Department of chemistry

Column Chromatography

Presented by: Prof. A. G. Sarode

Introduction Chromatography was invented by the Russian Botanist,

Mikhail Tswett in 1906.

The word Chromatography is derived from two Greek words Chroma meaning ‘color’ and graphein meaning to ‘write’.

One of the fixed phase is called as stationary phase.

Other phase is moving that is called as Mobile phase.

Chromatography

Principle Chromatography is based on the principle of the partition of the solute between

two phases/Solvents Chromatography usually of a mobile phase and a

stationary phase

1)Mobile phase:-The mixture of substances to be separated dissolved in liquid

or a gas.

2)Stationary phase:-Is a porous solid matrix through witch the sample

contained in the mobile phase percolates. The interaction between the mobile

and stationary phases result in the separation of the compounds from the

mixture.

Types of Chromatography There are following types of Chromatography

Paper Chromatography.

Thin layer Chromatography (TLC).

Ion exchange Chromatography.

Size Exclusion Chromatography.

Column Chromatography.

Gas Liquid Chromatography.

Gas Solid Chromatography.

Column

Chromatography

Content Principles.

Practical Requirement.

Technique.

Packing of column.

The washing of column.

Factor affecting column efficiency.

Advantages.

Disadvantages.

Principle When a mixture of components dissolved in the

mobile phase is introduced into the column the individual

components move with different rate demanding upon

their relative affinities.

The compounds with lesser affinity towards

stationary phase move faster and it is eluted out of the

column first. The one with greater affinity towards

stationary phase move slower down the column and hence

it is eluted latter . Thus the compound are separated.

Practical Requirement Glass tube with stopcock.

Sintered glass frit or glass wool.

Sample.

mobile phase(solid or liquid)

Stationary phase(solid or liquid)

Technique

Stationary phase:

Particles should have uniform size and spherical shape(60-200) microns.

Should have high mechanical stability, inert, insoluble in mobile phase used.

Colorless.

Should allow free flow of mobile phase.

Freely available and in expensive.

Mobile phase

To introduce the mixture into column as solvent.

To develop the zones for separation as developing agent.

To remove pure component out of the column as eluent.

Packing of column.

Importance of packing

The column must be packed as uniformly as possible to

minimize the distortion of the chromatographic boundaries

Channeling is usually caused by the inclusion of air bubbles

during packing .

Types of packing

Dry Packing: In this required quantity of adsorbent is poured as

fine dry powder in the column and the solvent is allowed to

flow through the column till equilibrium is reached.

Wet packing: This the ideal technique. The slurry of adsorbent

with the mobile phase is prepared and is poured in to the

column. The stationary phase settle uniformly. There is no

entrapment of air bubble. The bands eluted from the column

will be uniform and ideal for seperation.

The washing of column.

The process of washing the column is called as elution.

The mobile liquid is called eluent.

The solution coming out of the column is called elute.

The solvent must be eluted slowly, so that there is adequate time to

established continues equilibrium.

Elution process:

After the introduction of the sample, by the elution

techniques the individual component are separated out from

the column.

The elution technique, the same solvent or solvent system

of same polarity is use throughout the process of separation.

Factor affecting column

efficiency

Dimension of the column.

Particle size of the adsorbent.

Nature of the solvent.

Temperature of the column.

Pressure.

Advantages

Column chromatography is best suited to

separate active principle from plant materials.

In separation of compound after organic

synthesis to obtained desired molecule.

To separate or purify natural compounds

mixtures.

Inexpensive ; Flexible, suitable for

preparation

Wider choice of mobile phase.

Automation is possible.

Disadvantages

Time consuming method.

More amount of solvent are required which are expensive.

Automation may makes the technique more complicated and expensive.

Reference

1. Www.google.com

2. VOGEL’S Text books of quantitative Analysis

3. B .K. Sharma

Thank You..