column chromatography

1. Jeneesha Jude
MSc .chemistry

2. ⦿Useful method for the separation and
purification of both solids and liquids .
⦿Solid – liquid technique
⦿stationary phase – solid
⦿Mobile phase – liquid

3. Principle
⦿Adsorption
⦿ Mixture of compounds dissolved in
mobile phase is introduced into the
column .
⦿Separation occurs because of the
different rates at which the components
are carried over the stationary phase by
the mobile phase

4. ⦿Adsorption column chromatography , the
adsorbent packed in glass column .
⦿Solvent – mobile phase , moves slowly
through the packed column .
⦿Process of dissolving out the
components from the adsorbent using a
suitable solvent is called elution .
⦿This Solvent used as mobile phase –
eluent .

5. ⦿Compound attracted more strongly by
the mobile phase will move rapidly
through the column .
⦿ Compound more strongly attracted to
the stationary phase will move slowly
through the column

7. OF COLUMN
CHROMATOGRAPHY
⦿ADSORBENTS : silica ,alumina, calcium
carbonate , calcium phosphate ,
magnesia and starch
⦿Alumina suitable for chromatography of
less polar compounds .
⦿Silica gel suitable for compounds
containing polar functional groups .

8. ADSORBENTS – CRITERIA
⦿ Particles - spherical in shape , uniform in
size
⦿ Mechanical stability must be great enough
to prevent the formation of fine dust which
might deposit in the channels of packing .
⦿ Don’t react chemically either with eluent or
with sample .
⦿ Useful for separating wide variety of
compounds.
⦿Inexpensive and freely available

9. SELECTION OF STATIONARY
PHASE
⦿Removal of impurities
⦿ Number of components to be separated
⦿Length of column used
⦿Affinity difference between compenents
⦿Quantity of adsorbent used

10. SELECTION OF MOBILE
PHASE
⦿ Acts as solvent , developer and eluent
⦿As developing agent
⦿To remove pure component out of the
column – as eluent
⦿ Based on solubility – solvent selection
⦿Polarity – important factor

11. DIFFERENT MOBILE PHASE
USED
⦿Petroleum ether
⦿ Carbon tetrachloride
⦿Cyclohexane
⦿ Ether
⦿ Ester
⦿ Acetone
⦿ Benzene
⦿Water

12. COLUMN CHARACTERISTICS
⦿Support stationary phase
⦿Column must be of good quality –
neutral glass
⦿ column dimensions : length and
diameter ratio (10:1)
⦿Better separation : long narrow column
because number of plates will be more.

13. PREPARTION OF
COLUMN
⦿Consists of glass tube with bottom
portion of the column – packed with
cotton / glass wool
⦿Above which adsorbent is packed .
⦿After packing , paper disk is kept on the
top , so that the adsorbent layer is not
disturbed during the introduction of
mobile phase .

15. PACKING TECHNIQUES OF
ADSORBENTS
⦿DRY PACKING
⦿WET PACKING
THE PROCESS
⦿ An adsorbent column is prepared by packing
a long glass tube with suitable adsorbent
⦿solution of the mixture of the components
(say a,b,c) in suitable solvents is introduced
at the top of the column .

16. ⦿ It percolates through the adsorbent column ,
different components adsorbed to different
extend .
⦿ Most readily adsorbed constituent(say a) is
held at the top .
⦿ Other (say b and c) of decreasing
adsorbabilities are held up at different zones
or bands down the column in the same order
[say b and then c]
⦿ This partial separation is improvised by
adding some amount of original solvent –
development of chromatogram.

17. ⦿Individual components are extracted from the
adsorbent by a suitable solvent(eluent).
⦿Most weakly adsorbed component (c)
emerges first which is at the bottom of the
column .
⦿Most strongly adsorbed one (a ) comes out
last which was at the top of the column .
⦿Pure components are then recovered by
removing the solvent by suitable method .

18. APPLICATIONS
⦿Used for separation of two or more similar
organic components of a mixture
⦿Identification , separation and purification of
natural products.
⦿Used for identification of various industrial
products
⦿Separation of protein mixture.

19. Advantages of CC
⦿Any type and any quantity of mixture can be
separated .
⦿Wider choice of mobile phase
DISADVANTAGES OF CC
⦿Time consuming
⦿ More amount of mobile phase is required .

20. THANK YOU