Chromatography Column chromatography High performance liquid chromatography

1. HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
Ayshath safwana A
2nd Msc. Zoology

2. C h r o m a t o g r a p h y
1 H i g h p e r f o r m a n c e
liquid chromatography
2
CONTENTS

3. CHROMATOGRAPHY
• Chromatography is a physical method for separation
of compounds.
• Tswett, Russian botanist, father of chromatography, is
credited for the development of chromatography.
• Chromatography is based on a very simple principle.
The sample to be examined is allowed to interact with
two immiscible phases -a mobile phase and a
stationary phase.

4. • The stationary phase which may be a solid or a
liquid supported on a solid, doesn't move.
• The stationary phase may be packed in a column,
spread as a layer, or distributed as a film, etc.
• The mobile phase moves sample through the
stationary phase.
• The two immiscible phases could be a solid and a
liquid, or a gas and a liquid or a liquid and another
liquid.

5. • All chromatographic methods involve passing a
mobile phase through a stationary phase.
• The two phases are chosen so that the
components of the sample distribute themselves
between the mobile and stationery phases to
varying degrees.
• The mobile phase may be a liquid (liquid
chromatography)or a gas (gas chromatography).

6. High Performance Liquid
Chromatography
• High performance liquid chromatography is a column
chromatography. it is a liquid chromatographic technique.
i.e, mobile phase is liquid.
• Instead of a solvent being allowed to drip through a column
under gravity, it is forced through under high pressure of
up to 400 atmosphere.
• It yields high performance and high speed compared with
traditional column chromatography.

9. Principle of High-Performance Liquid Chromatography
(HPLC)
• The purification takes place in a separation column
between a stationary and a mobile phase.
• The stationary phase is a granular material made of
solid particles such as silica or polymers, with very
small porous particles in a separation column.
•

10. • The mobile phase is a solvent or solvent
mixture which is forced at high pressure
through the separation column.
• Via a valve with a connected sample loop, i.e.
a small tube or a capillary made of stainless
steel.
• The sample is injected into the mobile phase
flow from the pump to the separation column
using a syringe.

11. • Subsequently, the individual components of the
sample migrate through the column at different
rates because they are retained to a varying degree
by interactions with the stationary phase.
• After leaving the column, the individual substances
are detected by a suitable detector and passed on
as a signal to the HPLC software on the computer.

12. • At the end of this operation/run, a
chromatogram in the HPLC software on the
computer is obtained.
• The chromatogram allows the identification
and quantification of the different
substances.

14. The principle of HPLC are based on Van Deemter equation
which relates the efficiency of the chromatographic column
to the particle size of the column, molecular diffusion and
thickness of stationary phase.
The Van Deemter Equation is given as,
H or HETP = A + B / υ + C υ
A= represents eddy diffusion
B= represents molecular diffusion
C =represents rate of mass transfer
υ =represents flow rate

15. INSTRUMENTATON
• 1. Solvent delivery system
• 2. Pumps
• 3. Sample injection system
• 4. Column
• 5. Detectors
• 6. Recorders and Integrators

18. Column

19. Types of High-Performance Liquid Chromatography
(HPLC)
• Normal phase:
• Column packing is polar (e.g silica) and the mobile
phase is non-polar. It is used for water-sensitive
compounds, geometric isomers, cis-trans isomers, and
chiral compounds.
• Reverse phase:
• The column packing is non-polar (e.g C18), the mobile
phase is water+ miscible solvent (e.g methanol). It can
Based on modes of Chromatography

20. • Ion exchange:
• Column packing contains ionic groups and the
mobile phase is buffer. It is used to separate
anions and cations.
• Size exclusion:
• Molecules diffuse into pores of a porous medium
and are separated according to their relative size
to the pore size. Large molecules elute first and
smaller molecules elute later.
Based on principle of seperation

21. Applications of High-Performance Liquid
Chromatography
• Analysis of drugs
• Analysis of synthetic polymers.
• Analysis of pollutants in environmental
samples.

22. • Determination of drugs in biological
matrices.
• Isolation of valuable products
• Product purity and quality control of
industrial products and fine chemicals.
• Separation and purification of biopolymers
such as enzymes or nucleic acids.
•
•

24. Advantages of HPLC
•High Speed.
•High resolution
•High sensitivity
•High accuracy

25. Limitations
• Cost: Despite its advantages, HPLC can be costly,
requiring large quantities of expensive organics.
• Complexity
• HPLC does have low sensitivity for certain
compounds, and some cannot be detected as they
are irreversibly adsorbed.
• Volatile substances are better separated by gas
chromatography.

26. THANKS