How to Do Paper Chromatography With Leaves

Simple paper chromatography setup
Simple paper chromatography setup.

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You can use paper chromatography to see the different pigments that produce the colors in leaves. Most plants contain several pigment molecules, so experiment with many species of leaves to see the wide range of colors. This is a simple science project that takes about 2 hours.

Key Takeaway: Leaf Paper Chromatography

  • Chromatography is a chemical purification method that separates colored substances. In paper chromatography, pigments may be separated based on the different size of the molecules.
  • Everyone knows leaves contain chlorophyll, which is green, but plants actually contain a wide range of other pigment molecules.
  • For paper chromatography, plant cells are broken open to release their pigment molecules. A solution of plant matter and alcohol is placed at the bottom of a piece of paper. Alcohol moves up the paper, taking pigment molecules with it. It's easier for smaller molecules to move through the fibers in paper, so they travel fastest and move the furthest up the paper. Larger molecules are slower and don't travel as far up the paper.

What You Need

You only need a few simple materials for this project. While you can perform it using only one type of leaf (e.g., chopped spinach), you can experience the greatest range of pigment colors by collecting several types of leaves.

  • Leaves
  • Small Jars with Lids
  • Rubbing Alcohol
  • Coffee Filters
  • Hot Water
  • Shallow Pan
  • Kitchen Utensils

Instructions

  1. Take 2-3 large leaves (or the equivalent with smaller leaves), tear them into tiny pieces, and place them into small jars with lids.
  2. Add enough alcohol to just cover the leaves.
  3. Loosely cover the jars and set them into a shallow pan containing an inch or so of hot tap water.
  4. Let the jars sit in the hot water for at least a half hour. Replace the hot water as it cools and swirl the jars from time to time.
  5. The jars are "done" when the alcohol has picked up color from the leaves. The darker the color, the brighter the chromatogram will be.
  6. Cut or tear a long strip of coffee filter paper for each jar.
  7. Place one strip of paper into each jar, with one end in the alcohol and the other outside of the jar.
  8. As the alcohol evaporates, it will pull the pigment up the paper, separating pigments according to size (largest will move the shortest distance).
  9. After 30-90 minutes (or until the desired separation is obtained), remove the strips of paper and allow them to dry.
  10. Can you identify which pigments are present? Does the season in which the leaves are picked affect their colors?

    Tips for Success

    1. Try using frozen chopped spinach leaves.
    2. Experiment with other types of paper.
    3. You can substitute other alcohols for the rubbing alcohol, such as ethyl alcohol or methyl alcohol.
    4. If your chromatogram is pale, next time use more leaves and/or smaller pieces to yield more pigment. If you have a blender available, you can use it to finely chop the leaves.

    How Leaf Paper Chromatography Works

    Pigment molecules, such as chlorophyll and anthocyanins, are contained within plant leaves. Chlorophyll is found in organelles called chloroplasts. The plant cells need to be torn open to expose their pigment molecules.

    The macerated leaves are placed in a small amount of alcohol, which acts as a solvent. Hot water helps soften the plant matter, making it easier to extract the pigments into the alcohol.

    The end of a piece of paper is placed in the solution of alcohol, water, and pigment. The other end stands straight up. Gravity pulls on the molecules, while alcohol travels up the paper via capillary action, pulling pigment molecules upward with it. The choice of paper is important because if the fiber mesh is too dense (like printer paper), few of the pigment molecules will be small enough to navigate the maze of cellulose fibers to travel upward. If the mesh is too open (like a paper towel), then all of the pigment molecules easily travel up the paper and it's difficult to separate them.

    Also, some pigment might be more soluble in water than in alcohol. If a molecule is highly soluble in alcohol, it travels through the paper (the mobile phase). An insoluble molecule might remain in the liquid.

    The technique is used to test purity of samples, where a pure solution should only produce a single band. It is also used to purify and isolate fractions. After the chromatogram has developed, the different bands may be cut apart and the pigments recovered.

    Sources

    • Block, Richard J.; Durrum, Emmett L.; Zweig, Gunter (1955). A Manual of Paper Chromatography and Paper Electrophoresis. Elsevier. ISBN 978-1-4832-7680-9.
    • Haslam, Edwin (2007). "Vegetable tannins – Lessons of a phytochemical lifetime." Phytochemistry. 68 (22–24): 2713–21. doi:10.1016/j.phytochem.2007.09.009