ASSIGNMENT on Column Chromatography

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

th

Submission date: 8  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 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 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 t a faster rate than those with the greatest affinity for the adsorbent.

Preparation Principle

Figure: Parts of Column 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: І) A small portion of the stationery phase is mixed with it and packed into the column. ІI) 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 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.