Biochem Lab Report 1

To separate the given mixture of amino acids (glycine and valine) using radial
paper chromatography
APPARATUS REQUIRED
Square whatman filter paper, petri dish, cotton, fine capillary
CHEMICALS REQUIRED
Glycine, valine, butanol, acetic acid, distilled water, ninhydrin reagent.
THEORY
Chromatography is a physicochemical method for separation of complex mixtures and was

discovered in 1903 by Russian-Italian botanist M. S. Tswett. Chromatography is a modern and
sensitive technique used for rapid and efficient analysis and separation of components of a
mixture and separation of components of a mixture and purification of compounds.
The basis of principle of chromatographic technique is based on the differential migration of the
individual components of a mixture through a stationary phase under the influence of moving
phase.
Stationary phase – the phase that is immobilized on the support particles, or on the inner wall of
the column tubing.
Mobile phase – the phase that moves in a definite direction.

Paper chromatography – This is a type of partition chromatography which involves paper
placed in a jar containing a shallow layer of solvent and sealed. A small dot of sample solution is
placed onto a strip of chromatography paper. Components of the mixture are carried along with
the solvent up the paper to varying degrees, depending on the compound’s performance to be
adsorbed onto the paper v/s being carried along with the solvent. This paper is made of cellulose
to which polar water molecules are adsorbed while the solvent is less polar usually consisting of
a mixture of water and an organic liquid. The compounds within the mixture travel farther if they
are non-polar. More polar substances bond with the cellulose paper quickly and therefore, do not
travel so far.
Radial chromatography – It is also known as horizontal or circular chromatography. The

method radially from centre (mid point) towards the periphery of a circular chromatogram with
the help of a wick and petri dish. A circular chromatograph of suitable size is taken and two
perpendicular diameters are drawn to locate the centre of the chromatograph. The sample is
spotted at the centre of the circular paper and is placed in a peti dish containing the solvent
whose size is smaller than the paper. The horizontal development takes through the help of a
wick held at the centre of the paper. The components appear as concentric bands rather than
ovals or spots. Hence this mode gives a better resolution as compared to ascending or descending
chromatography.
FIGURE 1: RADIAL CHROMATOGRAPHY

Identification of metals by radial paper chromatography:-
In this experiment, dilute solutions of amino acids (glycine and valine) are applied or spotted in
the centre of the circular chromatograph. If a comparison is to made, then each solution should
be applied on the same place (centre). After the solutions have been applied, the paper is dried
and placed in a petri dish which contains a few milliliters of the liquid mobile phase. For
separation of amino acids, a mixture of solvents is employed as the mobile phase. As soon as the
paper is placed in the mobile phase, the solution (sometimes called the eluting solvent) will begin
to rise in the horizontal direction of the paper by the capillary action.
As the mobile phase rises on the paper, it will ultimately develop the amino acids as concentric
circles. The outcome of each amino acid in the mixture depends on the affinity for the mobile
phase than the stationary phase. If a amino acid has a higher affinity for the mobile phase than
the stationary phase, it will tend to travel with the solvent front and be relatively unobstructed by
the paper. In contrast, if the amino acid has a higher affinity for the paper than the solvent, it will
tend to stick to the paper and travel more slowly than the solvent front. It is these differences in
the amino acids affinities that lead to their separation on the chromatograph.
FIGURE 2: STRUCTURE OF GLYCINE
FIGURE 3: STRUCTURE OF VALINE

When the solvent reaches three-fourth of the paper, it is removed and the distance known as
solvent front is marked with a pencil. The circular bands corresponding to each amino acid can
be visualized by spraying the paper with visualizing agent called ninhydrin which reacts with
metals to form colored complex.
The further the circular band from the starting spot, the higher the affinity of the amino acids for
the mobile phase and faster is its migration. The relative distances are measured in terms of
retention factor (Rf).
DETECTION
If the components are of different color, the position of each component on the paper can be
seen.
If the components are colorless there are number of techniques used to detect each component.
For example:
a. Reactions with color producing reagent such as alizarin.

b. Place the sample under ultraviolet light.
FIGURE 4: STRUCTURE OF NINHYDRIN
FIGURE 4: REACTION OF AMINO ACIDS WITH NINHYDRIN
PROCEDURE
1. Take a square whatman filter paper and mark the centre by drawing two perpendicular
diameters. Make a hole with a pin at this point.
2. Make a wick of cotton and insert it through the hole of the chromatogram.
3. In a petri dish, place the solvent mixture (40-50 mL) and cover it with another petri dish.
4. Remove the wick from the chromatogram and apply the given mixture of amino acids
(glycine and valine) using a fine capillary to spot at the centre of centre of the
chromatogram.
5. Repeat the spotting two times and insert the wick back when the spot has dried.
6. Introduce the system of chromatogram and wick into the chromatography chamber
(containing the solvent system).
7. Ensure that the chromatogram rests on the rim of the petri dish and does not dip into it.
The chromatogram should remain in a stretched state and now cover it with another petri
dish.
8. Leave the system undisturbed to allow the development of chromatogram.
9. When the solvent front has almost reached three-fourth of the chromatogram, remove it
and immediately mark the solvent front with a pencil. Calculate Rf value of each
components at four different points.
OBSERVATIONS
S. No. Component Distance travelled at different Mean distance/cm

points/cm
A B C D
1 Solvent
2 Glycine
3 Valine
CALCULATIONS
A. Calculation of Rf value of glycine:-
B. Calculation of Rf value for valine:-

RESULT
1. Rf value of glycine =
2. Rf value of valine =
INTERPRETATION OF RESULT
Rf value of valine is greater than glucose. This is because valine is more soluble in mobile phase
than in stationary phase and has more affinity towards the mobile phase and hence travels
further. Glycine is less soluble in mobile phase than in stationary phase and has more affinity
towards the stationary phase and hence do not travels further.
PRECAUTIONS
1. The chromatogram should remain in the stretched position on the rim of the petri dish.
2. Do not disturb the system while the chromatogram develops.
3. The chromatogram should be dried properly before introducing it in the chromatography
chamber.
4. The solvent mark should be marked immediately with a pencil after removing it from the
chromatography chamber.

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To separate the given mixture of amino acids (glycine and valine) using radial

Square whatman filter paper, petri dish, cotton, fine capillary

Glycine, valine, butanol, acetic acid, distilled water, ninhydrin reagent.

Chromatography is a physicochemical method for separation of complex mixtures and was

Mobile phase – the phase that moves in a definite direction.

Radial chromatography – It is also known as horizontal or circular chromatography. The

FIGURE 1: RADIAL CHROMATOGRAPHY

FIGURE 2: STRUCTURE OF GLYCINE

FIGURE 3: STRUCTURE OF VALINE

a. Reactions with color producing reagent such as alizarin.

FIGURE 4: STRUCTURE OF NINHYDRIN

FIGURE 4: REACTION OF AMINO ACIDS WITH NINHYDRIN

S. No. Component Distance travelled at different Mean distance/cm

A. Calculation of Rf value of glycine:-

B. Calculation of Rf value for valine:-

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