Photosynthesis SPA

Topic: Photosynthesis
Practical 3E: Chromatography

Activity: To conduct a chromatographic investigation of the pigments present in a leaf extract.

Skill A – Planning

You are required to plan, but not carry out, a chromatographic investigation of the pigments present in a leaf extract.

A leaf extract can be made by mixing finely ground leaf with extraction solution.

A thin line of leaf extract is painted across the silica gel strip. The strip is a sheet of plastic coated with a thin layer of absorbent material, silica gel. The silica gel strip should be placed in the chromatography solvent to conduct the experiment.

Your planning must be based on the assumption that you have been provided with the following equipment and materials which you must use:
• fresh spinach leaves
• sharp knife
• mortar and pestle
• extraction solution
• chromatography solvent
• pure photosynthetic pigments e.g. chlorophyll, carotenoid and xanthophylls
• silica gel strip
• boiling tube with a rubber bung
• ruler
• brush
• a variety of different sized beakers, measuring cylinders, syringes and pipettes for measuring volumes

Your plan should: have a clear and helpful structure to include
• an explanation of theory to support your practical procedure
• a description of the method used including the scientific reasoning behind the method
• an explanation of the dependent and independent variables involved
• relevant, clearly labeled diagrams
• how you will record your results and ensure they are as accurate and reliable as possible
• proposed layout of results tables and graphs with clear headings and labels
• the correct use of technical and scientific terms









1. An explanation of theory to support your practical procedure
a. Reaction: a variety of pigments are involved in photosynthesis, i.e. chlorophyll a, chlorophyll b, carotenoids.

Chromatography is a means of separating one type of molecule from another. A strip of absorptive silica gel strip carrying a concentrated line of the leaf extract is dipped into a suitable solvent. The solvent moves up the silica gel strip sweeping the pigments with it. The pigments have different solubilities in the solvent and will therefore travel at different speeds and become separated from each other.

b. Relate to method of measurement: The identification of the molecule is made on the basis of the distance travelled by the substance in relation to the distance moved by the solvent. Each molecule can then be refferred to by its Rf value expressed as:
Rf = Distance travelled by a compound
Distance travelled by solvent front

2. A description of the method used including the scientific reasoning behind the method
a. Preparation of material:
• chloroplast extraction, use of knife/pestle and mortar/sharp sands
• use of extraction solution
• filter to remove the debris
b. Experiment set up: method of spotting leaf extract on silica gel strip, e.g. paint a thin line across the strip and allow this to dry; place the strip in a beaker filled with 10 cm3 of solvent; the spot should be above the solvent; cover the beaker;
c. Other variables to be controlled for replicates: mass of leaves used;

d. Standard: carry chromatography using pure photosynthetic pigments, one strip for each pigment;

e. Taking measurement: Measure the distance traveled by the pigments and solvent; calculate Rf value (formula); compare Rf with standard to identify the pigments;

3. an explanation of the dependent and independent variables involved relevant, clearly labeled diagrams
N.A.

4. Relevant, clearly labeled diagrams

5. How you will record your results and ensure they are as accurate and reliable as possible
a. At least 3 replicates

6. Proposed layout of results tables and graphs with clear headings and labels
Rf value of pure photosynthetic pigments
Pigment Rf

A
B
C
D
E








Table showing the Rf value of the unknown pigments

Pigment Distance traveled, d/cm Rf Identity of pigment
D1 D 2 D 3

1
2
3
4
5
7. The correct use of technical and scientific terms