Plant Pigments
Introduction:
The color found in plant leaves is called pigments. Most of these are utilized in the process of
photosynthesis. Pigments, like those found in paint, are complex chemicals that reflect and absorb
various wavelengths of light from the visible spectrum. There are over 600 naturally occurring
carotenoids, as well as 7,000 flavonoids, including 500+ anthocyanins. Biological pigments such as
chlorophyll owe their color to the presence of unsaturated bonds. Chlorophyll absorbs light in the red
(long wavelength) and the blue (short wavelength) regions of the visible light spectrum. Because of
this absorption, pigments play an important role in photosynthesis. They capture light energy and
enable the plant to split water into hydrogen and oxygen. The oxygen is given off as a waste product
(much to our delight), while the hydrogen is combined with carbon dioxide to form the simple sugar
glucose.
Chlorophyll breaks down in bright sunlight, so plants constantly synthesize more chlorophyll.
Many green leaves contain pigments that are not seen until autumn and winter because they are
masked (hidden) by chlorophyll which is also destroyed by cold in many plants. A few plants mask
their chlorophyll by having large amounts of red, orange, or yellow pigments all year long.
Photosynthesis begins with the light dependent reaction, where the light is absorbed by the
green pigment chlorophyll, a photoreceptor. Even though a plant leaf may appear green, it still contains
many different colored pigments. A method of separating these pigments is called paper
chromatography. In paper chromatography, pigments are placed on the bottom of a piece of
chromatography paper and then the paper is placed in a solvent, ether/acetone. As the solvent moves
up the paper, it carries the pigments different distances due to their weight and polarity.
To quantitatively measure the absorption/reflection of light in various pigments, a
spectrophotometer can be used. This machine is a “photometer” that can measure intensity as a
function of the light source wavelength.
Purpose:
In this laboratory exercise, you will use paper chromatography to observe the different
pigments in a green leaf and show that chlorophyll exists in leaves that are not green in color.
Methods:
Materials: 2 strips of chromatography paper
non-green leaf
2 test tubes
a dime
ether/acetone solvent
spinach leaf
pencil
Caution: do not breath in the solvent and maintain proper ventilation
1. Observe your leaf and predict the pigments and wavelength that will be visible at the
completion of the lab (p.96 in text). You will draw what it looks like while you wait for the
chromatography run to finish.
2. Cut a strip of chromatography paper so that is just fit in the chromatography tube then cut a
point.
3. With a pencil, draw a faint base line at the top of the point. (see arrow)
4. Place a spinach leaf over the pencil line and run a coin over the leaf so a green
line is transferred to the paper. Move the leaf around to get a fresh area and roll the
dime over the same spot until you have one deep, green line on the paper.
5. Put enough solvent in the test tube so that it rises halfway up the point.
Base Line
6. Carefully place the strip of paper, colored line down first into the test tube. Be
careful not to let the paper touch the side of the test tube or allow the pigment
to be submerged in the solvent!!!
7. Leave it undisturbed for about 10 minutes or until the solvent has moved 2/3
up the paper.
8. Use forceps to remove the strip and place it on a paper towel.
9. Repeat procedures using another leaf if there is time.
10. Locate and identify all visible pigments on the paper. List the pigments on a chart
in your data section. Pigment 1 will be the closest to the baseline.
Color
Pigment
Yellow-Orange
Carotene
Yellow
Xanthophyll
Bright Green-Blue Green
Chlorophyll a
Yellow Green-Olive Green
Chlorophyll b
Red
Anthocyanin
11. Using a CLEAN mortar and pestle, grind the rest of the leaf to extra the pigments in a liquid.
Add 5 ml of water as you grind.
12. Filter the pigment through cheese cloth into a beaker before placing liquid into a clean cuvette.
13. The cuvette should be 2/3 full of liquid when placed into the spectrophotometer. Record the
wavelength(s) of light where your pigment has the maximum absorption.
Results: (do not write on this – recreate table on a separate sheet of paper)
Sketch your leaf.
Chromatography Data
Leaf Name
Overall
Color
Color of Chromatography
Band
Corresponding
Pigment
Wavelength
Absorption
Create two tables if examining two leaves.
Discussion:
1. Which pigment is the most important in photosynthesis?
2. What gas is needed to make glucose?
3. What color of wavelengths of light are absorbed by green plants? Reflected?
4. What are the accessory pigments functions?
5. Many trees have leaves that are green in the summer and red, yellow, and orange
in autumn. Where were these colors during the summer?
6. Besides photosynthesis, what might be another function of color in a plant?
(thought question)