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         Plant pigments include green chlorophylls, orange carotenes, yellow xanthophylls, and red and
blue anthocyanins. Plant pigments can be isolated by chromatography. The process of chromatography
separates compounds from one another because of their different solubilities in water or other solvents. In
paper chromatography, a piece of filter paper is used to separate and indicate the individual substances. The
substances appear as colored bands or colored spots at different points on the filter paper. The filter paper and
spots are called a chromatogram. Plant pigments function as absorbers of different wavelengths of lights
during photosynthesis.
         Green plants synthesize their own food from carbon dioxide and water. In the process of
photosynthesis, green plants produce food in the form of carbohydrates. Another product of photosynthesis is
oxygen. The energy for photosynthesis comes from the sun. Sunlight, a form of radiant energy, is absorbed by
the pigments in the leaves of green plants. The radiant energy is converted into chemical energy and stored in
the bonds of carbohydrates. The chemical energy can be used by the plants.
         The sun light needed for photosynthesis, as well as the light from artificial sources such as light bulbs,
is white light. White light is composed of all the different colors of the visible spectrum. By using a prism, a solid
triangular piece of glass, white light can be separated into its many wavelengths. The wavelengths become
visible as different colors. Not all wavelengths are absorbed and used by green plants fro photosynthesis.
Some wavelengths are reflected by the plant. For example, green light is reflected away from the plant. This
reflection is why plants containing chlorophyll appear green. The pigment chlorophyll absorbs and, therefore,
uses mostly red and blue light. Only the colors of light absorbed by a plant are used for photosynthesis.

The purpose of this investigation is to separate plant pigments. You will also learn how to construct and run a
paper chromatogram.

Filter paper                             Pencil                                    Test tube holder
Scissors                                 Spinach/Plant leaves                      Pipette
1 test tube                              Coin                                      70% isopropyl alcohol

  1. Cut the filter paper into an oblong strip wide enough to fit into the test tube without touching the sides of
     the test tube. The strip should be 5 cm longer than the length of the test tube. Cut one end into a point.
     Using a pencil, draw a light line across the strip about 2 cm from the tip of the point. Note: be sure your
     hands are clean and dry in order to prevent any contamination of the filter paper.
  2. Place a spinach leaf on the strip of filter paper. The leaf should cover the pencil line. Press down and
     roll the edge of the coin across the portion of the leaf directly on top of the pencil line. Move the leaf
     slightly and roll another layer of plant pigment directly on top of the first. Repeat this procedure three or
     four more times. When you are finished, there should be a thick band of plant pigment above the pencil
  3. Fold the filter paper, so it can hang over the top of the test tube without touching the bottom of the test
  CAUTION: Isopropyl alcohol is highly flammable and toxic if ingested. Do not have any open flames or
  heating devices in the room. Keep the container of isopropyl alcohol and test tube sealed at all times.
  4. Using a pipette carefully add the isopropyl alcohol to the test tube, until just the tip of the filter paper
     would be in contact with the alcohol.
  5. carefully insert the filter paper into the test tube. Only the pointed end of the strip should come in
     contact with the alcohol. Be careful not to splash the alcohol all over the strip.
  6. Let filter paper sit for 10 minutes. Observe the movement of the pigments up the filter paper strip. The
     pigments that are most soluble in the alcohol will move the farthest up the strip.
  7. Remove the chromatogram from the test tube. Allow the chromatogram to air dry. Carefully pour the
     alcohol down the sink and flush it with water. Rinse the test tube out with water.
Problem: How many different pigments does a spinach leaf have? What about a
leaf found outside?

Hypotheiss: Make an educated guess as to how many pigments are in a spinach
leaf .

Data And Observation:
Draw a colored picture of your group’s chromatogram. Label the pigments you
see: green chlorophylls, orange carotenes, yellow xanthophylls, and red and blue
anthocyanins. Be sure to use appropriate colors for the various pigments found in
your leaf.

Summary Questions (answer in complete sentences):

1. What was the significance of the pencil line?

2. What does the appearance of more than one band indicate?

3. Which pigment was the most soluble in alcohol? The least soluble? Support your
   answer with evidence.

4. What would cause a pigment not to move up the chromatogram?

5. How can you explain the difference shades of green found in various species of
   trees and other plants?

6. Why is it necessary for plants to have more than one pigment?

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