1. 1 Pigments in plants Separation of chloroplast pigments by paper chromatography Refer to the Practical Manual Unit 5: Cell Biology Practical 16

2. 2 Aims: 1.To remove pigments from plant material. 2.To separate the pigments using paper chromatography. 3.To identify the pigments by their colours and relative positions on the chromatograph. 4.To determine relative amounts of each pigment.

3. 3 Theoretical basis: (Textbook reference pp.361–367) Various pigments found in the chloroplasts of plants are used in the process of photosynthesis. The specific pigments used depend to some extent upon the amount of light normally present and the wavelengths of the light that normally falls on the plant. Other pigments are not used in photosynthesis. There are five pigments commonly found in chloroplasts. Although leaves usually appear green, the chlorophylls present may mask the appearance of other pigments. Some leaves are red and here a red pigment is masking the chlorophylls. Variegated leaves, those which are green with white areas, only photosynthesise in the green areas. The white areas are devoid of chlorophylls.

4. 4 Theoretical basis: (Textbook reference pp.361–7) The chloroplast pigments can be separated and identified by paper chromatography. Absorptive paper containing a concentrated spot of chloroplast extract is dipped into a suitable solvent. The various pigments have different sized molecules, with the result that as the solvent ascends the absorptive paper it carries the pigments with it at different rates, the smaller molecules being more mobile than the larger ones. In this way they can become separated from each other and can be identified by their different colours and positions.

5. 5 Requirements: water tweezers solvent metric ruler hot plate glass rod scissors drawing pin parsley or spinach mortar and pestle boiling tube ethyl alcohol test tube holder rubber stopper chromatography paper centrifuge & tube Solvent = 1 part of 90% acetone to 9 parts of petroleum ether (BP= 80 - 100 °C)

6. 6 Equipment

7. 7 Method: Part A PREPARATION OF LEAF PIGMENTS 1.Quickly immerse leaves in boiling water. 2.Place killed leaves in mortar and pestle and grind these up with a small amount of alcohol. 3.Decant the dark green liquid produced into a centrifuge tube and settle out using a centrifuge.

8. 8 Quickly immerse leaves in boiling water Immersing the leaves in boiling water kills the leaves and so the cell membranes Do this process VERY quickly

9. 9 Place killed leaves in mortar & pestle

10. 10 Add a small amount of alcohol

11. 11 Grind the leaves with the alcohol

12. 12 Decant dark green liquid into centrifuge tube Centrifuge tube

13. 13 Settle out using the centrifuge Lid Speed control Run Brake

14. 14 Place tube in centrifuge * Remember its position

15. 15 Balance the centrifuge Remember the position of your tube e.g. 3a Place the tubes opposite each other If you have three tubes to do, fill a blank tube with a similar amount of fluid to the other tubes being spun and add to the centrifuge NEVER OPEN A CENTRIFUGE UNTIL YOU ARE SURE THAT IT HAS STOPPED SPINNING

16. 16 Method: Part B PREPARING THE CHROMATOGRAPH CHAMBER 1.Cut a length of chromatography paper of sufficient length to almost reach the bottom of a large test tube (width should be less than that of the tube so that the paper does not touch the sides of the tube).

17. 17 Method: Part B PREPARING THE CHROMATOGRAPH CHAMBER 2.Rule a pencil line across the strip of paper 30 mm from one end. Make a cut from each side of the line to the centre bottom to form an arrow head. Fold the other end through 90 o and by means of a drawing pin, attach it to the cork stopper. The bottom tip of the strip should almost reach the bottom of the tube when the cork is inserted.

18. 18 Setup of chamber and appearance of pigments

19. 19 Cut to form an arrow head

20. 20 Measure distance for line

21. 21 Rule pencil line 30 mm from end

22. 22 Fold other end 90 o and attach to cork stopper with drawing pin

23. 23 Fitting the paper in tube Tip of the strip should almost touch the bottom of tube with the cork in place Paper should not touch the sides of the tube as this will upset the running of the solvent and pigments

24. 24 Method: Part B 3.Place the chromatography paper on the desk, dull side up. Add chloroplast extract to the centre of the drawn line by dipping the fine glass pipette into the extract and then quickly touching it to the centre spot on the paper. Allow the spot to dry. Add more pigment to the same spot. Repeat this procedure (allowing the spot to dry before adding more pigment) about 20 times, until there is a small spot of concentrated pigment.

25. 25 Add chloroplast extract

26. 26 Finished pigment dot

27. 27 CAUTION SOLVENT IS HIGHLY FLAMMABLE Make sure all flames are turned off and the room is well ventilated

28. 28 Method: Part C SEPARATING THE PIGMENTS 1.Add the solvent to a depth of 15 mm in the test tube. 2.Carefully place the tube into the test tube holder. 3.Place the chromatography paper into the tube. Do not let the spot of extract touch the solvent. 4.Do not shake or move the tube for at least 15 minutes. Remove the chromatograph from the tube when the solvent has almost reached the top of the paper.

29. 29 Add solvent to 15 mm depth

30. 30 Carefully place in test tube holder then place chromatography paper into tube.

31. 31 Do not let the spot of extract touch the solvent. Solvent front

32. 32 Leave the tube for 15+ minutes Solvent front

33. 33 Remove the chromatograph

34. 34 Method: Part D ANALYSIS OF THE CHROMATOGRAPH 1.Quickly measure the distance travelled by the solvent. Record. (The solvent rapidly evaporates in air.) 2.Examine the chromatograph for the presence of different bands of colour. Each colour band will be a different pigment. Some bands are very faint. 3.Measure the distance from the pencil line to the leading edge of each clearly detectable pigment. 4.Record.

35. 35 Mark distance solvent travelled Mark and measure distance travelled by solvent The solvent rapidly evaporates in air

36. 36 Look for different colours Mark and measure furthest distance travelled by each different pigment Record Draw the outline of each of the pigment spots These fade very quickly

37. 37 Close-up of strip

38. 38 R f values The ratio of the rate of movement of the pigment to solvent (the R f value) can then be calculated for each pigment R f = distance moved by the substance from the original position distance moved by solvent from the same position Use table to identify leaf pigments Measure the width and depth of each of the pigment bands Estimate the relative quantities of each pigment present

39. 39 Identify the leaf pigments NAMECOLOURRf Caroteneyellow0.95 Phaeophytinyellow-grey0.83 Xanthophyllyellow-brown0.71 Chlorophyll ablue-green0.65 Chlorophyll b green 0.45

40. 40 Student results 1 Solvent front

41. 41 Student results 2: R f values

42. 42 Student results 3: areas

43. 43 Interpretation of results: Write a discussion of your results including the following points: The pigments present in the leaf. Phaeophytin is a breakdown product of chlorophyll. Suggest functions for the other pigments. Chlorophyll a and b are easily broken down in cool temperatures. Explain how leaf colour change can come about in deciduous trees in autumn. Possible differences in types and/or concentrations of pigments present between plants with very dark green leaves and those with light green leaves. Some rainforest trees and ferns have young leaves that are red in colour, as do the red algae that are found at greater depths than the green and brown. Suggest a scientific reason based on pigments that could explain these observations.

44. 44 Red rainforest leaves