assignment on column chromatography

assignment on column chromatography

1. Column chromatography

COLUMN CHROMATOGRAPHY | INTRODUCTION
It is a technique employed for separation of components of mixture by continuous
distribution of components between two phases i.e. one phase moves (mobile)over
the other(stationary) in a continuous manner. when chromatography is carried out in
column it is called column chromatography.
COLUMN CHROMATOGRAPHY | PRINCIPLE
 when a mixture of components dissolved in the mobile phase is introduced
into the column, the individual components move with different rates
depending upon their relative affinities.
 The compound with lesser affinity towards stationary phase moves faster and
it is eluted out of the column first. The one with greater affinity towards
stationary phase moves slower down the column and hence it is eluted latter.
Thus the compounds are separated.
COLUMN CHROMATOGRAPHY | TYPES
Gravity columns:
The mobile phase move through the stationary phase by gravity.
Flash column:
The mobile phase is pushed by stream of air or nitrogen using special
adaptor.
Pumped column:
The movement of mobile phase is accelerated by using pumps that
generate low or medium pressure.
Vacuum column:
The adsorbent is applied drying into sintered glass funnel. The sample
is applied by drying method or as solution.
Then the mobile phase is added portion by portion and vacuum is
applied after each portion to collect each fraction.
High pressure columns:
High pressure pumps are used to push the solvent through the column
which in this case must be made of stainless steel.

2. Ideal properties of 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.
Preparation of stationary phase:
 Adsorbents require activation before use.
 This can be achieved by heating, where adsorbent losses
water and other adsorbed materials.
 Generally there is an optimum temperature for activation.
 Alumina -400o C silica gel- 100o C for 1hr.
 Long time heating leads to loss of its activity.
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.
 Different mobile phases used: Ex; In increasing order of polarity or elution
strength.
Cyclohexane < carbon disulphide< ether < Benzene < toluene < esters <
alcohols < chloroform < acetone <water < pyridine < organic acids.

Packing of column:
 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 the 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.

3. DEMERITS:
 Air bubbles are entrapped between mobile phase and stationary phase and the
column may not be uniformly packed.
 Cracks appear in the adsorbent present in the column. Hence flow
characteristics and clear band of the separated component may not be
obtained.
WET PACKING:
 This is the ideal technique.
 The slurry of adsorbent with the mobile phase is prepared and is poured into
the column.
 The stationary phase settles uniformly in the column and there is no
entrapment of air bubbles.
 There will not be any crack in the column of adsorbent.
 The bands eluted from the column will be uniform and ideal for separation.
Introduction of the Sample:
• The sample which is usually a mixture of components is dissolved in minimum
quantity of the mobile phase.
• The entire sample is introduced into the column at once and get adsorbed on the
top portion of the column.
• From this zone, individual sample can be separated by a process of elution.

Elusion:
After the introduction of the sample, by the elution techniques the individual
components are separated out from the column.
Two types of elution techniques.
Isocratic elution technique:
Iso means same/ similar. In this elution technique, the same solvent or solvent
system of same polarity is used throughout the process of separation. Ex-
chloroform only, petroleum: ether=1:1
Gradients:

4. The solvents of gradually increasing polarity or increasing elution strength are
used during the process of separation. Initially low polar solvent is used followed
by gradually increasing the polarity.
Ex-Initially Benzene than Chloroform, Ethyl acetate, Methanol etc.
COLUMN CHROMATOGRAPHY | DETECTION OF COMPONENTS
• If the compounds separated in a column chromatography procedure are colored,
the progress of the separation can simply be monitored visually.
• If the compounds to be isolated from column chromatography are colorless. In
this case, small fractions of the eluent are collected sequentially in labelled tubes
and the composition of each fraction is analyzed by TLC.
FACTORS AFFECTING
1. Dimension of the column: column efficiency has been improved by increasing
length/width ratio of the column.
2. Particle size of column packing: separation to be improved by decreasing the
particle size of the adsorbent.
3. Activity of the adsorbent
4. Temperature of the column: The speed of the elution increases at higher
temperatures.
5. Packing of the column
6. Quality of solvents: solvents having low viscosities is giving better results.
COLUMN CHROMATOGRAPHY | APPLICATIONS
►Separation of mixture of compounds
►Purification process
►Isolation of active constituents
►Estimation of drugs in formulation
►Isolation of active constituents
►Determination of primary and secondary glycosides in digitalis leaf.
► separation of diastereomers

5. COLUMN CHROMATOGRAPHY
 Advantages of C.C
 Any type of mixture can be separated
 Any quantity of mixture can be separated
 Wider choice of Mobile Phase
 Automation is possible
 Disadvantages of C.C
 Time consuming
 more amount of Mobile Phase are required
 Automation makes the techniques more complicated & expensive.

CONCLUSION:
Column chromatography is a conventional tool for separation of phytochemicals,
removal of impurities and purification of drugs. Effective separation of constituents
from different sources in preparative scale (milligram to gram) can be achieved by
column chromatography. Understanding the basic principles of column
chromatography enables us to find solutions for current research problems.