Thin layer chromatography

Question 1

what leaves did we use and why

Answer 1

spinach as they easily squish and release a fair amount of pigment

Question 2

what are the main pigments in the leaves and what colours are they

Answer 2

chlorophylls = green
carotenoids = orange and yellow

Question 3

what are the pigments separated by in TLC

Answer 3

the basis of the strength of their interaction with the thin layer of silica gel (stationary phase) - pigments that interact weakly will travel further

Question 4

should you describe methods of practical’s in past, present or future tense

Answer 4

past

Question 5

what did we use to grind the spinach leave in

Answer 5

a pestle and mortar

Question 6

what did we add to the 5g of spinach to make extraction easier when grinding

Answer 6

10ml of methanol

Question 7

describe the method from after grinding until in the chromatography tank

Answer 7

1. the extract is filtered to remove any solids
2. 5ml of ether is added and swirled thoroughly
3. 0.5ml of 1 mM NaCl is dripped down the side of the tube
4. the mixture is allowed to stand until the two layers separate
5. pipette off the upper green layer and place into a small conical flask
6. add a small amount of anhydrous sodium sulphate to remove any water
7. pipette 2ml of the green extract into a boiling tube and dry the extract under nitrogen in the fume hood
8. dissolve the dry extract in 0.4ml cyclohexane and transfer into a small glass vial - makes using a capillary tube easier
9. add extract to TLC plate and place in chromatography tank that contains ethyl acetate: hexane (50:50)

Question 8

what do you use to add the extract to the TLC plate

Answer 8

a capillary tube

Question 9

how far up the plate should the baseline be drawn and in what

Answer 9

1.5cm from the edge and in pencil

Question 10

how many times should the extract be reapplied

Answer 10

4-5 times in 5 separate areas

Question 11

once the separation has finished what can you calculate

Answer 11

the Rf values of each pigment and the colour of the pigment - both can be used to identify which colour separation is which pigment

Question 12

name the 5 pigments and their colours, in order of most polar to least polar

Answer 12

Xanthophylls - yellow
chlorophyll b - light green
chlorophyll a - dark green
pheophytin - grey
beta-carotene - orange

Question 13

simply explain the separation in terms of polarity

Answer 13

the more polar a pigment is the less it will travel up the TLC plate because it has a high affinity for the non-polar silica stationary phase