Photosynthesis Experiments
Anglo European School, Ingatestone
Colorimetry and Photosynthesis
Colorimetry
Colorimetry is a widely used technique in Biochemistry for measuring amounts of substances
present in solution, and for studying rates of change in the concentrations of substances in
solution. The technique works by measuring the absorbance of a solution for a particular
wavelength of light. Absorbance is a measure of the amount of light absorbed during its passage
through a sample.
The absorbance of a coloured solution is directly proportional to the concentration of the coloured
solute.
Colorimeters
All colorimeters contain a light source, a filter (or monochromator), a slit, a sample cell or cuvette
a light detector and a read-out device. The layout of a simple colorimeter is shown below. I0 is the
intensity of the light beam as it enetrs the sample, and I is that of the beam as it exits the sample.
Io
I
0.05
light
source
filter
transmits light
of selected
wavelength
slit
cuvette
containing
sample
photocell detects
transmitted light
digital read-out
Absorbance = A = log10(I0/I)
For the most accurate results, one should measure the sample’s absorbance of a wavelength of
light complementary to the colour of the solute.
Colorimetry and Photosynthesis
Bicarbonate indicator is an extremely sensitive pH indicator. As such, it can be used to determine
the levels of carbon dioxide in a solution. (Carbon dioxide will dissolve in water to form carbonic
acid)
As with all indicators, bicarbonate indicator contains a pigment molecule which can exist in two
different states according to the pH of its environment. At low pH, this pigment exists in a yellow
form; at high pH it is purple.
When bicrbonate indicator is equilibrated with atmospheric carbon dioxide it is orange/red. With
more carbon dioxide the solution becomes yellow. As carbon dioxide is removed, the solution
turns red and then purple as an increasing amount of the pigment is converted to the purple form.
Bicarbonate is not toxic to plants, and so we can carry out photosynthesis experiments with
aquatic plants immersed in the indicator. This experiment uses algae – single-celled
photosynthetic organisms. So that the algae do not interefere with our measurments of
absorbance, they will be immobilised in alginate beads.
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Photosynthesis Experiments
Anglo European School, Ingatestone
Making the Alginate Beads
Materials:
 5ml 3% sodium alginate
paste
 5 ml thick algal suspension
 2x 50 ml beakers
 3 ml plastic pipette
 50 ml 2% calcium chloride
solution
1. Place 5ml of the alginate paste and 5 ml of the algal
suspension in a small beaker. Mix thoroughly with the
glass rod. The resulting mixture should be dark green
pipette
algae-alginate
mixture
calcium
chloride
solution
algal beads
 glass stirring rod
 sieve
 distilled water
glass rod
alagal
suspension
2. Place 50 ml of the calcium
chloride in the second beaker.
Fill the pipette with the aglaealginate mixture. Dispense the
mixture drop-wise into the
alginate
calcium chloride solution. The
paste
calcium chloride will cause the
alginate to set, forming beads. Remember to apply even
pressure to the pipette so that all the beads are the same size.
3. After 10 minutes, place the
algal beads in a sieve and
wash them with distilled water.
sieve
Light intensity and the Rate of Photosynthesis
 180 algal beads
 84 ml bicarbonate indicator
 12x clean universal bottles
7 ml bicarbonate
indicator
15 algal beads
 150W halogen lamp
 colorimeter with 550nm
filter
 metre rule
 clean cuvettes
 distilled water
1. Into each of twelve clean universal bottles place 15 algal
balls, along with 7 ml of the bicarbonate indicator. Screw
the lid tightly onto the bottles so that they are sealed.
2. Place ten of the sealed bottles at five different distances
from a 150W halogen lamp, two at each position. Place
a beaker of water between the bottles and the lamp to
act as a thermal insulator. Place the remaining two
bottles in the
150W halogen
Heat shield
dark.
lamp
metre-rule
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Photosynthesis Experiments
Anglo European School, Ingatestone
3. Set up the colorimeter as follows:
- select the brilliant green filter (550nm) on the colorimeter.
