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.