pure.

The adsorbent say, silica is made into a slurry with a suitable

at the
solvent and placed in a cylindrical tube which is plugged
of the
bottom by glass wool. A porous plug is placed at the top
separated is dissolved in
a
adsorbent column. The mixture to be
suitable solvent and this solution is poured on the top of the column
of the adsorbent. Some components in the mixture are adsorbea
strongly while the others less strongly. Thus the components movin
d
down with different rate undergo separation and a number of coloui
bands or zones are obtained if the components are coloured.
strongly adsorbed component moves slowly and hence it form
nore

band near the top. The weakly adsorbed component moves mo

CHROMATOGRAPHY
33
Mobile phase
rapidlyand hence it forms
Sample
the band near the bottom. mixture

Progressive separation of a
Direction
of mobile
mixture is diagrammatically phase flow
Stationary phase
shown in the figure 11.
Then components are then
eluted out (dissolved out) by Column
suitable (metal
pouring gradually a
or glass)
Separated
sample
solvent (which acts a mobile bands

phase) down the column. The

process of dissolving out the
components from adsorbent
is called elution and the
solvent used is termed an Retaining plug- Eluted separated
sample bands
eluent. The weakly adsorbed
component will be eluted out O
more rapidly than the strongly Fig. 11. Column chromatography.
adsorbed component. Hence,
the components are washed down one by one and they are collected
separately. Pure components are obtained by the removal of the
solvents by suitable methods.

1 2 3

Band 2
Elution
Adsorbed mixture Band 1
Solution to be
Mixture placed Front of
chromatographed in column
band

Adsorbent
alumina

Polar compound
O Nonpolar
compound
34 ADVANCEDPRACTICAL ORGANIC CHEMISTRY
5 6 7

Band 2 Band 2

Gap

Band 1
Band 2

Compound A Compound B
collected collected
Fig. 12 Sequence of steps in a
chromatographic separation
Column chromatography
is used for the large scale
of of
separation
components mixtures. It is also used for the
separation of
complex mixtures of plant pigments and other natural products.
The separation of organic compounds by column
chromatography
Steps involved
(1) Selection of solvent system (eluent) using TLC
TLC technique is very easy and rapid to perform and requires
only extremely small amounts of the mixture to be separated. It
provides analysis of a mixture of compounds and can be used in
determining the best eluting solvent system for subsequent column
chromatography. The solvent migrates up the plate, carrying with
it the components of the mixture at different rates.
The rate of
movement of a compound with respect to the rate of moyement of
the solvent front, Rf, is a property of that
compound. If two
compounds have Rf values very close to each other, a
good separation
of them is not possible. It is possible to select suitable solvent
CHROMATOGRAPHY 35

of the given mixture with solvents of

system by performing TLC
different polarity. If any of the components is not coloured, we
can detect them either by UV irradiation or by formation of a
complex with iodine.
Table 2. Solvents (eluents) for chromatography

Petroleum ether

Hexane
Carbon tetrachloride
Toluene
Benzene
Dichloromethane Increasing polarity and
Chloroform solvent power (eluting power)
Diethyl ether towards polar functional groups
Ethyl acetate
Acetone
Propanol
Ethanol
Methanol
Water
Acetic acid
(2)Separation using column chromatographhy
Column
chromatography works on a much larger scale by
packing the same materials used in TLC into a vertical glass
column. This is a
solid-liquid technique in which the stationary
phase is a solid and the mobile phase is a liquid. The
column
chromatography is based on differential
principle of
Substances by the adsorbent. The adsorption of
usual adsorbents
column chromatography are silica and employea n
alumina. Selection of eluent
is based on
TLC.
The adsorbent is
made into slurry with a suitable solvent and
placed in a
cylindrical tube that is plugged at the bottom by
Oglass wool. The mixture a piece
to be separated is introduced at the top
36 ADVANCED PRACTICAL ORGANIC CHEMISTRY
of the column and is allowed to pass through the column using the
eluent. As the mixture moves down through the column, the
components are adsorbed in silica (or alumina) at different regions
depending on their ability for adsorption. The component with greater
adsorption power will be adsorbed at the top and the other will be
adsorbed at the bottom. The weakly adsorbed component will be
eluted more rapidly than the other. The different fractions are
collected separately. Distillation or evaporation of the solvent from
the different fractions gives the pure components.

Experiment No. 11
Column Chromatographic Separation of Organic Binary
Mixture No. 1

Aim
To carry out the column
chromatographic separation of a mixture
of 2-nitrophenol and
4-nitrophenol, and to determine the yield of
the separated components.

