Investigating the Rate of Photosynthesis:
Redox Indicators
• The light-dependent reactions of photosynthesis take place in the thylakoid membrane
and involve the release of high-energy electrons from chlorophyll a molecules.
• These electrons are picked up by electron acceptors and then passed down the electron
transport chain.
• However, if a redox indicator (such as DCPIP or methylene blue) is present, the
indicator takes up the electrons instead. This would prove the presence of photolysis
and electrons being used in photosynthesis.
• This causes the indicator to change colour
o DCPIP: oxidised (blue) → accepts electrons → reduced (colourless)
o Methylene blue: oxidised (blue) → accepts electrons → reduced (colourless)
o The colour of the reduced solution may appear green because the chlorophyll
have a green colour
• The rate at which the redox indicator changes colour from its oxidised state to
its reduced state can be used as a measure of the rate of photosynthesis
o When light is at a higher intensity, or at more preferable light wavelengths, the
rate of photoactivation of electrons is faster, therefore the rate of reduction of the
indicator is faster.
o Typically if the mixture is left in the dark it stays blue and when put in white light
of high intensity the blue colour disappaeras as the molecules of the blue dye are
reduced.
Method
Step 1:
Leaves are crushed in a liquid known as an isolation medium
•
o This produces a concentrated leaf extract that contains a suspension of intact and
functional chloroplasts
o The medium must have the same water potential as the leaf cells (so the
chloroplasts don’t shrivel or burst) and contain a buffer (to keep the pH constant).
It should also be ice-cold (to avoid damaging the chloroplasts and to maintain
membrane structure)
Step 2:
Small tubes are set up with different intensities, or different colours (wavelengths) of light
shining of them
•
o If different intensities of light are used, they must all be of the same wavelength
(same colour of light)
o If different wavelengths of light are used, they must all be of the same light
intensity
Step 3:
DCPIP of methylene blue indicator is added to each tube, as well as a small volume of the leaf
extract
Step 4:
The time taken for the redox indicator to go colourless is recorded
•
o This is a measure of the rate of photosynthesis
Aquatic Plants
• Investigations to determine the effects of light intensity, carbon dioxide concentration and
temperature on the rate of photosynthesis can be carried out using aquatic plants, such
as Elodea or Cabomba (types of pondweed)
• The effect of these limiting factors on the rate of photosynthesis can be investigated in
the following ways:
o Light intensity – change the distance (d) of a light source from the plant (light
intensity is proportional to 1/d2)
o Carbon dioxide concentration – add different quantities of sodium
hydrogencarbonate (NaHCO3) to the water surrounding the plant, this dissolves to
produce CO2
o Temperature (of the solution surrounding the plant) – place the boiling tube
containing the submerged plant in water baths of different temperatures
• Whilst changing one of these factors during the investigation (as described
below), ensure the other two remain constant
o For example, when investigating the effect of light intensity on the rate of
photosynthesis, a glass tank should be placed in between the lamp and the boiling
tube containing the pondweed to absorb heat from the lamp – this prevents the
solution surrounding the plant from changing temperature
Method
Step 1:
Ensure the water is well aerated before use by bubbling air through it
•
o This will ensure oxygen gas given off by the plant during the investigation
form bubbles and do not dissolve in the water
Step 2:
Ensure the plant has been well illuminated before use
•
o This will ensure that the plant contains all the enzymes required for
photosynthesis and that any changes of rate are due to the independent variable
Step 3:
Set up the apparatus in a darkened room
•
o Ensure the pondweed is submerged in sodium hydrogencarbonate solution
(1%) – this ensures the pondweed has a controlled supply of carbon dioxide (a
reactant in photosynthesis)
Step 4:
Cut the stem of the pondweed cleanly just before placing into the boiling tube
Step 5:
Measure the volume of gas collected in the gas-syringe in a set period of time (eg. 5 minutes)
Step 6:
Change the independent variable (ie. change the light intensity, carbon dioxide concentration
or temperature depending on which limiting factor you are investigating) and repeat step 5
Step 7:
Record the results in a table and plot a graph of volume of oxygen produced per minute against
the distance from the lamp (if investigating light intensity), carbon dioxide concentration, or
temperature
The set up of the experiment to measure the rate of photosynthesis of an aquatic plant (pond
weed) by measuring the rate of oxygen gas produced. All three limiting factors can be assessed
this way
Exam Tip
Learn the 3 limiting factors and how each one can be altered in an laboratory environment:
Light intensity – the distance of the light source from the plant (intensity ∝ 1/d2)
Temperature - changing the temperature of the water bath the test tube sits in
Carbon dioxide - the amount of NaHCO3 dissolved in the water the pondweed is in
Also remember that the variables not being tested (the control variables) must be kept constant.
