Use thin-layer chromatography to discover the variety of pigments that play a role in photosynthesis and give leaves their colour.
Looking out over a lush green valley or forest, it is fascinating to see the array of different shades. Leaves range from light to dark and even speckled. The colours are determined by the presence of different pigments, many of which are responsible for one of the most interesting and important metabolic reactions in living organisms: photosynthesis.
Photosynthetic pigments are located in the chloroplasts of the leaf. They capture energy from the visible light spectrum, which they use to synthesise carbohydrates from inorganic matter. There are many types of photosynthetic pigments, but the two main groups are chlorophylls and carotenoids (which are further split into two classes: carotenes and xanthophylls). Each type absorbs a different wavelength, so that together they capture more light.
Chlorophylls are the pigments primarily responsible for photosynthesis. They absorb red and blue light, and reflect green light, which is what gives leaves their green colour. Carotenoids, on the other hand, reflect yellow, orange and red – the colour of leaves during autumn. During this time of year, chlorophyll breaks down so the carotenoid pigments become visible.
Carotenoids assist with photosynthesis by absorbing wavelengths of light that chlorophylls cannot absorb. They transfer energy to chlorophyll molecules and also help to protect the leaf from excess light – they absorb surplus light energy and dissipate it as heat to prevent it from damaging the leaf.
Other non-photosynthetic pigments, such as anthocyanins or other flavonoids, determine the colour of flowers, so their absorption spectra vary. The function of these pigments is to attract insects or birds for pollination.
This article presents a simple laboratory experiment to understand leaf pigments. Students use thin-layer chromatography to separate the various pigments that are present in two different leaf extracts. They identify each pigment and determine whether the two extracts have any pigments in common. The experiment is suitable for students aged 11–16 and takes 1–2 hours to complete.
Note that we used leaves from Epipremnum aureum (commonly known as devil’s ivy) and Ficus benjamina (commonly known as weeping fig), but any species could be used for the leaf extracts. You might also like to carry out the experiment using a brightly coloured flower, such as those in the Petunia genus, and also a yellow or orange leaf.
For the thin-layer chromatography, we use a combined mobile phase of hexane, acetone and trichloromethane (3:1:1) as it provides the best separation result. However, it requires part of the activity to be carried out inside a fume hood by the teacher. This mobile phase separates the pigments most clearly, but you could adapt the activity to use mobile phases of hexane or ethanol alone, which the students can carry out themselves. Both hexane and ethanol successfully separate the pigments, but the distinction between each pigment is not as clear as when the combined solvent is used.
A lab coat, gloves and eye protection should be worn. The solvents used in this experiment are flammable, so they must not be used near flames. The combined solvent (hexane, acetone and trichloromethane) must only be used inside a fume hood due to the volatility, smell and health risks associated with it.
The following steps should be carried out by the students:
The following steps must be carried out by the teacher:
The following steps should be carried out by the students:
Record your results in a table. Compare these to the values in table 1: were your answers correct?
The different pigments in a leaf extract are separated based on their affinities for the stationary phase (the silica on the thin-layer chromatography plate – a polar substance) and the mobile phase (the solvent – a nonpolar substance). Compounds with a high affinity for the solvent (i.e. nonpolar compounds) will move much further than compounds with a high affinity for silica (i.e. polar compounds).
In our example (see figure 2), both leaf extracts contained four pigments. Pigment 4 moved a shorter distance than pigment 1, indicating that pigment 4 is more polar and pigment 1 is less polar. By looking at the chemical structures of different pigments and the polar and nonpolar groups, students can try to identify the pigments in each of the leaf extracts.
They will need to know that, of the functional groups present in the pigments in figure 1, alcohol groups are the most polar, ester and ether groups the least polar, and aldehyde and ketone groups are in between. From this, we can deduce that carotenes are the least polar pigments (no polar groups), and xanthophylls are the most polar (two alcohol groups, one at each end of the molecule). Therefore, pigments 1 and 2 are likely to be carotenes, and pigment 4 is likely to be a xanthophyll. Pigment 3 is likely to be chlorophyll, since it is more polar than carotenes but less polar than xanthophylls. You can observe the characteristic green colour from chlorophyll on the chromatogram.
Now look at the Rf values, which range between 0 and 1, with 0 being a pigment that does not move at all, and 1 indicating a pigment that moves the same distance as the solvent. The Rf value varies depending on the solvent used, but the general order of the pigments (from the highest to the lowest Rf value) usually remains the same, because the nonpolar compounds move further than the polar compounds. Rf values for various pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are shown in table 1.
After the experiment, you can ask your students some of the following questions to gauge their understanding of plant pigments and thin-layer chromatography.