3. Introduction
HPLC stands for high performance liquid
chromatography or high pressure liquid
chromatography .
HPLC is a chromatographic technique used to
separate a mixture of compounds in analytical
chemistry and biochemistry with the purpose to
identifying, quantifying, the purifying the
individual component of the mixture .
It is having high resolution and separation
capacity or can be used both as qualitative and
quantitative analysis.
4. Chromatography
• Chromatography is a physical method of
separation of components between two
phases :
• Stationary phase : the substance on which
adsorption of analyte ( the substance to
be separated during chromatography ) take
place .It can be a solid gel or a solid liquid
combination.
• Mobile phase : the solvent which carry
analyte ( a liquid or a gas )
5. Type of Chromatography
• On the basis of physical state of mobile
phase there are two type of
chromatography:-
I. Gas chromatography
II. Liquid chromatography
6. HPLC
HPLC is a type of liquid chromatography
where the sample is forced through the
column that is packed with stationary phase
composed of irregularity or spherically
shaped particle , a porous monolithic layer or
a porous membrane by a liquid ( mobile
phase) at high pressure.
11. Classification
• Depending upon the phase system (stationary phase and
mobile phase ) HPLC is classified into following types:
1. Normal Phase HPLC:
It has polar stationary phase and non –polar
mobile phase.
The stationary phase is usually silica and typical
mobile phase are hexane, methylene chloride ,
chloroform, diethyl ether and mixture of these.
Polar samples are thus retained on the polar
surface of the column packing longer than les
polar materials.
12. 2) Reverse Phase HPLC
The stationary phase is non-polar
(hydrophobic) in nature, while the mobile phase is
a polar liquid , such as mixtures of water and
methanol or acetonitrile.
It works on the principle of hydrophobic
interaction hence the more non-polar the material
is, the longer it will be retained.
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17. • Solvent reservoir : it holds the solvent called mobile
phase which moves through the various parts of the
system.
• Pumps: the role of the pump is to force the liquid ( called
the mobile phase) through the liquid chromatography at a
specific flow rate , expressed in mililitres per min
(ml/min).
Normal flow rate in HPLC are in the 1 to 2ml/min
range.
Typical pumps can reach pressures in the range of
6000-9000 psi (400to 600bar).
18. • During the chromatography experiment a pump can deliver
a constant mobile phase composition (isocratic) or an
increasing mobile phase composition (gradient).
• Sample injector :- the injector serves to introduce the
liquid sample into the flow stream of the mobile phase.
Typical sample volumes are 5 –to -20 , microlitre.
The injector must also be able to withstand the high
pressure of he liquid system.