Procedure
A column (glass tube) of approximately 20 cm length and diameter
of 2 cm (whose stopcock is without
lubricating grease) is prepared
as follows. The tube is washed
thoroughly (if
necessary, with
chromic acid, followed by distilled water and acetone) and then
dried. Using a glass rod, a small plug of cotton wool is inserted as
a support base
just within the narrow neck of the tube. (If the
column has a built-in filter disc, it is not necessary to prepare a
support base before the adsorbent is added). The column is then
clamped in a vertical position, using two clamps.
About 50 g of activated silica
gel is mixed a little at a time
with about 200 ml solvent (hexane) in a conical
flask until a slurry
of suitable consistency is
obtained Enough adsorbent is added to
the solvent, and mixed by
swirling the conical flask, to form a
thick but flowing slurry. The conical flak flask
should be swirled
until the slurry is homogeneous and
air bubbles.
relatively free of entrapped
CHROMATOGRAPHY 37
When the slurry has been prepared, the column is filled with
solvent to half level. The slurry is then poured carefully in portions
into the column (using a wide necked funnel) keeping the stopcock
and gently on the
slightly open. The column is tapped constantly
with the fingers. The solvent is
side during the pouring operation
silica column settles in the tube. The
allowed to run slowly as the
bottom of the tube is collected in a
solvent emerging from the
the top of the column should be
conical flask. It is essential that
and that the latter passes uniformly
kept immersed in the solvent,
down the column without 'channelling'.
(A disc of filter paper
column to prevent
can, if desired,
be fitted over the top of the
disturbance of the surface).
and 4-nitrophenols (0.2 g each) is
Meanwhile the mixture of 2-
of ethyl
in minimum volume (about ml)
5
dissolving it
prepared by amount of silica gel
mixture is adsorbed on a small
The the
The solvent level in
acetate.

and the solvent is completely evaporated.

adsorbent column by drawing
tube is lowered to the top of the the silica
glass The mixture adsorbed dn
column.
the solvent from the of the column and
a
transferred to the top
gel is then carefully filter is inserted on top
of it..
or a disc of paper
piece of cotton a c e t a t e : 94/6)
the eluent (hexane/ethyl
The column is filled
with eluent flow
immediately. Adjusted the
and the stopcock is opened of the eluent is continued,
minute. Running from
rate to about 10 ml per collecting the eluent
is developed by of the
and the chromatogram
it again through the top
he bottom of the
tube and running bands have
become
until the two yellow of
tube. Continued in this way to the lower portion
their passage each
and separate during fresh eluent until
istinct continued washing
with that
column. Then 150 ml of the solvent,
ne eluted in about chromatographic
has been separately tube. (The
Dand emerging first
from the
Never allow
or the 2-nirophenol operation.
in one
Separation should
be completed
column to run dry). about 20
even the top of the eluates) to
solutions (or
each of the
two
eluent, the distilling
the
part of
oncentrated the
off the greater Then poured
n by distilling water
bath. and
flask being i m m e r s e d in
the boilin e v a p o r a t i n g - b a s i n ,

water
pre-weighed heated
solution into a
electrically
entrated an and
eluent on desiccator

evaporated the remaining from the

eluates in a
residues
bath. Drled the
each component.
recorded the yield of
38
ADVANCED PRACTICAL ORGANIC CHEMISTRY
Result
Yield of 2-nitrophenol =
Yield of
4-nitrophenol = ---

8
Note : Some mixtures and
used for column solvents (eluents) that can be
chromatographic separation
Stationary phase: Silica gel
Eluent Hexane/ethyl acetate: 94/6
(1) 2-Nirophenol (Rf 0.2) and
=

(2) Benzil (Rf 0.2) and

=
4-Nitrophenol( Rf =
0.4)
(3) Benzil
2-Nitrophenol (R 0.4) =

(Rf 0.2) and Naphthalene (Rf

=

(4) Anthracene (R= 0.3) and =0.6)

(5) Benzophenone (R
Anthraldehyde (Rs =
0.5)
0.4) and Naphthalene (Rf
=
=
0.6)
Conclusion
Chromatography has grown over the
central separation science. It has becomepast the
century to be the
common denominator) for "bridge" (or the
of only one or several analytical methods. Instead of measurement
facilitates the separation,components
in a
sample, chromatography
detection, identification, and
measurement with selective detectors of quantitative
usually all the components
in a sample. Its
characteristics of sensitivity,
and quantitative features on micro,
selectivity, versatility,
macro, and
have led to its rapid
expansion. The preparative scales
include the persistence and driving forces of chromatography
creativity of scientists, their
experimental
investigations, their interrelated seminal
concepts, their
journals and other research
publications, and the relevant scientific
organizations.
References
1. Pavia, Lampman, Kriz, Engel;
Manual, Cengage Learning, 2009.
Organic
Chenistry - A Lab
2. Furniss, Hannaford, Rogers, Smith,
of Practical Organic Chemistry, 5 Tatchell; Vogel's Textbook
ed., Pearson Education,
2005.
3. Wixom, Gehrke (Eds);
Discovery, Wiley, 2010.
Chromatography- A Science of