Title:
Separation of Amino Acids by Paper Chromatography
Theory:
A mixture of unknown amino acids can be separated and identified by means of paper
chromatography. The positions of the amino acids in the chromatogram can be detected by spraying
with ninhydrin, which reacts with amino acids to yield highly coloured products (purple).
Paper chromatography is a method of separating and analysing mixture. For example, simple paper
chromatography can be used to separate a mixture of dyes.
The filter paper, which contains a thin film of water trapped on it, forms the stationary phase. The
solvent is called the mobile phase or eluant. The solvent moves up a piece of filter paper by capillary
action. As the solvent moves past the simple spot of dye, there is a competition between
1. the ability of dyes to attach to the adsorbed water, and
2. the ability of dyes to dissolve in the solvent.
This is a type of partition between the stationary phase (the adsorbed water) and the mobile phase (the
solvent). As different dyes have different partition between the mobile and the stationary phases, they
would be carried forward to different extents. Dyes which are more soluble in the solvent than in
water are carried further up by the mobile phase. Fresh solvent is continuously moving up and there is,
in fact, an enormous number of successive solvent extractions occur.
At a suitable time, the experiment is stopped by removing the filter paper from the solvent. The
retardation factor, Rf , can be calculated as
Rf = distance traveled by spot / distance traveled by the solvent (Note: Rf value is always less than one)
The Rf of component A and B can be determined as:
Rf of component A = d2 / d1
Rf of component B = d3 / d1
Solvent front
The Rf value of any substance may be about the same
whenever we use that particular solvent at a given
temperature. However, the Rf value of a substance
d1
differs in different solvents and at different
d2
d3
temperatures.
base line
Chemical:
A
B
2% ammonia, propan-2-ol, aluminium foil, ninhydrin
spray (2% solution of ninhydrin in ethanol), four separate test tubes containing respectively 0.05M
glycine, tyrosine, leucine, and aspartic acid in 1.5% HCl, an unknown containing one to four of the
above amino acids at a concentration of about 0.05M each 1.5% HCl.
Apparatus:
Capillary tube, chromatography paper, beaker, oven
Hazard Warming:
Propan-1-ol is flammable. The ninhydrin solution should be kept off the body because it reacts with
proteins in the skin to form a rather long-lasting purple discoloration. Students should wear laboratory
gowns, gloves and safety spectacles in carrying the experiment.
Procedures:
1. 10 cm3 of 2 % ammonia solution was mixed with 20 cm3 of propan-2-ol in a clean, 500 cm3 beaker,
and covered tightly with a piece of aluminium foil. This would be used as the solvent for the
experiment.
2. On a clean sheet of chromatography paper with size about 12 cm by 22 cm, a light pencil line was
marked to the bottom and about 1.5 cm away. Along this line ten light crosses (“x”) was marked at
intervals of about 2 cm. Each cross was labelled. (“U” was represented the unknown amino acid
mixture .)
3. Capillary tubes was used and placed a small amount of each appropriate solution was placed on its
two positions along the line on the chromatography paper. Getting the spot was avoided on the paper
larger than about 2 mm in diameter. The paper was let dry for a few minutes in air. A second portion
of the unknown was added to one of its two positions to make certain that sufficient quantities of
each component of the unknown was presented for good visual observation when the paper was
developed.
4. The paper was rolled into a cylindrical form. The ends was stapled together in such a fashion that
they was not touched each other. Otherwise the solvent will be flowed more rapidly at that point and
was formed an uneven solvent front.
5. When the spots on the cylindrical paper was dry, it might be necessary to place the paper in an oven
at about 100oC for a short time. It was placed carefully in the beaker of solvent, and was covered
carefully and tightly with the aluminium foil. The paper was carefully placed in order not to touch
the wall of the beaker.
6. The solvent was let to rise up the paper for at least 1.5 hours. If the time was shorter, the component
might not be sufficiently separated for easy identification. The paper was removed and placed it
upside down on the desk top to dry. When most of the solvent had evaporated, the cylinder was
opened by tearing it apart where it was stapled and hanged it in a fume cupboard. The paper was
lightly sprayed but completely with a solution of ninhydrin, and was left the paper in the fume
cupboard until the spray solution was dry.
7. The paper was placed in an oven at 100o-110oC for about 10 minutes, or until all the spots had been
developed.
8. Each spot was circled with a pencil, and was measured the distance each spot traveled. The center of
the spot was used for measurement. The distance was measured the solvent traveled at each position,
and was calculated the Rf values for each amino acid. The composition of the unknown was
determined by visual comparison of spot colours and by comparing the Rf values.