Chapter 12

Chromatographic Separations

12.1 Separation and Identification of Amino Acids

by Descending Paper Chromatography

12.1.1 Principle

Amino acids in a given mixture are separated on the basis of differences in their
solubilities and hence differential partitioning coefficients in a binary solvent
system. The amino acids with higher solubilities in stationary phase move slowly
as compared to those with higher solubilities in the mobile phase. The separated
amino acids are detected by spraying the air dried chromatogram with ninhydrin
reagent. All amino acids give purple or bluish purple colour on reaction
with ninhydrin except proline and hydroxyproline, which give a yellow coloured
product. The reactions leading to the formation of purple complexes are given
below (Fig. 12.1).

12.1.2 Reagents

1. Whatman No. 1 filter paper sheet.

2. Micropipette.
3. Oven (105
C)
4. Drier
5. Sprayer
6. Chromatographic chamber saturated with water vapours.
7. Developing solvent: Butanol, acetic acid and water in the ratio of 4:1:5 in
a separting funnel and mix it thoroughly. Allow the phases to separate out
completely. Use the lower aqueous phase for saturating the chamber. The upper
organic phase is used as mobile phase.

R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology, 233

DOI 10.1007/978-1-4419-9785-2_12, # Springer Science+Business Media, LLC 2011
234 12 Chromatographic Separations

O O
R
OH H
+ H 2N C H + RCHO + NH3 + CH2
OH OH
COOH
O O
Ninhydrin Amino Acid Hydrindantrin

O O O O O
OH H H
+H N + C=N C + 3H2O
OH H HO
O O O O
Ninhydrin Ammonia Hydrindantin Purple coloured product

Fig. 12.1 Reaction for ninhydrin test

8. Ninhydrin spray reagent: Prepare fresh by dissolving 0.2 g ninhydrin in 100 ml

acetone.
9. Standard amino acids: Prepare solutions of authentic samples of amino acids
such as methionine, tryptophan, alanine, glycine, threonine, etc. (1 mg/ml of
10% iso-propanol).
10. A sample containing mixture of unknown amino acids.

12.1.3 Procedure

1. Take Whatman No. 1 filter paper and lay it on a rough filter paper. Throughout
the experiment care should be taken not to handle the filter paper with naked
hands and for this purpose either gloves should be used or it should be handled
with the help of folded piece of rough filter paper.
2. Fold the Whatman No. 1 filter paper about 2.0–2 cm from one edge. Reverse
the paper and again fold it 2 cm further down from the first fold.
3. Draw a line across the filter paper with a lead pencil at a distance of about 2 cm
from the second fold. Put circular marks along this line at a distance of 2.5 cm
from each other.
4. With the help of a micropipette or micro-syringe apply 20 ml of solution of each
standard amino acid on a separate mark. Also apply spot of the sample or
mixture to be analyzed, preferably on the mark at centre of this base line. The
size of the spot should be as small as possible so that the developed spots are
compact and do not overlap.
5. Hang the filter paper in a chromatographic chamber which has previously been
saturated with aqueous phase of the solvent system. This is done by keeping
Petri plates containing the aqueous phase at the bottom of the chamber. The
paper is hung from the trough/tray and a glass rod is kept to hold it in place.
Care should be taken to ensure that the base line should not get submerged
12.1 Separation and Identification of Amino Acids by Descending Paper . . . 235

when the mobile phase is added to the trough otherwise the spotted material
would get dissolved in the solvent.
6. Close the chamber firmly so that it is airtight. Allow sufficient time for
cellulose fibres of the paper to get fully hydrated.
7. Pour the mobile phase through the holes provided on the lid of the chamber into
the trough. Replace the rubber bungs in the hole and allow the mobile phase to run
down the paper till the solvent front reaches about 5 cm from the opposite edge.
8. Remove the paper and mark the solvent front with lead pencil and let it dry at
room temperature.
9. Spray the filter paper (chromatogram) with ninhydrin reagent and after drying it
at room temperature, transfer it to an oven at 105
C for 5–10 min.
10. Blue or purple coloured spots would appear on the paper. Mark the boundary of
each spot with lead pencil.
11. Measure the distance between the centre of the spots and also the distance of
the solvent front from the base line.
12. Calculate the Rf value of standard amino acids as well as those in the given
mixture or sample.

Distance traveled by unknown amino acid

Rf ¼ :
Distance traveled by the solvent system

13. Identify the amino acids in the mixture or sample by comparing their Rf values
with those of the reference standards.

