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• Paper Chromatography Resources

Project Summary

Difficulty	1
Time required	Very Short (a day or less)
Prerequisites	None
Material Availability	Readily available
Cost	Very Low (under $20)
Safety	No issues

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Sponsored by a generous grant from Monsanto Fund

Abstract

Objective

In this project, you will uncover the changing colors of fall leaves by separating plant pigments with paper chromatography.

Introduction

Everyone loves the beautiful colors of fall, but where do they come from? What happens to make the leaves turn from green to yellow, orange, and red? Part of the answer is that tree leaves have pigments or colorful molecules, inside them. These pigments make food for the tree during warm, sunny months through the process of photosynthesis. Photosynthesis is when the energy of the sun is absorbed by the plant pigments and turned into food energy, which keeps the leaves green. But winter brings colder, shorter days and without as much sunlight, the tree shuts down the food-making leaves for the winter. So what you see in the changing autumn colors is the gradual change of these pigments.

• Chlorophyll (green) - Chlorophyll is necessary for photosynthesis, which is the chemical reaction that enables plants to use sunlight to manufacture sugars for their food. Trees in the temperate zones store these sugars for their winter dormant period and so do not need to actively make food during the winter. Tree leaves must constantly make chlorophyll when they need it for photosynthesis, so once they stop, the green goes away.
  
• Xanthophylls (yellow) and Carotenoids (orange) - These pigments are also used for photosynthesis, and are there all summer long, you just don't see them because they are usually masked by the strong color of the green chlorophyll. These pigments do not break down as fast as chlorophyll, so they hang around longer during the fall. These pigments are also commonly found in such things as corn, carrots, and daffodils, as well as rutabagas, buttercups, and bananas.
  
• Anthocyanins (red) -The anthocyanins are different, because they begin to appear after the plant stops making chlorophyll. So these pigments are not there during the summer, and they appear latest in the fall succession of color. Anthocyanins are very intense color molecules, and they also give color to such familiar things as cranberries, red apples, concord grapes, blueberries, cherries, strawberries, and plums. They are water soluble and appear in the watery liquid of leaf cells.

When you look at a leaf, you see the result of all of these colors mixing together. But if you separate the pigment molecules, you can see each individual pigment color on its own. One method for separating molecules is called paper chromatography. In this method, a solvent is used to dissolve the molecules of interest. Then the solution containing the dissolved molecules is passed through a strip of strong paper. The fibers of the paper trap the molecules as the solvent carries them through the paper. Larger molecules get trapped by the paper fibers first, and smaller molecules can travel farther along through the paper fibers. With this method, a colorful mixture of pigment molecules can be separated by size.

In this project you will use paper chromatography to separate the colored pigment molecules from fall leaves. By collecting leaves at different stages of turning, you will be able to capture all of the colors of fall. Will you be able to uncover the hidden colors, and tell the full fall story?

Terms, Concepts and Questions to Start Background Research

To do this type of experiment you should know what the following terms mean. Have an adult help you search the Internet or take you to your local library to find out more.

• Pigment
• Photosynthesis
• Chlorophyll
• Xanthophyll
• Carotenoid
• Anthocyanin
• Paper chromatography
• Solvent

• What makes a leaf look so colorful?
• Can I extract the pigment from a leaf?
• Why do leaves turn fall colors?
• Are plant pigments involved in leaves turning fall colors?

Bibliography

• This project was adapted from "How To Do Paper Chromatography With Leaves" at About.com: Chemistry:
  Helmenstine, A.M. (2007). How To Do Paper Chromatography With Leaves. About.com: Chemistry. The New York Times Company. Retrieved December 6, 2007 from http://chemistry.about.com/cs/howtos/ht/paperchroma.htm
• The Science Buddies project deals with HOW leaves turn colors, but if you want to know WHY, then ask Bill Nye:
  Nye, B. (2007). Ask Bill Nye. MSN Encarta. Retrieved December 6, 2007 from http://encarta.msn.com/weblink_701879545/Encarta_Column_Falling_Leaves.html
• You can find out more from the SUNY College of Environmental Science and Forestry:
  Palm Jr., C.E. (2007). Why Leaves Change Color. SUNY College of Environmental Science and Forestry (ESF) E-Center. Retrieved December 6, 2007 from http://www.esf.edu/pubprog/brochure/leaves/leaves.htm
• Then find out more than you ever thought you could know at Harvard:
  O'Keefe, J., and Lee, D. (2004). Autumn Foliage Color. The Fisher Museum, Harvard Forest, Faculty of Arts and Sciences of Harvard University; and Florida International University, Department of Biological Sciences. Retrieved December 6, 2007 from http://harvardforest.fas.harvard.edu/research/leaves/autumn_leaves.html

Materials and Equipment

• Leaves at different stages of turning colors (30-40, 10 per color group)
• Scissors
• Good, strong glasses (3-4)
• Rubbing alcohol (isopropyl alcohol)
• Wooden spoon
• Fork
• Very small bowls for evaporating and concentrating extract
• Strong, white, heavy-weight, ultra-absorbent paper towels
• Ruler
• Pencil
• Toothpicks
• Plate
• Tall mason jars (3-4)
• Clothes pins (3-4)

