Photosynthetic pigments with chromatography A Level Biology
During this practical, students are going to separate the coloured pigments found in leaves or grass. An area of grass in full sun should be selected, as well as an area which is in the shade, to select the plant from.
Subject: Biology | Level: A Level |
You will need:
Method
Students set up two vials labelling one ‘Shade’ and one ‘Light’ using a marker pen, each containing approximately 3cm³ of running solvent, placing the lid back on top of the bottle.
Two TLC Plates are cut to size so that they fit into the McCartney Bottles. A pencil line is drawn approximately 2cm from the bottom of the plate. This is the origin line.
A pinch of grass from the shaded area is taken and laid across the pencil line on one of the TLC Plates. A coin or glass rod can be used to roll the pigment onto the TLC Plate by rolling the side of the coin across the grass, tracing the pencil line through with the grass sample. The plate should be labelled, using a pencil, with ‘Shade’ at the top.
A pinch of grass from the shaded area is taken and laid across the pencil line on one of the TLC Plates. A coin or glass rod can be used to roll the pigment onto the TLC Plate by rolling the side of the coin across the grass, tracing the pencil line through with the grass sample. The plate should be labelled, using a pencil, with ‘Light’ at the top.
Students then place each of the two TLC Plates into separate McCartney Vials, ensuring the solvent line is just below the pencil / pigment line. They should now leave the vials to stand undisturbed so that the chromatogram can develop.
When the solvent line reaches the top of the TLC Plates, remove them from the vial and make a pencil mark to show how far the solvent has travelled on each plate. This is the solvent front. Place the lid back onto the McCartney Vials to prevent further fumes from escaping.
Using a pencil, the students now mark off each of the coloured pigment spots on the TLC Plates.
The Rf values for each coloured pigment spot is worked out using the following equation:
Rf Value = Distance moved by pigment from the origin line
Distance from origin line to the solvent front
Technician tips
Thin Layer Chromatography plates are expensive. Students can be provided with small strips of chromatography plate to cut to size themselves, or pre-cut TLC plates can be provided by the technician that already fit into the McCartney bottles or chosen vessel.
Running Solvent Recipe (100mls): Propanone / Petroleum Ether (100-120°C) needs to be mixed in a ratio of 1:9. 10mls of Propanone mixed with 90mls of Petroleum Ether (100-120°C)
Glass measuring cylinders should be used to dispense the solvent.
