Experiment O06
Chemicals:
Separation of amino acids by paper chromatography
2% ammonia, (20 cm3)
propan-2-ol, (20 cm3)
aluminium foil,
ninhydrin spray (2% solution of ninhydrin in ethanol),
four separate test tubes containing respectively: 0.05 M glycine, 0.05 M tyrosine,
0.05 M leucine, and 0.05M aspartic acid in 1.5% HCl,
an unknown containing one to four of the above amino acids at a concentration of
about 0.05 M each in 1.5% HCl.
Apparatus:
Capillary tube,
chromatography paper, (12 cm × 22 cm)
500 cm3 beaker,
oven.
Introduction:
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.
P.1
Experiment O06
Separation of amino acids by paper chromatography
Procedures:
1.
Mix 10 cm3 of 2% ammonia solution with 20 cm3 of propan-2-ol in a clean, 500 cm3 beaker,
and cover tightly with a piece of aluminium foil. This would be used as the solvent for the
experiment.
Hazard Warning: Propan-2-ol is flammable.
2.
On a clean sheet of chromatography paper with size about 12 cm by 22 cm, mark a light
pencil line parallel to the bottom and about 1.5 cm away (Figure 1). Along this line mark ten
light crosses ("x") at intervals of about 2 cm.
Label each cross as shown in Figure 1.
("U" represents the unknown amino acid mixture.)
3.
Using capillary tubes, place a small amount of each appropriate solution on its two positions
along the line on the chromatography paper. Avoid getting the spot on the paper larger than
about 3 mm in diameter. Let the paper dry for a few minutes in air. Add a second portion of
the unknown to one of its two positions, to make certain that sufficient quantities of each
component of the unknown will be present for good visual observation when the paper is
developed.
4.
Roll the paper into a cylindrical form. Staple the ends together in such a fashion that they do
not touch each other (Figure 2). Otherwise the solvent will flow more rapidly at that point
and form an uneven solvent front.
5.
When the spots on the cylindrical paper are dry (it may be necessary to place the paper in an
oven at about 100oC for a short time), place it carefully in the beaker of solvent, and cover
carefully and tightly with the aluminium foil. Make sure that the paper does not touch the
wall of the beaker.
P.2
Experiment O06
6.
Separation of amino acids by paper chromatography
Let the solvent rise up the paper for at least 1.5 hours. If the time is shorter, the components
may not be sufficiently separated for easy identification. Remove the paper and place it
upside down on the desk top to dry. When most of the solvent has evaporated, open the
cylinder by tearing it apart where it was stapled and hang it in a fume cupboard. Spray the
paper lightly but completely with a solution of ninhydrin, and leave the paper in the fume
cupboard until the spray solution is dry.
Hazard warning: 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.
The teacher should ensure that student wear laboratory gowns, gloves and safety spectacles
in carrying the experiment.
7.
Place the paper in an oven at 100-110oC for about 10 minutes, or until all the spots have
developed.
8.
Circle each spot with a pencil, and measure the distance each spot traveled (use the centre of
the for spot measurement). Measure the distance the solvent traveled at each position, and
calculate the Rf values for each amino acid.
Determine the composition of the unknown by visual comparison of spot colours and by
comparing the Rf values.
P.3
Experiment O06
Separation of amino acids by paper chromatography
Name:
Seat No.:
Date:
Grade:
Chromatogram
Rf1 =
Rf2 =
Rf3 =
Rf4 =
Conclusion: The unknown contains
P.4