Lab Exercise 8 - Photosynthesis
Objectives
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Provide evidence for the importance of light and chlorophyll in photosynthesis
Separate plant pigments by paper chromatography
Introduction
If a person on the street were asked to provide an example of an endangered species,
he/she would most likely name some organism that flies, crawls, squeaks or howls. Rarely will
someone mention a member of the plant kingdom. Plants generally aren’t the first living things
to pop into a person’s head when thinking about organisms on this planet. However, without
plants and other organisms capable of photosynthesis, all other life on this planet would cease.
Plants, algae, and cyanobacteria are the producers in every food chain, and all living organisms
depend on these producers.
Energy is defined by its ability to change the property of matter. Energy can exist in
different forms: mechanical, heat (or radiant), light, electrical, and chemical. Light energy from
the sun supplies life with enough energy to exist, grow, and reproduce. Plants, algae, and
cyanobacteria can extract this ultimate energy from the sun through photosynthesis. These
producers convert or metabolize light energy into chemical energy by building complex organic
molecules, such as glucose, which is stored as starch. Carbon dioxide and water are also needed
in photosynthesis. Other organisms then extract the chemical energy stored in starch. Producers
make over 200 billion tons of food a year!
Photosynthesis occurs in chloroplasts, where plant pigments absorb solar energy. The
process consists of two reactions: the light-dependent reaction and the light-independent
reaction. The light-dependent reaction produces oxygen and ATP and NADPH. The lightindependent reaction reduces carbon dioxide and makes PGAL, utilizing the ATP and NADPH
produced during the light-dependent reaction. PGAL may be converted to glucose, fatty acids,
or proteins.
What does a plant leaf have in common with photographic paper? They both can be used
to make a picture. In ordinary photography, illuminating a piece of photographic paper, through
a negative, makes a print. A chemical reaction occurs where the paper is illuminated, and fine
grains are deposited. A starch picture is made in the same way by illuminating a leaf, which
contains starch. If the leaf is illuminated only in certain areas, similar to a negative, a picture is
formed. This picture will become clear when the starch is stained, and the green chlorophyll is
bleached. What would happen if we removed the plant from light for several hours? Would
there be chemical energy available for the plant?
Pigments are defined as substances that absorb visible light. In a leaf, several pigments
are present, though we may not notice all the pigments at one time. The principal pigment in
advanced plants is chlorophyll a. Chlorophyll b, carotenes, and xanthophylls play a secondary
role by transferring energy they absorb to chlorophyll a. Chromatography is a technique that
separates molecules from each other on the basis of their solubility in particular solvents. The
solvent used in the following chromatography procedure is nonpolar (petroleum ether and
acetone). As a nonpolar solvent moves up the chromatography paper, the pigment moves along
with it. The most nonpolar pigment is most soluble in the solvent and will move faster and
farther up the chromatography paper.
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Lab Exercise 8 - Photosynthesis
8.1 Importance of chlorophyll in photosynthesis
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Retrieve a variegated Coleus leaf. The white area does not contain chlorophyll.
Make a drawing of the leaf on the QUESTIONS page. Label areas of chlorophyll and
areas without chlorophyll.
Set up a double boil system where a small beaker containing a small amount of 95 %
denatured ethanol is placed inside a beaker with boiling water. [Do NOT set a beaker
with 95 % denatured ethanol directly on the hot plate.]
Once the alcohol boils, place the leaf inside the alcohol beaker.
Leave the leaf in the alcohol until it has lost its pigments.
Carefully remove the leaf from the alcohol and place it in the petri dish.
Drop IKI solution onto the leaf, generously.
Gently swirl the petri dish until the starch picture appears. You may have to wait about
5-15 minutes for results to appear.
Draw a picture of the leaf, indicating which areas have starch.
8.2 Importance of light in photosynthesis
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Retrieve the Coleus leaf with the aluminum foil that you had prepared during last lab.
Make a drawing of the leaf, labeling the area with the aluminum foil.
Remove the aluminum foil.
Place the leaf into the beaker containing boiling 95 % denatured ethanol.
Leave the leaf in the alcohol until it has lost its pigments.
Carefully remove the leaf from the alcohol and place it in the petri dish.
Drop IKI solution onto the leaf, generously.
Gently swirl the petri dish until the starch picture appears. You may have to wait about
5-15 minutes for results to appear.
Draw a picture of the leaf, indicating which areas have starch.
8.3 Chromatography
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Obtain a graduated cylinder and strip of chromatography paper.
Place the chromatography paper on a paper towel.
Fill a capillary tube by placing it into the pigment extract. This pigment extract was
prepared by the instructor who boiled many leaves in 95 % denatured ethanol to extract
the pigment.
Apply one small drop of pigment on the chromatographic strip at the appropriate notch.
Wait 10 seconds. Apply another small drop of pigment on top of the drop previously
made. Wait 10 seconds. Apply another small drop of pigment on top of the drop
previously made. Wait 10 seconds. Repeat this procedure until you have a nice,
concentrated area of pigment.
Fill the graduated cylinder with a small amount of chromatography solvent such that
when you put the chromatography strip into the tube, the solvent DOES NOT touch your
pigment spot.
Allow 5-15 minutes for the solvent to travel up the strip. The time may vary.
Remove the chromatogram when you see the band of beta-carotene pigments.
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Lab Exercise 8 - Photosynthesis
8.1 QUESTIONS
1.
Draw your variegated Coleus leaf. Label areas of chlorophyll/no chlorophyll on the
“Before” leaf and starch/non-starch on the “After” leaf.
Variegated Coleus leaf before
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Variegated Coleus leaf after staining
Usually, we write the balanced equation for photosynthesis as:
Light energy + 6 H2O + 6 CO2 → 6 O2 + C6H12O6 (which is glucose)
However, throughout this entire lab, we use IKI to measure for the presence of starch, not
glucose. Explain the relationship between glucose and starch, such that we can measure
starch as in indirect evidence for photosynthesis.
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Use your results as evidence to explain if chlorophyll is necessary for photosynthesis.
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Lab Exercise 8 - Photosynthesis
8.2 QUESTIONS
4.
Draw your Coleus leaf that was covered with foil. Label areas of foil/no foil on the
“Before” leaf and starch/non-starch on the “After” leaf.
Coleus leaf before with the foil
Coleus leaf after staining
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Use your results as evidence to explain if light is necessary for photosynthesis.
6.
Based on your understanding of the importance of photosynthesis, discuss how a giant
meteor hitting the earth may contribute to the extinction of all the large dinosaurs.
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Lab Exercise 8 - Photosynthesis
8.3 QUESTIONS
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Staple/tape your chromatogram here.
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Label the following pigment bands on your chromatogram. The bright orange-yellow
band closest to the top represents beta-carotenes. Xanthophylls are yellow and may be
represented in multiple bands. The blue-green band is chlorophyll a; and the lowest,
olive-green (yellow-green) band is chlorophyll b.
9.
There are four pigment bands on the chromatogram. Two of the four pigment bands are
the chlorophyll pigments.
a. What general color are these pigments?
b. Therefore, why do most leaves appear green for most of the year?
10.
What happens to the chlorophyll pigments in autumn such that we see more yellow,
orange, and red pigments in many plant leaves?
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