Plant Pigment Chromatography Exploration

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. 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 fibers in the paper through the formation of intermolecular bonds, such as hydrogen bonds. Since the solvent in non-polar and the paper is polar, molecules that are non-polar tend to move further since they are more dissolved in the solvent and polar molecules don't move as far because they are more attracted to the paper.

Beta carotene is carried the furthest because it is highly soluble in the solvent and because it forms no hydrogen bonds with the chromatography paper fibers. Xanthophyll contains oxygen and does not travel quite as far with the solvent because it is less soluble than beta carotene and forms some hydrogen bonds with the paper. Chlorophylls are bound more tightly to the paper than the other two, so they travel the shortest distance.

DRAWING OF SET UP:

PROCEDURE:
Obtain and wear goggles! Caution: The solvent in this experiment is flammable and poisonous. Be sure there are no open flames in the lab during this experiment. Avoid inhaling fumes. Wear goggles at all times. Notify your teacher immediately if an accident occurs.

1. Obtain a glass vial with 2 mL of solvent in the bottom.
2. Cut the chromatography paper so that it is long enough to reach the solvent. Cut one end of the paper into a point.
3. Draw a pencil line 2.0 cm above the pointed end of the paper.
4. Use the coin to extract the pigments from the spinach leaf. Place a small section of the leaf on top of the pencil line. Use the ribbed edge of the coin to push the plant cells into the chromatography paper. Repeat the procedure 10 times making sure to use a different part of the leaf each time.
5. Place the chromatography paper in the cylinder so the pointed end just touches the solvent. Make sure the pigment is not in the solvent.
6. Stopper the cylinder and wait until the solvent is approximately 1 cm from the top of the paper. Remove the chromatography paper and mark the solvent front before it evaporates.
7. Allow the paper to dry. Mark the bottom of each pigment band. Measure the distance each pigment moved from the starting line to the bottom of the pigment band. Record the distance that each of the pigments and the solvent moved, in millimeters.
8. Identify each of the bands and label them on the chromatography paper.
    - beta carotene: yellow to yellow orange
    -xanthophyll: yellow
    -chlorophyll a: bright green to blue green
    -chlorophyll b: yellow green to olive green
9. Staple the chromatogram to the front of your lab sheet.
10. Discard the solvent as directed by your teacher.

DATA TABLES: PART A

Table 1: Distance Moved by Pigment Band (mm)

Distance solvent front moved ________mm

ANALYSIS: PART A

1.The ratio of the distance moved by a pigment to the distance moved by the solvent is a constant, Rf. Each type of molecule has its own Rf  value. Rf =distance traveled by pigment/distance traveled by solvent. Calculate the Rf  for each band.

3. Would you expect the Rf value to be different with a different solvent?

5. What type of chlorophyll does the photosystem contain? What are the roles of the other pigments?