1. Thin Layer Chromatography at Higher and Advanced Higher
Biology Summer School 2013
Thin Layer Chromatography at Higher and Advanced Higher
Kath Crawford, Paul Beaumont, Lorraine Bruce

2. Unit: Sustainability and Interdepedence
1 The science of food production
(b) Plant growth and productivity
Photosynthesis
Energy capture by photosynthetic pigments....

3. Unit: Cells and Proteins
Laboratory techniques for biologists
(c) Separation techniques
Unit: Investigative Biology
2. Experimentation

4. Thin Layer Chromatography
Separation of complex mixtures between two phases:
a stationary phase
a mobile phase

5. Thin Layer Chromatography
In TLC:
the stationary phase is a thin layer of absorbent particles, in this case silica gel
the mobile phase is the solvent

6. Fisher Scientific UK. Tel: 01509 231166
Cat No: TLC X
Price £35 for pack of 50 (5 x 20 cm)

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

9. Method Place ca. 1 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
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

11. 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

12. Method
Repeat process until a second line is produced

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

14. Student results

15. Results 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

16. Coleus