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ASSIGNMENT ON

Column Chromatograph

Course name: Advanced Pharmaceutical Analysis
Course code: PHRM 409
Course Instructor: Dr. Shamsun Nahar Khan
Section: 1


SUBMITTED BY:
Ferdousi Hoque
ID: 2010-3-70-021



Submission date: 8
th
May, 2014

Chromatography
Chromatography according to USP can be defined as a procedure by which solutes are separated
by a differential migration process in a system consisting of two or more phases, one of which
moves continuously in a given direction and in which the individual substances exhibit different
motilities by reason of differences in absorption, partition, solubility, vapour pressure, molecular
size, or ionic charge density.
Column Chromatography
Column chromatography may be defined as the selective absorption and separation of a mixture
of chemical compounds on a column of stationery phase, through which a suitable mobile phase
(solvent) has been passed under the influence of gravity.
Types of Column Chromatography
1) Liquid-solid chromatography: Here stationery phase is solid and mobile phase is liquid
2) Liquid- liquid chromatography: Here both stationery and mobile phase is liquid
3) Thin layer chromatography: Here stationery phase is a plane in the form of a solid
supported on an inert plate.
4) High performance liquid chromatography: This system relies on pumps to pass a
pressurized liquid solvent containing the sample mixture through a column filled with
a solid adsorbent material.
Principle of Column Chromatography
The principle underlying the separation of the compounds is adsorption at the solid-liquid
interface. For successful separation the compounds of a mixture must show different degree of
affinity for the solid support (or adsorbent) and the interaction between the compounds and the
adsorbent must be irreversible.
As the adsorbent is washed with fresh solvent, the various components will, therefore, move
down through the column in order of their affinity for the adsorbent. Those with least affinity
move down the column at a faster rate than those with the greatest affinity for the adsorbent.
Preparation Principle

Figure: Parts of Column Chromatography
1) Preparation of the column: At first a plug of cotton wool is placed in the bottom of the
column and pressed down evenly. Then some cleaned washed stand can be poured on the top of
the plug to form a thin layer. It gives a flat base. Then the adsorbent may be introduced into the
column either as a dry powder or as a slurry suspended in the solvent and firmly packed into the
tube. After setting the absorbent, the excess solvent is allowed to drain out by the duct but a
head on layer of solvent should always cover the absorbent.
2) Sample application: The sample is placed at the top of the column as a band either by two
ways:
) A small portion of the stationery phase is mixed with it and packed into the column.
I) A small portion of the mobile phase is dissolved with it and deposited in the column packing.
3) Development of the Chromatogram: The solvent is added continuously from the reservoir
to the top of the column at a sufficient rate to ensure a head of liquid on the column. As the
chromatography develops, the constituents of the sample will pass down the column at varying
rates.
4) Detection and recovery of the compounds: By visual examination using fluorescent eluting
the compounds with solvents.
Normal Phase and Reverse Phase Chromatography
In the normal phase mode, the stationery phase is a polar substance such as, polyethylene glycol
or silica and the mobile phase is non-polar (e.g. hexane). Under these conditions, polar
compounds are retarded preferentially ad non-polar substances elute more quickly.
In reversed phase, the stationery phase is non-polar such as octadecyl silyl (ODS) and the mobile
phase is polar, usually a mixture of water and methanol.

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