5A - Photosynthesis experiments

Question 1

What methods can be used when investigating photosynthesis?

Answer 1

Chromatography, TLC, calorimetry.

Question 2

What can you investigate in plants using chromatography?

Answer 2

The pigments in leaves.

Question 3

What method can you use to investigate the pigments in leaves?

Answer 3

Chromatography

Question 4

Explain differences in the photosynthetic pigments in a leaf and what this means for the plant

Answer 4

All plants contain several different pigments in their leaves. Each pigment absorbs a different wavelength of light, so having more than one type of pigment increases the range of wavelengths of light that a plant can absorb.

Question 5

Other than photosynthetic pigments, why do leaves of a plant also contain other pigments?

Answer 5

As they play other essential roles, such as protecting the leaves from excessive UV radiation.

Question 6

How are photosynthetic pigments and other pigments different from plant to plant?

Answer 6

Different species of plants contain different proportions and mixtures of pigments.

Question 7

What 2 things does TLC involve?

Answer 7

A mobile phase and a stationary phase.

Question 8

What does TLC stand for?

Answer 8

Thin layer chromatography.

Question 9

What is a mobile phase? TLC?

Answer 9

Where molecules can move. In TLC, this is a liquid solvent.

Question 10

What is a stationary phase? TLC?

Answer 10

Where molecules can’t move. In TLC, this consists of a solid (e.g. glass) plate with a thin layer of gel (e.g. silica gel) on top.

Question 11

Explain how TLC can be used to investigate the pigments in leaves

Answer 11

• A sample of pigments can be extracted from the plant and put on the TLC plate.
• When the plate is placed vertically in the solvent, the solvent moves upwards through the gel, carrying the dissolved pigments with it.
• Some pigments will travel faster or further through the gel than others, which separates them out.

Question 12

How can you identify a certain pigment from a leaf using TLC?

Answer 12

By calculating its Rf value and looking it up in a database - the Rf value is the distance a substance has moved through the gel in relation to the solvent. Each pigment has a specific Rf value.

Question 13

How do you calculate Rf value/what is it? (In TLC)

Answer 13

Rf value is the distance a substance has moved through the gel in relation to the solvent.

Question 14

Describe the process to compare the pigments present in shade-tolerant (ST) plants and shade-intolerant (SI) plants using TLC

Answer 14

• Wear a lab coat, eye protection and gloves before starting as many of the chemicals are toxic and highly flammable.

1) Grind up several leaves from the ST plant with some anhydrous sodium sulfate, then add a few drops of propanone.
2) Transfer the liquid to a test tube, add some petroleum ether and gently shake the tube. Two distinct layers will form in the liquid (top layer is the pigments mixed in with the petroleum ether).
3) Transfer some of the liquid top layer into a second test tube with some anhydrous sodium sulfate.
4) Draw a horizontal pencil line near the bottom of a TLC plate. Build up a single concentrated spot of the liquid from step 3 on the line by applying several drops and ensuring each one is dry before the next is added. This is the point of origin.
5) Once this point is completely dry, put the plate into a small glass container with some prepared solvent (e.g. a mixture of propanone, cyclohexane and petroleum ether) - just enough so that the point of origin is a little bit above the solvent. Put a lid on the container and leave the plate to develop. As the solvent spreads up the plate, the different pigments move with it, but at different rates - so they separate.
6) When the solvent has nearly reached the top, take the plate out and mark the solvent front (the furthest point the solvent has reached) with a pencil and leave the plate to dry in a well-ventilated place.
7) There should be several new coloured spots on the chromatography plate between the point of origin and the solvent front. These are separated pigments. You can calculate their Rf values and look them up in a database to identify what pigments they are.
8) Repeat the process for the SI plant and compare the pigments present in their leaves.

Question 15

What is the equation for working out RF value?

Answer 15

Rf value = B/A = distance travelled by spot/distance travelled by solvent.

Question 16

How are shade tolerant and shade intolerant plants different in terms of their structure?

Answer 16

Shade tolerant possess a different proportion of photosynthetic pigments allowing the plant to make the best use of the light available to it.

