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Isolation of Plant Pigments by Column Chromatography


Preparation of the crude extract:


  1. 10 grams of dry spinach leaves are to be weighed. (All stems and veins should be removed from the leaves before weighing).
  2. The spinach leaves are then crushed and ground using a mortar and pestle and made into a fine paste by adding 10mL (50:50) mixture of hexane with acetone.
  3. The filtrate from the well crushed paste should be transferred into a conical flask through a funnel that is plugged with a small piece of cotton.


Removing the traces of water present in the crude:


  1. Add 2-3 spatula of anhydrous sodium sulfate to remove traces of water that may be present in the crude extract.
  2. Filter off sodium sulfate from the crude extract using a funnel.


Adsorbing the Crude filtrate to Silica gel:


  1. Add about 3g of silica gel (for column chromatography 60-120 mesh) to the crude extract.
  2. Gently heat the mixture using a hair dryer until the silica gel becomes free flowing.


Packing the Column for Chromatography:


  1. Take a cylindrical glass column and plug in a small piece of cotton.
  2. Mount the column on the stand.
  3. Take 25g of fresh silica gel (for column chromatography 60-120 mesh) in a 250 mL beaker.
  4. Pour 100mL of hexane into the beaker and stir well using a glass rod to make slurry of the silica.
  5. Pour the slurry into the column.
  6. Place a conical flask below the mounted column and drain out the excess solvent (hexane).
  7. Close the stop cock when the level of the solvent reaches just above the settled silica gel.


Loading the Crude material on to the Column:


  1. Transfer the adsorbed crude material into the solvent layer above the silica gel in the packed column.


Elution using Hexane:


  1. Continue filling the column with hexane and elute it until the yellow colored β- carotene runs down the column.
  2. As the elution progresses, β-carotene will elute out of the column, collect this in a conical flask.


Elution using Acetone: 


  1. After collecting the yellow pigment, fill the column with acetone.
  2. Place another conical flask below the mounted column.
  3. As elution with acetone progresses, the green pigments start moving down the column.
  4. Collect this compound in the conical flask.


Removal of Solvent:


The yellow and green pigments collected from the column are then concentrated by removing the solvents using a rotary evaporator.
The pigments left behind in the round bottomed flask after rotary evaporation are transferred into watch glasses using spatula.





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