the hill reaction

• IV


  Light intensity reaching the chloroplast samples
• DV


  Amount of DCPIP reduced (can be judged based on the colour of the mixture)
• CVs

  • Use chloroplasts from the same species of plant – chopped spinach leaves can be used
  • Amount of chloroplast/buffer solution added to each tube – 5cm³ to each tube
  • Amount of DCPIP added to each tube – 10cm³ will be added
  • Amount of time – both tubes will be left for the same amount of time before comparing the colour of the mixture
  • Same pH used – buffer solution will be added to maintain pH of 7.0
• DCPIP

  • The Hill Reaction depends on electrons released during the light-dependent stage of photosynthesis being picked up by the blue electron acceptor DCPIP.
  • DCPIP is blue when oxidised (at pH 7.0) and colourless when reduced, so it is possible to monitor the loss of blue colour as an indication that DCPIP has accepted electrons
  • In this experiment the DCPIP takes the place of NADP, allowing photolysis to continue
• method

  1. grind up leaves with 20cm3 isolation solution in a pestle and mortar(The isolation medium will prevent cell damage due to osmosis or extreme pH)
  2. Filter the resulting liquid into a clean beaker using a funnel and some filter paper or cloth
  3. Transfer the filtered liquid into a centrifuge tube and centrifuge for 10 minutes=pellets of chloroplast
  4. Discard the liquid in the centrifuge tube and keep the pellet
• method 2

  5. Place 2 cm3 fresh isolation medium and the chloroplast pellet into a clean test tube, stirring with a glass rod- chloroplast extract

  6.Transfer the chloroplast extract to an ice-cold water bath. temp slows down activity

  7.Place a test tube containing 0.5 cm3 chloroplast extract into a test tube rack set up at a set distance from a lamp-

  • A beaker of water can be placed in between the lamp and the rack here to prevent a temperature increasedue to heat from the lamp

  8.Add 5 cm3 DCPIP solution to the chloroplast extract and mix
• method 3

  9.Use a pipette to immediately remove a sample of the DCPIP-chloroplast mixture and place the sample into a clean cuvette

  10.Place the cuvette into a colorimeter and take a reading for absorbance
• results

  he absorbance reading of the colorimeter should decrease over the 10 minute test period as the DCPIP indicator is reduced by electrons in the chloroplasts

  • The solution changes from blue to colourless as the DCPIP is reduced, reducing the amount of light that is absorbed by the solution