Name:__________________________
LAB 7. Photosynthesis
The process of photosynthesis is critical for the existence of most forms of life on
earth. Photosynthesis involves an organized set of biochemical reactions wherein light
energy (electromagnetic energy) is converted to chemical potential energy (energy stored
in bonds between atoms of a molecule). Implicit in this definition of photosynthesis is
the fact that some kind of molecule is being made, that is, a molecular compound
“charged” with chemical potential energy. In general terms this substance is glucose
(C6H12O6), and in plants it is built from H2O and CO2. An important “by-product” of
photosynthesis is O2, a gaseous compound required by most forms of life, and the
precursor to ozone (O3), a substance that protects life at the surface of the earth from the
damaging effects of ultra-violet (U.V.) radiation.
While organisms in the plant Kingdom are the most familiar of photosynthetic organisms,
“algae”, both microscopic and macroscopic are important too.
The process of photosynthesis is summarized by the following chemical equation:
6 CO2 + 12 H2O   C6H12O6 + H2O + O2
This relatively simple equation involves many inter-connected sets of chemical
reactions that take place in a spatially and temporally organized system. You just don’t
add CO2 molecules to water (H2O) turn on the lights and stir!
The organelles known as chloroplasts are the site of photosynthesis.
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Turn in entire lab and worksheet for next time!
Procedures:
Part 1. Extraction and examination of photosynthetic pigments
The most abundant photosynthetic pigment is chlorophyll. However, various
other molecules help in the absorption of light energy: these are known as accessory
pigments. Accessory pigments are visible to our eyes after chlorophyll is removed from
plant leaves in the autumn, providing the glorious fall colors. In this lab we will employ
a technique known as paper chromatography to separate various pigments involved in
photosynthesis. The separation of different substances via paper chromatography is based
on slight differences in molecular weight and solubility.
Walk-thru
Note!! Wear gloves throughout entire procedure!! One setup per table!
1. Take fresh piece of chromatography paper. Hook paper
to paperclip on stopper. Place stopper in large test-tube.
Mark 0.5 cm above bottom of paper slip on outside of
test tube.
2. Take a capillary tube, grab some spinach extract (place
thumb on top of tube and set in solution – hold finger
on the top – release finger to release solution), drop a
bit of spinach solution in the center of the
chromatography paper 2 cm from the bottom. Do this 3
more times. You’re trying get a concentrated dot of
spinach solution on the paper.
3. Put test tube in beaker. Fill chromatography solvent
solution to the line that you marked earlier on the test
tube. Place stopper with paper in solution. Wait
approximately 20 minutes and watch solution rise.
Fig. 1 The spinach pigment
4. Careful inspection of strip should yield at least 3-4
experimental setup.
pigment bands, depending on quality of extract and
your technique. Note that the colors will fade. The
pigment bands are various pigments involved in the process of photosynthesis,
including chlorophylls and various accessory pigments. The various colors are
owing to the presence the following pigments:
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Color
Pigment
Blue-green
chlorophyll a
Yellow-green chlorophyll b
Light yellow xanthophylls
Yellow-orange
carotenes
Part 2. Production of oxygen gas (O2) during photosynthesis
As will be discussed in lecture, the overall process of photosynthesis involves 2 major
sets of interdependent chemical reactions: the Light Reactions and the Calvin Cycle.
As a part of the light reactions, O2 (oxygen gas) is produced from water.
Essentially, water is oxidized, releasing O2. The oxidation of water molecules occurs
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because its electrons (which are removed from the bonds between O and H) are necessary
for the continued function of the light reactions. A general equation for the process is as
follows. Note that 2 water molecules are oxidized to yield 2 oxygen atoms, which then
combine to form O2.
H2O  2 H+ + O + 2 electrons
The rate of oxygen production is a direct indication of the rate of the overall
process of photosynthesis. In this part of the lab we will measure the rate of oxygen
production (volume/time) under various experimental conditions. Here,
photosynthetically derived oxygen will displace water, and we’ll measure that
displacement. We will use the common aquatic plant Elodea. Another method to measure
rate of photosynthesis, employed by plant scientists and other researchers, is
measurement of the rate of CO2 uptake, but this requires a very sensitive electronic
device known as an infra-red gas analyzer, or IRGA.
