8.1.1 Required Practical 7

Equipment:

Filter paper

Leaves from different plants (e.g. shade-tolerant, shade-intolerant)

Distilled water

Chromatography solvent (e.g. propanone or acetone)

Pestle and mortar

Capillary tube

Pencil and ruler

Beakers and glass rods

Risk Assessment:

Hazard	Risk	Safety Precaution	In Emergency	Risk Level
Chromatography solvent	Flammable, harmful by inhalation, causes skin and eye irritation	Work in a fume cupboard, wear protective clothing, avoid open flames	Wash from skin with water, seek medical assistance	Low
Acetone	Flammable, inhalation may cause dizziness, harmful to eyes	Use in a well-ventilated area, avoid skin contact, wear eye protection	Wash with cold water, seek assistance if symptoms persist	Low

Method:

1. Draw a light pencil line 1 cm above the bottom of a strip of filter paper. Ensure the line is straight, as this will serve as the baseline for spotting pigments.
2. Grind a leaf sample from a chosen plant using a pestle and mortar, adding approximately 20 drops of acetone to extract the pigments.
3. Use a capillary tube to apply a small dot of the pigment extract onto the pencil line. Allow it to dry and repeat several times to concentrate the spot.
4. Suspend the filter paper in a beaker containing chromatography solvent, ensuring the solvent level is below the pencil line. Cover the beaker and allow the solvent to rise up the paper.
5. Once the solvent has moved near the top, remove the paper and immediately mark the solvent front with a pencil. Allow the paper to dry.

6. Observe and mark the different pigment bands that have separated.
7. Calculate the Rf value for each pigment spot using the formula:

$$Rf = \frac{\text{Distance travelled by solute (pigment)}}{\text{Distance travelled by solvent front}}$$

Factors that affect the rate of mobility

1. Affinity- pigments have different affinities to the chromatography paper; those with lower affinities will travel further up the paper.
2. Solubility- pigments that are more soluble

Conclusion:

• Compare the Rf values of the pigments to known values in a database to identify the specific pigments present in the leaf samples.
• Factors such as affinity to the paper and solubility in the solvent affect the movement of pigments. Pigments that are more soluble in the solvent or have less affinity for the paper will move further up, leading to a higher Rf value.