Download Chromatography

A SEPARATION TECHNIQUE.…….
CHROMATOGRAPHY
Chromatography is a technique of separation
of different dissolved constituents of a
mixture by absorbing them over an
appropriate adsorbent material .It is a
versatile method of separating different
kinds of chemical mixtures .
TYPES OF CHROMATOGRAPHY
Paper chromatography
 Thin Layer Chromatography(TLC)
 Column Chromatography
 Adsorption Chromatography
 Gel Filtration Chromatography
 Ion Exchange Chromatography
 Affinity Chromatography
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PAPER CHROMATOGRAPHY
The technique in which the filter paper is used
as an adsorbent material for the separation of
various constituents of a mixture is called
paper chromatography.
In Paper Chromatography, the sample to be
analyzed is applied in the form of a spot near
one of the edges of a “Whatman Filter Paper”.
DEPENDING UPON THE DIRECTION OF FLOW OF MOBILE PHASE ,
THE TWO COMMONLY USED SYSTEMS ARE : ASCENDING &
DESCENDING PAPER CHROMATOGRAPHY
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In the ascending method, the solvent is kept at the base of the
chamber & the edges of the paper where the sample has been
applied is immersed in the solvent taking care that the sample
spots do not get dipped in the solvent but remain just above
the surface of the solvent. The solvent moves up or ascends
the paper by capillary action & the separation of different
components ocuur on the basis of differences in the partition
coefficients.
In the descending method, the paper is hung in such a way that
the side where the sample has been spotted sips in a trough
which is fitted at the top of the chamber & contains the mobile
phase & this solvent travels down the paper under the force of
gravity.
TYPES OF PAPER CHROMATOGRAPHY
IDENTIFICATION OF COMPOUNDS
The separated compounds are identified on the
basis of Rf values which denotes relative to
front. The Rf value is calculated as follows:
Rf = dist. Travelled by the component from base line
dist. Travelled by the solvent from the base line
APPLICATIONS OF PAPER CHROMATOGRAPHY
It is used in separation of amino acids , sugar
from urine , drugs from the samples of blood
and colors from dyes .
 It is used for the detection of adulterants and
contaminants in foods and drinks .
 It is used for the detection of drugs and dopes
in animals & humans .
 It is used to control the purity of
pharmaceuticals .
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THIN LAYER CHROMATOGRAPHY
This technique is similar to paper chromatography
and is more convenient & less time consuming.
Instead of paper, the supporting material is a glass
plate, a plastic sheet or a piece of foil. The layer
may be 250µm for analytical separations & 2-5
mm for preparative separations. A agent such as
Calcium sulphate or a Gypsum are incorporated
into the chromatographic media to facilitate a firm
adhesion of adsorbent to plate. Most widely used
adsorbent in TLC is Silica Gel which contains 13%
CaSO4 .
THIN LAYER CHROMATOGRAPHY
APPLICATIONS OF TLC
It is used as pilot technique to quickly
determine the complexity of a mixture .
 Thin layer techniques are used to identify
drugs, contaminants and adulterants .
 It has also been used to resolve plant extracts
and many other biochemical preparations .
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COLUMN CHROMATOGRAPHY

Paper and thin layer chromatography described
in proceeding sections use a flat bed or surface
for the separation of molecules. In column
chromatography, separation is achieved by the
passage of sample through vertical fixed
tubular glass or polypropylene column which is
packed with an chromatography media.
COLUMN CHROMATOGRAPHY
APPLICATIONS OF COLUMN CHROMATOGRAPHY
It is used to separate or purify natural
compound mixtures like alkanoids , glycosides .
 It is used for the removal of impurities .
 It is used for the isolation of active constituents
 It is used for the estimation of drugs in
formulations .
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ADSORPTION CHROMATOGRAPHY

Adsorption is a phenomenon in which
compounds are held on to the surface of a
solid adsorbent, having specific adsorption
sites, though weak non-ionic interactions such
vander waal forces and H-bonding. For good
resolution, selection of right type of the
adsorbents includes charcoal, silica, alumina
etc.
APPLICATIONS OF ADSORPTION
CHROMATOGRAPHY
It is widely used for biochemical separations
that includes amino acids , mono &
disaccharides , neutral lipids , phospholipids ,
cholesterol easters , carotenoids , etc
 It is used for the clarification of sugar as the
sugar is decolorized by treating sugar solution
with charcoal powder . The latter adsorbs the
undesirable colors present .
 It is also used in paint industry .
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GEL FILTRATION OR EXCLUSION COLUMN
CHROMATOGRAPHY

The chromatographic media used in this
technique are porous, polymeric organic
compounds with molecular sieving properties.
These are cross-linked polymers which swell
in water forming a gel of 3-D networks.
GEL FILTRATION
APPLICATIONS OF GEL FILTRATION
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Removal of salts and small molecules from
macromolecules can be easily performed by
using gel filtration .
It is used in the determination of molecular
weight of macromolecules .
It is used for the purpose of separation of
biological molecules leading to their ultimate
purification .
ION EXCHANGE CHROMATOGRAPHY

Ion exchange chromatography is a type of
adsorption chromatography in which retention
of a solute occurs due to its reversible
electrostatic interaction with the oppositely
charged groups on an ion exchanger. Hence,
this technique is useful for separation of
compounds which bear a net electric charge
such as proteins, amino acids, nucleic acids
etc.
ION EXCHANGE CHROMATOGRAPHY
APPLICATIONS OF ION EXCHANGE
CHROMATOGRAPHY
The most stimulating use of ion exchange
chromatography is in amino acid analysis .
 It is used to determine the base composition of
nucleic acids .
 It is the fast and effective method of water
purification .
 It is also used for the separation of many
vitamins , other biological amines and organic
acids .
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AFFINITY CHROMATOGRAPHY
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Affinity chromatography is a type of adsorption
chromatography in which the substance to be is
specificially and reversibly bound to a
complementary binding substance immobilized
on an insoluble chromatographic bed material
(matrix).
AFFINITY CHROMATOGRAPHY
APPLICATIONS OF AFFINITY CHROMATOGRAPHY
It is used to purify a large variety of
macromolecules such as enzymes ,
immunoglobins , nucleic acids etc .
 It is also used to separate cells that include fat
cells , T and B lymphocytes , spleen cells ,
lymph node cells , oocytes and chick embryo
neural cells .
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SUBMITTED TO: DR.SHIFFALI SINGLA
SUBMITTED BY: ISHA DHIMAN
BSc.BT (hons.) – I
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