Earlier we assumed that the yellow carotenoids are present but masked in green leaves, whereas the red anthocyanins are only made in the fall. Let’s test that assumption.

First, we’ll grind up three leaves: a green leaf, a yellow leaf, and a red leaf. Next, we’ll extract the pigments from the leaves with alcohol. Alcohol is a good choice of solvent because it dissolves molecules with low water-solubility such as chlorophyll, as well as water-soluble molecules like the anthocyanins.

We’ll spot the alcohol extracts near the bottom of a sheet of filter paper and allow them to dry. Then we’ll place the filter paper in a chromatography chamber with a mixture of alcohol and water, and cover it.

Because leaf pigments have different chemical structures and physical properties, the technique of chromatography can be used to separate them from a mixture. Paper chromatography separates compounds on the basis of their relative attraction to two phases—a mobile phase and a stationary phase. In our experiment, the filter paper is the stationary phase and the alcohol-water mixture is the mobile phase.

Test your understanding of chromatography by dragging your choice to fill in the blank. Then click Submit.

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The greater the solubility in the mobile phase and the weaker the attraction for the paper, the greater the distance the pigment will migrate.

Once the solvent has reached the top of the filter paper, we’ll remove the paper from the chromatography jar. The ratio of the distance moved by a pigment to the distance moved by the solvent is a constant called Rf. The Rf value can be used to identify the separated plant pigments. Approximate Rf values for this experiment are listed in the table. Use them to identify the separated pigments.

Identify the separated plant pigments by dragging the pigment name next to the corresponding migration distance at the side of the paper. Click Submit when you’re finished.

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The water-soluble anthocyanins have greater solubility in the alcohol-water mobile phase and migrate farther than the chlorophylls, which have a greater attraction for the stationary paper phase and migrate a shorter distance.

Based on our results, what can we conclude about the presence of accessory pigments in leaves of different colors? Select your answer, and then click Submit.

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The equal intensities of the carotene spots (yellow) tells us that the carotene pigments are present at equal levels in the green, yellow, and red leaves. The decrease in chlorophyll intensity in the extracts from the yellow and red leaves tells us that the chlorophyll levels are lower in these leaves than in the green leaf. The decrease in chlorophyll causes the leaves to lose their green color. A spot corresponding to anthocyanin is observed only in the red leaf extract. This indicates that unlike carotenes, the anthocyanins are not unmasked when the chlorophyll levels decrease. Rather, they are made only in the fall.

The results from our chromatography experiment support our assumptions about pigment levels and leaf color.

Copyright 2006 The Regents of the University of California and Monterey Institute for Technology and Education