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