12.1.4 Observations and Calculations

Distance travelled by the solvent front from base line ¼ x cm

Distance travelled by glycine from base line ¼ a cm
Distance travelled by alanine from base line ¼ b cm
Distance travelled by threonine from base line ¼ c cm
Distance travelled by methionine from base line ¼ d cm
Distance travelled by spot no.1 in sample from base line ¼ a cm
The sample contains glycine since Rf value of spot no. 1 is identical to that of
glycine standard.

12.1.5 Note

• The chromatography should be carried out in temperature controlled room

because any fluctuation in the temperature would cause the uneven flow of the
solvent and may alter the Rf value.
236 12 Chromatographic Separations

• The paper should not be touched with naked hands because sweat on hands
contains significant amount of amino acids.
• The spots of the applied sample should be as compact as possible. Larger the
spot, poorer will be the resolution.
• At the time of fixing paper in chromatography chamber, it should be ensured that
the base line on which the sample has been applied does not dip into the solvent
otherwise the sample might get washed away in the solvent.
• Allow sufficient time for filter paper to absorb sufficient water (which will act as
a stationary phase) before pouring the solvent into the tank. Inadequate condi-
tioning or equilibration will result in improper or poor quality resolution.
• Solvent front should advance in a straight line and should not be zig-zag or
sloping but should be parallel to the base line.
• Dry the paper thoroughly before spraying with the detection reagent. Wet paper
may interfere with the appearance of evenly shaped compact spots.

12.2 Separation and Identification of Amino Acids

by Ascending Paper Chromatography

12.2.1 Reagents and Materials

Same as given in above experiment except that cylindrical chromatography

chambers are needed for this experiment.

12.2.2 Procedure

1. Take Whatman No. 1 filter paper sheet of appropriate size so that it can
be rolled into a cylinder and can be accommodated in the cylindrical
chromatographic jar.
2. Draw a base line 2 cm from one of the breadthwise edge of the paper. Put small
circular marks along the base line in such a way that the distance from the edge
of the paper and the first spot and the distance between the adjacent spots is not
less than 2.5 cm.
3. Apply 20 ml aliquots of the standard amino acids and of the sample on different
spots. Diameter of the spotted material should be as small as possible and, if
required, the applied solution may be dried prior to loading additional volume.
4. Roll the paper into a cylinder; fasten its edges with a paper clip. Pour sufficient
volume of the mobile phase into the chromatographic jar which has been earlier
saturated with water vapours by lining the tank with filter paper saturated with
aqueous phase of the solvent system.
12.3 Separation and Identification of the Amino Acids by Two-Dimensional. . . 237

5. Gently place the rolled filter paper upright in the jar ensuring that it does not
touch the sides of the chamber and at the same time taking care that the base
line where the spots have been applied does not dip into the solvent.
6. Close the tank with n airtight lid or a glass plate to which sufficient amount of
silicon grease has been applied.
7. Leave the set up undisturbed and allow the solvent to move up fill it reaches
about 5 cm from the upper edge.
8. Remove the chromatogram from the chamber and air dry it.
9. Spray the paper with ninhydrin reagent and let it dry again at room temperature
prior to transferring it to an oven at 105
C for 5–10 min. Locate the position of
amino acids from the bluish or purple coloured spots on the chromatogram.
10. Calculate the Rf values of the standard amino acids and those is the sample or
mixture as described in above experiment.
11. Identify the amino acids in the mixture or sample by comparing Rf values with
those of applied standard amino acids.

12.3 Separation and Identification of the Amino Acids

by Two-Dimensional Paper Chromatography

12.3.1 Principle

Amino acids having very close Rf values in a particular solvent system may appear
as a single or overlapping spots in a single dimensional chromatography and may be
mistaken as one component. They can be separated into individual components by
developing the chromatogram again in a direction perpendicular to the first run in a
second solvent system in which they have different Rf values. The main limitation
of this method is that only one spot either of the sample or of a standard amino acid
can be applied on each filter paper sheet necessitating running of a large number of
chromatograms for the standard amino acids.

12.3.2 Reagents

1. As in previous experiment with an additional chromatographic chamber for the

second solvent system.
2. Solvent system No.2: Phenol: water (80:20, w/v) is used as second solvent
system. Add 125 ml of water to 500 g to phenol. Add a few drops of ammonia
(0.88%) to this mixture just before use (CAUTION-Phenol is corrosive and can
cause burns on the skin).
3. Standard amino acids: Prepare 1% solution of standard amino acids such as
aspartate, glycine, serine, arginine in 10% iso-propanol (u/v).