department of chemistry column chromatography
TRANSCRIPT
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..