Laboratory Exercises on Photosynthesis

Laboratory Exercises on Photosynthesis

1.  Paper Chromatography of Photosynthetic Pigments

In this part of today’s lab, you will be separating the pigments contained in plant extract using the process of paper chromatograph. Green plants contain the following pigments

1. Chlorophyll a—this will appear as a blue-green band on the chromatography strip.
2. Chlorophyll b—this will appear as a yellow-green band on the chromatography strip.
3. Carotenes—these will appear as a yellow band on the chromatography strip.
4. Xanthophylls—these will appear as a yellow orange band on the chromatography strip.

Follow Procedure 12.1 in your lab manual.

Draw and label the pigment origin, the solvent front and each of the pigments as they appear on your chromatography strip using Figure 1. Include the name of the pigment, its relative position, and its distance from the pigment origin.

Each pigment has a characteristic Rf value.

Rf = distance moved by pigment/distance from pigment origin to solvent front.

Table 1: Rf values for Plant Pigments

Pigment / Distance
Pigment
Moved (mm) / Rf
Value
Carotene
Xanthophylls
Chlorophyll a
Chlorophyll b
Distance moved by solvent = _____ mm

Questions

1.  What two characteristics determine the distance moved by each of the pigment?

2.  Of the above two characteristics, which one decreases the distance moved? Which one increases the distance moved?

3.  What three characteristics determine the pigments solubility in the pigment and adsorption to the paper strip?

Assume Pigment A has an Rf value of .4 and Pigment B has an Rf value of .7.

4.  Which of the above pigments traveled the fastest?

5.  Which of the above pigments was most soluble in the solvent?

6.  Which of the above pigments was most closely adsorbed to the paper?

7.  Would you expect the Rf value of a pigment to change if a different solvent was used? Why or why not?

8.  Is it possible to have an Rf value greater than 1? Why or why not?

2.  Absorption of Light by Chlorophyll

A spectroscope separates white light into its component colors. Using the spectroscope provided look at the visible spectrum. Then place a sample of chlorophyll extract between the light and the end of the scope allowing the chlorophyll to act as a filter. The chlorophyll absorbed those parts of the spectrum that disappear when the chlorophyll extract is viewed.

Figure 2: Place an X on the graph showing the relative visibility of each color visible through the chlorophyll extract. Remember that as absorbance increases, visibility decreases.

Questions

1.  What colors do you see when you look through the spectroscope?

2.  What color do you expect to be visible when you look through the chlorophyll using the spectroscope? Why?

3.  What color(s) do you see when you place the chlorophyll in front of the spectroscope? Why?

4.  Which component (color) of the spectrum would be worst for growing plants? Why?

4. Fluorescence

When light strikes chlorophyll molecules, electrons are excited or raised to a higher energy level. During photosynthesis an electron acceptor picks up these high-energy electrons. In the chlorophyll extract you will be using in this experiment, the electron transfer system is disrupted. The electron is not picked up by another compound, but simply falls back to its original position. The energy released by the falling electron is released in the form of a photon. This photon is lower in energy than the photon that initially excited the electron. These photons are visible, but they have a longer wavelength and lower frequency than the initial photons. This emission of light by a substance is called fluorescence.

Place a tube of chlorophyll in front of a bright light and view the extract from the side.

Questions

1.  What color do you see when you view the green chlorophyll extract from the side?

2.  Could green chlorophyll extract fluoresce blue light? Why?

5.  Electron Transport in Chloroplasts

Follow the steps in procedure 12.2 (page 126) of your lab manual.

IMPORTANT—READ CAREFULLY—In preparing test tube four, it is vital that the tube be completely encased in foil before you add the chloroplasts and DCPIP. After adding the chloroplasts and DCPIP quickly cover any parts of the tube not already covered by foil. You do not want the contents of this tube exposed to light. Check the contents of this tube as soon as tube 2 shows a color change. It is important that you look quickly.

Record your data in Table 2.

