High

Performance
L iquid
C hromatography
The principle of separation in normal phase mode
and reverse phase mode is adsorption. When a
mixture of components are introduced into a
HPLC column, they travel according to their
relative affinities towards the stationary phase.
The component which has more affinity towards
the adsorbent, travels slower. The component
which has less affinity towards the stationary
phase travels faster. Since no 2 components have
the same affinity towards the stationary phase, the
components are separated.
HPLC - Modes
Normal Phase.
- Polar stationary phase and non-polar
solvent.

• Reverse Phase.
- Non-polar stationary phase and a polar solvent.
Mobile phase:
 The choice of mobile phase is very important in HPLC.
 Mixing unit is used to mix the solvents in different
proportions and pass through the column. There are 2 types of
mixing units.
 Several gases are soluble in organic solvents. When solvents
are pumped under high pressure, gas bubbles are formed which will
interfere with the separation process, steady base line and the shape
of the peak. Hence degassing of solvent is important. This can be
done by using Vacuum filtration, Helium purging, Ultrasonication.
 In normal phase chromatography hexane, iso octane, di ethyl
ether are the mobile phases. In reverse phase chromatography water,
methanol, acetonitrile are the mobile phases.
Injector:
In order to introduce a sample onto the column for
analysis, a special valve called the injector must be
used to transfer the sample into the pressurized
system
Stationary Phase(Column):
Detector:
Detectors used depends upon the property of the compounds to
be separated. Different detectors available are:
 1. Refractive index detectors
 2. U.V detectors
 3. Fluorescence detectors
 4. Electro chemical detectors
 5. Evaporative light scattering detectors
 6. IR detectors
 7. Photo diode array detector:
 Injector Chromatogram

Separations Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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 Injector Chromatogram

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The
t -
Chromatogram
elution time of unretained peak
o
tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

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