SEPARATION OF LEAF PIGMENTS BY COLUMN CHROMATOGRAPHY - Computer Programming

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Friday, April 1, 2011

SEPARATION OF LEAF PIGMENTS BY COLUMN CHROMATOGRAPHY


SEPARATION OF LEAF PIGMENTS BY COLUMN CHROMATOGRAPHY
Aim:
To separate the various pigments of a concentrated leaf extract using adsorption column chromatography.
Principle:
A solid stationary phase and a liquid mobile phase are used and the principle of separation is adsorption. When a mixture of components dissolved in the mobile phase is introduced in to the column, the individual components move with different rates depending upon their relative affinities. The compound with lesser affinity towards the stationary phase (adsorbent) moves faster and hence it is eluted out of the column first. The one with greater affinity towards the stationary phase (adsorbent) moves slower down the column and hence it is eluted later. Thus the compounds are separated. The type of interaction between the stationary phase (adsorbent) and the solute is reversible in nature. The rate of movement of a component (R) is given as follows:
This equation can be simplified as follows:
When a liquid mobile phase is use, the equation is written as
Where 𝛼 is the partition co-efficient
Am is the average cross section of mobile phase
As is the average cross section of stationary phase.
Requirements:
1.      Chromatography (20cm X 1 cm)
2.      Fresh green leaves
3.      Alumina
4.      Calcium carbonate
5.      Sucrose
6.      Sodium Sulphate
7.      Petroleum ether
8.      Methanol
9.      Benzene
10.  Mortar and pestle
Procedure:
Homogenize 5gm of green leaves in a mortar and pestle, then the extract by shaking with a mixture of petroleum ether, methanol and benzene (45: 15: 5). Remove the residue by filtration and wash the filtrate four times with water to remove the methanol. Avoid vigorous shaking or an emulsion will form. Remove the last traces of water by adding anhydrous sodium sulphate, filter to remove the solid, and concentrate the extract to a few milliliters by careful evaporation in a fume chamber.
Preparation of column:
Prepare slurries of the column materials ( alumina, calcium carbonate and sucrose) in petroleum ether and pack the column with alumina (5cm) ,calcium carbonate (7cm) and sucrose(7cm), inserting a filter paper disc between each adsorbent . Gentle suction may be applied to the bottom of the column to assist packing. Wash the column with several volumes of the eluting solvent, a mixture of benzene and petroleum ether (1:4).
Separation and elution of the pigments:
When the top of the column is almost dry, add the extract and elute the solvent. If the flow rate is too slow, apply gentle pressure to the top of the column.
Observation:
Column details:
Column adsorbent:
Column height:
Eluting agent:
Flow rate:
Numbers of bands………………..
Result:
The number of bands obtained in this experiment was…………….
Note:
The color chart for the pigments is as follows:
Chlorophyll ‘a’ and chlorophyll ‘b’ – green
Xanthophylls- yellow
Carotenes – orange
By continuously adding the eluting solvent, collect the factions and plot the absorption spectrum of each colored peak.

  

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