2. Definitions
• Tswett (1906) stated that " chromatography is a
method in which the components of a mixture are
separated on adsorbent column in a flowing system”.
• IUPAC definition (International Union of pure and
applied Chemistry) (1993): Chromatography is a physical
method of separation in which the components to be
separated are distributed between two phases, one of
which is stationary while the other moves in a definite
direction.
• The stationary phase may be a solid, or a liquid
supported on a solid or gel, the mobile phase may be
either a gas or a liquid.
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5. Chromatography is a physical process.
Any Chromatography system is composed of
three Components:
Stationary phase
Mobile phase
Mixture to be separated
The stationary phase may be a solid, or a liquid
supported on a solid or gel,
the mobile phase may be either a gas or a liquid.
We can only control stationary and mobile phase as
mixtures are the problem we have to deal with.
Chromatography is a dynamic process in which the
mobile phase moves in definite direction. 5
8. Types of Chromatography
• Liquid Chromatography – separates liquid samples
with a liquid solvent (mobile phase) and a
column composed of solid beads (stationary phase)
• Gas Chromatography – separates vaporized samples
with a carrier gas (mobile phase) and a column
composed of a liquid or of solid beads (stationary
phase)
• Paper Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a
paper strip (stationary phase)
• Thin-Layer Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a
glass plate covered with a thin layer of alumina or 8
9. Planar Chromatography
• Planar chromatographic methods:- two-dimensional
chromatography, include
thin-layer chromatography (TLC),
paper chromatography (PC), and
electrochromatography.
• Each makes use of a flat, relatively thin layer of
material that is either self-supporting or is coated on a
glass, plastic, or metal surface.
• The mobile phase moves through the stationary phase
by capillary action, sometimes assisted by gravity or
an electrical potential.
•
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10. Sample Application
• Sample application is perhaps the most critical
aspect of thin-layer chromatography.
• Usually the sample is applied as a spot 1 to 2 cm
from the edge of the plate.
• Manual application of samples is performed by
touching a capillary tube containing the sample
to the plate or by use of a syringe.
• Mechanical dispensers, which increase the
precision and accuracy of sample application, are
available commercially.
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11. Plate Development
Plate development is the process by which a
sample is carried through the stationary phase
by a mobile phase.
After applying a spot and evaporating the
solvent, the plate is placed in a closed container
saturated with vapors of the developing solvent.
One end of the plate is immersed in the
developing solvent, with care being taken to
avoid direct contact between the sample and
the developer.
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12. After the developer has traversed one half or
two thirds of the length of the plate, the plate
is removed from the container and dried.
The positions of the components are then
determined in any of several ways.
Ascending-flow Developing
chamber Thin layer
chromatography
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13. Paper Chromatography
• Separations by paper chromatography are
performed in the same way as those on thin-layer
plates.
• The papers are manufactured from highly
purified cellulose with close control over porosity
and thickness.
• Such papers contain sufficient adsorbed water to
make the stationary phase aqueous.
• Other liquids can be made to displace the water,
however, thus providing a different type of
stationary phase.
13
14. • For example, paper treated with silicone or
paraffin oil permits reversed-phase paper
chromatography in which the mobile phase is
a polar solvent.
• Also available commercially are special papers
that contain an adsorbent or an ion-exchange
resin, thus permitting adsorption and ion-
exchange paper chromatography
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15. Capillary Action – the movement of liquid within the
spaces of a porous material due to the forces of adhesion,
cohesion, and surface tension.
The liquid is able to move up the filter paper because its
attraction to itself is stronger than the force of gravity.
Solubility – the degree to which a material (solute)
dissolves into a solvent. Solutes dissolve into solvents that
have similar properties. (Like dissolves like)
This allows different solutes to be separated by different
combinations of solvents.
Separation of components depends on both their solubility
in the mobile phase and their differential affinity to the
mobile phase and the stationary phase.
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16. Thin-Layer Chromatography
The stationary phase is a thin layer on the surface of an
appropriate plate. The mobile phase is drawn over the
surface by capillary action.
TLC has found widespread use in clinical laboratories and
is the backbone of many biochemical and biological
studies.
It also finds extensive use in industrial
laboratories.Because of these many areas of application;
TLC remains a very important technique
Typical thin-layer separations are performed on a glass
plate that is coated with a stationary phase, which
consists of a thin and adherent layer of finely divided
particles
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21. 8. Gas Chromatography (GC)
A common type of chromatography used in
analytical chemistry for separating and
analyzing compounds that can be vaporized
without decomposition
The samples are also required to be salt-free;
they should not contain ions.
Very minute amounts of a substance can be
measured, but it is often required that the
sample must be measured in comparison to a
sample containing the pure, suspected
substance.
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22. Typical uses
Testing the purity of a particular
substance
Separating the different
components of a mixture(the
relative amounts of such
components can also be
determined).
Identifying a compound.
Preparing pure compounds from
a mixture in preparative
chromatography
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23. Advantages of Gas Chromatography
Requires only very small samples with little
preparation
Good at separating complex mixtures into
components
Results are rapidly obtained (1 to 100 minutes)
Very high precision
Only instrument with the sensitivity to detect
volatile organic mixtures of low concentrations
Equipment is not very complex (sophisticated
oven)
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24. Disadvantages of Gas
Chromatography
Low sensitivity
Affected by fluctuations in temperature and
flow rate
Biological samples cannot be analysed
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25. Separation theory
• Chromatographic separation involves the use
of a stationary phase and a mobile phase.
• Components of a mixture carried in the
mobile phase are differentially attracted to
the stationary phase and thus move through
the stationary phase at different rates.
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27. 27
Instruments for
GC
Components
1. Gas Supply: (usually N2 or
He)
2. Sample Injector: (syringe /
septum)
3. Column: 1/8” or 1/4” x 6-
50’ tubing packed with small
uniform size, inert support
coated with thin film of
nonvolatile liquid
4. Detector: TC - thermal
