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COLUMN CHROMATOGRAPHY

Column of stationary phase is used


Solid S.P
Liquid M.P

PRINCIPLE
Adsorption
Mixture of components dissolved in the M.P is
introduced in to the column.
Components moves depending upon their
relative affinities.

PRACTICAL REQUIREMENTS
1.STATIONARY PHASE
Adsorbent in C.C should meet following criteria
Spherical in shape
Mechanical stability must be high
They shouldnt react chemically
It should be useful for separating for
wide variety of compounds
It should be freely available & inexpensive

SELECTION OF S.P
Success of chromatography proper
selection of S.P, it depends on the
following.
1. It should have the property of removal
of impurities
2. It should have the capacity to
separate more no. of components
3. Length of column is more
4. Its affinity differs b/w components
5. Good quality of adsorbent is used

MOBILE PHASE
They act as
Solvent
Developer
Eluent
COLUMN CHARACTERISTICS
Column - Neutral glass
Column dimensions - length & diameter ratio
(10:1,30:1 or 100:1)

PREPARATION OF THE COLUMN


Bottom portion of the column packed with
glass wool/cotton wool or may contain asbestos
pad,
Above which adsorbent is packed
After packing a paper disc kept on the top

Two types of packing techniques are


there.
1.Dry packing
2.Wet packing

DRY PACKING

Dry Packing Technique


Adsorbent is packed in the column in dry form
Fill the solvent, till equilibrium is reached
DEMERIT: Air bubbles are entrapped b/w M.P & S.P
cracks appear in the adsorbent layer.

Wet Packing Technique

ideal & common technique


Adsorbent + M.P in a beaker & poured in to
column
S.P settles uniformly & no crack in the column of
adsorbent

INTRODUCTION OF THE SAMPLE


Samples dissolved in M.P & introduced into
the column at once eluted
ELUTION PROCEDURES
Two techniques
1)Isocratic elution techniques
2)Gradient elution techniques
Isocratic elution techniques
(Iso means same)
Same solvent composition or solvent of same
polarity used throughout the process of
separation

Gradient elution techniques

( gradient gradually)
Solvents of gradually polarity or elution
strength are used during the process of
seperation.
E.g. initially benzene, then chloroform, then ethyl
acetate then chloroform
DETECTION OF COMPONENTS
Colored components-Visually
Colorless components- Different properties
which can be used are uv / visible detector,
flourescence detector, RI detector, Flame
ionization detector

RECOVERY OF COMPONENTS
ELUTION
FACTORS AFFECTING COLUMN
EFFICIENCY
1) Nature of solvents
2) Dimension of columns
3) Particle size of column packing
4)Temperature of the column

APPLICATIONS
Seperation of mixture of components
Purification process
Isolation of metabolites
Estimation of drugs in formulations

Advantages of C.C
Any type of mix. can be separated
Any quantity of mix. can be separated
Wider choice of M.P
Automation is possible

Disadvantages of C.C
Time consuming
amounts of M.P required

Automation makes the techniques more complicated &


expensive