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Why are most plants green? Why do the leaves of some trees turn from green to orange or yellow in autumn? This experiment will help shed some light on this subject. Although most plant leaves appear green to our eyes, several pigments of different color are usually present in the chloroplasts. In the flowering plants (angiosperms), chlorophylls a and b provide the green color and absorb the light energy needed for photosynthesis. However, other accessory pigments, such as yellow xanthophylls and orange carotenes are also present in the chloroplasts and collect additional light energy for photosynthesis. The purpose of this experiment is to identify plant pigments by separation and isolation of the pigments using thin layer paper chromatography.

Paper chromatography is a useful technique in the separation and identification of different plant pigments. In this technique, the mixture containing the pigments to be separated is first applied as a spot or a line to the paper about 1.5 cm from the bottom edge of the paper. The paper is then placed in a container with the tip of the paper touching the solvent. Solvent is absorbed by the chromatographic paper and moved up the paper by capillary action. As the solvent crosses the area containing plant pigment extract, the pigments dissolve in and move with the solvent. The solvent carries the dissolved pigments as it moves up the paper. The pigments are carried along at different rates because they are not equally soluble. Therefore, the less soluble pigments will move slower up the paper than the more soluble pigments. This is known as developing a chromatogram.

The distance traveled by a particular compound can be used to identify the compound. The ratio of the distance traveled by a compound to that of the solvent front is known as the R_f value; unknown compounds may be identified by comparing their R_f's to the R_f's of known standards.

Day before the lab - Tear fresh spinach leaves and place in a glass container, cover with acetone to extract lipid soluble pigments. Cover the glass container to keep the acetone from evaporating. Set aside until day of the lab experiment.

Day of the lab – Pour acetone and spinach solution through filter paper. Place pigment solution in a corked bottle. Follow directions listed in the lab worksheet on the next page.

CAUTION: Acetone is flammable (even the amount found in nail polish remover), keep it away from sparks or open flame and wear eye protection. Lab may be done under a vent hood, but beakers containing acetone must remain covered and students should avoid direct inhalation of fumes.

1. What reference numbers (R_f) did you calculate for chlorophyll a and chlorophyll b?

Answer. Numbers will vary according to solvent used, pigment concentration, experiment results, and calculations. However, results should be similar to the following typical Rf values:

2. With what you have discovered about pigments, what conclusions can you
make regarding the changing color of leaves in autumn?

Answer. Accessory pigments, such as carotenes and xanthophylls are present in leaves that appear green and are responsible for the color change of leaves associated with many species in the autumn. For more information, refer to the module section on leaf abscission.

Answer. Accessory pigments absorb additional portions of the light spectrum which increases the light energy available for photosynthesis.

Answer. Pigments, such as carotene and chlorophyll a are more soluble than chlorophyll b and xanthophylls, and move farther up the chromatogram.

Additional applications: A comparison could be made of the pigments found in plants that grow in the sun and those that grow in the shade to determine if there is any difference in the concentration of pigments.

Tomkins, S. P. and Miller, M. B. (1994). A rapid extraction and fast separation of leaf pigments using thin layer chromatography. School Science review75 (273), 69 - 72.

1. Each lab group (or individual if not working in groups) will need
one strip of filter paper, approximately 6 inches long and 1 inch wide, cut
to a point at the bottom and a glass beaker that will serve as a chromatography development chamber, a large rubber band (able to stretch lengthwise around the chamber from the mouth to the bottom of the vessel), enough acetone (nail polish) to completely cover the bottom of the chamber.

2. Place the acetone in the beaker and stretch a rubber band length-wise around each vessel. The rubber band will be the mechanism for hanging the chromatography strips.

3. Make a pencil mark on the chromatography strip, in the center, directly above the point of the strip, 1.5 cm from the tip of the paper. Using a capillary tube, or a small paint brush, apply pigment solution to the strip. This is done by touching capillary tube or the brush which has been dipped in the pigment, to the pencil mark. Make an application then wave the paper gently to dry it a little (or use a hair dryer) before the next application. Be patient, you will need 12 to 15 applications.

4. After you have applied the pigment solution, suspend the paper in the beaker. Make sure that the paper does not touch the side of the beaker. You can attach it to the rubber band with a paper clip, or simply fold over a portion of the end and it should hang in place. The tip of the strip should just touch the solvent. Cover the beaker with aluminum foil and place under a vent hood to minimize fumes and protect the experiment.

5. Wait 20 to 30 minutes for the chromatogram to develop. Remove the
chromatogram. Be careful to handle the paper using the paper clip or by the top fold.

1. Mark with a pencil (NOT a pen) where the solvent stopped as it moved up the chromatogram. This is called the solvent front. Also mark in pencil where each pigment stopped moving up the chromatogram.

2. Assign a reference number for each pigment band on the chromatogram. You should see greens, yellow, and yellow-orange. Using a metric ruler, measure the distance each band and the solvent moved from the initial line of pigment and record data in Table 1.

Band Number	Distance (mm)	Band Color
1
2
3
4
5

3. The relationship of the distance moved by a pigment to the distance moved by the solvent is a constant called R_f. It can be calculated for each of the four pigments using the formula.

___________________________	= R_f for carotene (yellow to yellow -orange)
___________________________	= R_f for xanthophyll (yellow)
___________________________	= R_f for Chlorophyll a (bright green to blue green)
___________________________	= R_f for Chlorophyll b (yellow green to olive green)

1. What reference numbers (R_f) did you calculate for chlorophyll a and chlorophyll b?

2. With what you have discovered about pigments, what conclusions can you
make regarding the changing color of leaves in autumn?

4. Why do some Why do some pigments move farther up the chromatogram than others?