C1.3.4 Separation and identification of photosynthetic pigments by chromatography Notes

Chromatography Separates and Identifies Photosynthetic Pigments by Their Solubility

1. Plants contain a variety of photosynthetic pigments that absorb light energy.
2. These pigments can be separated and identified using chromatography, a technique that relies on differences in solubility and affinity for a stationary phase.
3. These can then be identified by their colour and Rf values.
4. This process is particularly useful for studying the pigments involved in photosynthesis, such as chlorophyll a, chlorophyll b, and carotenoids.

There Are Two Common Types of Chromatography

1. Thin-Layer Chromatography (TLC)

1. A TLC plate coated with a thin layer of silica gel or alumina is prepared.
2. The sample (e.g. a plant extract) is applied as a small spot near the bottom of the plate.
3. The plate is placed vertically in a solvent inside a TLC chamber.
4. The solvent (mobile phase) moves up the plate by capillary action, carrying the compounds with it.
5. Compounds separate based on their polarity: less polar substances travel farther than more polar ones because they interact less with the polar stationary phase.
6. The relative distance each compound travels can be measured using its Rf value:

$$R_f=\frac{\text{distance travelled by compound}}{\text{distance travelled by solvent front}}$$

2. Paper Chromatography

1. A strip of chromatography paper (usually cellulose) is used as the stationary phase.
2. The plant extract is spotted near the bottom of the paper.
3. The bottom edge of the paper is placed in a solvent (e.g. water or a mixture like propanone and petroleum ether).
4. As the solvent travels up the paper by capillary action, it carries the pigments at different rates depending on their solubility and interactions with the paper.

How Chromatography Works

Chromatography involves two main components:

1. Stationary Phase: A solid or liquid surface that remains fixed.
2. Mobile Phase: A solvent that moves through the stationary phase, carrying the pigments with it.
3. As the solvent travels up the paper or TLC strip, it dissolves the pigments.
4. Thse then move at different rates depending on their solubility in the solvent and their affinity for the stationary phase.

Steps to Perform Chromatography

1. Extract Pigments: Tear a leaf into small pieces and grind it with sand and acetone to dissolve the pigments.
2. Prepare the Chromatography Strip: Use a brush to apply a concentrated spot of pigment extract near one end of the strip.
3. Develop the Chromatogram: Place the strip in a container with a solvent, ensuring the pigment spot is above the solvent level. Allow the solvent to move up the strip, separating the pigments.
4. Analyze the Results: Remove the strip when the solvent is near the top and mark the solvent front. Identify the separated pigments by their colors and calculate their $R_f$ values.

Calculating Rf Values

1. Once the solvent has traveled up the paper, the paper is removed and dried.
2. The pigment bands are measured, and the Rf value (Retention Factor) for each pigment can be calculated using the formula:

$$R_f = \frac{\text{Distance moved by pigment}}{\text{Distance moved by solvent}}$$

Interpreting the Results

1. Chlorophyll a: The primary pigment involved in photosynthesis, usually appears as a dark green band.
2. Chlorophyll b: A secondary pigment that assists chlorophyll a in capturing light, typically appears as a yellow-green band.
3. Carotenoids: Yellow and orange pigments that protect the plant from excess light, appearing as yellow or orange bands.