7. Chromatography of Photosynthetic Pigments

Question 1

What is the purpose of chromatography?

Answer 1

To separate different components in a sample.

Question 2

State the factors affecting the rate of migration of different pigments.

Answer 2

Solubility
Mass
Affinity to the paper

Question 3

What is the formula of the RF value?

Answer 3

Distance moved by pigment / Distance moved by solvent

Question 4

What is the purpose of finding the RF value of a pigment?

Answer 4

Experimental RF value can be compared to a standard value in a database to identify the pigment.

The standard value should be measured
using the same paper and solvent.

Question 5

Outline the procedure of using chromatography to separate photosynthetic pigments.

Answer 5

1. Draw a horizontal pencil line 1cm above the bottom of the filter paper. (Do not use a pen as the ink will obscure the results)
2. Add some acetone and use the mortar and pestle to grind up the leaf sample
   and release the pigments.
3. Use a capillary tube to transfer the pigment onto the pencil line.
4. Suspend the paper in the solvent so that the level of the liquid does not lie above the pencil line and leave the paper until the solvent has run up the paper to near the top.
5. Remove the paper from the solvent and draw a pencil line marking where the
   solvent moved up to.
6. Calculate the Rf value for each spot. (always measure to the centre of each spot).

Question 6

State the hazards and precautions in this practical.

Answer 6

Solvents are irritant and flammable.

Keep away from naked flames, wear eye protection and avoid contact with skin.

Leaf extract may be a biohazard. Wash hands after use.

Question 7

How does affinity affect the rate of mobility?

Answer 7

Pigments have different affinities to the chromatography paper; those with lower​​ affinities will ​travel further​​ up the paper

Question 8

How does solubility affect the rate of mobility?

Answer 8

Pigments that are more soluble ​travel faster​​ up the paper and will end up closer to the top ​​at the ​solvent front​​.
Pigments that travel further up the paper will have a ​higher Rf value​​.