introduction and principles of chromatography

2. CHROMATOGRAPHY
 Chroma- colour and Graphein- to write
 Separation of mixture of components into individual
compounds.
 Discovered by Mikhail Tswett (1903)- Russian scientist
 He 1st discovered column chromatography- plant pigment
separation through a column (calcium carbonate) using the
mobile phase petroleum ether.

3. PRINCIPLE
1.ADSORPTION
 Uses stationary phase- static and a mobile phase- moving/
dynamic
 Adsorption is a surface phenomenon where interaction takes
place on the surface.
 Mobile phase liquid/gas will run over the stationary phase
solid.
 Compounds travel acc. to the relative affinities towards s.p.
 The compound which has more affinity towards s.p travels
slower and eluted .
 The compound which has less affinity towards s.p travels
faster and eluted .

5. 2. PARTITION
 Uses stationary phase- liquid and a mobile phase-
liquid/gas.
 Mixture of components dissolved in m.p and passed through a
column of liquid stationary phase.
 Here the components are separated depending upon
.
 The compound which is more soluble in m.p travels faster
and eluted .
 The compound which is less soluble in m.p travels slower and
eluted .

7. 3. ION- EXCHANGE
Ion exchange may be defined as the reversible
reaction in which free ions of a solid (called ion
exchange) are exchanged for different ions of similar
charge present in solution.
S.P- polymeric matrix, M.P- liquid
Here ion exchange resin is used for separation.
Cation exchange resin is used to separate cations and
vice versa.

10. 4. SIZE EXCLUSION
 Size-exculsion chromatography (SEC), gel-filtration
or gel-permeation chromatography (GPC), uses
porous particles to separate molecules of different
sizes.
 It is generally used to separate biological molecules
and usually applied to large molecules or
macromolecular complexes such as proteins and
industrial polymers.

11. • A mixture of molecules dissolved in liquid (the mobile phase)
is applied to a chromatography column which contains gel /
gel beads (the stationary phase). •
• The beads act as “traps” or “sieves” and function to filter the
particles.
• small molecules temporarily trapped within the pores of the
gel and Larger molecules pass around or are “excluded” from
the gels.
• Large sample molecules cannot or can only partially penetrate
the pores, whereas smaller molecules can access most or all
pores.
• Thus, large molecules elute first, smaller molecules elute later

13. 5. AFFINITY CHROMATOGRAPHY
Is a sample purification technique, used
primarily for biological molecules such as
proteins.
 It is a method of separating a mixture of
proteins or nucleic acids (molecules) by
specific interactions between these molecules.
E.g to separate AG , we use AB and to separte
enzyme , we use substrate.

15. CLASSIFICATION
1. BASED ON PRINCIPLE
 Adsorption
 Partition
 Ion exchange
 Affinity
 Size exclusion

16. 2. POLARITY OF PHASES
Normal phase- TLC
s.p- polar (silica gel)
m.p- nonpolar (CCL4,Benzene)
qus- which compond eluated 1st?
Reverse phase- HPLC
s.p- non-polar (ODS)
m.p- polar
qus- which compond eluated 1st?

17. 3. ACC. TO M.P-
s.p- liquid, m.p- liquid (liquid- liquid chr)
s.p- liquid, m.p- Gas (liquid- gas chr.)
s.p- solid, m.p- liquid (solid- liquid)
4. Acc. To geometry
 Plannar- run in a single plane.
e.g- Paper, TLC
 Column- run in column.
e.g- HPLC, size exclusion, column