6.

SEPARATION AND IDENTIFICATION OF AMINO ACIDS BYASCENDING

Aim: To separate and identify the given amino acids by Ascending Paper Chromatography.
Requirements:
Apparatus and glass ware: Chromatographic chamber, spraying gun, capillary tubes
and whatman grade filter paper.
Chemicals:
Solvent System: n-butanol, Glacial acetic acid, Distilled water (4:1:5),
Visualizing Agent: Ninhydrin solution
Standard references: Amino acids (Tryptophan and threonine),

Reference:

Principle:
The principle involved is partition, where the substances are distributed or partitioned between
to liquid phases. One phase is the water which is held in pores of filter paper used and other
phase is that of mobile phase which moves over the paper. The compounds in the mixture get
separated due to differences in their affinity towards water (in stationary phase) and mobile
phase during the movement of mobile phase under the capillary action of pores in the paper.
The principle can also be adsorption chromatography between solid and liquid phases, where
in the stationary phase is the solid surface of paper and the liquid phase is of mobile phase. But
most of the applications of paper chromatography work on the principle of partition
chromatography i.e. partitioned between to liquid phases. Identification of amino acids in the
given mixture is determined by Rf value. Rf value is less than one for all compounds.

Theory:
The chromatographic techniques used to separate out mixtures of different substances into their
discrete components. All forms of chromatographic techniques work on the same principle.
They all have basic requirements of stationary phase (a solid or a liquid supported on a solid)
and a mobile phase (a liquid or a gas). The mobile phase tides through the stationary phase and
carries the components of the mixture with it. Different components travel at different rates
based on their affinities toward stationary phase and mobile phase. In paper chromatography,
the stationary phase is a very uniform adsorbent paper and the mobile phase is a suitable liquid
solvent or mixture of solvents.
Retention (or) retardation factor (Rf): Retention factor value (Rf Value) is defined the ratio
of the distance travelled by the solute to the distance travelled by solvent. The distance travelled
relative to the solvent is called the Rf value.
Rf = Distance travelled by solute / Distance travelled by solvents
Procedure:
Preparation of solutions:-
Solution A: 0.1 gm of Methionine in required quantity of suitable solvent
Solution B: 0.1 gm of Valine in required quantity of suitable solvent
Solution C: 0.1 gm of Tryptophan in required quantity of suitable solvent
Solution D: unknown mixture
Mobile phase: N-butanol: Acetic acid: Water (4: 1: 5)
Ninhydrin Solution: 0.2% (200mg in 100ml of N-butanol)
Solvents system and methods of preparation – For solvents system and methods preparation
n-butanol and water are taken in ratios of 4:5 in a flask and allow it to saturate for 24 hours, by
using the separating funnel separate out the n - butanol and water. The saturated n - butanol
and glacial acetic acid both are taken in the 4:1 ratio which can be used as a solvent system (or)
mobile phase.
Paper chromatography: - For paper chromatography method is quite simple as compared to
other methods of chromatography. The chromatography paper is cut into rectangular strips and
marks a line on the paper with pencil at about 2 cm from the bottom. With the help of capillary
tube, the samples are applied at different points on the starting line and place the
chromatography paper in the developing chamber, which contains the mobile phase. While
placing the paper, it is important that the solvent level should not reach the starting line or the
sample spots and paper shouldn’t touch the walls of the developing chamber. After sometime
the solvent rises up the paper or the stationary phase by capillary action and dissolves the
sample. The components of the sample move along with the solvent in upward direction.
Checkered if the solvent has reached near the top level of chromatography paper, then the paper
is removed when it reaches the top and marked the level with pencil. This level (or) height is
called the “solvent front”. By using UV light, Ninhydrin or iodine vapors examined the
different spots of varied colors.

(OR)
Procedure:-
a. Preparation of mobile phase: 40 ml of N-butanol, 10 ml of acetic acid and 50 ml of water
were taken in a beaker and mixed well.
b. Preparation of sample: 0.1 gm of ariginine, 0.1 gm of valine and 0.1 gm of methionine
were taken separately and dissolved in required quantity of ethanol.
c. Application of sample: One drop of individual sample solutions were applied to
chromatographic paper with capillary tube.
Procedure of development of chromatogram:-
• Take a whatman filter paper and draw a thin straight line of about 2cm from the bottom of
the paper.
• Mark four points with equidistance on the straight line and number them.
• Prepare sample solutions and place a drop of these solutions on the straight line by using
capillary tube.
• Hang the chromatographic paper in chamber containing mobile phase, run the chromatogram
till the mobile phase travels on ¾th of the chromatographic paper.
• Remove the paper from the chamber and mark the solvent distance with a pencil and dry in
air for 15 minutes
• Now spray ninhydrin solution to the chromatogram and dry it in oven for 10-15 minutes
• Measure the distance of purple colour spots from the baseline and also the distance of
travelled by mobile phase.
• Calculate the Rf values for the identification of amino acids in the given mixture.

Rf = Distance travelled by solute/ Distance travelled by solvent

Observation:
Distance travelled Distance travelled
S. No. Amino acids Rf value
By amino acid By solvent front
1. Methionine
2. Valine
3. Tryptophan
4. Unknown mixture
 Sample

Report/ Results: The distance moved by Methionine, Valine and Tryptophan is …… cm,
…… cm and ….. cm respectively, and the solvent is ……cm.
Rf value of Methionine is ……
Rf value of Valine is ……..
Rf value of Tryptophan is ……..

Rf value of unknown mixture is ….. & …..