BACKGROUND OF CHROMATOGRAPHY

Chromatography literally “color writing” was first employed by Russian

scientist MIKHAIL TSVET in 1900. He continued to work with
chromatography in the first decade of 20th century, primarily for the
separation of plant pigment such as chlorophyll, carotenes and
xanthophylls.

Some important terms

Mobile Phase: The solvent system which carried the mixture to be

separated .

Stationary Phase: Immobile surface which is particulate in nature. This

is the region over which the component gets separated

HPLC ( High Performance Liquid Chromatography)

INTRODUCTION:
HPLC is the form of liquid chromatography which is use to separate the
compound that are dissolved in the solution. Each component in the sample
interacts slightly differently with the adsorbent material, causing different flow
rates for the different components and leading to the separation of the
components as they flow out the column.

INSTRUMENT AND WORKING

HPLC instrument consist of a reservoir of mobile phase, a pump, an injector,a

separation column and a detector. The sampler brings the sample mixture into
the mobile phase stream which carries it into the column. The pumps deliver the
desired flow and composition of the mobile phase through the column. The
detector generates a signal proportional to the amount of sample component
emerging from the column, hence allowing for quantitative analysis of the sample
components. A digital microprocessor and user software control the HPLC
instrument and provide data analysis. Some models of mechanical pumps in a
HPLC instrument can mix multiple solvents together in ratios changing in time,
generating a composition gradient in the mobile phase. Since the technique is
slow and time taking so it is improved by the use of pump which pressurized the
liquid in the range of 5000-10000

Various type and applications of HPLC

The various types of HPLC and their characteristics are summarized in the table below.

Table 1. Various Types and Applications of HPLC

TYPE SAMPLE MOLECULAR STATIONARY MOBILE PHASE
POLARITY WEIGHT PHASE
RANGE
Adsorption non-polar to some 100 - 104 silica or alumina non-polar to polar
what polar
Partition non-polar to some 100 - 104 non-polar liquid relatively polar
(reversed-phase) what polar adsorbed or
chemically bonded to
the packing material

Partition (normal- Some what polar to 100 - 104 highly polar liquid relatively non-polar
phase) highly polar adsorbed or
chemically bonded to
the packing material
Ion Exchange highly polar to ionic 100 - 104 ion-exchange resins aqueous buffers with
made of insoluble, added organic
high-molecular solvents to moderate
weight solids solvent strength
functionalized
typically with
sulfonic acid (cationic
exchange) or amine
(anionic exchange)
groups

Size-Exclusion non-polar to ionic 103 – 106 small, porous, silica polar to non-polar
or polymeric particles
USES OF HPLC:

This technique is used for-

 Chemistry and biochemistry research analyzing

complex mixtures
 Purifying chemical compounds
 Developing processes for synthesizing chemical
compound
 Isolating natural products, or predicting physical
properties
It is also in quality control to ensure the purity of raw
material