Practical manual

M.Sc. II Sem

Dr. Ranjana Shrivastava

Professor and Head Department of Botany

Aim: Separation of chloroplast pigments by thin layer chromatographic technique.

Materials required: Spinach leaves, separatory funnel, thin glass plates, silica gel,
mortar and pestle, beakers, capillary tube, chromatographic chambers, sterilized
sand, distilled water, acetone, petroleum ether, n-butanol, acetic acid etc.

Principle:

Chromatography consists of separation of constituents of a mixture. Separation of

these components is a function of their different affinities for a stationary phase
such as a solid or liquid and their differential solubility in a moving phase such as
liquid or gas. The separation of compounds is controlled by their character to
distribute themselves between the solution in the liquid and absorption on the solid
surface e.g. paper, silica, etc. if bulk solids are employed as a thin layer on
supporting glass plate, the procedure is known as thin layer chromatography
(TLC).

When a mixture to be separated is applied as a spot on support medium, the

compound travels a particular distance under specific set conditions, this develops
a chromatogram. This is a characteristic feature of a compound and is used for its
identification. Rf value is calculated as follows

Rf = distance from origin travelled by compound

distance of solvent front from origin
Rf values are constant and thus used for an identification of a particular compound.
The unknown is chromatographed with series of known compounds and Rf values
are then compared to determine and identify the compound.

The major are major steps-

i) Preparation of plates-
 1. Slurry is prepared by suspending 2g of silica gel in 10 ml of
distilled water.
2. The homogenous slurry is spread uniformly over the clean
glass plate. The gel is allowed to set and is oven dried
overnight at 40o C.

ii) Preparation of extract-

1. Cut fine strips of spinach leaves, place in a clean mortar and pestle to
reduce it to pulp.
2. Add 50 ml of precooled 80% acetone to leaf pulp to which small
quantities of acid washed sand and a small amount of CaCO3 is added.
3. The clear supernatant green coloured liquid to 10 ml of ethyl ether in
a separatory funnel.
4. 60ml of distilled water was gradually added while rotating the funnel
slowly without shaking.
5. Two layers now separated- lower acetone-water layer and upper ether
layer.
6. Allow the lower acetone layer to run off and discard it.
7. Water is added till two layers are formed and lower layer is discarded.
Repeat it thrice to discard all the acetone.
8. 30 ml of methyl alcoholic KOH is added and the separating funnel is
allowed to stand for 15 minutes.
9. Some amount of water (20ml) and ether (5ml) are again added and the
separating funnel is shaken vigorously before keeping it at rest for
some time.
10. Two layers appear, upper contains chlorophyll and lower contains
carotenoids.
iii) Application
1. A spot of the extract is put with a capillary tube, ½ inch from the
bottom and ½ inch from left margin of the plate.
iv) Development
1. The solvent used is petroleum ether-95%, acetone in ratio 100:12 or
n-butanol, acetic acid and water (5:1:4)
2. Sufficient solvent is poured into chromatography jar filling an inch
from the bottom.
 3. The spotted paper is placed in the jar vertically so that spot is just
above the solvent level.
4. The lid of the jar is closed and allowed 1-2 hours for development.
5. The chromatogram is removed when the solvent reaches the top and
allowed to dry.

Result:

The sequence of pigments from top to bottom shall be-

Carotenes: orange yellow
Xanthophylls: yellow bands
Chlorophyll a: blue green
Chlorophyll b: yellow green

The Rf value is determined by the formula-

Rf = distance from origin travelled by compound

distance of solvent front from origin
The components can be identified by comparing the results with standard values.

Precautions:
1. Pigment spots should be applied carefully.
2. The solvents should be prepared accurately.
3. Observation should be done carefully.