EXPERIMENT 7

COLUMN CHROMATOGRAPHIC
SEPARATION OF PIGMENTS FROM
GREEN LEAVESo

Structure

7.1 Introduction
Objective
7.2 Prin~iple
7.3 Requirements
7.4 Procedure
7.5 Result and discussion

7.1 INTRODUCTION
In the last three experiment you have studied about paper and thin layer
chromatography. In this experiment you will learn how to separate the pigment from
green leaves through column chromatography.

Column chromatography is a technique which can be applied to the separation of

many complex mixtures. Thgsample solution is applied to the top of the column.
The mobile phase flows down through the column filled with the stationary phase
material.

The green leaves of plants contain a number of pigments viz: chlorophyll-a,

chlorophyll-b, xanthophylls and carotenes. You will learn the separation of these
pigments by column chromatography in this experiment.

Objective:

After studying and performing this experiment you should be able to:

explain the basic principle of column chromatography,

prepare the extract (of leaves) for the experiment,
prepare the calcium carbonate column, and
separate green pigment (of leaves) by column chromatography.

7.2 PRINCIPLE
The success of a separation by column chromatography depends on the choice of the
stationary and mobile phases. The stationary phase material is filled in a column.
Any of the three possible mechanisms: partition, adsorption o r ion exchange can be
employed by the use of a particular type of the stationary phase inside the column.
For example, for the separation based on adsorption a n adsorbent is packed in the
column.

The choice of the mobile phase depends on the nature of the substance and how
strongly it is adsorbed. In a number of cases such as alumina and silica gel as the
adsorbent, the mobile phase is generally a non-polar solvent such as petrol and
benzene because polar groups such as hydroxyl-(OH) group in water and in ethanol
would cause desorption. Eluents containing two or more solvents may be used for
 Column Chromatographic
better results. In such cases the polarity is increased by adding a polar solvent with a
Separation of Pigments From
non-polar one. Green Leaves

7.3 REOUIREMENTS
Apparatus Chemicals
Chromatography column 1 Calcium carbonate
Glass wool Anhydrous Sodium
Cotton wool Sulphate anhydrous
Beaker (100 cm3) 2 Benzene
Conical flask (250 cm3) 1 Petroleum ether
Mortar 1 Ethyl alcohol
China dish 1
Separatory funnel 1
Graduated cylinder (100 cm3) 1
Wash bottle 1

7.4 PROCEDURE
Proceed according t o the following steps

1) Preparation of the Extract: Take 5-10 g of fresh grass (or leaves of a green
plant) cut it up into fine pieces in a mortar, grind for about 3 0 seconds, add
1 0 cm 1of ethyl alcohol and 2 0 cm3 of petroleum ether, grind again. Decant the
liquid into a separatory funnel after filtering through glass wool placed in an
ordinary funnel. Add 1 0 cm3 alcohol and 20 cm3 petroleum ether again to the
mortar containing grass, grind and transfer the liquid after decantation to the
separatory funnel containing the first fraction. Shake gently. A light green
- S o l ~ dand solvent
emulsion may form, if shaken vigorously. Allow to settle the layers. T h e
bottom layer is water-ethanol layer and the upper layer of petroleum-ether
contains grass extract. Remove the bottom layer and wash the petroleum layer
-Glass wool
with water for 3 o r 4 times until the layer is clear. Remove the aqueous layer.
T h e extract is now free from alcohol but contains water in very small amount.
Transfer the upper layer containing the extract to a dry conical flask. To this,
add anhydrous sodium sulphate (dried by heating in an ovenlhot plate before
use), shake the flask and leave it over for about 15 minutes to remove any
water present with the extract. Transfer the extract to a clean and dry test tube,
cover it and take it for chromatography.
2) Preparation of column: Take a glass column o r a burette of about 20 cm in
length and 7-8 m m diameter tube. Place a small wad of cotton wool a s the
column support. Pack the column with anhydrous calcium carbonate (dried by
Chsomatogmphic column
heating in a china dish over a burner), tap it regularly with a glass rod. Add
t h e adsorbent in small portions and gently press down until a column of 8-10
c m has been uniformly packed. Place a small wad of cotton wool at the top of
the calcium carbonate column and use it for chromatography. The physical state of the column
packing material should be such
3. T a k e t h e uniformly packed column containing calcium carbonate and fix it in a that it allows uniform packing of
stand vertically. the column and a free flow of the
solvent through it.
4. Take 1-2 cm3 of dried extract o f leaves, drip into the column in the form o f a
thin layer of solution, allow t o run evenly into the adsorbent unti: a green The extract from green leaves
zone 3-4 mm deep is formed a t the top of the column. This is known as the should be completely free from
water since the presence of a polar
loading o f the sample. substance can alter the course of
5. Add the developer (benzene) t o the column and a l l a y the developer through
the column packing till separate bands a r e observed.
6. N o t e the colour of different bands and their order.
41
Chemistry Lab -V 7. If extra time is available, continue the passage of developer and collect the
different coloured substances in fractions, noting the volume eluted by a
measuring cylinder.

7.5 RESULT AND DISCUSSION

The bands observed on the column are of different colours. The uppermost thin
yellowish green zone is chlorophyll-b, below this the bluish green zone of
chlorphyll-a, next orange-yellow zone contains xanthopylls and the lowest orange
zone contains carotenes. The carotenes are least adsorbed by the adsorbent and can
be easily washed out of the column.

Three main interactions are to be considered in column chromatography: the activity

of the adsorbent, the polar behaviour of the substance and the polarity of the eluting
solvent.

This experiment is based on the results of the inventor of the technique of

chromatography, M. Tswett, who applied the technique to separate various plant
pigments using calcium carbonate as the stationary phase packed in a column.