Thin Layer Chromatography Investigations.ppt

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