Year 11 Pre-Diploma BiologyChlorophyll pigments occur in the chloroplasts. They are a mixture of different pigments, all absorbing light at different wavelengths. The pigments can be separated and identified by chromatography.Chromatography is a technique which is based upon the theory that insoluble molecules of different molecular densities and/or masses, will be pulled at different speeds by a solvent, along absorbent paper (chromatography paper). It is then possible to determine which pigments are present by looking at their colours and also by evaluating how fast they are pulled along the chromatography paper.

What pigments are present in a leaf? There are 3 steps in this investigation:

AExtracting the chlorophyll pigments from fresh leavesA.1Dip the leaves in boiling water to kill the cells and make the membranes permeable.

A.2Finely cut up a small handful of leaves, excluding the stalks and petioles, and put them into a mortar.A.3Add about 20 cm3 of propanone (acetone) and grind up the leaf matter to make a concentrated extract.

A.4Carefully transfer the liquid you do not want any solids into a centrifuge tube and centrifuge it for two or three minutes. When you complete this stage, do not shake the tube or mix the separated solids with the liquid on top.A.5Keep this chlorophyll extract in the dark, in ice, until required. (The pigments are very unstable away from the chloroplasts and degrade quickly in light.)

B Separation of the pigments by paper chromatography

B.1Set up a simple chromatography tank, with petroleum ether/propanone (the chromatography solvent in the bottom to a depth of about 1cm. Cover the tank and leave it to saturate.

B.2Cut a piece of chromatography paper to fit into the tank, so that it does not touch the sides and the bottom of the paper will be dipping into the solvent when the tank lid is on. Make sure that you do not touch the sides of the chromatography paper while you are working.

B.3Draw a pencil cross in the middle of the paper, at a height of 1.5 cm so that the cross will not be submerged when the paper is put into the jar.B.4Use a fine pipette to extract a drop of the pigment solution from the pigment extract and place this drop on the cross. Let it dry completely. Repeat this as many times as you can do, until the extract becomes concentrated.B.5Very carefully attach the chromatography paper to the lid and place the lid on the jar, so that the paper is suspended in the solvent, and the pigment extract is above the solvent level.

B.6Watch as the solvent rises up the chromatography paper, passing through the pigment extract and carrying the pigments with it. The pigments travel at different speeds.

B.7When the solvent has reached almost to the top of the chromatography paper, remove the paper, mark exactly the point where the solvent reached, and dry the chromatogram.CIdentifying the pigments

You can identify the most obvious pigments by their colour, but this does not enable us to identify all of what could be as any as 12 different pigments. Another and much more accurate way is to calculate how fast or far the pigments have moved in relation to the distance the solvent moved. This ratio is called the Rf value and is ALWAYS the same.

Pigment

Spot colour

Rf value

Carotene

orange

0.95

Phaeophytin

yellow-grey

0.83

Xanthophyll

yellow-brown/orange0.71

Chlorophyll a

blue-green

0.65

Chlorophyll b

yellow-green

0.45

You may well have more than these 5 pigments. For instance there are 4 different xanthophylls. Try to identify the pigments which you have obtained. You might need to consult a textbook or the internet to find other Rf values. Remember that the colours you obtain are not a reliable way to identify the pigments. Nevertheless the colours may well confirm a pigment if its Rf value is not very precise. FOR THE LAB TECHNICIAN

EQUIPMENT

CHEMICALS

Small beaker for boiling water

Solvent A: acetone - about 20 cm3Test tube rack

Pestle and mortar

Solvent B: 15:1 Petroleum ether:acetone

Centrifuge

about 20 cm3Centrifuge tubes

Small specimen tubeAluminium foil (to cover test-tube)

Chromatography jar with lidChromatography paper

Chromatography capillary dropping tubes

Forceps

Scissors

John Osborne

May 2015 PHOTOSYNTHETIC PIGMENTS AND CHROMATOGRAPHY