Thin layer chromatography practical

Question 1

Lipids

Answer 1

water insoluble molecules that are soluble in a variety
of non-polar organic solvents

Question 2

Pigments

Answer 2

> The main pigments are chlorophylls (green) and carotenoids (orange and yellow).
The chlorophylls are involved in light harvesting and the primary reactions of photosynthesis,
while the carotenoids are associated with reducing free radical damage to membranes. > Both groups are important in human diet, and there is much interest in their potential anti-cancer activity.

Question 3

Pigment interactions

Answer 3

Those pigments that interact weakly with the stationary phase will be carried easily up the plate by the solvent (the mobile phase), and thus travel further than pigments that interact strongly with the stationary phase and are retarded.

Question 4

Rf

Answer 4

Distance moved by pigment
Rf = —————————————-
Distance moved by solvent

Question 5

Materials

Answer 5

> Spinach leaves
Methanol
diethyl ether
1mM NaCl
Anhydrous sodium sulphate
cyclohexane;
ethyl acetate:hexane (50:50 v/v)

Question 6

Step one

Answer 6

Cut 5 g spinach leaves into small pieces and grind them into a fine paste using the mortar and pestle provided, with 10 ml methanol.

Question 7

Step two

Answer 7

Filter the green extract into a test tube and add 5 ml ether. Swirl the tube thoroughly
then drip 0.5 ml of 1 mM NaCl down the side of the tube. Allow the mixture to stand until two separate layers appear.

Question 8

Step three

Answer 8

Pipette off the upper green layer and place it in a small conical flask. Add a small amount (a pinch is enough) of anhydrous sodium sulphate to remove any water.

Question 9

Step four

Answer 9

Pipette 2 ml of the green extract from the conical flask into a boiling tube. Dry the extract
under nitrogen.

Question 10

Step five

Answer 10

Dissolve the dry extract in 0.4 ml
cyclohexane. Transfer the extract from the boiling tube into the small glass vial provided.

Question 11

Step six

Answer 11

The silica plate has a white front and silver back: use the white surface for the next step.

Question 12

Step seven

Answer 12

Draw a line 1.5 cm from the shorter edge of a silica plate using a pencil (pen ink will run up the plate). Try not to scratch the silica off the plate.

Question 13

Step eight

Answer 13

Using a capillary tube, spot about 0.2 ml of the extract as shown. Allow to dry then re-apply the extract to the spots several times (drying in between each application).

Question 14

Step nine

Answer 14

Place in a chromatography tank without submerging the sample in the ethyl acetate:hexane (50:50).

Question 15

Step ten

Answer 15

An orange line should be visible near the solvent front. When this and the remaining pigments are well separated, remove the plate from the solvent and mark the position of the solvent front immediately. Allow to air dry. Keep in the dark as pigments fade.

Question 16

Analysis of results

Answer 16

1. Draw a diagram of the plate, showing the positions of each band of pigment and also
   the colour of the band.
2. Calculate the Rf value for each band of pigment.
3. Using the information given in the table below, identify each band of pigment.
4. Briefly explain the basis of the separation.

Question 17

Pigments in order of decreasing polarity and their colour

Answer 17

> Xanthophylls (most popular)- yellow
Chlorophyll b- light green
Chlorophyll a- dark green
Pheophytin- grey
beta-Carotene (least polar)- orange