Leaf Chromatography
You can bring science alive for students with these lessons on leaf pigments.
By Lynsey Peterson
By autumn, I have taught my Biology class many of the underlying cellular processes of organisms. Now, a wonderful coincidence occurs, we begin discussing photosynthesis just as the leaves are turning color. I use this as a ‘teachable moment’, and take my classes outside.
We conduct a leaf hunt to find the variety of shapes and colors of leaves present in our schoolyard forest. The students and I love to go outside and enjoy the fleeting beauty of autumn. Then, we return inside to investigate the plant pigments found in the leaves.
My students know that plants contain chlorophyll, a green pigment. It is fun to explain to them that chlorophyll hides the presence of other pigments in the leaf. Sunlight destroys the chlorophyll over time, but the plant continues to manufacture it throughout the growing season. In autumn, deciduous plants stop making chlorophyll as the days grow shorter and the plant prepares for the dormancy of winter. As chlorophyll breaks down, the carotenoids show through. Carotenoids are pigments that range in color from red to orange and yellow. They include carotenes (orange), xanthophylls (yellow), and anthocyanin (red).
The leaves that we collect belong to a variety of species and show an assortment of colors. We use the leaves for a photosynthesis lab. In addition to viewing the stomata of the leaves, we also use paper chromatography to separate and view the pigments present. Chromatography means "color writing." Different pigments have different densities and move up the paper at different rates. Students may have completed a paper chromatography lesson previously with inks or food dyes.
The supplies used in paper chromatography are relatively simple. Filter paper or coffee filters are cut into inch wide strips a few inches long. Using a coin, students rub the leaf onto the paper until a thick line of pigment is visible. Alternately, you can use a mortar and pestle to mash the leaf and deposit small drops of the pigmented solution onto the paper. Then, students tape the paper onto a pencil and suspend it over a beaker that contains a small amount of isopropyl alcohol. Make sure that the students do not submerge the pigment line as it will dissolve in the alcohol. After the experiment is set up, I have the students work on viewing stomata or some other assignment, as it takes a few minutes for the solution to travel up the paper. After as much as 20 or 30 minutes, students should see that the alcohol has moved up the paper, taking the pigments with it. The more dense pigments will be lower on the chromatogram and the lighter ones will be higher. The colors and positions of the pigments will allow students to identify chlorophyll a (yellow-green), chlorophyll b (blue-green), and the carotenoids. The distances that the pigments travel will allow students to calculate the retention factor (Rf) of the pigment, the speed at which it moves over the paper compared with the speed of the solvent, with Rf = Distance moved by the pigment / Distance moved by the solvent.
After you try this and the following lessons, your students will be able to explain to their parents why leaves change color in autumn!
Leaf Chromatography:
Students investigate the separation of mixtures using plant pigments. Using everyday items, they demonstrate the separation of pigments in plants through the process of chromatography.
Students perform a paper chromatography separation of pigment mixture and analyze its separate parts by determining the Rf (retention factor). Using leaf chromatography, students show that the Rf is a constant.
Chromatography of Photosynthetic Pigments
Students explore the technique of chromatography to separate compounds in a mixture. In small groups, they separate by paper chromatography and identify the major and accessory photosynthetic pigments.
Students identify and define chlorophyll as the pigment in plants that captures sunlight energy and is used to drive photosynthesis. Then, they conduct an experiment in which they use paper chromatography to separate the many pigments from one another. Finally, students compare chlorophyll extracted from the uncovered half of the leaf with chlorophyll extracted from the covered half.
