Hill Reaction Practical – DCPIP and Isolated Chloroplasts
BiologyPlant PhysiologyAges 16–17Virtual lab
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Next in Plant Physiology
Blend leaves in cold isolation medium, filter and centrifuge to get chloroplasts, then add blue DCPIP and read the absorbance as it turns colourless. Run dark and no-chloroplast controls, and plot the rate against lamp distance or temperature.
Lesson: Light-dependent reactions: the Hill reaction, DCPIP as electron acceptor, chloroplast isolation, effect of light intensity and temperature
What it shows
In 1938 Robin Hill showed that isolated chloroplasts in light can reduce an added dye. A dehydrogenase in the chloroplast normally passes electrons from the light-dependent reactions to NADP; here DCPIP accepts them, blue when oxidised and colourless when reduced. You prepare a chloroplast extract, run five tubes that show what the colour change depends on, and then measure the rate at different lamp distances and temperatures with a colorimeter. The tube set and the reading plan follow the school practical; the rates, the filter factors and the effect of each preparation choice are estimates that give the usual result.
How to use
On Extract, choose the Medium, Kept and Centrifuge settings, then press Next step or Run all steps. On Five tubes, set the Lamp distance and press Start: readings every 2 minutes fill the table. On Rate, press Measure for one tube, or Distance series or Temperature series, then choose the graph's x-axis with Plot rate against.
Parameters you can change
- Experiment Extract, Five tubes, Rate
- Grinding medium Isolation medium, Distilled water
- Kept On ice, On the bench
- Centrifuge High speed, Low speed
- Lamp distance 10–100 cm
- Temperature 5–60 °C
- Water beaker as heat shield
- Colorimeter filter Red, Green, Blue
- Plot rate against Distance, Light intensity, Temperature
Questions to explore
- Which tubes stay blue, and what does each of them show about the conditions the reaction needs?
- When light intensity rises four times, from 60 cm to 30 cm, does the rate rise four times? Why or why not?
- How do the rates at 15 cm and 100 cm respond to temperature, and which factor limits the rate in each?
