11 - Chloroplast pigments

What is chromatography used for?

To separate out different components in a sample:

• the photosynthetic pigments of a plant sample are separated into bands of colour by paper chromatography

• rates of migration of individual pigments will depend on their solubility, mass & affinity to the paper 

What equipment is needed for this practical?

• Chromatography paper & leaf sample

• Distilled water

• Pestle & mortar

• Boiling tube with bung

• Boiling tube rack

• Measuring cylinder & spatula

• Pencil & ruler

• Capillary tube

• Chromatography solvent

• Propanone 

What is the method for this practical?

1. Draw a straight line in pencil 1cm above the bottom of the filter paper (don’t used pen as ink will obscure the results)

2. Cut a section of leaf & place in a mortar, then add 20 drops of propanone & use the pestle to grind up the leaf sample to release the pigments

3. Use a capillary tube to extract some of the pigment & blot it onto the centre of the pencil line drawn (allow to dry & then blot again)

4. Suspend the paper in the solvent so the liquid is below the pencil line & leave the paper for 10mins or until the solvent has run up the paper to near the top

5. Remove the paper from the solvent & draw a pencil line marking where the solvent finished

6. Calculate the Rf value for each spot (always measure to the centre of each spot)

What is the risk, safety precaution & in emergency for the hazard: biohazard

Risk:

• allergies

• soil bacteria & contamination

Safety precaution:

• wash hands after use

In emergency:

• seek assistance

What is the risk, safety precaution & in emergency for the hazard: chromatography solvent

Risk:

• flammable

• causes irritation to the eyes & skin

• harmful by inhalation

Safety precaution:

• avoid contact with solvent 

• wear eye protection

• keep solvent in a fume cupboard

• make sure the room is well ventilated

• keep away from naked flames

In emergency:

• wash from the skin & eyes with cold water

• put out fires

• seek medical assistance

What is the risk, safety precaution & in emergency for the hazard: acetone

Risk:

• flammable 

• causes irritation to the eyes

• inhalation may lead to dizziness & drowsiness

Safety precaution:

• avoid contact 

• wear eye protection

• ensure the room is well ventilated

• keep away from naked flames

In emergency:

• wash from the skin & eyes with cold water

• put out fires

• seek medical assistance

What is the conclusion from this practical?

Pigments that travel further up the paper will have a higher Rf value

What are the factors that affect the rate of mobility?

• Affinity: pigments with a lower affinity for the paper (stationary phase) travel further up the paper, as they are less strongly attracted to it

• Solubility: pigments that are more soluble in the solvent (mobile phase) travel further up the paper, as they spend more time dissolved in the moving solvent