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 which when separated formed distinct coloured
rings. These zones or rings are named chromatograms. These days, it is
applied to coloured as well as colourless substances for analysis.

The chromatography
paper is made up of
cellulose fibers and
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 a
chromatography paper and a small spot of the mixture to be separated is
dabbed on the paper. This is placed in a beaker of water ensuring that the water
does not touch the spots. As the water 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.
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
gases in industrial plants that’s
are harmful if leaked.
 It is used to reduce the
emissions from a power
generation industry.

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