Year 11 Pre-Diploma Biology

PHOTOSYNTHETIC PIGMENTS AND

CHROMATOGRAPHY

Chlorophyll
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:
A
A.1
A.2
A.3
A.4
A.5
B
B.1
B.2
B.3
B.4
B.5
B.6
B.7

Extracting the chlorophyll pigments from fresh leaves

Dip the leaves in boiling water to kill the cells and make the membranes permeable.
Finely cut up a small handful of leaves, excluding the stalks and petioles, and put them into a
mortar.
Add about 20 cm3 of propanone (acetone) and grind up the leaf matter to make a concentrated
extract.
Carefully 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.
Keep this chlorophyll extract in the dark, in ice, until required. (The pigments are very
unstable away from the chloroplasts and degrade quickly in light.)
Separation of the pigments by paper chromatography
Set 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.
Cut 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.
Draw 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.
Use 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.
Very 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.
Watch 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.
When 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.

C
Identifying 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
Phaeophytin
Xanthophyll
Chlorophyll a
Chlorophyll b

orange
yellow-grey
yellow-brown/orange
blue-green
yellow-green

0.95
0.83
0.71
0.65
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
Small beaker for boiling water
Test tube rack
Pestle and mortar
Centrifuge
Centrifuge tubes
Small specimen tube
Aluminium foil (to cover test-tube)
Chromatography jar with lid
Chromatography paper
Chromatography capillary dropping tubes
Forceps
Scissors

John Osborne
May 2015

CHEMICALS
Solvent A: acetone - about 20 cm3
Solvent B: 15:1 Petroleum ether:acetone
about 20 cm3