Column chromatography

Chromatographic Method

1. COLUMN
CHROMATOGRAPHY
by
Mr. Shaise Jacob
Faculty, Nirmala College of Pharmacy
Muvattupuzha
Kerala, India

2. 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.

3. PRACTICAL REQUIREMENTS
1.STATIONARY PHASE
Adsorbent in C.C should meet following criteria

◘ Spherical in shape
◘ Mechanical stability must be high
◘ They shouldn’t react chemically
◘ It should be useful for separating for
wide variety of compounds
◘ It should be freely available & inexpensive

5. SELECTION OF S.P
Success of chromatography → proper
selection of S.P, it depends on the
following.
Removal of impurities
No. of components to be separated
Length of the column used
Affinity differences b/w components
Quality of adsorbent used

6. ◊ 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)

7. 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

8. DRY PACKING

10. 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

13. U

15. 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

16. 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…

17. 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

18. 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

19. THANK YOU