CHEMISTRY

FA-2 AOL-2
Chromatography
The project at a glance:
1. What is chromatography?
2. Principle (How it works)
3. Types of chromatography and their Applications
i. Column chromatography
ii. Paper chromatography
iii. Thin layer chromatography
iv. Gas chromatography
v. Liquid chromatography

What is chromatography?
Chromatography is a set of techniques used for the separation of
mixtures. It deals with the interaction between two different phases,
the mobile phase and the stationary phase. The technique of
separation is achieved by the differential movement of individual
components through the stationary phase under the influence of the
mobile phase.
The name chromatography means colour writing, since in its original
applications by Michael Tswett in 1906, it involved separation of
mixtures containing
coloured substances
when separated
The chromatography
paperwhich
is
formed distinctmade
coloured
rings.
These fibers
zones or rings are named
up of
cellulose
chromatograms.
days,
is applied
and These
cellulose
is aitpolymer
of to coloured as well as
colourless substances
forsugar,
analysis.
simple
cellulose.

Principle
The principle on which chromatography works is that the pulp in the
filter paper used for chromatography is composed of thin capillaries.
When this paper is suspended in a beaker of water, touching the
water surface, water ascends through these capillaries. Different
soluble dyes move upwards at different speeds and to different
levels.
In another type of chromatography, the adsorbent medium used is
generally magnesium oxide, alumina or filter paper. The solvent
generally used for dissolving a mixture of two or more constituents is
water or alcohol.
The different constituents get absorbed differently on the
same adsorbent material, because they have different rates
of movement. The rate of movement of each absorbed material
depends upon:
The relative solubility of the constituents of mixture in a given
solvent.
The relative affinity of the constituents of mixture for the
adsorbent medium.

Types of Chromatography
The different types of chromatographic techniques used are:

Column Chromatography
Column chromatography is a type of chromatographic technique,
wherein the stationary phase is present inside a column and the
mobile phase containing the mixture to be separated is introduced
into the column.
In the bottom of a glass column, cotton and the stationary phase
(silica gel, alumina etc.) are kept. On top of this, sand is kept to
support the stationary phase. The mixture
to be separated is dissolved in alcohol and
introduced into the column. The
components of the mixture separate and
form thin bands. The component that is
more weakly absorbed will pass faster
through the column and gets separated
first and the component that is more
strongly absorbed will get separated last.
Application
Column chromatography is best
suited to separate active principle from plant materials.
In separation of compounds after organic synthesis to obtain
desired molecule.
To separate or purify natural compound mixtures like alkaloids,
glycosides.

Paper Chromatography
To separate components by this
method a pencil line is drawn on
chromatography paper and a
spot of the mixture to be
separated is dabbed on the
paper. This is placed in a beaker
water ensuring that the water
not touch the spots. As the
is absorbed it separates the multiple
components of the mixture. The
chromatography paper is called the stationary
phase as it does not move and the water is
called the mobile phase as it moves up the
chromatography paper.

a
small

of
does
water

Applications
It is an inexpensive and rapid method that
provides graphic and clear results.
The separated spots on the finished and dried chromatogram can
be cut out and re-dissolved to obtain a pure sample of
component of the sample mixtures.
In forensic studies paper chromatography is used in crime scene
investigation and DNA and RNA sequencing along with other
studies.

Thin Layer Chromatography (TLC)

Thin layer chromatography (TLC) is a widely employed laboratory
technique and is similar to paper chromatography. However, instead
of using a stationary phase of paper, it involves a stationary phase of
a thin layer of adsorbent like silica gel, alumina, or cellulose on

a glass plate. If the separated components are colourless Ninhydrin is

sprayed on the dried chromatogram which can turn some
components purple.
Applications
TLC is extremely useful in isolation or separation of biochemical
metabolites or constituent from its body fluids, blood plasma,
serum, urine etc.
Various medicines like hypnotics, sedatives, anticonvulsant
tranquillizers, antihistaminics, analgesics, local anesthetics,
steroids have been tested qualitatively by TLC method.
One of the most important applications of TLC is in separation of
multicomponent pharmaceutical formulations.
Gas Chromatography
Gas chromatography is a separation technique in which the mobile
phase is a gas. The mobile phase is inserted through the injector, as a
liquid, and gets vapourised. As it is in the gaseous state an inert
carrier gas is supplied to push these through the column. The longer
the column the better separation we will get. Inside the column, the
walls are coated with a liquid (stationary phase), and one of the
components clings to the walls and travels slowly while the other
moves faster in the middle and reaches the detector first. The
detector picks up the number of particles of the components found
and displays it on a data system in the form of a graph which is
understandable to us.
Applications
It is used in gas processing
plants.
It is used for monitoring the
in industrial plants thats
harmful if leaked.
It is used to reduce the
emissions from a power generation industry.

gases
are

Liquid Chromatography
Liquid chromatography (LC) is a separation technique in which the
mobile phase is a liquid. It can be carried out either in a column or a
plane. Present day liquid chromatography that generally utilizes very
small packing particles and a relatively high pressure is referred to
as high performance liquid chromatography (HPLC).
Applications
It is used in the field of analytical toxicology.
It is used in biochemical screening for genetic disorders.
LC is relevant to several areas of steroid biochemistry.

Done by
Sindhuja
9G