Maraj & Samai 2009

Chromatography
Chromatography is used to analyze small quantities
of a mixture of substances which are chemically
similar to each other.

 It involves the partition of the components of the

mixture between a stationary phase and a mobile
phase.

The mixture to be separated is introduced on to the

stationary phase which stays still.
 Chromatography
The mobile phase is then allowed to move over the
stationary phase for separation.

Partition depends on the different solubilities of the

components in the mobile phase and the different
adsorption forces of the components with the stationary
phase.

Adsorption is the temporary attraction of molecules of a

gas or liquid to a solid surface. Components with greater
solubilities will dissolve in the mobile phase and move
along with it readily.
 Chromatography
Components with stronger adsorption forces will be
held on the Stationary phase and not move along
readily with the mobile phase.

The differences in solubilities and adsorption bring

about separation.
 Paper Chromatography
In paper chromatography a piece of filter or
chromatography paper is used which consists of
stationary water molecules embedded in a cellulose
matrix.

 The water molecules act as the stationary phase.

 The mobile phase consists of a suitable solvent that

travels up the stationery phase.
 Paper Chromatography
The mixture to be separated is spotted a short
distance from one end of the paper (the base line).

 The end below the spot is placed in the solvent.

As the solvent moves along the paper it carries the

mixture with it.

The distance the solvent moves from the baseline is

called the solvent front.
 Paper Chromatography
Components of the mixture will separate readily
according to how strongly they adsorb on the
stationary phase and how readily they dissolve in the
mobile phase.

Paper Chromatography System

 Paper Chromatography
If separated components are colourless, then a
visualizing agent can be used to convert them into
coloured spots.

The positions of certain substances can also be

determined by fluorescing under a UV lamp.
 Paper Chromatography
The ration of the distance moved by a component of the
mixture to the distance is moved by the solvent is called
the retention factor.
Rf = distance moved by a component
distance moved by solvent

Each component has a characteristic Rf value for a given

solvent under controlled conditions.

Thus Rf values of known substances can be used to

identify components of a mixture.
 Paper Chromatography
Paper chromatography is used to analyze mixtures
such as dyes in ink, colouring in food additives and
amino acids from protein hydrolysis.

A visualizing agent such as ninhydrin is used to

detecting amino acids and amines.
 Thin Layer Chromatography (TLC)
This method is similar to paper chromatography.

The stationary phase is a thin layer of powered alumina or

silica gel which is fixed on to a glass or plastic plate.

Plates can be coated with a slurry of the powdered

adsorbent and then oven-dried.

The mixture to be analyzed is spotted near the bottom of

the plate. The end below the spot is placed in suitable
solvent.
 Thin Layer Chromatography (TLC)
This solvent is the mobile phase and moves up the plate
causing the components of the mixture to partition
between the adsorbent on the plate and the moving
solvent.

The separated components may be recovered for further

analysis by scrapping off the plate.

Thin layer chromatography has the advantage that a

variety of adsorbents can be used for separation.

It is commonly used to separate amino acids in blood

samples and for analysis of food dyes.
 Column Chromatography
This method is similar to thin layer chromatography
however the stationary phase is packed into a vertical
glass column (diameter 1-2cm) instead of being
coated on to a plate.

A slurry of silica gel or alumina is commonly used for

column chromatography.

The mixture to be analyzed is applied to the top of

the column.
 Column Chromatography
The mobile phase is a suitable solvent which is added
to the top of the loaded column.

The solvent flows down the column under gravity

causing the components of the mixture to partition
between the adsorbent and the solvent.

 Each component emerges from the column at

different times and can be collected separately.
 Column Chromatography
The time between addition of the sample at the top of the
column and the emergency of a component at the bottom
of the column is called the retention time of that
component.

Identical substances will have the same retention time

under the same conditions thus retention times can be
used to identify substances.

Column chromatography has the advantage that larger

quantities can be separated and therefore can be used to
prepare compounds in addition to analyzing them.
 Column Chromatography
This method is used in biochemical research and in
hospitals to identify amino acids, peptides and
nucleotides.
High Performance Liquid Chromatography
This technique is similar to column chromatography
however instead of gravity feed; high pressure is sued
to force the solvent through the column.

Columns are smaller than those used in column

chromatography, some being 10 cm to 30 cm long and
4 mm in diameter.

Retention times are shorter thus rapid analysis of

substances can be made.
High Performance Liquid Chromatography
HPLC is used in the industry and hospitals.

It is also used to identify suspected stimulants, doping and

drugs that may be present in athletes and racehorses.

Components of a HPLC system

Gas-Liquid Chromatography (GLC)
GLC uses a longer column than HPLC.

It is usually packed with the stationary phase which is

an inert powder coasted with an involatile oil.

The column is maintained at a constant, pre-set

temperature in an oven.

The mobile phase is an unreactive gas, usually

nitrogen or helium and is referred to as the carrier
gas.
Gas-Liquid Chromatography (GLC)
The sample to be analyzed has to be in the vapour state at
the temperature at which the column is operated.

The vapourized sample is carried through the column by

the mobile phase.

The sample is partitioned between the oil and the carrier

gas.

A detector records each component as it leaves the

column at different times.
Gas-Liquid Chromatography (GLC)
Emerging components can also be fed directly into a
mass spectrometer for identification.

GLC method of analysis is very sensitive and can be

used in forensic testing, to monitor air and water
pollution, to detect and identify traces of pesticides or
agricultural chemicals in foodstuff and to check
dosage of drugs in blood or urine samples.
Gas-Liquid Chromatography (GLC)

Components of a GLC system