Photosynthesis Investigation
Excerpts from: Carolina and Vernier Lab
Purpose: In this investigation you will use spectroscopy to compare the light reactions of photosynthesis of chloroplasts under varying conditions.
In this experiment, you will
• Use a Spectrometer to measure color changes due to photosynthesis.
• Study the effect of light on photosynthesis.
• Study the effect that the boiling of plant cells has on photosynthesis.
• Compare the rates of photosynthesis for plants in different light conditions.
Background: In the light reactions of photosynthesis, light energy is absorbed by chlorophyll and used to excite electrons. The excited electrons then enter one of the two electron transport chains. One chain consists of enzymes that convert adenosine diphosphate (ADP) and another phosphate to adenosine triphosphate (ATP). The other electron transport chain converts nicotinamide adenine dinucleotide phosphate (NADP+) and a hydrogen to NADPH. In this activity, you will use DPIP (2,6 – dichlorophenol-indophenol – a blue dye) as an indicator of the light reaction. The DPIP, when added to a suspension of chloroplasts, will react like NADP+ as follows: DPIP + H à DPIPH. DPIPH is colorless, so as the light reactions take place, the blue color of the solution diminishes. The rate of the color change indicates the rate of the light reaction.
Spectrophotometers
You will use a spectrophotometer to measure the color change of DPIP. You will prepare a sample by adding a chloroplast suspension, DPIP, and a buffer to water in a vial called a cuvette. The instrument works by shining a beam of light of known intensity through the cuvette. On the opposite side from the light source is a photoresistor that detects the intensity of light that has passed through the sample. DPIP will absorb some of the light. Since DPIP absorbs light most strongly at orange-red wavelengths, you will set the spectrophotometer to read the amount of light transmitted in that part of the spectrum (605nm).
Prelab Questions
1. How does DPIP measure the rate of the light reactions?
2. What will happen to the solutions of DPIP and chloroplast suspension if the chloroplasts are actively carrying out light reactions?
3. What is your hypothesis for each of the conditions you will test?
4. Which cuvette serves as a control for this experiment? Explain your answer.
5. What variables are tested in this experiment? Describe how each variable is tested.
6. Create data table.
Procedure:
1. Obtain and wear goggles.
2. Obtain four plastic Beral pipets, four cuvettes and one aluminum foil covered cuvette. Mark one Beral pipet with a U (unboiled), one with a B (boiled), one with DPIP, and one with W(water).
3. For the cuvettes, mark:
1. Blank
2. Aluminum foil one D (dark, unboiled)
3. U (light, unboiled),
4. B (light, boiled),
5. No Chloroplasts
4. Turn-on the spectrophotometer. Some require a warm-up period. Calibrate by preparing a cuvette ¾ filled with distilled water. To correctly use a cuvette remember the following:
a. Wipe the outside of each cuvette with a lint-free tissue
b. Handle cuvettes only by the top edge of ribbed sides
c. Dislodge any bubbles by gently tapping the cuvette on a hard suface
d. Always position the cuvette so the light passes through the clear sides
Next connect the Spectrometer to your Veneer device. Choose NEW FILE from the menu. Place the blank cuvette in the slot and choolse Calibrate. The dialog box will display: “ Waiting 90 sec for lamp to warm-up” After 90 seconds, the message will change to warm up complete and then click finish calibration.
5. Determine the optimum wavelength for examining DPIP (should be close to 605nm).
a. Empty blank and fill it with 1mL of phosphate buffer, 1 mL of distilled water,1mL of DPIP, and 3mL of the unboiled chloroplast. Place in spectrometer.
b. Click collect, a full spectrum graph of the solution will be displayed – click stop.
c. To select a wavelength for analysis, click the Configure Spectrometer Data Collection icon, , in the toolbar.
d. Select Absorbance vs. Concentration (under the Collection Mode). The wavelength of maximum absorbance (λ max) will be selected. (It should be close to 605 nm.) Click . Remove the cuvette from the Spectrometer and dispose of the solution as directed.
6. Obtain a 600 mL beaker filled with water and a flood lamp. Arrange the lamp and beaker as shown in Figure. The beaker will act as a heat shield, protecting the chloroplasts from warming by the flood lamp. Do not turn the lamp on until Step 10.
7. Locate the unboiled and boiled chloroplast suspension prepared by your instructor. Before removing any of the chloroplast suspension, gently swirl to re-suspend any chloroplast which may have settled out. Using the pipet marked U, draw up ~1 mL of unboiled chloroplast suspension. Using the pipet marked B, draw up ~1 mL of boiled chloroplast suspension. Set both pipettes in the small beaker filled with ice at your lab station to keep the chloroplasts cooled.
8. Add 1 mL of phosphate buffer solution to each of the cuvettes and 3mL of distilled water (4mL to the blank cuvette). Then add 1mL of DPIP to all the cuvettes except the blank. Important: Perform the following steps as quickly as possible and proceed directly to Step 9.
a. Cuvette U: Add 3 drops of unboiled chloroplasts. Place the lid on the cuvette and gently mix; try not to introduce bubbles in the solution. Place the cuvette in front of the lamp as shown in Figure 2. Mark the cuvette's position so that it can always be placed back in the same spot.
b. Cuvette D: Add 3 drops of unboiled chloroplasts. Place the lid on the cuvette and gently mix; try not to introduce bubbles in the solution. Place the foil-covered cuvette in front of the lamp as shown in Figure 2 and mark its position. Make sure that no light can penetrate the cuvette.
c. Cuvette B: Add 3 drops of boiled chloroplasts. Place the lid on the cuvette and gently mix; try not to introduce bubbles in the solution. Place the cuvette in front of the lamp as shown in Figure. Mark the cuvette's position so that it can be placed back in the same spot.
d. Cuvette No Chloro: do not add any chloroplast solution (should just be the phosphate buffer, water, and DPIP. ADD 3 additional drops of water.
9. Take absorbance readings for each cuvette. Invert each cuvette two times to resuspend the chloroplast before taking a reading. If any air bubbles form, gently tap on the cuvette lid to knock them loose.
a. Cuvette U: Place the cuvette in the device (close the lid if using a Colorimeter). Allow10 seconds for the readings displayed in the meter to stabilize. Click and record the absorbance value in Table. Remove the cuvette and place it in its original position in front of the lamp.
b. Cuvette D: Remove the cuvette from the foil sleeve and place it in the device (close the lid if using a Colorimeter). Wait 10 seconds and record the absorbance value in Table 2. Remove the cuvette and place it back into the foil sleeve. Place the cuvette in its original position in front of the lamp.
c. Cuvette B: Place the cuvette in the device (close the lid if using a Colorimeter). Wait 10 seconds and record the absorbance value in Table 2. Remove the cuvette and place it in its original position in front of the lamp.
10. Turn on the lamp.
11. Repeat Step 9 when 5 minutes have elapsed.
12. Repeat Step 9 when 10 minutes have elapsed.
13. Repeat Step 9 when 15 minutes have elapsed.
14. Repeat Step 9when 20 minutes have elapsed.
Data
Turn-in all observations, data table, and graph.
Plot the data from your table. Title the graph and label the info.
a. Independent Variable is: ______
b. Dependent Variable is: ______
Post Lab Analysis:
1. Why was DPIP not added to the blank cuvette?
2. Why were 3 drops of water added to the No Chloroplast cuvette?
3. Is there evidence that chloroplasts were able to reduce DPIP in this experiment? Explain.
4. What effect did boiling have on the chloroplast solution?
5. Were chloroplasts able to reduce DPIP when kept in the dark? Explain.
6. What conclusions can you make about the photosynthetic activity of spinach?