PRACTICAL 2

PHOTOSYNTHESIS
Skill to be assessed: ORR
Extraction and identification of the photosynthetic pigments in a leaf

Instructions
1. Using a sharp pencil and a ruler draw a horizontal line 20 mm from one end of the strip of filter
paper provided. Handle the paper as little as possible as the oils from your fingers can affect the
movement of the solvent.
2. Place the leaf directly over the pencil line. Use a coin or a blunt pencil to rub across the leaf along
the line (green pigment should leak onto the paper). Remove the leaf and allow the pigment to dry.
Repeat using another part of the leaf or another leaf until a dark green line is produced.

Solvent front

Figure 1 Figure 2

3. Carefully pour the solvent into the boiling tube without getting any solvent on the sides of the tube
(use a dropping pipette to help if necessary). Pour the solvent to a depth of 15 mm.
4. Stopper the tube and leave it to sit for 3 minutes. Remove the stopper and slowly lower the
chromatography paper into the boiling tube (ensure that the extract line is not submerged into the
solvent).
5. Tightly stopper the tube and place in the test tube rack until the solvent rises to 5 mm below the top
of the chromatography paper or until the solvent front stops moving (Figure 2).
6. Remove the paper and immediately use a pencil to mark the position of the solvent front and the
leading edge of each pigment (colour). Place the paper to dry.
7. Affix the chromatogram to your report using the clear tape provided and label accordingly.
 8. You should be able to see at least three colours/pigment on the paper. Record your results in a
table. Include the colour of the pigment, the distance the solvent front travelled, the distance each
colour travelled, the calculated Rf value, the name of the pigment
9. Use the table below to identify each of the pigments in your sample.

Pigment Colour
β Carotene Orange-yellow
Phaeophytin Yellow-grey
Xanthophylls Yellow-brown
Violaxanthin yellow
Lutein yellow
Chlorophyll a Blue-green
Chlorophyll b Green

10. Measure the distance from the original line to the “leading edge” of each colour/pigment. Record
these results in your table.
11. Use the results to calculate the Rf value for each pigment in your extract by using the formula
R=a/b where a is the distance moved by the pigment and b is the distance moved by the solvent.
12. Compare your results to the theoretical values presented below.

Rf Values:
β-carotene - 0.99
Phaeophytin 0.83
Xanthophylls 0.71
chlorophyll a - 0.30
chlorophyll b - 0.13
violaxanthin - 0.40
lutein - 0.68
NB β carotene, violaxanthin, and lutein are carotenoids

Write up
Write up the experiment using the standard reporting format.
Secure your chromatograph into your report using transparent tape and label it.
In your discussion: Define relevant terms
Describe the theory related to the use of chromatography to separate pigments
Why are two solvents used in the extraction process?
Identify each of the pigments visible on your chromatograph
Compare the calculated Rf values to the theoretical values and suggest possible
reasons for any discrepancies seen
Discuss the importance of the knowledge of the Rf values of different pigments.