You are on page 1of 3


To investigate the effect of different temperatures on the rate of light-dependent reaction of photosynthesis in spinach leaves using the dye DCPIP.


In the light-dependent reaction of photosynthesis, NADP+ acts as the final electron acceptor of cyclic photophosphorylation. DCPIP can also accept electrons from ETC and be reduced, turning from blue to colourless. Hence, DCPIP competes with NADP+ for electrons. Rate of photosynthesis (of the light-dependent reaction) is determined by measuring the amount of DCPIP present in the spinach chloroplast cells after 1 minute, using a spectrophotometer.


1) Independent variable: temperature of the solution at 5 different temperatures (5ºC, 15ºC, 25ºC, 35ºC, 45ºC) 2) Dependent variable: absorbance of the chloroplast solutions after 1 minute 3) Controlled variables:

• Carbon dioxide concentration (atmospheric CO2) • pH of solution (buffer is added to ensure constant pH throughout the experiment) • volume of DCPIP added (5 drops) • volume of chloroplast solutions in each test tube (1cm3 ) • Time of exposure (1 minute) • light intensity (distance between the lamp & capillary tube at 5cm)


  • 1. Cut 5 fresh spinach green leaves with a sharp knife and homogenize with cold buffer solution

with mortar and pestle and fine sand.

  • 2. Cover the chloroplast extract with a light-proof aluminium foil and placed in a petri dish.

Extract has to be maintained at low temperature using ice.

  • 3. Using pipette, add 5 drops of DCPIP to the leaf extract in the petri dish. Rock the petri dish to

mix the liquids and replace the foil cover

4.Take 5 test tubes.

  • 5. Pipette out about 1 cm3 of leaf extract/DCPIP mixture into each test tube and cover the test

tubes quickly in aluminium foil to prevent exposure to light.

  • 6. Cool the first test tube by placing them in a water bath containing ice, and maintain the

temperature at 5ºC for 1 minute.

  • 7. Place the test tube 5 cm away from the lamp (measured by a ruler), remove the aluminium

foil from the tube and immediately turn on the lamp.

  • 8. After one minute of exposure to the lamp, measure and record the absorbances of the

chloroplast solutions in the test tube using a spectrophotometer at a wavelength of 600 nm.

9. Steps 6-8 are repeated for the subsequent 4 test tubes with a water bath ensuring temperatures of 15ºC, 25ºC, 35ºC and 45ºC respectively. 10. Set up in replicates of 3 for each temperature.







Absorbance after 1 minute/ %


Rate of






1st trial

2nd trial

3rd trial
























Evaluation: A curve graph that should show an increasing gradient from 5ºC to 25ºC, peaking at


A curve graph that should show an increasing gradient from 5ºC to 25ºC, peaking at 35°C, before steeply decreasing after 35°C.

This is as temperature increases,the rate of light dependent reaction of photosynthesis increases, with the optimal temperature at 35°C. After 35°C, enzymes involved in the light-dependent reactions are denatured by the relatively high temperature, and hence, this decreases the rate of photosynthesis.