IV.
CHROMATOGRAPHY
Chromatography is a technique to separate almost all the biological substance, like
aminoacids carbohydrates, and lipids . In addition, this can be used to
1. establish the identity
2. establish the molecular mass
3. concentrate solute molecules
4. purify a substance
5. elucidate structure.
Types:
1. Column Chromatography
a) Partition Chromatography
b) Adsorption Chromatography
c) Ion exchange Chromatography
d) Gel filtration Chromatography
e) Affinity Chromatography
f) High Pressure Liquid Chromatography
2. Paper Chromatography
a) Ascending
b) Descending
3. Thin layer Chromatography
4. Gas –Liquid Chromatography
PAPER CHROMATOGRAPHY (Separation of Aminoacids)
MATERIALS REQUIRED:
1) Chromatography tank and lid
2) Chromatography paper
3) Capillary tubes or Pauster pipettes.
4) Amino acis samples in 1 molar hydrochloric acid
5) SOLVENT : Butanol : Acetic Acid : Water ( 4: 1: 1 )
6) Spray : 1 % Ninhydrin in butanol.
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Principle: Compound to be separated is placed over the stationary phase.i.e filter paper. A mobile
phase i.e organic solvent moves over the stationary phase..Interaction between the stationary phase
and mobile phase occurs. Depending on the partition co efficient, redistribution occurs . The
biological substance gets separated.
PROCEDURE:
1. Prepare the solvent mixture of butanol, acetic acid and water in the proportion of 4 : 1 : 1 ml
( 20 ml : 5ml : 5 ml ).
2. Take a whatmann no.1 filter paper of dimensions(14”* 12”) appropriate for the
chromatographic chamber.
3. Draw a line with a pencil 2 cm above the lower edge of the chromatography paper .Make
small marks at 1.5 cm intervals along the pencil line.
4. With the help of a Pipette, Spot (apply ) the test sample at one end of the pencil line on the
Chromatography Paper.Spoting should be very small, roughly of about 5 mm area. Allow it
to dry .
5. Spot the standards on the pencil line at the marked 1.5 cm intervals.Allow to dry .
6. Place the chromatography paper in the tank such the lower edge of the chromatography paper
touches the solvent . The sample spots should not touch the solvent.The remaining paper
should not touch the tank.
7. Close the chromatography chamber.
8. The solvent moves up on the paper by capillary action, carrying with it the amino acids..The
run is stopped after 18- 20 hours.
9. Using gloved hand , take the damp paper . Mark the solvent front with a pencil.
10. Dry yhe paper in the fume cupboard.
11. Spray the whole paper with 0.5 % Ninhydrin in acetone. Aminoacids appear as distinct
purple spots.
12. Dry the paper in the drying oven for 1-2 mnts.
13. Calculate the Rf values for amino acids in the given mixture and identify them by comparing
with the Rf values.
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PAPER CHROMATOGRAM
(b) (b) (b) Solvent front
(a)
()() (b)
(b)
ss
…
2cm from the lower edge
sample
Standard
Rf = Distance travelled by the solute(aminoacid) (a)
Distance travelled by the solvent (b)
( RF VALUE MEANS RATIO OF FRONTS )
CONCEPT MADE SIMPLE: Standard is a known amino acid. Due to capillary action when the
mobile phase ascends the filter paper, the standard amino acid also ascends up. Suppose the
standard amino acid Phenylalanine ascends(travels up) 4 cms. Test containing unknown amino
acid also travels up 4 cms.then the unknown test sample is also Phenylalanine.
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