Without pigments we’re nothing!

Modified from:
Demonstrating that Light is dissipated as Chlorophyll a Fluoresces by H. Alexandria Bodha
Michigan State Sample Core Curriculum, Lesson 8, True Colors

By Rachel Hughes and Kirstin Bittel

Time: 1 class period
Preparation Time: 30 minutes plus time for solution to chill
Materials: 4 pieces of chromatography paper or coffee filters
Coffee stirrers (1 per group)
Cups (6 ounce plastic, two per group)
Denatured alcohol (15 ml per group, 100 ml for teacher demonstration)
Leaves (fresh spinach or geranium, and a variegated leaf like Coleus or red maple)
Mortar and pestle with sand, or other device for mashing leaves
Ruler (cm, per group)
Scissors (per group)
Water (about 15 ml per group)
Activity sheet

Safety Precautions: Alcohol is a toxic and flammable substance. Its use should be carefully controlled. Students may associate the denatured ethyl alcohol with consumable alcohol, or fail to understand the process of “denaturing” ethyl alcohol. Boil alcohol in a protected, ventilated area (preferably a fume hood) using a double boiler. Have MSDS sheet available.

Students have, in previous experiments, established that light is necessary for photosynthesis to happen, and that light drives the reaction, but how? In this exercise students find out about the pigments present using paper chromatography and explore their role in providing energy for the combination of carbon dioxide and water to produce sugar.

Students will be able to:-
1. Identify chloroplasts as the primary site of photosynthesis
2. Identify chlorophyll pigments as the primary pigments that absorb light as part of photosynthesis
3. Explain the role chlorophyll plays in ‘capturing’ the light energy of the sun in a written form

National Science Education Standards
Plant cells contain chloroplasts, the site of photosynthesis. Plants and many microorganisms use solar energy to combine molecules of carbon dioxide and water into complex, energy rich organic compounds and release oxygen to the environment. This process of photosynthesis provides a vital connection between the sun and the energy needs of living systems.

Teacher Background
Photosynthesis depends upon the energy supplied by the sun. Electrons within pigment molecules, such as chlorophyll a and b, are excited from a ground state to a higher energy state when photons of light hit the molecule. In the leaf these high energy electrons are transferred from the chlorophyll to another molecule via a carrier molecule ex. NADP+ with a hydrogen ion to areas where the energy is used to create different types of molecules such as glucose. At the beginning of this lesson, acetone extracted chlorophyll a receives light that is not used in the photochemical processes of photosynthesis. Instead, the electrons return to the ground state and energy is re-emitted as fluorescent light. Students use a flashlight to observe this fluorescence.

Resource Websites




The day before prepare the following solution, being sure to use gloves. To a large test tube add 5 leaves of spinach. Add 50ml of acetone and 1 tablespoon of sand. Mix well and place carefully in the freezer overnight. In the morning you should see a dark liquid at the top of the test tube; this is chlorophyll that has been extracted from the chloroplast. In the morning carefully remove the test tube.

Before the students arrive make sure that the room can be darkened effectively.

1. On the overhead or board have the basic photosynthesis equation we have been using displayed with the new addition of light driving the reaction. Ask students what ideas they have about how light helps the photosynthetic process. Students often connect chlorophyll, the green color of most leaves with photosynthesis. Guide the students back to the role of energy in chemical reactions and that light is energy.

2. Explain that the sun’s energy is captured by pigments in the leaves and used in a number of ways including making glucose through photosynthesis, but also explain that some of the energy is reemitted as red fluorescence. Explain that you have extracted a pigment, chlorophyll a, from spinach leaves. As the pigment has been extracted from the leaves ask the students what they think will happen to energy that hits the pigments now. Have a student hold the test tube, turn off the lights, and have another student shine a flash light at the tube. You should see a red line of fluorescence around the top of the tube. Hopefully this is a tangible experience for the student that connects pigments with the conversion of sunlight into other forms of energy.

3. Ask students if a plant has to be green to photosynthesize? Can they think of other colors of leaves? If it is a different color can it contain pigments that will be useful for photosynthesis? Have students add light to their photosynthesis equation if they haven’t already.

