Procedures:

 

• Cut a piece of green leaf approximately 5 cm x 5 cm (square) and drop it into a small amount of boiling water and boil for 2 min.

 

• Remove the leaf from the water and pat it dry between pieces of paper towel.

 

• Grind the leaf with few pellets (3 – 5) of CaCl2 in a pestle and mortar and add 3 ml of petroleum ether and 10 ml of acetone (SAFETY ALERT: do it inside the fume-hood).

 

• Let stand for a few minutes until most of the pigments are dissolved and the leaf has lost most of its colour.

 

• Transfer the liquid into a test-tube, add another 3 ml of petroleum ether and 10 ml of DI water, and gently shake the test-tubes several times. Let stand until the two phases separate.

 

• With a transfer pipette, transfer the upper dark green layer to a clean test-tube. Add 10 ml of water and gently shake the test-tube several times. Again let stand until the two phases separate. The pigments are now in the petroleum ether phase and the water has removed most of the acetone.

 

• Prepare the developing fluid. Pipette 1 ml of n-butanol into a 100 ml volumetric falsk and add with petroleum ether. Shake well. Caution: keep away from flame. (Prepared)

 

• Using a strip of the filter paper, make fold 1.2 cm from one end of the strip, and with a pencil place a dot in the centre and 1.2 cm from the other end of the strip.

 

• Pour the aliquot of the developer into the free standing test tubes to a depth of 1.2 cm and covered with stopper cap.

 

• With a micropipette or capillary tube, apply a small drop of the petroleum ether extract of the pigments to the filter paper strip at the position marked by the dot. Keep the spot small. Make four to eight applications at the same spot, but allow the extract to dry for a few seconds between applications. Practice spotting with a scrap of filter paper first.

 

• With a paper clip, attach the strip of filter paper to it. Make sure the strip hangs straight.

 

• Lower the strip into the free standing tubes that contain developer. The developing fluid should reach the end of the filter paper strip, but it should not touch the spot of the pigments.

 

• Let the chromatogram develop until the pigments are separated.

Coon Mistakes Made:

• Students have to ensure that sufficient pigments have dissolved and the leaf has lost most of its colour before transferring the liquid into a test-tube.

 

• Ensure that the spot of the pigments on the filter paper strip is dried before applying the next drop. You can shake or blow at the filter paper strip to aid in drying the extract.

 

• Ensure that the spot on the filter paper strip is small.

 

• Ensure that the spot does not touch the developing fluid when placed in the standing tubes. You should try placing the filter paper into the standing tube before spotting on the filter paper.

 

• Avoid moving the standing tubes that contain developing fluid and the filter paper strip.

 

• Watch the chromatography to prevent over-run.

 

• Draw on the filter paper strip after chromatography as the pigments will dry out. Once it dries out, the separation of pigments cannot be noticed. 

 

ATTENTION:

 

• Appearing on the chromatogram of extracts from most green leaves are carotenes. A faint grey band of Pheophytin a may be observed in front of chlorophyll a. One or more spots or bands of yellow xanthophylls may appear on the chromatogram

 

• Draw a diagram of the chromatogram and label the spots.

 

• Students have to ensure that sufficient pigments have dissolved and the leaf has lost most of its colour before transferring the liquid into a test-tube.

 

• Ensure that the spot of the pigments on the filter paper strip is dried before applying the next drop. You can shake or blow at the filter paper strip to aid in drying the extract.

 

• Ensure that the spot on the filter paper strip is small.

 

• Ensure that the spot does not touch the developing fluid when placed in the standing tubes. You should try placing the filter paper into the standing tube before spotting on the filter paper.

 

• Avoid moving the standing tubes that contain developing fluid and the filter paper strip.

 

• Watch the chromatography to prevent over-run.

 

• Draw on the filter paper strip after chromatography as the pigments will dry out. Once it dries out, the separation of pigments cannot be noticed.