Pigments and the spectra of light

Why is a leaf green?

White light holds all the colours of the rainbow. A leaf looks green because its pigments take up the blue and red parts of the light and let much of the green part go. We see the part that is not absorbed.

A leaf has more than one pigment. Let us separate them.

Separating the pigments

You can do this with paper chromatography.

1. Crush some leaves in a solvent such as acetone, and filter. This gives a green extract.
2. Put a small spot of the extract on a line near the bottom of a strip of chromatography paper.
3. Stand the strip with its lower edge in a solvent, so that the spot is above the liquid.
4. The solvent climbs the paper and carries the pigments with it. Each pigment travels a different distance.

Four pigments are found.

A pigment that dissolves well in the solvent and clings less to the paper travels farther. The Rf value compares the two journeys.

Here $d_{\text{pigment}}$ is the distance moved by the pigment, and $d_{\text{solvent}}$ is the distance moved by the solvent front.

Rf is a pure number. It is always less than $1$. On the strip, the carotenoids travel the farthest and chlorophyll b the least.

Absorption and action spectra

An absorption spectrum is a graph of how much light a pigment absorbs at each wavelength. An action spectrum is a graph of how fast photosynthesis goes at each wavelength.

• Chlorophyll a absorbs best in the blue region, near $430\ \text{nm}$, and in the red region, near $662\ \text{nm}$.
• Chlorophyll b absorbs near $453\ \text{nm}$ and $642\ \text{nm}$.
• Carotenoids absorb in the blue part, roughly from $400$ to $520\ \text{nm}$.
• The action spectrum has peaks in the blue and red regions, like chlorophyll a. But it is higher than the chlorophyll a curve in the blue-green region.

Why? Chlorophyll a is the main pigment that traps light for photosynthesis. The other pigments, chlorophyll b, xanthophylls and carotenoids, are accessory pigments. They absorb light at wavelengths where chlorophyll a absorbs less, and they pass the energy on to chlorophyll a. They do two good things.

• They let a wider range of wavelengths be used for photosynthesis.
• They protect chlorophyll a from photo-oxidation, which is damage caused by too much light.

This also explains Engelmann's result. His bacteria gathered where the alga made the most oxygen, in the blue and red regions. That graph is an action spectrum.

You should find that at $450\ \text{nm}$ chlorophyll a and b both absorb strongly, and the carotenoids absorb too. At $550\ \text{nm}$, in the green, all three absorb weakly. So green light is used least, and it is the colour that leaves are not good at keeping.

Try it in Python

The program turns the distances on a chromatogram into Rf values and finds the pigment that travels the farthest.

You should see that the carotenoids have the highest Rf, $0.92$, and chlorophyll b the lowest, $0.45$. With the new distance, chlorophyll b passes chlorophyll a, and it is no longer the pigment that travels least.

Practise with new numbers

Common mistakes

• Mixing up the colours of the two chlorophylls. Chlorophyll a is bright or blue green. Chlorophyll b is yellow green.
• Calling every pigment a chlorophyll. Xanthophylls and carotenoids are not.
• Dividing Rf the wrong way. The pigment distance goes on top. Rf is always below $1$.
• Mixing up the two spectra. Absorption spectrum: light absorbed. Action spectrum: rate of photosynthesis.
• Thinking only chlorophyll a takes part. The accessory pigments absorb light too, and pass the energy to chlorophyll a.

Remember these

• Four pigments: chlorophyll a (blue green), chlorophyll b (yellow green), xanthophylls (yellow), carotenoids (yellow to yellow-orange).
• $R_f$ is the pigment distance divided by the solvent distance.
• Chlorophyll a absorbs in the blue and red. The action spectrum follows it.
• Accessory pigments widen the range of light used and protect chlorophyll a from photo-oxidation.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Name the four pigments found in a leaf extract.

2. What is the Rf value of a pigment that moved $3\ \text{cm}$ when the solvent moved $10\ \text{cm}$?

3. Which colours of light does chlorophyll a absorb best?

4. What is the difference between an absorption spectrum and an action spectrum?

5. Give two jobs of the accessory pigments.