Separation of photosynthetic pigments by paper chromatography.

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Separation of photosynthetic pigments by paper chromatography

Introduction

Chlorophyll is in fact only one pigment in a group of closely related pigments commonly found in photosynthesising plants called photosynthetic pigments. This can be demonstrated by extracting the pigments from leaves with acetone and separating them by means of paper chromatography. With a bit of luck five pigments can be identified: chlorophyll a (blue-green), chlorophyll b (yellow-green), xanthophylls (yellow), carotene (orange) and phaeophytin (grey, it is a breakdown product of chlorophyll).

Absorptive paper with a concentrated spot of leaf extract is used in this experiment. When dipping in a suitable solvent, the pigments ascend the absorptive paper at different rates because they have different solubilities in the solvent. In this way they become separated from one another and can be identified by their different colours and positions.

Requirements:

  • Large test tube (24 * 150 mm);
  • Stopper to fit test tube;
  • Pin;
  • A small glass tube to transfer pigment solution;
  • Chromatography paper or filter paper;
  • Rack of test tube;
  • Pigment solution;
  • Solvent (5 cm3).

Procedure:

  • A strip of absorptive paper has been prepared. It has such a length that it almost reaches the bottom of a large test tube and such a width that the edges do not the sides of the tube;
  • Draw a pencil line across the strip of paper 30 mm from one end. The paper has been folded at the other end through 90 degrees and attached to the stopper using a pin. Take care not to let the lower end of the paper touch the bottom of the tube or edges touch the sides;
  • Remove the paper from the boiling tube and use the small glass tube provided, place a drop of the pigment solution at the centre of the pencil line. Dry the spot under the heat from a hairdryer or let it dry naturally. Place a second small drop on the first. Repeat this process for about 15 minutes and a small area of concentrated pigment has been set up. N.B. the smaller and more concentrated the spot is, the better;
  • While preparing the pigment spot, pour a mixture of propanone and petroleum ether into the boiling tube to a depth of no more than 15 mm. Seal the tube with a stopper for about 10 minutes so that the inside of the boiling becomes saturated with vapour;
  • Suspend the strip of paper in the boiling tube. The lower end of the paper should dip into the solvent but the pigment spot should not be immersed;
  • The solvent will ascend rapidly carrying the pigments and in about 10 minutes the pigments can be separated. When the solvent is about 20 mm from the top of the paper remove the strip, rule a pencil line to mark the solvent front and dry the paper;
  • Detectable pigments can be identified by their colours and the Bf values;
  • Measure the distance from the pencil line to the leading edge of each clear pigment and work out the Rf value for each one using this formulae:
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Rf = a / b

Where a = distance moved by substance from its original position;

      b = distance moved by solvent from the same position.      

Table 1

Colours and Rf values of the pigments found in a typical leaf

(Rf values for propane/ ether mixture).

Result and calculation:

b = 8.4 cm

For the first pigment: a = 8.1 cm           Rf = 8.1 / 8.4 = 0.965

For the second pigment: a = 7.2 cm        Rf = 7.2 / 8.4 = 0.857

For the third pigment: a = 5.9 cm           ...

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