1. Bio 103 - Introduction to Biology
Ch. 7: Photosynthesis: Using Light to Make Food
Chromatography
Background: Paper chromatography is a useful technique for separating and identifying pigments and other
molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by
capillary action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction
of solvent molecules to one another. As the solvent moves up the paper, it carries along any substances
dissolved in it, in this case pigments. The pigments are carried along at different rates because they are not
equally soluble in the solvent and because they are attracted, to different degrees, to the cellulose in the paper
through the formation of hydrogen bonds.
Beta-carotene, the most abundant carotene in plants, is carried along near the solvent front because it is very
soluble in the solvent being used and because it contains no atoms that form hydrogen bonds with cellulose.
Another pigment, xanthophyll. differs from carotene in that it contains oxygen. Xanthophyll occurs further
from the solvent front because it is less soluble in the solvent and has been slowed down by hydrogen bonding
to the cellulose. Chlorophylls contain oxygen and nitrogen and are bound more tightly to the paper than are
the other pigments.
A large number of different chromatographic systems are used to separate various molecular components of
cells. They differ in the type of support (paper in this ease) and in the type of solvent system used. In most
cases special stains are needed in order to visualize the molecules that have been separated.
Chlorophyll a is the primary photosynthetic pigment in all plants. A molecule of chlorophyll a is located at the
reaction center of photosystems. Other chlorophyll a molecules, chlorophyll b, and the carotenoids (i.e.,
carotenes and xanthophylls) capture light energy and transfer it to the chlorophyll a at the reaction center.
Carotenoids also protect the photosynthetic system from the damaging effects at bright sunlight.
Objectives:
• understand the principles of chromatography
Materials:
• Leaves (Most plants contain several pigment molecules, so experiment with different leaves to see the
wide range of pigments.)
• Baby Food Jars with Lids
• Rubbing Alcohol
• Coffee Filters (the thicker the better)
• Hot Water
• Shallow Pan
• Kitchen Utensils
Procedure:
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.
1
success = preparation + execution

2. Analysis:
1. What two factors are involved in the separation of the pigments?
2. Explain the significance of the pigments with regards to photosynthesis, especially the light reactions.
Tips:
• Try using frozen chopped spinach leaves.
• Experiment with other types of paper.
• You can substitute other alcohols for the rubbing alcohol, such as ethyl alcohol or methyl alcohol.
• If your chromatogram is pale, next time use more leaves and/or smaller pieces to yield more pigment.
2
success = preparation + execution