Walk-thru
1. Obtain a medium test tube and a stopper to fit
with an inserted, bent glass pipette. This will
make a simple device for measuring volume of
oxygen produced.
2. Fill the tube half-way with 3% sodium
bicarbonate (NaHCO3) solution. The sodium
bicarbonate will dissociate (separate) to yield CO2
for the plant in this closed system. Otherwise,
photosynthesis would not proceed very long, as
the CO2 is used up during the Calvin Cycle of
photosynthesis.
3. Insert 2-3 freshly cut Elodea stems, cut end up,
into the (NaHCO3) solution. Add (NaHCO3)
solution to fill volumeter to about 2 cm from the
top.
4. Insert stopper/bent pipette into test tube. Be sure
the solution rises in the tube, at least as far as the
bent part of the tube, but not much further. If it
doesn’t rise this far, add solution, if it’s past it,
dump some out. Make sure your seal is airtight!
Fig. 2. Experimental setup for
oxygen water displacement
procedure.
5. Place the tube in test tube rack, next to a beaker of tap water. Place lamp and
water flask next to beaker such that the beaker serves as a heat sink. Allow your
setup to come into thermal equilibrium for about 10 minutes.
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6. Note solution level in pipette, mark with a grease pencil. Wait to observe the
production of small bubbles from the cut end of the Elodea. These are bubbles of
O2 being released from the light reactions of photosynthesis.
7. Wait 5 minutes and allow the Elodea to acclimate. Then make volume change
measurements at 5 minute intervals for at least 15 minutes from zero time.

Make a simple graph of your data with time as the x-axis and volume as
the y-axis. How has the volume changed over time?
8. Now gently wrap the volumeter in aluminum foil, so as to prevent any light from
reaching the Elodea. Do not jar the stopper or glass tube. Keep the volumeter in
the same location as when performing step 7.
9. After 10 minutes mark the solution front again.
Part. 3 Uptake of CO2 during photosynthesis
Before starting the next experiment note the following: when CO2 dissolves in water it
reacts with water to form carbonic acid: H2CO3. The carbonic acid then dissociates
forming HCO3- (bicarbonate ion) and hydrogen ion (H+). Changes in H+ concentration
affect solution acidity: more H+ ions = increased acidity (lowered pH).
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CO2 + H2O ⇌ H2CO3
H2CO3 ⇌ HCO3− + H+
Thus, we can measure how much CO2 the plants use during photosynthesis by measuring
changes in the pH of the solution.
Walk-thru
1. Obtain a 500 ml beaker. Rinse it and fill to 200 ml with tap water.
2. Add 6 drops of phenol red. This compound changes color with changes in
solution pH. Your solution should be slightly pink at this point of the experiment.
3. With a soda straw, exhale into the solution until it turns yellow. Your solution
should turn yellow. Based on the above chemical equations, does phenol red turn
yellow under increasingly acidic or increasingly basic conditions?
4. Obtain 2 medium test tubes and add a fresh steam of Elodea to one.
5. Partially fill each tube with the yellow solution – covering the Elodea stem.
6. Place a stopper on each tube – make sure it’s on tight!
7. Place the 2 tubes, side by side in test tube rack and illuminate with the light
source. The tubes should be protected from overheating with the water bath heat
sink.
8. Monitor changes in color over an approximate 20 minute period
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Lab7 Worksheet
Pigments
1. What is the function of chlorophyll?
2. Light has different wavelengths that result in different colors. Chlorophyll absorbs
only certain colors. Which color does chlorophyll NOT absorb? Why?
3. What is the purpose of the accessory pigments?
4. The proportion of chlorophyll to accessory pigments varies depending on the
environment. Provide an example where leaves will have a much higher
proportion of chlorophyll relative to accessory pigments.
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Evolution of Oxygen
5. Why would the water front recede when you placed aluminum foil over the
volumeter apparatus?
6. Why do plants need O2, just like animals?
Evolution of CO2
7. Based on the chemical equations described in the text, does phenol red turn
yellow under increasingly acidic or increasingly basic conditions?
8. What does a gradual change from yellow to pink indicate in terms of pH
change?
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9. What does the change in pH suggest about changes in CO2 concentration in
the test tubes?
10. Where did the CO2 in the solution go?
11. Would you predict that the pH of a small pond or aquarium might vary
when it is dark?
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12. Why did we include a test tube without Elodea in the experimental set-up?
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