1. Commercially available open glass column, 19 mm i.d. x 20 cm, is used. A small wad
of cotton wool is tamped down in the bottom of the column and then covered with a layer of
sand.
2. Mix 10ml ethanol and 5g silica gel to obtain slurry. Transfer the slurry into prepared
column containing 5ml of ethanol with concurrent tapping on the side of the column to free air
bubbles. After the silica gel is settled, it is then covered with another layer of sand. Allow the
solvent to drain to the level of silica.
3. 1ml of mixture is gently added to the top of the column. Allow the mixture to adsorb
into the top of the silica.
4. Carefully add ethanol to the column and allow it to drip through. Eluant from the
column is collected in test tubes, held in a rack. It is important to keep these tubes in order.
5. When the first dye is either completely or nearly emerged from the column, increase
the polarity of solvent using 2:2:1 ethanol: water: acetic acid in order to elute the second dye.
5. The contents of each test tube are checked by TLC. Several tubes can be spotted on
the same TLC plate. It helps to mark the spotting points lightly with a pencil. Record the Rf of
each component.