4. Share with students a picture of a plant cell that shows chloroplasts. Remind them that this is the site of photosynthesis and that it contains special structures with clusters of pigments.

5. Students are going to explore the variety of pigments found in plants using paper chromatography.
Give each student team two clear drinking cups, 1 very green leaf and 1 variegated leaf, a strip of chromatography paper (a piece of coffee filter that can be cut into two 2 x 10 cm strips will also work), a pencil, 15 mL of rubbing (denatured) alcohol, 15 ml water, 1 coffee stirrer, mortar and pestle to shred or grind the leaf.

Provide the students with the following instructions:

a. Grind, tear, and squeeze leaves until a liquid is obtained. (A little sand can make the grinding more efficient.) (Another way to do this is to lay the leaf over the chromatography paper and rub the leaf with the eraser of a pencil. The leaf tears, but if you keep going, you transfer enough chlorophyll to the paper to do the chromatography.).

b. Being careful to touch the paper only on the edges, draw a faint line using pencil across the narrow part of the paper, approximately 2 cm from the bottom. Use a coffee stirrer to place juice onto the chromatography paper strips 2 cm from bottom in the center. Place a drop on each strip; let it dry, and then place another drop until the spot is very dark.

c. Add 15 mL alcohol to one drinking cup, 15 ml water to the other.

d. Place paper clips through each strip towards the top at a height that will allow the bottom of the filter to just touch the alcohol and water without submerging the pigment spots. The paper clips can be clipped to a coffee stirrer and hung across the top of the cup. Do not let the spot wash off in the alcohol. The alcohol should be about 1 cm below the spot.

e. Observe as alcohol and water rise through the strips, causing pigments to separate (approximately 15 minutes). Pull out the strips when the colors have stopped spreading or just before solvent reaches the top, and let them dry without touching them. Record the results quickly as the pigments will quickly fade.

Your students should see a variety of colors. In the alcohol solution, they should find two shades of green, a blue green chlorophyll a, a yellow green chlorophyll b, a faint yellow band of xanthophylls, and a thin orange band of carotenes. In the water solution, blue, red, or brown pigments may appear. You may want to use this opportunity to discuss and use Rf values.

7. Ask students how many colors they saw. Why might the plant have so many pigments? Guide the students to establishing that it might be to catch more light by asking “What is the “color” we see when we look at something?” You may need to review this. It is the color that bounces off the leaf and isn’t absorbed. “What color light would be the least useful to a plant?” That would be green, because it bounces off. Explain: “Chlorophyll is a lot like a battery.” The chlorophyll gets charged in the sun, and then the plant can use its energy.

8. Referring back to yesterday’s concept ask the students “What does the plant do with the chlorophyll’s energy?” If the students can respond, “Make food,” remind them that food contains energy that the plant can use to grow.

9. Yesterday students saw the effect of no light on food production for the leaf when using the geraniums. How would they design an experiment to test different frequencies of light? Set this writing challenge: A new planet was discovered. The sun is red. It is very hot and dry on the planet. Most of the sunlight that reaches the planet is red, orange, and yellow. Imagine that there is life on the planet. Draw a picture in the color of the imaginary plants on this alien planet. Then write a paragraph about why you imagined the plants looking as they do. (Students should realize that the plants could not be red or yellow, because those colors would bounce back the light they are getting. Plants should be blue to purple. An additional great extension is for students to realize that the plants would have to have small leaves so they wouldn’t dry out easily, like desert plants.

Social Studies
Students can compare cultures when investigating plant pigments and photosynthesis. Students can read about ancient cultures’ use of natural pigments to create art and decorate clothing. (Dyes have been derived from plants for many years. African tie-dyes and Old English lichen dyes are good resources.)

Embedded Assessment
The writing challenge provides an opportunity for students to demonstrate an understanding of form and function. Knowing the relationship between different frequencies of light and the capture of energy is important in understanding food production for the plant.

PULSE is a project of the Community Outreach and Education Program of the Southwest Environmental Health Sciences Center and is funded by:

NIH/NCRR award #16260-01A1
The Community Outreach and Education Program is part of the Southwest Environmental Health Sciences Center: an NIEHS Award


Supported by NIEHS grant # ES06694

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Last update: November 10, 2009
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