Separation of Pigments

by Adsorption Column
Chromatography
Hina Fatima
Principle
• Different pigments get adsorbed to alumina to different extents.
• They can be selectively desorbed by using mobile phase of increasing
polarity in a stepwise manner.
Materials and Reagents
1. Leaves/flowers
2. Pestle and mortar
3. Glass column
4. Whatman No.1 filter paper
5. Alumina
6. Benzene: methanol (2:1)
7. Sodium sulphate (anhydrous)
8. Acetone
Procedure
(a) Preparation of extract:
1. Homogenize 5 g leaves or flowers in a pestle and mortar, using sand
as an abrasive in 20 ml of benzene: methanol (2:1) adding a small
amount of this extractant at a time.
2. Filter the extract through Whatman No.1 filter paper and transfer the
filtrate to a separating funnel.
3. Add 10 ml of water to the filtrate and after shaking the contents and
allowing the phase to separate out, drain out the lower aqueous
methanol layer. Repeat this step. Avoid very vigorous shaking.
4. Collect the benzene layer in a beaker and add small amount of solid
anhydrous Na2SO4 to remove the traces of moisture.
5. Decant the clear benzene layer to another beaker and concentrate the
extract by evaporating the solvent on a boiling water bath.
(b) Column preparation:
1. Mount a burette or a glass column vertically on a burette stand with
the help of clamps.
2. Place lightly a plug of glass wool at the base of the burette and close
the stopcock or outlet at the bottom of the column.
3. Take 5 g of alumina (adsorbent) previously dried at 120ºC for 8 h
and prepare its slurry in benzene. Pour the slurry carefully into the
column or burette, by gentle tapping of the column so that no air
bubbles get trapped in the adsorbent.
4. Allow the adsorbent to settle by opening the outlet. After the
adsorbent has completely settled, add 20 ml of benzene and let it
pass out of the column.
5. Care should be taken not to let the adsorbent to get dried. When
about 1 cm layer of the solvent remains at the top of the
chromatographic bed, close the outlet.
Sample application:
• Allow the solvent at the surface of the column to drain out slowly and
transfer the leaf or flower extract with the help of pipette without
disturbing the surface of the column adsorbent.
• Let it enter into the column and then add a few drops of benzene to
wash the traces of the extract sticking to the wall of the column.
• Add 20 ml more benzene to wash out the column of any unadsorbed
material.
Column development:
1. For desorption of the adsorbed substance change the polarity of the
solvent in a stepwise manner.
2. After 20 ml of benzene has passed through the column add 10 ml of
5% acetone (u/v) in benzene and let it percolate through the column
and collect 1–2 ml fractions of the effluent from the outlet.
3. Continue increasing the concentration of acetone in benzene at every
succeeding step. Finally pass pure acetone through the column.
4. Note the change in the color of the collected fractions. In case of the
leaf extract, the initial fractions are colorless followed by yellow
colored and then by the green colored ones.
5. The colorless fractions do not contain any pigments but it is quite
possible that these fractions may contain some UV absorbing
materials.