- switch on the colorimeter.
- fill a clean cuvette with distilled water
- place the cuvette in the sample holder in the colorimeter.
- press the ‘zero’ button.
(The colorimeter should be zeroed each time before you take a reading)
4. After 30 minutes, remove the indicator from its universal bottle and pour it into a clean
cuvette. Measure the absorbance of the indicator, the place the indicator back into its
bottle and reposition the bottle in the experiment. Measure the absorbance of the indicator
in each bottle in this way.
5. Repeat the absorbance readings at 60 and 90 minutes.
Results: Plot a graph of absorbance (y-axis) against relative light intensity (x-axis). Relative light
intensity can be estimated as 1/(distance)2.
Action Spectrum of Photosynthesis
 105 algal beads
 49 ml bicarbonate indicator
 7x clean universal bottles
 colorimeter with 550nm
filter
 coloured gels (red, orange,
yellow, green, blue, indigo,
violet)
 150W halogen lamp
 scissors
 metre rule
 clean cuvettes
 distilled water
 sellotape
1. Into each of seven clean universal bottles place 15 algal balls, along with 7 ml of the
bicarbonate indicator. Screw the lid tightly onto the bottles so that they are sealed.
2. Cut a rectangle of coloured gel of sufficient size to completely cover a bottle when
wrapped around it. Cut one such rectangle from each gel.
3. Wrap each bottle in a gel, holding the gel in position with
a piece of sellotape.
7 ml bicarbonate
indicator
15 algal beads
Coloured gel
4. Place the bottles at an equal distance from a 150W
halogen lamp, placing a large beaker of water between
the lamp and the bottles to act as a heat shield. Leave
the bottles for 60 minutes.
5. Measure the absorbance of the indicator as before,
remembering to zero the colorimeter.
Results: You will need to plot a suitable graph of your results. (Hint: you may wish to make
estimates for the main wavelength of light transmitted by each gel.)
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Photosynthesis Experiments
Anglo European School, Ingatestone
Rate of Photosynthesis and Carbon Dioxide Concentration
 1x plastic straw
 1x 10ml syringe
 4x 25ml beakers
 stopclock
 tin foil
 1 piece black liner
 150W halogen lamp
cotyledons
1. Use the plastic straw to punch out 20 leaf
discs from the radish cotyledons.
2. Cover the sides of a beaker with foil,
keeping the top clear, and place 5cm3 of
0.0125 mol dm-3 hydrogen carbonate
solution into it.
 Radish cotyledons
 hydrogen carbonate
solutions (0.0125, 0.1000,
0.2500 & 0.5000 mol dm-3)
Seedling
first
leaves
straw
3. Remove the plunger from the syringe.
Place your finger over the nozzle of the
syringe and add 5 cm3 of the 0.0125 mol
dm-3 hydrogen carbonate solution.
4. Add 5 of the leaf discs to the solution in the syringe and replace the plunger. The discs
should float near the surface of the solution.
5. Invert the syringe and, using the plunger, remove all the air.
finger
6. Place your finger over the nozzle of the syringe and pull
the plunger down. The leaf discs should sink to the bottom
(plunger end) of the syringe. You may need to repeat this
step a number of times to get all the leaf discs to sink.
leaf discs
7. Place the contents of the syringe into the prepared beaker,
and cover with the black liner.
hydrogen
carbonate
solution
8. Place the beaker under the
150W halogen lamp. The
bottom of the lamp should be
10 cm from the top of the
beaker.
9. Remove the black liner and
start the stop clock. Time how
long it takes each leaf disc to
rise to the surface of the
solution.
10 cm
foil
10. Repeat the experiment using the 0.1000, 0.2500 & 0.5000 mol
dm-3 hydrogen carbonate solutions.
Write up - your write-up should include the answers to the following
questions:
- Why did the leaf discs sink at stage 6?
- Why did the discs rise when illuminated?
- Why did some discs take longer to rise than others?
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