Various Leaves (Without Thick

Materials

Various leaves (without thick

cuticles)

Chromatography Vessel Pencil

Ruler Coin Chromatography paper strips

Chromatography solvent (90/10PetEt/Acetone)or (isopropyl alcohol)

Procedure

1. Obtain a strip of chromatography paper. Cut the bottom of the paper into a V shape.

2. Measure 2 cm vertically from the bottom of each strip and with a pencil, draw a horizontal line across the chromatography paper strip (this is the origin).

3. Using a Coin, extract the pigments from leaf cells as shown in the picture at right. Place a small section of leaf on top of the pencil line but do not cover the entire strip of chromatography paper with the leaf. Use the ribbed edge of the coin to crush

the cells. Be sure that the pigment line is on top of the pencil

line. You should repeat this procedure 8 to 10 times being sure to use a new portion of the leaf each time.

4. In the fume hood, pour chromatography solvent to a depth of 1 cm into a chromatography vessel.

5. Place the chromatography paper in the graduated cylinder, being careful not to submerge the pigment in the solvent. Place the paper so that only the tip touches the surface of the solvent. Cap the chromatography vessel immediately after placing your paper

6. Place your chromatography chamber in a location where you will not disturb it, and let your chromatogram develop for about 15 minutes, or until the solvent front is less than 1 cm from the top of the paper. DO NOT let the solvent move all the way to the top of the paper.

7. Once you have removed your chromatogram, immediately mark the solvent front with a pencil. Use the pencil to mark the outlines of the pigment bands (they will fade very fast). You will measure the distance each pigment travels from the origin to the bottom of the pigment band to see how far each pigment traveled.

8. Record your data. To interpret what colors each pigment represent, use the guide below:

Carotenes: yellow, yellow orange (Rf = 0.95 – 0.99)

Xanthophyll: yellow (Rf = 0.4)

Chlorophyll a:bright green to blue green (Rf = 0.2)

Chlorophyll b: yellow green to olive green (Rf = 0.1)

1. What factors are involved in the separation of pigments?

2. How does hydrogen bonding affect how pigments travel up the chromatography paper?

3. How would the Rfvalue for each pigment change if a different solvent system were used? Why?

4. How would the Rfvalue for each pigment change if your chromatography strip was twice as long?

What if it were half as long? Why? (CAREFUL THOUGHT REQUIRED!)

5. Which plants that you chose had the most accessory pigments? What is the role of these pigments in

photosynthesis?

6. Which pigments in your chromatogram were most polar? Were least polar?

7. THINK: Predict what you think a chromatogram for a plant with red leaves would look like. Explain

your prediction. Make a sketch of your predicted chromatogram alongside your explanation.

8. How would you expect your results to differ if the primary pigment color of the leaf was purple?