B.

Tech : Analytical Techniques in Biotechnology Lab

EXP. No:
SEPARATION OF AMINO ACIDS USING PAPER CHROMATOGRAPHY

Aim: Separation and identification of amino acids using paper chromatography.

Introduction:
Chromatography is the most powerful technique to separate chemically closely related
substances into the individual components on the basis of their physiochemical properties.
The compounds are separated on the basis of their partition coefficients between two
immiscible phases. The static phase may be a solid or liquid while the mobile phase may be
liquid or gas. Depending on the stationary and mobile phases, a variety of chromatographic
techniques are available. These include paper, thin layer, gel filtration, ion exchange, HPLC
and GC.
Principle:
Paper chromatography is a type of partition chromatography in which a mixture of solute is
partitioned between two solvents. While one solvent is stationary and held in the fibres of
the paper (stationary phase), the other solvent (developing solvent) moves along the paper
and is called mobile phase. The components of the mixture to be separated migrate at
different rates depending on the solubility of either phases and appear as spots at different
points on the paper. The distance moved by the individual solutes is denoted as retention
factor (R f) (This is defined as ratio of distance moved by solute front to that of solvent front).
For a given developing system, this Rf is characteristic of the solute.
In this experiment, paper chromatography is used to separate amino acids. Amino acids will
be placed on a sheet of paper and the solvent allowed to move along the paper by capillary
action. Since amino acids are colorless, a means of identifying their position at the end of
the experiment is necessary. The ninhydrin test for amino acids will be used to develop a
spot of color at the point where the amino acid is located. Amino acids react with ninhydrin
in the presence of heat to produce a purple colored spots.
Chemicals required:
1. Standard Amino acid solution (1mg/ml in water).
Dissolve different individual amino acids in distilled water at a concentration of 1mg/ml.
Use very dilute (0.05N) HCl to dissolve the free amino acids tyrosine and phenylalanine.
Dissolve tryptophan in very dilute (0.005N)NaOH.
2. Mobile phase: Mix n-butanol, glacial acetic acid and distilled water in the ration of 4:1:1
(v/v).
3. Spray Reagent (Ninhydrin): Dissolve 100mg ninhydrin in 100mL of acetone.

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B. Tech : Analytical Techniques in Biotechnology Lab
EXP. No:
Procedure:
1. Cut out the strip of Whatman No.1 filter paper (12 x 10cm).
2. Draw a thin line with pencil approximately 2cm from one end of paper.
3. Using a capillary tube spot the given amino acid sample, keeping a distance of at least
1.5 cm between individual spots.
4. Ensure also the first and the last spots are at least 1 cm away from the either end.
5. The size of the spot should be as small as possible for better resolution
6. In a tall beaker pour developing solvent to above 0.5cm deep.
7. Suspend the chromatographic strip in the beaker in such a way the edge of the paper is
just below the solvent level.
8. The paper should not be kept in slanting position nor should it touch the sides.
9. Cover the set-up with a glass plate and leave the step-up undisturbed until the solvent
rises up and reaches almost the top edge.
10. Once the solvent reaches almost the top of the edge, remove the paper and mark the
solvent front quickly and dry the chromatogram free of solvent in a fume chamber.
11. Spray the chromatogram with the ninhydrin reagent using an automiser.
12. Dry the paper for about 5 min at room temperature and keep the chromatogram at 100
°C for a few minutes or until the individual amino acids appear as purple spots.
13. Mark all the spots and calculate their Rf values by the formula.
Retardation Factor =

14. Compare the values you obtain with reference Rf values. Different solvents and different
types or makes of chromatogaphy papers will give slightly different results.

Result:
The mixture of amino acids was separated and individual amino acids identified by
comparison of Rf with standards.
Distance traveled by the solvent = ------------------------cm
Distance traveled by gylcine = ----------------------cm
Distance traveled by tyrosine = -------------------------cm
Distance traveled by leucine =--------------------------cm
Distance traveled by aspartic acid = -------------------------cm
Distance traveled by components in the unknown =------------------ cm

Components identified:…………………………………………….

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B. Tech : Analytical Techniques in Biotechnology Lab
EXP. No:
Rf values of amino acids:

Amino acid Rf value Amino acid Rf value

Histidine 0.11 Cysteine 0.4
Glutamine 0.13 Proline 0.43
Lysine 0.14 Tyrosine 0.45
Arginine 0.20 Asparagine 0.5
Aspartic acid 0.24 Methionine 0.55
Glycine 0.26 Valine 0.61
Serine 0.27 Tryptophan 0.66
Glutamic acid 0.30 Phenylalanine 0.68
Threonine 0.35 Isoleucine 0.72
Alanine 0.38 Leucine 0.73