5.1.8

Photosynthesis Experiments

Test yourself

Using Chromatography to Investigate Photosynthesis

Chromatography is a technique that can be used to identify which pigments are in the leaves of different plants. This allows us to identify what wavelengths of light a plant can absorb.

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1) Extract the chlorophyll

  • Take some leaves from a plant that grows in the shade.
  • Place boiling water from a kettle into a beaker and dip a few leaves to kill them.
  • Tear up the leaves and grind with 5-10 cm3 of acetone in a pestle and mortar until you obtain a dark green chlorophyll extract. Add a pinch of sand to break up the cells.
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2) Add the chlorophyll

  • Use a strip of chromatography paper, which fits into a boiling tube without touching the sides. Mark a line 1.5 cm from the bottom in pencil and in the centre put a cross.
  • Mark another line 2 cm from the top of the paper.
  • Pick up your chlorophyll solution using a capillary tube and place on the cross. Allow to dry.
  • Repeat 4-5 times to have a small, dense green spot.
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3) Suspend the paper

  • Put 1.5 cm of acetone/petroleum solvent into the bottom of the boiling tube, using a funnel to avoid wetting the sides.
  • Suspend the paper using a drawing pin so that it just touches the solvent and leave.
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4) Repeat the experiment

  • Repeat steps 1-3 using leaves from a plant that grows well in direct sunlight.
  • Remove the paper from each boiling tube when the solvent reaches the line at the top of the papers. Mark the positions of the pigments before they fade.
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5) Calculate Rf

  • Calculate the Rf values using:
    • Rf = distance travelled by spot ÷ distance travelled by solvent
  • Find the pigment that corresponds with the given Rf value.

Investigating Dehydrogenase Activity in Chloroplasts

During the light-dependent reaction, NADP is reduced to NADPH. A dehydrogenase enzyme catalyses this reaction. This experiment will monitor dehydrogenase activity by using DCPIP, a redox indicator.

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DCPIP

  • In this investigation, a blue dye (DCPIP) is used to monitor the rate of dehydrogenase activity. DCPIP is a redox indicator.
  • This means that it is blue in the oxidised state and colourless in its reduced state.
  • When electrons are released by the chlorophyll, DCPIP will change from blue to colourless.
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1) Extracting chloroplasts

  • Put 50 cm3 of isolation medium into a beaker.
  • Tear eight spinach leaves into small pieces and put the pieces into the isolation medium in the beaker.
    • Do NOT put pieces of the midrib or the leaf stalk into the beaker.
  • Half fill a large beaker with ice and place a small beaker on top of the ice.
  • When carrying out this step, all solutions and apparatus should be kept as cold as possible and the extraction should be carried out as quickly as possible.
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2) Suspending chloroplasts

  • Put 3 layers of muslin over the top of the filter funnel and wet with the isolation medium. Rest the filter funnel in the small beaker on the ice.
  • Pour the spinach and isolation medium into the blender and blend for 15 seconds. Pour the blended mixture back into the beaker.
  • Pour your blended mixture through the muslin in the filter funnel. Carefully squeeze the muslin to assist the filtering process.
  • Label this filtrate which is in the small beaker on ice as ‘chloroplast suspension’.
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3) Set up tubes A and B

  • Label five test tubes A, B, C, X and Y and stand them in the large beaker. Put the lamp about 10 cm away so that all tubes are illuminated. Set up tubes A and B as follows:
    • Tube A - 5 cm3 DCPIP solution + 1 cm3 water + 1 cm3 chloroplast suspension. Immediately wrap the tube in aluminium foil to exclude light.
    • Tube B - 5 cm3 DCPIP solution + 1 cm3 water + 1 cm3 isolation medium.
  • Tubes A and B are control experiments. Leave both tubes until the end of your investigation.
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4) Set up tube C

  • Set up tube C as follows:
    • Tube C - 6 cm3 water + 1 cm3 chloroplast suspension.
  • Tube C is for you to use as a standard to help you to determine when any colour change is complete.
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5) Set up tube X

  • Set up tube X as follows:
    • Tube X - 5 cm3 DCPIP solution + 1 cm3 water in the tube.
  • Add 1 cm3 chloroplast suspension to tube X, quickly mix the contents and start the timer.
  • Record in seconds how long it takes for the contents of tube X to change colour from blue-green to green. Use tube C to help you determine when the colour change is complete.
    • Repeat this step four more times.
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6) Set up tube Y

  • Set up tube Y as follows:
    • Tube Y - 5 cm3 DCPIP solution + 1 cm3 ammonium hydroxide.
  • Add 1 cm3 chloroplast suspension to tube Y, quickly mix the contents and start the timer.
  • Record in seconds how long it takes for the contents of tube Y to change colour from blue-green to green. Use tube C to help you determine when the colour change is complete.
    • Repeat this step four more times.
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7) Record the results

  • Record your data in a suitable table.
  • At the end of your investigation, record the colour of the mixtures in tubes A and B.

Jump to other topics

1Biological Molecules

2Cells

3Substance Exchange

4Genetic Information & Variation

5Energy Transfers (A2 only)

6Responding to Change (A2 only)

7Genetics & Ecosystems (A2 only)

8The Control of Gene Expression (A2 only)

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