Chromatography

INTRODUCTION = a colour; graphein

Chroma
=to wwrite) is
chromatography
(Gr. separate individ
term that help to
The techniques
a collective set of laboratory 1s the method of
chromatography
separation in
applied for
from a mixture. Hence two phases, one
ne is
components are distributed between
to be separated mobile phase which mova
which the c o m p o n e n t s termed a s
phase and second is
called as stationary d e f i n i t e d i r e c t i o n . The component
immiscible) in a
(immobile, two phases by the process
o n stationary phase
t h e m s e l v e s between
redistribute
of the mixture (analyte) s i z e e x c l u s i o n . The stationary
partition, ion exchange o r
which may be adsorption, can be
or bonded coating and the nmobile phase (eluent)
be or chosen such that components of
phase c a n solid a liquid The phases are
or critical fluid. which is quite
Iquid. gas a super in each phase. A component
the mixture have differing solubility
to pass through it than a
will take longer time
soluble in stationary phase
phase but very soluble in the
component which is not very soluble in stationary

mobile phase. and the

As a result of these the differences in mobility of sample components
of the compounds
stationary phases separation
the mobile and
interaction between phase.
achieved a s they travel through the stationary
from the mixture in
scientists
a r e used by the
Presently different versions (types) of chromatography
to
examine a mixture, its components and their relations o
1. Analyse (to
another) on
sed
(to determine the identity of a mixture or components bast
2. Identify
known components)

3. Purify (to sperate compounds of interest for further study)

o n e n t s

4. Quantify (to determine the amount of a mixture and/or the comp

present in the sample)

a b l e

Principle
Thesampld(analytg is subjected to flow by mobile phase onto or througi t h e r

stationary phase. The sample components are separated into fraction basea

(206)
 tive affinity towards the two
phases during their travel. The fractlon le
edter afinity to stationary travels slower and at a shorter distance, while
that v
gevith less affinity travelsphase
faster and longer distance.
History In botanist Mikhail Tswett for the first
1906, Russian
introduced chromatography. He employed a technique to separate various plant
time
igments like chlorophylls and xanthophylls by passing the solution of these
DOunds into the glass column which was packed with finely divided caleium
rhonate. Later on Thompson and Way reported the Ion Exchange properties ot
oil. In 1935 Adams and Homes observed ion exchange characteristics in crusned
soil.
nhonograph and opened to the field for preparation of lon Exchange resins. The
concept of Gas-Liquid chromatography was first introduced by Martin and synge
of
1944 separation
in 1941, they also introduced liquid-liquid chromatography. In
amino acid by paper chromatography was done. In 1930 Arne Wilhelm Tiselius
in
that is
Nobel Prize 1948) developed Affinity Chromatography based
(won the Nobel Prize
biological interactions. In 1952 Synge and Martin were awarded
on
was
for their work on chromatography. In 1959 Gel Filteration Chromatography
introduced to separate low molecular weight substances from high molecular
substances. In 1960, further improvement in liquid
chromatography led to the
(HPLC). A n e w
development of High Performance Liquid Chromatography
that is a hybrid of gas and liquid
technique Supercritical fluid chromatography both
combined advantageous features of
chromatography was also developed; It has
gas and liquid chromatography.
can be classified as follows
On the basis of different parameters chromatography
Fig. 1).
Classification of Chromatography
On the basis of On the basis of physical state
On the basis of interaction of
chromatographic bed shape of mobile phase
solute to the stationary phase |
Three Liquid Super Critical|
Two
lon Exchang | |Dimensional Dimensiona Chromato- Fluid
Adsorption
Chromato- Chromato0
Chromato graphy
graphy graphy
Eraphy
Gas
Partition Size Exclusion Column Chromatgraphy
Chromato Chromato Chromatogruphy
graphy graphy
Thin Layer Paper
Chromatography
Chromatography
or a n o r e n t types of
Flow Chart Dlagram Chromatography
Fig. 1. Chromatography:
 Modern Phytotechniques and
208
Biostatistcsa
Classification of Chromatography
. On the Basis of interaction of Solute to the Stationary Phase
(i) Adsorption Chromatography: In this technique the stationary phasei.
while mobile phase is liquid. The compound travels along the solid sI
solid
under the influence of mobile liquid. The separation depends on the extent
pnysical adsorption to the solid surface. e.g.: Column chromatography, TLC and
gas
solid chromatography.
(ii) Partition Chromatography: In this mode, both phases are liquid. The
components have an afinity based on their partition coefificientsinto the individual
layer. The component with the greater partition to mobile phase has a higher
affinity to it. e.g., : Liquid-liquid chromatography, gas-liquid chromatography.
(iii) lon--Exchange Chromatography: It is a process that allows the
separation of ions and polar molecules based on their affinity to the ion exchanger.

Civ) Size Exclusion Chromatography: SEC is a chromatographic method in

which molecules in a solution are separated by their size or in some cases by their
molecular weight.
2. Based on the Physical State of the Mobile Phase
) Liquid Chromatography
(ii) Gas Chromatography
(iii) Super Critical Fluid Chromatography
3. Based on the shape of Chromgraphic bed
(i) Planner Chromatography: In this
type the stationary phase is a plane
surface (two dimensional surface where only length and breadth are taken as area)
on which chromatograms are
developed.
It is further divided into two main types
(a) Paper Chromatography
(b) Thin layer chromatography or high performance thin layer chromatography
(ii) Columnar or Three dimensional
Chromatography : In this advanceu
mode of chromatography we use a column, which is packed with stationary
while mobile phase is run through the column. pna
Columns may be of two types:
(a) Packed Column: In this type of column Se or
particles of solid
the support coated with a
liquid stationary phase is filled stationary pide
in the whole
volume of the column.
(b) Open Tubular Column: Here are
particles of stationary
concentrated on or along the inside column tricted
path for the mobile phase in the middle of wall
the
leaving an open, unre
tube.
Thus the basic of any
chromatographic technique is tionary
solid, thick, that there is a
staplace and
phase usually a
liquid or bonded coating that
mobile phase (eluent) that can be a stays fixed in one

liquid or
gas moves S It.
through it or acrU
Paper Chromatography
It 1s most simple of
analytical technique for the separation and identificAO
the components
It
Irom a mixture. The components De "
can he coloured or can
coloured. 1s a liquid
partition chromatography used to separate amino acids, plat
pigments, Sugars, sugar derivatives and peptides etc. It separates dried liquid
samples with the help of a liquid solvent (mobile phase) and a paper stP

