Photosynthetic Pigments

What is chromatography?

Chromatography is a laboratory technique used by biologists and biochemists. It allows for the separation of a mixture, which is achieved by passing it in a solution or suspension in which the mixture's components can move at different rates. It is often used to separate a product from any unreacted solution.

There are two primary types of chromatography: thin-layer chromatography (sometimes shortened to TLC) and column chromatography.

The function of both methods are the same, but each uses different equipment to separate a mixture into its various components. You are most likely to use thin layer chromatography during your studies; however, at university-level or a professional laboratory, you will make use of both. The differences between the two are illustrated in Figure 1 below.

How do you investigate photosynthesis using chromatography?

For your A-level course, you will be asked to use chromatography to investigate the pigments isolated from the leaves of different plants. You might, for example, be asked to compare the leaves of shade-tolerant vs shade-intolerant plants or leaves that are different colours from the same plant. To do this, you will need to carry out the experiment as detailed below.

Equipment:

• Leaf samples

• Filter paper

• Distilled water

• Pestle and mortar

• Ruler

• Capillary tube

• Chromatography solvent

• Acetones

• Pipette

• Pencil

Method:

1. Using the pencil, draw a straight line around 1 cm above the bottom of the filter paper. It is important to use a pencil for this step, not a pen, as the ink will get into your solution and mess up your results.

2. Take a section of the leaf, and place it into the mortar. Add 20 drops of acetone, and grind up the leaves with the acetone using the pestle. This will release the pigments in the leaf.

3. Using the capillary tube, extract the pigment and drop it onto the centre of the pencil line on the filter paper.

4. Suspend the filter paper with the pigment in the solvent; however, make sure that the liquid level does not lie above the pencil line.

5. Leave the paper until the solvent has risen close to the top. Make sure that the solvent does not soak through the paper.

6. Remove the paper from the solvent, and draw a pencil line marking where the solvent has moved up. You should notice that pigment has separated into different compounds, which should all be placed at different heights above the first pencil line.

Fig. 1 - Example of Thin Layer Chromatography

Although this won't look exactly like your experiment, it will be set up similarly.

How do you calculate an Rf value?

Once you have collected your filter paper with the different pigments separated, you will calculate the Rf value of each spot. Rf stands for retention factor, and this formula is used to help identify the pigments present on your chromatography sample.

The equation for the Rf value is as follows:

$Rf=\frac{dis\tan cetravelledbythecompound}{dis\tan cetravelledbythesolventfront}$

When you have your set of Rf values, you can then compare them to the known Rf values (provided by a teacher) to identify which pigments are present in your leaf. If you need to find your own database, make sure that they are specifically for paper chromatography. Use the same solvent as you since these variables will affect the distance travelled by certain pigments!

Which photosynthetic pigments are in chloroplasts?

You are likely to find the following pigments in a leaf during chromatography:

• Chlorophyll a and Chlorophyll b: These pigments are green in colour. Chlorophyll a, the more abundant of the two pigments, absorbs blue and red light and reflects green light. Chlorophyll b is the less abundant of the two; however it can absorb a wider wavelength of light energy than chlorophyll a. Chlorophyll b absorbs blue and red light and reflects green light, like chlorophyll a; however, the green light reflected is a different shade of green. At a molecular level, chlorophyll has a porphyrin ring, which helps absorb light energy more efficiently.

• Carotenoids: These pigments reflect orange, yellow and red light waves. In the chloroplast, carotenoid pigments cluster next to chlorophyll molecules to efficiently hand off any absorbed photons.

• Xanthophylls: like carotenoids, xanthophylls pass along light energy to chlorophyll a and act as antioxidants. Xanthophylls reflect yellow light waves.

• Anthocyanin: a pigment that absorbs blue and green light. Anthocyanin molecules are stored in the vacuole of the plant cell.

Why are photosynthetic pigments important in photosynthesis?

Photosynthetic pigments are crucial to photosynthesis as they absorb photons (waves of light) at certain wavelengths. As you will see in the diagram below, different pigments can absorb and reflect different wavelengths of light, which allows a plant to get as much energy as possible from a single light source.

After absorbing photons, the electrons contained in the pigments become excited and increase their energy level. Accessory pigments such as carotenoids, anthocyanins, and xanthophylls can then pass on this energy to primary pigments like chlorophyll a, which can oxidize and donate an electron to the electron transport chain in the light-dependent reaction.