Thin Layer Chromatography Investigations Leighton Dann and Paul

Thin Layer Chromatography Investigations Leighton Dann and Paul Beaumont Science & Plants for Schools www. saps. org. uk

TLC One of a number of types of chromatography: • gas/liquid • affinity • paper

TLC Separation of complex mixtures between two phases: - a stationary phase and a mobile phase

TLC Phases • the stationary phase is a thin layer of absorbent particles, in this case silica gel • the mobile phase is the solvent

Fisher Scientific UK. Tel: 01509 555500 Cat No: TLC-410 -610 X

Thin Layer Chromatography Separation of: • photosynthetic pigments in leaves • anthocyanins in leaves, flowers, fruit and vegetables

• Place ca. 3 cm 2 leaf in mortar • [If tissue is coarse, silver sand may be added] • Grind until all tissue broken up • Add ca. 750 ml propanone • Pour off supernatant into a container and seal Method

Method • Supernatant and residue from grinding leaf in propanone

Method • Mark (with a pencil) a plate 1. 5 cm up from the base on the edge of both sides • Use a ruler across these marks to mark two spots 2 cm apart in the middle of the plate

Method • Use Pasteur pipette to place small drops of supernatant • Dry off before adding another lot of drops • Continue until there is a concentrated line of colour

Method • Repeat process until a second line is produced (or one line across the whole plate)

Method • Plate into a 600 cm 3 beaker, replace foil • Remove plate when solvent nearly at top • Immediately mark solvent front

Solvent system Cyclohexane : propanone : petroleum ether (low boiling point) 5: 3: 2

Pigment Colour RF value carotene pheophytin a pheophytin b chlorophyll a chlorophyll b xanthophylls yellow-orange grey light grey blue green yellow 0. 91 0. 75 0. 63 -0. 75 0. 63 0. 58 0. 53 0. 47 0. 32

Coleus

Anthocyanin results