Photosynthesis Experiments

Test yourself on Photosynthesis Experiments

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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.

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.

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.

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.

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.

5) Calculate R_f

Calculate the R_f values using:
- R_f = distance travelled by spot ÷ distance travelled by solvent
Find the pigment that corresponds with the given R_f 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.

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.

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.

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’.

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.

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.

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.

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.