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Thin Layer Chromatography

Investigations

USING PRE COATED PLATES


© akashmarathakam
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


Here using Pre coated TLC plates
Thin Layer
Chromatography
Separation of:

• photosynthetic pigments in leaves

• anthocyanins in leaves, flowers, fruit and


vegetables
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 µ l propanone
• Pour off supernatant into a
container and seal
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
Student results
Results
Pigment Colour RF value
carotene yellow-orange 0.91
pheophytin a grey 0.75
pheophytin b light grey 0.63-0.75
chlorophyll a blue green 0.63
chlorophyll b green 0.58
xanthophylls yellow 0.53
xanthophylls yellow 0.47
xanthophylls yellow 0.32
Coleus
Anthocyanin results