POB
PHOTOSYNTHESIS
LAB
Amanda Matsumura
Nathan Marcy
Alisa Craig
Tim Waldrip
Dr. Ellen Turner, Instructor
Introduction
The purpose of experiment one is to
demonstrate the reactants and products of
photosynthesis. The purpose of experiment
two is to demonstrate that energy is stored in
plants in the form of starch. Experiment three
demonstrates which colors of light are
absorbed by plant pigments. Experiment four
clearly demonstrates visually the different
primary pigments and accessory pigments
stored in the chloroplasts of spinach.
Experiment I
• Pour 50ml of bromothymol blue solution in a
beaker
• Gently blow into the solution until it turns
yellowish-green
• Place a sprig of Elodea into a vial with the
yellowish-green bromothymol solution
• Create a control vial with the yellowish-green
solution alone
• Place under a bright light (overhead light i.e.)
• Observe changes you see in colors and also the
presence of bubbles
Experiment II
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Pour 350 ml of water into a 600ml and bring to a boil on a hot
plate
Get one of the coleus leaves that have been kept away from light
and one that has been photosynthesizing. Observe the
differences between the two
Boil the leaves for two minutes
Place the leaves in a 250ml beaker and pour 50ml of ethanol over
them
Place the 250ml beaker with the leaves in the 600ml on the hot
plate
Boil the leaves in the alcohol in the water until the leaf turns pale,
due to a loss of chlorophyll
Carefully remove the chlorophyll-containing alcohol from the
water bath and allow it to cool under the ventilation hood
Experiment III
• Place a sample of dried spinach leaves in a
mortar, add 5ml of ethanol and grind into a fine,
watery pulp
• Put some of the ethanol-ground extract into a
centrifuge tube and place it in one of the slots in
the centrifuge head. Balance with a tube directly
across from the extract
• Centrifuge the sample for 5 minutes
• Decant the chlorophyll extract into a
Spectrophotometer curette. Read directions for
use of the spectrophotometer (which is at the
end of the lab)
Experiment IV
• Use a lead pencil to lightly mark a straight line across a strip
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of chromatographic paper 1cm from the end
Make cuts from the 1cm mark to the center of the end of
the strip (diagram in lab explains)
Use a Pasteur pipette to make a line of chlorophyll extract
across the paper strip about 2cm above the point
Place the chromatography paper strip into a bottle
containing a small amount of petroleum ether solvent so
only the tip is touching the liquid
Bend the top of the paper over the lip of the bottle and
lightly cap the bottle to slow the evaporation
Measure the distance from the origin at the chlorophyll
extract line to the center of each band and record
Results
 Experiment 1:
 Acidic solution converts to basic solution as CO2 is consumed and
oxygen is produced.
 Experiment 2:
 The leaf that has been starved contains no starch. The leaf that
has been nourished has abundant starch.
 Experiment 3:
 Spectrophotometer readings confirm the absorption spectrums
of pigments in chloroplasts. Plants absorb all wavelengths of light
except green.
 Experiment 4:
 The pigments found in spinach clearly form different bands of
color.
EXPERIMENT 1
These are the materials needed for experiment one.
On the left is the bromothymol blue solution (basic) and on the right is the elodea leaves.
EXPERIMENT 1
Addition of CO2
to Bromothymol
solution makes
the solution
acidic. A pH
indicator in the
solution causes
the color to
change from
blue green to
yellow.
EXPERIMENT 1
The bromothymol blue solution in the presence of carbon dioxide becomes acidic. The
indicator in the solution turns yellow to indicate a low pH.
EXPERIMENT 1
Place the acidic bromothymol solution and the elodea in a sealed container. It should look
similar to the above.
EXPERIMENT 1
Place the sealed containers in a source of heat and light. For the purposes of this
experiment, an overhead projector can be used. The class set up one control group and
four experimental groups.
EXPERIMENT 1
The results here clearly show that the acidic bromothymol solution turns back to a basic
solution. Here we see the target experimental group next to the control group.
EXPERIMENT 2
Plant one versus plant two. Differences in appearance are apparent.
EXPERIMENT 2
Boiling the two leaves to remove waxy coating from the exterior.
EXPERIMENT 2
Boiling the leaves again in alcohol to remove the chlorophyll.
EXPERIMENT 2
The leaves, after boiling. Remove the leaves and dye them with iodine. Shine a light on the
remaining solution.
EXPERIMENT 2
The leaves after iodine staining. On the left, the malnourished leaf contains very little
starch. On the right, the nourished leaf has abundant starch.
EXPERIMENT 2
The alcohol
containing
the
pigments
from the
leaves
fluoresces
red when a
light is shone
on it.
EXPERIMENT 2
The pigments emit a red light when light is shone on them and no electron acceptor is
present.
EXPERIMENT 3
On the left, ethyl alcohol. On the right, spinach placed in a mortar.
EXPERIMENT 3
Use the mortar and pestle to grind the alcohol soluble pigments put of the spinach leaves.
EXPERIMENT 3
Decanting
the primary
and
secondary
pigments
using
alcohol in
combination
with a mortar
and pestle.
EXPERIMENT 3
Place two test tubes, one with the chlorophyll containing solution and the other with a
ballast solution of water or alcohol on opposing sides of the centrifuge to properly balance
it.
EXPERIMENT 3
A centrifuge
uses
gravitational
and inertial
forces to
separate
components of
a solution by
weight. The
pigments settle
to the bottom
while anything
else rises to the
top.
EXPERIMENT 3
Using curettes and alcohol, zero the spectrophotometer. Then, place the pigment solution
in the machine to obtain a reading. Record the data as instructed, and be sure to zero the
machine between each reading.
EXPERIMENT 4
Using the pigment solution from experiment 3, use a capillary tube or pipette to place a
line on chromatography paper shaped as shown on the right.
EXPERIMENT 4
Place the paper so that it is able to soak in petroleum ether. Be very careful, as petroleum
ether is quite flammable.
EXPERIMENT 4
Placing the
chromatography paper
inside the bottle of
ether. Place just the tip
inside the ether and
allow the rest to sit
above. Please note: put
the cap back on the
bottle of ether after
the paper is in place or
the ether will
evaporate.
EXPERIMENT 4
The results of the experiment. Several lines each indicative of a separate pigment can
clearly be seen.