Exercise 7: Pigment Separation, Starch Production, and CO2 Uptake
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OBJECTIVES:
1. Describe the differences between the light-dependent and light-independent reactions of
photosynthesis
2. Separate photosynthetic pigments using paper chromatography
3. Describe fluorescence and its role in photosynthesis
4. Describe the process of electron transport in chloroplasts and its role in photosynthesis
5. Describe the change in pH that occurs as plants take up CO2 from their environment
6. Describe the distribution of starch in leaves relative to the amount of light they receive
and the distribution of pigments
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INTRODUCTION
Photosynthesis is a very important series of chemical reactions that convert light energy
to chemical energy and leads to the production of food and oxygen necessary for life:
6 CO2
+
Carbon Dioxide
Light
12 H2O

C6H12O6
Water
Chlorophyll Sugar
+
6 H2O
Water
+
6 O2
Oxygen
Thus photosynthesis is a light- and chlorophyll-dependent conversion of carbon dioxide and
water to sugar, water, and oxygen. Oxygen is released to the environment and sugar is used for
growth or is stored as starch. Water is present on both sides of the equation, but they are not the
same. The reactant water (left side) is split to release electrons during the photochemical (lightdependent) reactions. The product water (right side) is assembled from hydrogen and oxygen
released during the photochemical and biochemical (light-independent) reactions (Calvin cycle).
Photochemical "Light" Reactions
Fast (almost instantaneous)
Light-dependent
Splits water to release oxygen, electrons, and protons
Biochemical "Light-Independent"
Reactions
Slower, but still fast
Light-independent
Converts carbon dioxide to sugar
In today’s lab you will investigate some of the major aspects of photosynthesis.
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Task 1 - PAPER CHROMATOGRAPHY
Light must be absorbed before it can be used, and a pigment is a substance that absorbs
light. The primary photosynthetic pigments that absorb light for photosynthesis are chlorophyll a
and b. However, chlorophylls are not the only photosynthetic pigments; accessory pigments
such as carotenes and xanthophylls also absorb light and transfer energy to chlorophyll a. Paper
chromatography is a technique for separating dissolved compounds, such as chlorophylls,
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carotene, and xanthophylls. When a solution of these pigments is applied to strips of paper, they
absorb into the fibers and the distance a pigment travels dependents on the size, polarity, and
solubility of the pigments within the solvent - each pigment has a characteristic rate of
movement. In the following procedure, up to four bands of color should appear on the strip - a
yellow band of xanthophylls, a yellow-orange band of carotenes, a blue-green band of
chlorophyll a, and a yellow-green band of chlorophyll b. The relationship (Rf number) of the
distance moved by the pigment compared to that of the solvent is specific for a given set of
conditions:
Rf
=
Distance moved by pigment
Distance between pigment origin and solvent termination (i.e. where solvent
stopped on paper)
Paper chromatography can be used to identify each pigment by its characteristic R f number,
which is constant for each pigment in a particular solvent-matrix system.
Procedure:
1. Obtain a strip of chromatography paper and make sure to only touch the edges so that
oils from your skin do not contaminate it.
2. Apply a strip of plant extract ~2cm from the tip of the paper. Blow the strip dry and
repeat 15 times - for the application to work you must start with a highly concentrated
application of extract on the paper.
3. An alternative is to place a fresh leaf directly on the paper, then press and roll the
edge of a coin over the leaf to crush the cells and form a stripe on the paper. Your lab
instructor will tell you which procedure to follow.
4. Place the chromatography paper in a test tube containing 2 mL of chromatography
solvent. Position the chromatography strip so that the tip is submerged in the solvent,
but not the strip of extract. Make sure the test tube is capped to prevent the solvent
from evaporating.
5. Place the tube upright and watch as the solvent and the extract move up the paper.
6. Remove the paper before the solvent reaches the top. Mark the position of the solvent
front with a pencil and set the strip aside to dry. Observe the bands of color and draw
the results on Figure 1, including pigment labels for each band. Use a ruler to
measure the distance (in cm) from the pigment origin to the solvent front and from
the origin to each pigment band. Use these values to calculate the Rf number for each
pigment, and record this in Table 1.