(stationary paper)

Principles of Paper Chromatography

(A) Capillary Action : The movement of liquid within the spaces of a porous
material celllose paper) take place due the forces of adhesion, cohesion'and surface
is able to move up the paper strip because its attraction to
tension.1he hquid
stationary phase 1s stronger than the force of gravity.
(B) Solubility : It is the degree to which a mixture (solute) dissolves into a
solvent. The solute dissolves that have similar properties like dissolves like). It
allows ditferent components to be separated by different combination of solvents. i n

paper chromatography separation is depends on both solubility in the mobile phase

and their differential affinity to the mobile phase and the stationary phase.
In paper chromatography stationary base is water held on a solid support of
cellulose paper and mobile phase is a mixture of immiscible solvents of water, a
non polar solvent (acetone, petroleum ether) and an acid or a base e.g. Butanol,
acetic acid water or phenol-water-ammonia etc.
Method: First of all a sample of mixture is prepared from which components to
be separated in a suitable mobile phase. A small spot of sample is applied on the
strip of chromatography paper about two centimeter away from the base of the strip.
This sample is adsorbed onto the stationary phase or may form interactions with it.
The paper is then dipped into a tube or a jar having suitable solvent, taking care
that the spot is above the surface of the solvent and placed in a sealed tube or a jar
(air tight container) (Fig. 2).
The solvent went up the paper by capillary action and dissolved the samples
mixture, which will then travel up the paper with solvent-solute sample. In paper
chromatography different components in the sample mixture travel at different rate

dueto differences in solubility in the solvent and also due to difference in their
altraction to the fibers in the paper.
Now remove the strip from the container when ascending solution front is about
2 cm below from the top of the strip and let it dry. Some components show distinct

colour (plant pigments), while other may be colourless (amino acids) these can be
made coloured by using (spraying) some chemicals (inhydrin) and exposing to UV
light or treatment with iodine vapours Paper chromatography further
classified as
(a) Ascending Chromatography : where solvent is in the pool at the bottom
and rises up on the paper against the gravitational force.
 210 Moden Phytolochniqu and
Bontatistica
(b) Descending Chromatography : where solvent is keep ima through
top of the container and allow to flow down the paper. the
Chromatogruphy Jur
Watann paper
(Stationury phase)
Chromatogram
Separated
plant pigments
Sample loading point
Solvent (Mobile phase)
Fig. 2. Chromatography: Paper chromatography
developing the chromatogram In a sealed jar.
Substance that cannot be separated ascending method can be separated by the
descending method.
(c) Radial Development Technique : Here the sample is applied at the
centre of the paper and it moves outward forming concentric circle of increasing
diametérs.
VAnalysis : After development of chromatogram, the retention factor (RF Value)
is calculated.
Retention Factor : The components that have been separated differ in
their
retention factor. Retention factor is the ratio of distance travelled from the spot of
origin by the solute components to that of the distance travelled by the solvent.
RF Value : It can be calculated as
Rf of component +ADistance travelled by the component A
'A'= Distance travelled the by solventt
Retention factor can never be greater than 1. If Rf value is zero (0), the
remains in the stationary phase and thus it is immobile. If Rf value is one (1)
solute
Une
solute has no affinity for the stationary phase and travels along with the solvene
front.
Two Dimension Paper Chromatography When it becomes
:
separate a complex mixture of components by a single run with one solvent, a second
dificult
is car
run is carried out with difterent solvent and rotating the paper at 90° in between
f. t s isO Called double way paper chromatography and is useful for eompie
mixtu ot compounds having similar polarity eg. anmino acids.
Paper
Drop
Lid (a) mixture
Solvent
Some
hours
Paper later
Solvent
Front
Turn paper 0 ° cloxkwise
and use a ditterent solvent
Some
Solvent (c) hours
later
Fig. 3. Paper chromatography: Representation of two dimensional paper
chromatography
Paper Used Either pure cellulose. Whatmann N. 1 and N. 3 or modified
cellulose paper like DEAE cellulose. CM cellulose and Resin impregnated paper
Amberlite can be used for the purpose of paper chromatography.
Solvent Used The choice of solvent depends upon the mixture to be separated
eg.. solvent system for sugar generally used is butanol (4): Acetic acid (1) : Water (5).
Significance of Paper Chromatography
is very easy. simple. rapid and highly efficient
1. Paper Chromatography a
method of separation.
of the sample
2. It can be applied in microgram quantities
It is useful to separate a wide variety ot components like plant pigments,
3.
amino acids, oligopeptides, sugars, oligosaceharides. glyeosides. purines and
pyrimidines, steroids, vitamins and some alkaloids like peneillin. tetracyelin
and steroptomyein.
But this technique is comparatively less eflicient than thin lava
chromatography as it has low resolving power and it also can not separate proteins ayer
ECause of their insolubility in many solvents.