Experimental Procedure

1. Go on a nice walk with an adult and collect some leaves from different stages of color change during fall. It is best for all of your leaves to come from the same tree, so look for a tree with a variety of leaves at different stages. Here are some leaves I collected from a tree in my neighborhood:

   

2. Separate and group the leaves into color groups, with ten good leaves in each group (unless you are using a tree with small leaves, like aspen or birch, then you should use a higher number of leaves). Try to form groups from colors that are as different as possible. For example, I made a green group, a yellow group, and a red group. In each group, I chose leaves in the deepest colors possible:

   

3. Cut the leaves into small pieces with your scissors and put each group into the bottom of a good, strong glass:

   

4. Add 1 Tbsp. of rubbing alcohol to each glass.
5. Using the blunt end of a wooden spoon, macerate (soften) the chopped leaves by squashing them into the rubbing alcohol at the bottom of the cup.
6. As you squish the leaves, you will notice that the rubbing alcohol will start to turn the color of the leaves. This is called extraction, and the rubbing alcohol is called the solvent.
7. Continue until the liquid turns a deep shade of the color of the leaves, about 5 minutes per glass.
8. Let the macerated suspensions sit for 30 minutes in a dark, room-temperature place to allow the color molecules to fully extract.
9. Using a fork, lift out the bits and pieces of leaf material and set them aside. Take care to remove any liquid by gently pressing the leafy bits against the glass before you remove them. You should be left with a dark suspension of leafy color in rubbing alcohol at the bottom of your cup.
10. Pour each extract into a very small bowl (I used tealight candle holders), and leave in a dark room-temperature place to evaporate off some of the rubbing alcohol. This will concentrate your extract, and make the color even more intense. In the meantime, you should prepare your paper towel strips.

    

11. Cut up a good, thick piece of paper towel into long, 1-inch thick strips. Make sure they are long enough to reach the bottom of the mason jars and still drape over the top. You will need a few strips (2-3) for each color group.
12. Measure up from the bottom of each strip and using a pencil, gently draw a line that is 1 inch from the bottom of the strip. This will be where you apply the color extract to the paper towel strip for your separation.
13. When your color extracts have concentrated, they will be gooey when stirred by a toothpick. Stir each color thoroughly with a toothpick to blend and loosen any bits of dried up pigment from the side of the bowl. Be sure to use a different toothpick for each separate color so you don't mix them!
14. Using the toothpick, "paint" some of the colored extract onto the pencil line on your paper towel strip. Some plant pigments can stain, so you should do this on a plate so that the color won't seep through and stain your work surface. Try to apply the extract as smoothly and evenly as you can along the line. Repeat with 2-3 more strips, using the same color extract, so that you have duplicates for each color pigment.
15. Repeat with the other colored extracts. Be sure to use a new toothpick for each different color! Allow the strips to dry.
16. While the strips are drying, pour a small amount of rubbing alcohol into each mason jar, just enough to cover the bottom. About 2 Tbsp. will usually do the trick. You will need one jar for each color extract you have.
17. When the strips are dry, carefully lower the pigmented end of the strip down into the jar until the bottom edge of the strip just touches the alcohol. Drape the top of the strip over the mouth of the jar and secure it with a clothes pin. Hang the other strips similarly. You can put strips of the same color extract together in the same jar, just be sure to keep them from touching each other and drape the tops separately over the mouth of the jar, each strip secured with a different clothes pin. However, different colored extracts should be in different jars.
18. Make sure that the strips do not come into contact with the sides of the jar, except for at the top where they are secured, so that they are all hanging freely. Leave the jars undisturbed.

    

19. Set the glasses aside for about 30 minutes, and watch as the colors separate along the length of the strip. Stop when one of the colors reaches the top. As soon as one of the colors reaches the top of a strip, remove ALL of the strips and allow them to dry.
20. Compare the colors found in the different strips. What happened to the colors? Did the different groups of leaves have unique colors, or shared colors, or both? Is each color found in the same place along each strip, or in different places? Are the colors in the same order, or in a different order of separation along the strip?

Variations

• If you find a really good tree, you can include all of the intermediate stages of leaf turning in your experiment. An especially good source of a wide variety of colors are aspen trees!
• There are many other natural sources of color, and you can use the same rubbing alcohol extraction technique to see them. How do the color molecules of different plant sources compare? Some ideas to try are: red cabbage, blueberries, cranberries, carrots, beets, spinach, flowers, and practically any other intensely colored plant you can get a hold of.
• This experiment uses paper towels to separate the colors, but if you want a more precise and advanced way of separating the colors you can use laboratory filter paper or thin layer chromatography. Here are some Science Buddies resources and additional projects for paper chromatography:
  • Paper Chromatography Resources
  • Paper Chromatography: Basic Version
  • Paper Chromatography: Advanced Version 1
  • Paper Chromatography: Advanced Version 2
  • Candy Chromatography: What Makes Those Colors?

• For more science project ideas in this area of science, see Plant Biology Project Ideas.

Credits

Sara Agee, Ph.D., Science Buddies

Last edit date: 2008-02-20 22:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Plant Biology.

Soil and Plant Scientist With a growing world population, making sure that there is enough food for everyone is critical. Plant scientists work to ensure that agricultural practices result in an abundance of nutritious food in a sustainable and environmentally friendly manner.

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