Preparation of the crude extract:
- 10 grams of dry spinach leaves are to be weighed. (All stems and veins should be removed from the leaves before weighing).
- 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.
- 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:
- Add 2-3 spatula of anhydrous sodium sulfate to remove traces of water that may be present in the crude extract.
- Filter off sodium sulfate from the crude extract using a funnel.
Adsorbing the Crude filtrate to Silica gel:
- Add about 3g of silica gel (for column chromatography 60-120 mesh) to the crude extract.
- Gently heat the mixture using a hair dryer until the silica gel becomes free flowing.
Packing the Column for Chromatography:
- Take a cylindrical glass column and plug in a small piece of cotton.
- Mount the column on the stand.
- Take 25g of fresh silica gel (for column chromatography 60-120 mesh) in a 250 mL beaker.
- Pour 100mL of hexane into the beaker and stir well using a glass rod to make slurry of the silica.
- Pour the slurry into the column.
- Place a conical flask below the mounted column and drain out the excess solvent (hexane).
- 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:
- Transfer the adsorbed crude material into the solvent layer above the silica gel in the packed column.
Elution using Hexane:
- Continue filling the column with hexane and elute it until the yellow colored β- carotene runs down the column.
- As the elution progresses, β-carotene will elute out of the column, collect this in a conical flask.
Elution using Acetone:
- After collecting the yellow pigment, fill the column with acetone.
- Place another conical flask below the mounted column.
- As elution with acetone progresses, the green pigments start moving down the column.
- 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.