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Pigment
Carotene
Xanthophyll
Chlorophyll a
Chlorophyll b
Rf
Table 1: Rf numbers
Figure 1: Chromatography paper results
Questions
a. What does a small or large Rf number tell you about the characteristics of the moving
molecules?
b. Based on your results, which are more soluble in chromatography solvent
xanthophylls or chlorophyll a?
c. Would you expect the Rf number of a pigment to change if you altered the
composition of the solvent? Why?
d. If yellow xanthophylls were present in the abstract, why did the extract appear green?
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Task 2 - FLUORESCENCE
Light produces reactions only if it is absorbed by a molecule. When sunlight strikes a
plant, the chlorophyll absorbs some of the light and reflects some of the light. The green light is
reflected and is responsible for the plant’s green color. The absorbed light “excites” the
chlorophyll by boosting electrons to a higher-energy orbital. During photosynthesis, the energy
of these excited electrons from chlorophyll and chlorophyll’s central magnesium atom are passed
to another pigment molecule and photosynthesis proceeds. To observe these energy electrons we
can disrupt the photosynthetic system by blending the cells during the preparation of the plant
extract. The chlorophyll electrons in the extract are still energized if you shine light on them, but
they are left with nowhere to go. They quickly release their energy by falling back to their
original orbitals rather than continuing photosynthesis as they fall back, they emit a photon of red
light. This release of energy is fluorescence. The wavelength of reemitted light is determined by
the structure of the molecule reemitting the light.
Procedure:
1. Place a glass test tube of chlorophyll extract in front of a bright light. View the
extract from the side.
Question
a. What color light does the extract fluoresce?
Task 3 - ELECTRON TRANSPORT IN CHLOROPLASTS
The photochemical reactions of photosynthesis transfer electrons among various
compounds within chloroplasts, but do not require CO2 if provided with an alternate or artificial
electron-acceptor. Thus, electron transport does not require CO2-fixation to occur and each
involve separate sets of reactions. A dye called 2, 6-dichlorophenol-indolphenol (DCPIP) can be
used to detect electron transfers during photosynthesis. In its oxidized state, DCPIP is blue, but
after accepting electrons it becomes reduced and colorless, and the rate of decoloration depends
on its concentration and the rate of electron flow. By measuring decoloration of DCPIP we can
indirectly measure the rate of some photosynthetic reactions.
Procedure:
1. Prepare four test tubes according to Table 2.
2. Mix the contents of each tube well and place tubes 1-3 approximately 15 cm in front
of a high-intensity light bulb. Wrap tube 4 in aluminum foil and place it with the
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other three test tubes. Put chloroplasts in the test tube last. Do not position tubes
behind each other.
3. Observe the contents of the tubes intermittently and describe the changes in color you
see:
Tube Chloroplasts
0.1 M PO4 Buffer (pH 6.5)
H20
0.2 mM DCPIP
1
0.5 mL
3 mL
1.5 mL
0
2
0.5 mL
3 mL
0.5 mL
1 mL
3
0
3 mL
1.0 mL
1 mL
4
0.5 mL
3 mL
0.5 mL
1 mL
Table 2: Solutions for comparison of photosynthetic reaction rates
Questions
a. What was the purpose of each tube used?
Task 4 - UPTAKE OF CARBON DIOXIDE DURING PHOTOSYNTHESIS
Phenol red is a pH-indicator that turns yellow in an acidic solution (pH < 7) and becomes
red in a neutral to basic solution (pH > 7), and can be used to detect the uptake of CO2 during
photosynthesis. To detect CO2 uptake, you will put a plant into an environment that you have
made slightly acidic with your breath. CO2 in your breath will dissolve in water to form carbonic
acid, which lowers the pH of the solution:
H2O +
CO2
Water
Carbon dioxide
↔
H2CO3
Carbonic Acid
↔
H+
+
HCO3Hydrogen Ion
Bicarbonate Ion
As the plant fixes CO2 the pH will rise, and the solution will change from yellow to red.
