1. Thin Layer Chromatography Investigations
Leighton Dann and Paul Beaumont
Science & Plants for Schools

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

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

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

5. Fisher Scientific UK. Tel: 01509 555500
Cat No: TLC X

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

9. Method Place ca. 3 cm2 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

10. Method
Supernatant and residue from grinding leaf in propanone

11. 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

12. 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

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

14. Method Plate into a 600 cm3 beaker, replace foil
Remove plate when solvent nearly at top
Immediately mark solvent front

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

16. Pigment
Colour
RF value
carotene
yellow-orange
0.91
pheophytin a
grey
0.75
pheophytin b
light grey
chlorophyll a
blue green
0.63
chlorophyll b
green
0.58
xanthophylls
yellow
0.53
0.47
0.32

18. Coleus

21. Anthocyanin results