Shade tolerant plants also have a different mixture of non-photosynthetic pigments.

Shade tolerant plants have chloroplasts that are adapted for photosynthesis in low light conditions, but really sensitive to higher levels of light - these plants sometimes produce ark red and purple pigments called anthocyanins, which are thought to protect their chloroplasts from brief exposure to higher light levels.

Question 17

Why is it possible to investigate the activity of dehydrogenase in chloroplasts/where in the reaction is dehydrogenase present?

Answer 17

In photosystem I, during the LD stage of photosynthesis, NADP acts as an electron acceptor and is reduced - this reaction is catalysed by a dehydrogenase enzyme.

Question 18

What can you investigate the activity of in chloroplasts?

Answer 18

Dehydrogenase.

Question 19

Explain briefly how you can investigate the activity of dehydrogenase in chloroplasts

Answer 19

• The activity of the dehydrogenase enzyme can be investigated by adding a redox indicator dye to extracts of chloroplasts.
• Like NADP, the dye acts as an electron acceptor and gets reduced by the dehydrogenase in the chloroplasts.
• As the dye gets reduced, you’ll see a colour change (for example, the dye DCPIP changes from blue to colourless when it gets reduced).
• You can measure the rate of the dehydrogenase activity by measuring the rate at which DCPIP loses its blue colour.
• To do this, you need a colorimeter (this measures how much light a solution absorbs when a light source is shone directly through it. A coloured solution absorbs more light than a colourless solution).

Question 20

Explain a practical to investigate the effect of light intensity on dehydrogenase

Answer 20

1) Cut a few leaves (spinach works well) into pieces and remove any tough stalks.
2) Using a pestle and mortar, grind up the leaf pieces with some chilled isolation solution (a solution of sucrose, potassium chloride and phosphate buffer at pH7). Filter the liquid you make into a beaker through a funnel lined with a muslin cloth.
3) Transfer the liquid to centrifuge tubes and centrifuge them at high speed for 10mins. This will make the chloroplasts gather at the bottom of each tube in a ‘pellet’.
4) Get rid of the liquid from the top of the tubes, leaving the pellets in the bottom.
5) Re-suspend the pellets in fresh, chilled isolation solution. This is your chloroplast extract. Store it on ice for the rest of the experiment.
6) Set up a colorimeter with a red filter and zero it using a cuvette (a cuboid-shaped vessel used in colorimeters) containing the chloroplast extract and distilled water.
7) Set up a test tube rack at a set distance from a bench lamp. Switch the lamp on.
8) Put a test tube in the rack, add a set volume of chloroplast extract to the tube and a set volume of DCPIP. Mix the contents of the tube together.
9) Immediately take a sample of the mixture from the tube and add it to a clean cuvette. Then place the cuvette in your colorimeter and record the absorbance. Do this every 2mins for the next 10mins.
10) Repeat steps 7 to 9 for each distance under investigation (e.g. 15cm, 30cm and 45cm).

Question 21

If you are doing an experiment investigating the effect of light intensity on dehydrogenase activity what will you see in your results?

Answer 21

If dehydrogenase activity is taking place, the absorbance will decrease as the DCPIP gets reduced and loses its blue colour.

The faster the absorbance decreases, the faster the rate of dehydrogenase activity.

Question 22

What can you do with the results from an experiment investigating the effect of light intensity on dehydrogenase activity?

Answer 22

You can plot a graph of absorbance against time for each distance from the light source.

Then compare your results to determine how light intensity affects the rate of the dehydrogenase enzyme.

Question 23

What controls should be present when doing an experiment investigating the effect of light intensity on dehydrogenase activity?

What should the results be from these controls?

Answer 23

You should also check whether the absorbance changes at each distance in two negative control tubes.

The first should contain only DCPIP and chilled isolation solution (no chloroplast extract).

The second should contain both DCPIP and chloroplast extract, but it should be wrapped in tin foil (so no light reaches the contents of the tube).

No change in absorbance should be seen for these two controls.