It will cover basic terms, process, applications of Column Chromatography.

1. Column Chromatography
Presented by-
Aishwarya Thete.
GIFSA
Department of ‘Forensic
Chemistry & Toxicology’..
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2. Outline:
Introduction
Basic term
Principle
Experimental setup
adsorbent
Solvents
Working
Factors affecting column efficiency
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3. Basic terms
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4. Introduction
 Also known as adsorption chromatography.
 Developed by- D.T. Day(1900) & M.S. Tswett (1906)
 Experiment on separation of plant pigment
Solid - liquid technique
Stationary phase -solid & Mobile phase - liquid.
Generally used for separation of lipid molecule.
2 types of Column Chromatography are generally used:
a) Adsorption chromatography:
Procedure which separates lipid mixtures mainly according to their relative
polarities. Ex: mix of organic comp.
b) ion exchange chromatography:
Procedure which is based on the importance of ionic groups present in some
lipid molecules. Ex: mix of proteins, amino acid
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5. Principle
Selective Adsorption/differential adsorption.
 The component -
-which has greater adsorbing powder is adsorbed in the upper part of the
column.
- The next component is adsorbed in the lower portion of the column
which has lesser adsorbing power than the first component.
- This process is continued & as a result, the materials are partially
separated & adsorbed in the various parts of the column.
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6. Adsorbent
• Uniform size particle, spherical shape
• Good adsorbent, good filtration properties
• Chemically inert
• Neutral surface except in case of ion exchanger.
• Good mechanical stability so that their will not be formation of dust
particle.
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7. Silica gel Activated
Charcoal
Calcium
Carbonate
(Magnesium Carbonate)
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8. Selection of solvent
* Functions to perform:
a) act as developer.
b) introduces the mixture to the columns.
c) act as eluents
*Solvent selection:
a) Solubility of sample.
b) Boiling point b/w 45-85˚ C.
c) Pet ether having-most widely used B.P.<80 ˚ C.
d) Mobile phase can be single solvent or mixture of solvent. It depends on
eluting power.
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9. Pet ether Cyclohexane TCM
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11. Column preparation & procedure
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• Keep the supporting material(glass wool/cotton plug).
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• Clamp the column vertically.
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• Add the adsorbent material by making slurry in a suitable medium to the 2/3rd
of the column.
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• If adsorbent material is solid, pack the glass tube uniformly. After adding each
portion press it with flattened glass rod.
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• Cover the surface again with supporting material.
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• Add the sample to b separated & cover the surface with glass wool.
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• Add the eluent.
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12. Experimental Setup
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13. Dissolve the sample in suitable solvent
Preparation of column
Selection of eluent
Introduce the sample
Collection of eluent
STEPS
Video 1
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14. Process
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15. Factors Affecting Column Efficiency
1. Nature of solvent
-Low viscosity
-Rate of flow α 1
Viscosity
2. Pore diameter
- <20A˚
3. Temperature of the column:
- Generally we use room temp.
- Samples which are difficult to soluble, we use high temperature
-
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16. 4. Partical size of column packing:
- Usually it ranges from 100-200 mesh.
- Column efficiency= 1
Particle size
5. Dimensions of column:
- Ratio of length: diameter > 20:1
- Ideal dimension is 40:1
Peak resolution= RtA-RtB/ WA+WB
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17. Applications
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Separatio
n of
Proteins
Amino acids
Carbohydrates
Peptides
Mixture of
compounds
Geometrical isomers
Alkaloids

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Other applications
Determination of primary & sec. glycosides in
digitalis leaf.
Isolation of active constituents
Synthesizing or isolating novel
compounds
Isolation of metabolites from biological fluids.