11. Investigate the presence of different chloroplast pigments using chromatography.

Question 1

What is the purpose of chromotography?

Answer 1

Chromotography is used to separate out different components in a sample.

In this experiment, the photosynthetic pigments of a plant sample are separated into bands of colour by paper chromotography.

Question 2

What does the rate of movement of individual pigments depend on?

Answer 2

• Solubility
• Mass
• Affinity to the paper

Question 3

Method:

Answer 3

1. Draw a straight line in PENCIL, approximately 1cm above the bottom of the filter paper being used.
2. Cut a section of leaf and place inn a mortar. Add 20 drops of propanone and use the pestle to grind up the leaf sample and release the pigments.
3. Use the capillary tube to extract some of the pigment and blot it onto the centre of the pencil line drawn. Allow to dry and then blot again.
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 for approximately 10 mins/ until the solvent has run up the paper near to the top.
5. Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to. The pigment should have separated out and there should be different spots on the paper at different heights above the pencil line.
6. Calculate the Rf value for each spot. Always measure to the CENTRE of each spot.

Question 4

How is Rf value calculated?

Answer 4

Rf= distance travelled by solute/ distance travelled by solvent.

Question 5

Risks:

Answer 5

Biohazard: Allergies, soil bacteria, contamination

• wash hands after use
• seek assistance

Chromatography solvent: flammable, causes irritation to eyes and skin; harmful by inhalation

• avoid contact with solvent
• wear goggles
• keep solvent in fume cupboard
• make sure room is well ventilated
• keep away form naked flame
• wash from skin and eyes using cold water, seek medical assistance

Acetone: flammable, causes irritation to eyes, inhalation may lead to dizziness and drowsiness

• avoid contact
• wear eye protection
• make sure room is well ventilated
• keep away from naked flame
• wash from skin and eyes using cold water, seek medical assistance

Question 6

Analysis:

Answer 6

• Rf values should be compared to the Rf known values in database to identify pigment.
• When looking at databases, ensure they are for PAPER CHROMATOGRAPHY and USE THE SAME SOLVENT as these variables will make results differ.

Question 7

What factors can affect the rate of movement?

Answer 7

Affinity: pigments have different affinities to the chromatography paper; those with lower affinities will travel further up the paper.

Solubility: pigments that are more soluble travel faster up the paper and will end up closer to the top at the solvent front.

Question 8

Which pigments will have higher Rf values?

Answer 8

pigments that travel further up the paper.