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Procedure:
1. Fill one test tube one third (1/3) full with the dilute solution of phenol red (~2mL of
phenol red + tap water).
2. Use a straw to gently blow your breath into the solution - stop blowing as soon as the
solution turns yellow.
3. Add pieces of Elodea (about 10 cm within each tube) and pour out excess solution
above the Elodea.
4. Cover the tubes with plastic film or foil to prevent gas exchange with the atmosphere.
Place each tube ~50 cm in front of a 100-watt light bulb for 30-60 minutes.
5. Observe the tubes every 10 minutes
Questions:
a. What happens to the color of the indicator
b. What is the reason for the color change
Task 5 - USE OF LIGHT AND CHLOROPHYLL TO PRODUCE STARCH
The light-dependent reactions of photosynthesis occur on photosynthetic membranes. In
plants and algae, these membranes are called thylakoids and are located within chloroplasts.
Thylakoids are stacked to form columns called grana, and are held in place by lamellae. A
semiliquid stroma bathes the interior of the chloroplast and contains the enzymes that catalyze
the light-independent reactions of photosynthesis.
Sugars produced by photosynthesis are often stored as starch, thus starch production is
another indirect measure of photosynthesis. To produce starch, photosynthesis requires light as
an energy source and chlorophyll to capture the light energy (in the absence of light and/or
chlorophyll starch is not produced). In this task you will detect the presence of starch by staining
it with a solution of iodine and observe the requirement of light and chlorophyll for
photosynthesis.
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Procedure 1:
1. Place separate drops of water, glucose, and starch solutions on a glass slide.
2. Add a drop of iodine to each and describe your results.
Procedure 2:
1. Remove a leaf from a geranium plant that has been illuminated for several hours.
2. After immersing the leaf in boiling water for 1 minute, bleach the pigments from the
leaf by boiling it in methanol for 3-5 minutes - this will remove the pigments so that
you can see the color changes of the iodine starch test.
3. Place the leaf in a petri dish containing a small amount of water, and then add five to
eight drops of iodine.
4. Observe any color change in the leaf.
5. Record the color of the leaf after each successive treatment in Figure 2.
Questions
a. Was starch formed in the leaf?
b. Would you expect leaves to be the primary area for starch storage?
Procedure 3:
1. Obtain a geranium leaf that has been half or completely covered with foil or thick
paper for 3-4 days.
2. Repeat the bleaching experiment above.
3. Describe and explain any color change in the leaf:
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4. Record the color of the leaves after each successive treatment in Figure 2.
Question:
Does a leaf produce starch if it has been deprived of light.
Procedure 4:
1. Obtain leaves of a variegated coleus plant and a purple-leafed coleus plant. Make
sketches of their original pigmentation patterns
2. Extract the pigments and stain for starch as with the geranium leaves above - boiling
the leaves in water will remove the water-soluble pigments such as the red cyanins,
and boiling the leaves in alcohol will remove chlorophyll. These pigments must be
removed for you to see the color changes of the iodine starch test.
3. Record the color of the leaves after each successive treatment in Figure 2.
Questions
a. How does the pattern of starch storage relate to the distribution of chlorophyll?
b. Photosynthesis requires chlorophyll (green), but some of the coleus leaves that you
tested were purple. How do explain your results?
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Fresh geranium leaf
in light
Boiled in water
Boiled in methanol
Stained with iodine
Fresh geranium leaf
in dark
Boiled in water
Boiled in methanol
Stained with iodine
Coleus leaf in light
Boiled in water
Boiled in methanol
Stained with iodine
Purple Coleus leaf
in light
Boiled in water
Boiled in methanol
Stained with iodine
Figure 2: Color change in leaves as indicators of starch production during photosynthesis.
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