Paper Chromatography of Pigments in Chloroplasts

Objective

To use paper chromatography to observe the pigments in a solution of chloroplast (chlorophyll) extract.

Background Information

Chloroplasts contain several pigments. In terrestrial plants, chlorophylls a and b are usually found, along with carotenes and xanthophylls. In chromatography these pigments can be separated from one another based on their differing degrees of solubility in the chromatography solvent. A pigment that is very soluble will be moved higher on the chromatography paper.

Materials
large test tube
cork with paper clip hook
strip of chromatography or filter paper
ethyl alcohol
acetone
scissors
tweezers
green leaves such as carrot leaves (if available) or spinach (fresh or frozen)
clamp stand
capillary tube

Procedure

Part I: Leaf Pigment Preparation

1. Obtain a leaf from the desired plant and grind it using a mortar and pestle. A few grains of sand can be added to facilitate grinding. Add 8-10 drops of ethyl alcohol to extract the pigments from the leaf.

2. Continue grinding until you have a few drops of dark green liquid. It might be necessary to add a little more alcohol.

Part II Chromatogram Preparation:

3. Obtain a strip of chromatography paper which is 1/6 of a standard strip. Cut one end of the strip to form a taper.

4. Using a PENCIL, draw a line across the paper approximately 2 cm from the tapered tip. This is your baseline.

5. Using a capillary tube, apply a small dot of the pigment extract at the center of the line. Let dry and repeat four or five times. You should have a small, very dark spot of pigment. If the spot is not dark, apply more extract.

6. Pour the required amount of acetone into a large test tube so that the top of the solvent is below your baseline. Attach the hook to the other, non-tapered end of the paper. When the cork is in place, the tip of the paper should just touch the acetone. Hold your tube very still while proceeding.

7. Without moving or shaking the tube, attach the test tube to a clamp stand.

8. Allow the chromatogram to run until the solvent almost reaches the top of the paper strip.

9. Remove the paper and use a pencil to mark a line at the highest point the solvent reached. This is your solvent front.

10. Examine the chromatogram for the presence of different bands of color. Each color band is a different pigment. Chlorophyll a appears blue-green, chlorophyll b appears yellow-green, carotene appears bright yellow, and xanthophyll appears pale yellow-green. You may not see all of these bands.

11. Label your chromatogram and save it to attach to your lab report

Discussion Questions

1 What is the value of chromatography?

2 Which pigments are present in the smallest amounts in the leaf?

3 Which pigments are present in the greatest amount?

4 Many leaves change color in the fall. Explain. (Hint:

chlorophyll a and b are easily broken down in the cooler autumn temperatures.)

5 a) What is the role of chlorophyll a? b) What are the roles of carotene and xanthophyll?