Leaf chromatography is paper chromatography using leaves. Paper chromatography is a separation technique. When applied to leaves, it separates the pigment molecules mostly according to their size. The main pigment molecule in green leaves is chlorophyll, which performs photosynthesis in the plant. Other pigments also occur, such as carotenoids and anthocyanins. When leaves change color in the fall, the amount and type of pigment molecules changes. Leaf chromatography is a fun science project that lets you see these different pigments.
Leaf Chromatography Materials
You only need a few simple materials for the leaf chromatography project:
- Rubbing alcohol (isopropyl alcohol)
- Coffee filters or thick paper towels
- Hot water
- Small clear jars or glasses with lids (or plastic wrap to cover the jars)
- Shallow pan
- Kitchen utensils
You can use any leaves for this project. A single plant leaf contains several pigment molecules, but for the most colors, use a variety of leaves. Or, collect several of each kind of leaf and compare them to each other. Good choices are colorful autumn leaves or chopped spinach.
Perform Paper Chromatography on Leaves
The key steps are breaking open the cells in leaves and extracting the pigment molecule and then separating the pigment using the alcohol and paper.
- Finely chop 2-3 leaves or several small leaves. If available, use a blender to break open the plant cells. The pigment molecules are in the chloroplasts of the cells, which are organelles encased within the plant cell walls. The more you break up the leave, the more pigment you’ll collect.
- Add enough alcohol to just cover the leaves.
- If you have more samples of leaves, repeat this process.
- Cover the container of leaves and alcohol and set it in a shallow pan filled with enough hot tap water to surround and heat the container. You don’t want water getting into your container of leaves.
- Replace the hot water with fresh water as it cools. Swirl the container of leaves around from time to time to aid the pigment extraction into the alcohol. The extraction is ready when the alcohol is deeply colored. The darker its color, the brighter the resulting chromatogram.
- Cut a long strip of coffee filter or sturdy paper towel for each chromatography jar. Paper with an open mesh (like a paper towel) works quickly, but paper with a denser mesh (like a coffee filter) is slower but gives a better pigment separation.
- Place a strip of paper into jar, with one end in the leaf and alcohol mixture and the other end extending upward and out of the jar.
- The alcohol moves via capillary action and evaporation, pulling the pigment molecules along with it. Ultimately, you get bands of color, each containing different pigments. After 30 to 90 minutes (or whenever you achieve pigment separation), remove the paper strips and let them dry.
How Leaf Chromatography Works
Paper chromatography separates pigments in leaf cells on the basis of three criteria:
- Molecule size
Solubility is a measure of how well a pigment molecule dissolves in the solvent. In this project, the solvent is alcohol. Crushing the leaves breaks open cells so pigments interact with alcohol. Only molecules that are soluble in alcohol migrate with it up the paper.
Assuming a pigment is soluble, the biggest factor in how far it travels up the paper is particle size. Smaller molecules travel further up the paper than larger molecules. Small molecules fit between fibers in the paper more easily than big ones. So, they take a more direct path through the paper and get further in less time. Large molecules slowly work their way through the paper. In the beginning, not much space separates large and small molecules. The paper needs to be long enough that the different-sized molecules have enough time to separate enough to tell them apart.
Paper consists of cellulose, a polysaccharide found in wood, cotton, and other plants. Cellulose is a polar molecule. Polar molecules stick to cellulose and don’t travel very far in paper chromatography. Nonpolar molecules aren’t attracted to cellulose, so they travel further.
Of course, none of this matters if the solvent doesn’t move through the paper. Alcohol moves through paper via capillary action. The adhesive force between the liquid and the paper is greater than the cohesive force of the solvent molecules. So, the alcohol moves, carrying more alcohol and the pigment molecules along with it.
Interpreting the Chromatogram
- The smallest pigment molecules are the ones that traveled the greatest distance. The largest molecules are the ones that traveled the least distance.
- If you compare chromatograms from different jars, you can identify common pigments in their leaves. All things being equal, the lines made by the pigments should be the same distance from the origin as each other. But, usually conditions are not exactly the same, so you compare colors of lines and whether they traveled a short or long distance.
- Try identifying the pigments responsible for the colors.
There are three broad classes of plant pigments: porphyrins, carotenoids, and flavonoids. The main porphyrins are chlorophyll molecules. There are actually multiple forms of chlorophyll, but you can recognize them because they are green. Carotenoids include carotene (yellow or orange), lycopene (orange or red), and xanthophyll (yellow). Flavonoids include flavone and flavonol (both yellow) and anthocyanin (red, purple, or even blue).
- Collect leaves from a single tree or species of tree as they change color in the fall. Compare chromatograms from different colors of leaves. Are the same pigments always present in the leaves? Some plants produce the same pigments, just in differing amounts. Other plants start producing different pigments as the seasons change.
- Compare the pigments in leaves of different kinds of trees.
- Separate leaves according to color and perform leaf chromatography on the different sets. See if you can tell the color of leaves just by looking at the relative amount of different pigments.
- The solvent you use affects the pigments you see. Repeat the experiment using acetone (nail polish remover) instead of alcohol.
- Block, Richard J.; Durrum, Emmett L.; Zweig, Gunter (1955). A Manual of Paper Chromatography and Paper Electrophoresis. Elsevier. ISBN 978-1-4832-7680-9.
- Ettre, L.S.; Zlatkis, A. (eds.) (2011). 75 Years of Chromatography: A Historical Dialogue. Elsevier. ISBN 978-0-08-085817-3.
- Gross, J. (1991). Pigments in Vegetables: Chlorophylls and Carotenoids. Van Nostrand Reinhold. ISBN 978-0442006570.
- Haslam, Edwin (2007). “Vegetable tannins – Lessons of a phytochemical lifetime.” Phytochemistry. 68 (22–24): 2713–21. doi:10.1016/j.phytochem.2007.09.009
- McMurry, J. (2011). Organic chemistry With Biological Applications (2nd ed.). Belmont, CA: Brooks/Cole. ISBN 9780495391470.