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-410-610X
Thin Layer
Chromatography
Separation of:
• photosynthetic pigments in leaves
• anthocyanins in leaves, flowers, fruit and
vegetables
• 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
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
1.5 cm
1.0 2.0 3.0 4.0
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
1.5 cm
1.0 2.0 3.0 4.0
Method
• Repeat process until a second line is
produced (or one line across the whole
plate)
1.5 cm
1.0 2.0 3.0 4.0
Method
• Plate into a 600 cm3
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
xanthophylls
xanthophylls
yellow-orange
grey
light grey
blue green
green
yellow
yellow
yellow
0.91
0.75
0.63-0.75
0.63
0.58
0.53
0.47
0.32
Coleus
Anthocyanin results