(11) Investigating Photosynthesis Using Isolated Chloroplasts
Variables:
• The investigation can be modified to investigate the effects of different abiotic factors on the rate of
photosynthesis, i.e. light intensity, temperature
• E.g. the effect of light intensity can be investigated by altering the distance of the test tubes from the
light source
Method:
1. Cut a few leaves and remove any tough stalks
2. Grind the leaf pieces using a pestle and mortar with some chilled isolation medium
Isolation solution containing sucrose (to prevent water being drawn from the chloroplasts by
osmosis), potassium chloride and phosphate buffer (at pH 7)
3. Filter the liquid into a beaker through a funnel lined with muslin cloth
4. Transfer the liquid to centrifuge tubes and centrifuge at high speed for 10 minutes
Centrifuging will make the chloroplasts gather at the bottom of each tube as a ‘pellet’
5. Remove the liquid from the top of the tube, leaving the pellets in the bottom
6. Re-suspend the pellets in fresh, chilled isolation medium (this is the chloroplast extract)
7. Set up a colorimeter with a red filter and zero it using a cuvette containing the chloroplast extract and
distilled water
8. Add a set volume of chloroplast extract to the tube with a set volume of DCPIP and mix the contents
9. Every minute for 10 minutes, take a sample of the mixture from the tube, add it to a clean cuvette
and record the absorbance
Control Test:
• One control tube should contain only DCPIP and chilled isolation solution
This demonstrates that the DCPIP colour change occurs due to the action of the chloroplasts
• Another control tube should contain both DCPIP and chloroplast extract, but with no exposure to
light
This demonstrates that light is necessary for the light-dependent reaction, and DCPIP does not
simply decolourise over time
Theory:
• In photosystem I, during the light-dependent reaction of photosynthesis, NADP acts as an electron
acceptor, causing it to become reduced
• The reaction is caused the Hill Reaction
• The rate of the Hill reaction can be investigated by adding a redox indicator dye, e.g. DCPIP, to
isolated chloroplasts
• As NADP is reduced, there will be a colour change from blue to colourless
Conclusion:
• The rate of decrease in the absorbance of the solution is proportionate to the rate of the Hill reaction
Validity:
• It is difficult to keep all abiotic factors (that are not being investigated) constant
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