Table 2: Detecting Electron Transfer (Oxidation/Reduction Reactions)
Tube # / Chloroplasts / Buffer* / H2O / DCPIP
(.02mM) / Initial
Color / Predicted
Final Color / Final
Color / Time
Required for
Color change
1 / 0.5 mL / 3 mL / 1.5 mL / 0
2 / 0.5 mL / 3 mL / 0.5 mL / 1 mL
3 / 0 mL / 3 mL / 1.0 mL / 1 mL
4 / 0.5 mL / 3 mL / 0.5 mL / 1 mL
* The buffer maintains the pH of the solutions at approximately 6.5.

Questions

1.  What happens to the electrons in the chloroplasts when they are exposed to light?

2.  What is the function of the DCPIP in this experiment?

3.  If the DCPIP were not present what would happen to the excited electrons? What observable phenomenon would result (see experiment 3)?

4.  What is the purpose of the DCPIP?

5.  What color is DCPIP when it is oxidized? What compound is this analogous to in photosynthesis?

6.  What color is DCPIP when it is reduced? What compound is this analogous to in photosynthesis?

7.  Is the DCPIP that is initially added to the Tubes reduced or oxidized?

8.  Explain your prediction for Tube 1. Did Tube 1 behave as predicted? Why or why not?

9.  Explain your prediction for Tube 2. Did Tube 2 behave as predicted? Why or why not?

10.  Explain your prediction for Tube3. Did Tube 3 behave as predicted? Why or why not?

11.  Explain your prediction for Tube 4. Did Tube 4 behave as predicted? Why or why not?

6.  Uptake of Carbon Dioxide During Photosynthesis

Phenol Red is an indicator solution. It is red in neutral or basic solutions and yellow in acidic solutions.

Procedure

1.  Fill 3 beakers ½ full with a dilute solution of phenol red.

2.  Blow into each beaker until it turns yellow. Stop blowing as soon as the phenol red turns yellow. Additional blowing will increase the time required to complete the experiment.

3.  Place a sprig (about 10 cm in length) of Elodea in Beakers 1 and 3. Put Beaker 3 in a drawer.

4.  Put Beakers 1 (with Elodea) and 2 (without Elodea) in front of a bright light.

5.  Record the color in Beakers 1 and 2 at 10 minute intervals throughout the lab. Do not open the drawer and look at Beaker 1.

6.  When the solution in Beaker 1 changes from yellow to red, open the drawer and record the color of the solution in Beaker 3. Close the drawer quickly. Do not recheck this Beaker until the end of class. Then record the final color.

Table 3 CO2 Uptake by Plants

Time / Color of Solutions in Beakers
1 / 2 / 3*
Before
Blowing
After
Blowing
10
20
30
40
50
*The color of the solution in Beaker 3 will only be recorded twice. Once at the point where Beaker 1 shows a distinct color change and again at the end of class.
Table 3 CO2 Uptake by Plants (cont.)
Time / Color of Solutions in Beakers
1 / 2 / 3*
60
70
80
90
100
10
120
*The color of the solution in Beaker 3 will only be recorded twice. Once at the point where Beaker 1 shows a distinct color change and again at the end of class.

Questions

1.  Why did blowing into the phenol red solution cause it to turn yellow?

2.  In which Beakers, do you expect the solution to turn back to the original red color? Why?

3.  What causes the solution in the Beakers to turn from yellow, back to red?

4.  Is CO2 used in the light dependent or the light independent portion of photosynthesis?

5.  Is there any way that placing beaker 3 in a dark place could affect the uptake of CO2 by the plant? Give careful consideration to this question and explain your answer fully. Discuss it with you instructor if necessary.

6.  What must be provided to a plant for non-cyclic photophosphorylation to occur? (Think about the overall equation for photosynthesis)

7.  What are the 3 end products of non-cyclic photophosphorylation?

8.  What is each of these end products used for?

9.  What substances from the light dependent reactions are required by the light independent portion (Calvin Cycle) of photosynthesis?

10.  What activity (present in plants and animals) is the Elodea conducting that would counteract (to some extent), the removal of CO2 from the phenol red solution?

Author: Dr. Bette Jackson, FGCU, Ft. Myers, FL 33965