Column chromatography is a technique used to separate mixtures of compounds based on their interactions with a stationary and mobile phase. The mixture is dissolved in a mobile phase and passed through a column packed with a stationary phase. Compounds are separated as they travel through the column at different rates depending on how strongly they interact with each phase. Stronger interactions with the stationary phase cause slower movement through the column. Column chromatography can be used to purify compounds and is influenced by the selection of the stationary and mobile phases based on properties of the compounds.

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