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Thin Layer Chromatography Investigations
USING PRE COATED PLATES
© akashmarathakam
TLCTLC
One of a number of types of chromatography:
• gas/liquid• affinity • paper
TLCTLC
Separation of complex mixtures between two phases:
- a stationary phase
- and a mobile phase
TLCTLC
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 Thin Layer ChromatographyChromatography
Separation of:
• photosynthetic pigments in leaves
• anthocyanins in leaves, flowers, fruit and vegetables
MethodMethod• 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 l propanone
• Pour off supernatant into a container and seal
MethodMethod
• Supernatant and residue from grinding leaf in propanone
MethodMethod
• 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
1.0 2.0 3.0 4.0
1.5 cm
MethodMethod• 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
1.0 2.0 3.0 4.0
1.5 cm
MethodMethod• Repeat process until a second line is
produced (or one line across the whole plate)
1.0 2.0 3.0 4.0
1.5 cm
MethodMethod
• Plate into a 600 cm3 beaker, replace foil
• Remove plate when solvent nearly at top
• Immediately mark solvent front
Solvent systemSolvent system
Cyclohexane : propanone : petroleum ether (low boiling point)
5 : 3 : 2
Student resultsStudent results
ResultsResults
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
ColeusColeus
Anthocyanin resultsAnthocyanin results