Chromatography of Photosynthetic Pigments

Objective:

To use chromatography to separate the photosynthetic pigments found in leaves and determine their Rf values for identification.

Equipment List

• Filter paper
• Leaf sample
• Distilled water
• Pestle and mortar
• Pencil
• Ruler
• Capillary tube
• Chromatography solvent
• Acetone

Method

1. Draw a baseline:

• Using a pencil, draw a straight line approximately 1 cm above the bottom of the filter paper.
• Do not use a pen, as ink could interfere with the results.

2. Extract pigments:

• Cut a leaf section and grind it in a mortar with 20 drops of acetone to release the pigments.

3. Apply the sample:

• Use a capillary tube to extract pigment and blot it onto the pencil line on the filter paper.

4. Set up the chromatography:

• Suspend the paper in the chromatography solvent.
• Ensure the solvent level does not rise above the pencil line.
• Allow the solvent to run up the paper until it nears the top.

5. Mark the solvent front:

• Remove the paper and mark the solvent's highest point with a pencil.

6. Identify pigment spots:

• Mark the different pigment spots visible above the baseline.

7. Calculate Rf values:

• Measure the distance travelled by each pigment (centre of the spot) and the distance travelled by the solvent.
• Use the formula:

$$Rf = \frac{\text{Distance travelled by solute}}{\text{Distance travelled by solvent}}$$

Risk Assessment

Hazard	Risk	Safety Precaution	In Emergency	Risk Level
Biohazard	Allergies, soil bacteria contamination	Wash hands after use	Seek assistance	Low
Chromatography solvent	Flammable; irritant to eyes and skin	Wear eye protection; use in well-ventilated area	Wash skin/eyes with cold water; put out fires	Low
Acetone	Flammable; may cause dizziness if inhaled	Avoid contact; wear eye protection	Seek medical assistance; wash skin/eyes	Low