Plant Pigment and Photosynthesis Lab

(keep this page in notebook)

Background information
In this laboratory, you will separate plant pigments using paper
chromatography. Paper chromatography is a useful technique for separating and
identifying pigments and other molecules from cell extracts that contain a complex
mixture of molecules. A liquid solvent such as ethanol is used to separate the pigment
along the paper. The solvent moves up the paper by capillary action, which results
from the attraction of solvent molecules to the paper (adhesion), and the attraction of the
solvent molecules to one another (cohesion). As the solvent moves up the paper, it
carries along any substances dissolved in it. Pigments are carried along at different
rates for two reasons: (1) because they are not equally soluble in the solvent and (2)
because they are attracted, to different degrees, to the fibers of the paper through the
formation of intermolecular bonds, such as hydrogen bonds. See the example below
separating the pigments found in black ink.

Solvent front
Cyan pigment

Magenta pigment
Solvent front

Yellow pigment

Solvent level

In some solvents, beta carotene, the most abundant carotene pigment in plants,
is carried along near the solvent front because it is very soluble in the solvent being
used and because it does not form hydrogen bonds with the cellulose (fibers) in the
paper. Another pigment Xanthophyll differs from carotene in that it does not contain
oxygen. Xanthophyll is less soluble in the solvent and forms hydrogen bonds to the
cellulose in the paper. Chlorophylls contain oxygen and nitrogen and are bound more
tightly to the paper that the other pigments. Chlorophyll a is the primary
photosynthetic pigment in plants. Chlorophyll b is also common in green plants.

Materials
 Chromatography paper  Stopper
 Dropper pipette  Pencil
 Rubbing alcohol  Spinach leaf
 Test tube  Penny
Katy and Procedure
Brian
Per 1
DAY ONE
1. Obtain a strip of chromatography paper. Draw a line in PENCIL 1.5 cm from the
bottom of the paper like the figure at left. At the top end, write you group
members’ first names and your period, also in pencil.
2. Use a penny to extract the pigments from the spinach leaf cells by firmly rubbing
the ribbed edge of the coin across the leaf right on top of the pencil line. Do not
overload the line, a small amount should be fine.
3. Place 8 droppers full (not drops, droppers full) of rubbing alcohol into the test
tube. It should extend roughly 1 cm from the bottom. Place the test tube into the
rack at your table.
4. Carefully place the chromatography paper into the test tube. Do not allow the
pigment rubbings to go below the level of the solvent. If it is submerged or
solvent splashes over the line, you must start over.
5. Leave the paper in the solvent over night. The next day you will finish the lab.
6. Answer numbers 1- 8 in the post lab questions.

DAY TWO
7. Remove the paper from the solvent. Pour the solvent down the sink, rinse and
place upside down on the rack to dry.
8. Mark the solvent front and the bottom of each pigment line (different colors,
there should be four). Measure the distance the solvent and each pigment
migrated from the bottom of the pigment origin (original pencil line). Record the
distances traveled in Table 1.
9. Rf value is a ratio of how far the pigment travels to how far the solvent travels. It
is a number between 0-1 that communicates the pigments’ solubility and polarity.
It is figured by the equation below:

Rf = distance traveled by pigment

distance traveled by solvent

Record all the Rf values in Table 1.

10. Answer questions numbers 9 – 13 in the post lab questions.

11. Staple the chromatography paper to the front of the lab write up with all four
members in your group.

Name: __________________________________________________________________
 Period: ________

Plant Pigment and Photosynthesis Lab

Lab Write up- to turn in

Table 1: Distance traveled by Pigment Band (in mm)

Band # Distance Traveled (mm) Band Color Pigment name Rf value
Solvent ----------------- ---------------- 1
1
2
3
4

Graph the results of the different pigments and their Rf value in the space below.
Make sure to include all important parts of a graph and color the graph using the
colors of the actual pigments. Should you use a bar or a line graph?

Post Lab Questions:

Answer completely!

1. Does photosynthesis take place in the leaves or the roots?

2. What is the balanced formula for photosynthesis?

3. From where does the carbon dioxide gas for photosynthesis come?

4. What are the two products in photosynthesis? Are they useful to animals? What
molecule(s) do animals contribute to the photosynthesis equation?
5. From your notes or text, what purpose do pigments serve in photosynthesis?
What determines their color?

6. How is paper chromatography useful?

7. What process helps the solvent move up the paper? How does this work?

8. Why do the pigments move at different rates up the paper (2 reasons)?

9. Which pigment moved the furthest? Why (see the background information, but
you may have to change your answer based on the results)?

10. Which pigment moved the least far? Why (see the background information, but
you may have to change your answer based on the results)?

11. Often times plant growers will place a color filter on the light to help the plants
grow more efficiently.
a. What color light would most benefit this plant’s growth? At what
wavelength is this found? See figure 8-5 in text.

b. What color light would be of little use to this plant’s growth? At what
wavelength is this found? See figure 8-5 in text.

12. Which pigment was the most soluble in the solvent?

13. Would Rf values be the same ratio in a different solvent? Why?