Separate the leaf pigments from spinach leaves by column chromatography.

  Separate the leaf pigments from spinach leaves by column/adsorption chromatography

Theory: The juice of the leaves and flowers contains a number of colored compounds such as green, yellow, orange, or red. These can be separated by column chromatography. The leaves of spinach are crushed in a mortar with the help of a pestle and then extracted with acetone. The extract is added at the top of a chromatography column containing Alumina as adsorbent and acetone is allowed to percolate slowly. Different colored compounds are adsorbed in the column at different rates and form separate bands. These can be separated by Elution with suitable solvents. The pure pigments present in the leaves are recovered by evaporation of the solvent.

Requirements:

• Chromatography tube
• Aluminia (chromatography grade)
• Spinach leaves
• Pestle and mortar
• Acetone, benzene and petroluem ether
• Evaporating dishes

 Procedure:

•  Prepare a slurry of Alumina by mixing about 25.0 g of Alumina in acetone.
•  Pour the slurry into the chromatography tube and prepare about a 25 cm long column. Place a filter paper disc on the top of the column.
• Take 20-25 g of spinach leaves and crush them in a mortar.
• Mix the crushed paste of leaves with 25-30 ml. of acetone, stir with a glass rod, and filter. The filtrate is an extract of the spinach leaves. collect the extract in a boiling tube. 
• Open the top of the chromatography column to drain out the excess acetone. when the surface of the column is nearly dry, add about 10.0 ml of spinach leaves extract from the top of the column.
• Attach a tap funnel to the chromatography tube at the top and fill it with acetone.
• When the extract of the leaves has gone into the adsorbent column, start the addition of acetone from the tap funnel.
• The colored components present in the spinach leaves extract will start moving downward with the flow of acetone and get adsorbed at different points in the adsorbent column. These will appear as colored bands in the column.
• Separate the different bands by Elution with a mixture of acetone and petroleum ether (40-60) 
• Recover the leaf pigments by evaporation of the solvent.

Precautions:

• There should be no air bubble trapped in the column of alumina 
• Only chromatography-grade alumina should be used for filling the column
• For effective separation, the rate of the flow down the column should be about 10 drops per min.

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