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Photosynthesis Part I

Using a spectrophotometer with a DPIP indicator, we tested the dependence of the rate of photosynthesis on light and chlorophyll. Our data affirms our hypothesis that the rate of photosynthesis depends on both light and chlorophyll.

Introduction

The purpose of the Photosynthesis Lab was to find if the rates of photosynthesis depended on light and on the presence of chlorophyll. We hypothesized that the rate of photosynthesis depended both on the presence of chlorophyll and light.

The process of non-cyclic photosynthesis reduces NADP+ to NADPH. Using antenna pigments, chlorophyll a collects electrons and passes them down an electron transport chain. At the end of this chain, NADP+ is reduced by the free electrons.

Our detection mechanism for photosynthesis was DPIP (2, 6-dichlorophenol-idophenol). DPIP will become reduced before NADP+. DPIP, in its unreduced state, is blue in color but changes, when it is reduced, to clear, allowing a spectrophotometer to read the transmittance of the sample. Thus, if photosynthesis is occurring, DPIP will become clear and the percent transmittance will go up.

Percent transmittance is exponentially proportional to the concentration of the substance being tested. If we had data of different known concentrations of DPIP, we could interpolate to find the concentration of DPIP in an unknown concentration.

Procedure

The blank cuvette, used to calibrate the spectrophotometer before measurements was filled with 1mL phosphate buffer, 3mL distilled water, 3 drops of unboiled chloroplasts, and 1mL of distilled water to substitute for the DPIP. The dark sample had the 1mL DPIP and was kept in an aluminum foil jacket and cap to keep the solution in the dark. The unboiled sample was exactly like the dark sample except that it did not have a aluminum foil jacket. Finally, the boiled sample used 3 drops of boiled chloroplasts instead of unboiled chloroplasts. The boiled chloroplasts should have denatured cholorophyll which means there is no active chlorophyll present.

The cuvettes were loaded, mixed, and their "0 time" transmittance at 605nm measured. The light, behind a flask of water to stop infrared light, was then turned on a five minutes was timed. Readings were repeated at five minute intervals along with recalibration and mixing. The readings generally took about 20 seconds per a cuvette. The rack remained in front of the lamp even during the measurement and calibration procedure. The cuvette rack was shifted every minute or so to eliminate error from any focusing of the light by the water of the flask. The elapsed time from the loading of the cuvettes was measured so that the time disparity inherent in the serial measurement of the cuvettes could be compensated for.

We started the timer 1 minute and 35 seconds before the lamp was turned on. The timer was not stopped and the times of each measurement recorded to allow for further analysis.

Data


Time (min)  Dark    Unboiled   Boiled   

0           7%      7%         8%       

5           7%      19%        8.5%     

10          7%      33%        8.5%     

15          8%      47%        9%       



[See Appendix for Calculations and Detail of the Data Collected]

Results and Discussion

[See Appendix for Graph]

Neither the boiled nor the dark samples displayed any appreciable reactions. The unboiled sample showed significant photosynthetic reaction. This data does affirm our hypothesis that the rate of photosynthesis depends both on light and chlorophyll. The data is congruous with the established claim that chlorophyll is at the center of the photosystem and that photosynthesis requires light.

There is not enough data on various DPIP concentrations to make assertions about the DPIP concentrations from the percent transmittance data. Further tests could quantify the slight changes in percent transmittance in the dark and boiled samples.

Possible sources for error include the use of four different cuvettes. Each cuvette could have a slightly different scratch or imperfection that would impair readings. Timing in this lab was critical. Because we could not instantly measure all the cuvettes at once, our samples were not treated exactly equally. In order to measure percent transmission, we had to expose our dark sample to light. The precision of the spectrophotometer is 0.5%. Because we only took four readings, it is questionable whether we can use our data to make predictions of any significant accuracy. Most likely, all the errors present in our experiment fall within the tolerances of the spectrophotometer used.

Conclusions

  1. The dark sample did not reduce the DPIP as shown by the transmittance percentage. Had it reduced DPIP, it would have had increased transmittance, telling us that the unreduced, blue DPIP was being reduced.
  2. The boiled sample showed no appreciable reduction of DPIP. The transmittance percentage did not rise. Thus the concentration of unreduced DPIP remained rather high, thus blocking the passage of light.
  3. The difference in the percent transmittance of the unboiled sample and the dark sample shows the dependence of the reduction of DPIP on light.
  4. a) Tube 1 served as a calibration tube so that any change in the plant chlorophyll color could be compensated for, when we recalibrate the spectrophotometer. We are not interested if chlorophyll changes color or transmittance over time. This tube allows us to factor those changes out.
    b) It contained everything (buffer and chlorophyll) so that if any of the contents (buffer and chlorophyll) changed color, we could use this tube to set the spectrophotometer to ignore the changed color or transmittance.
    c) If tube 1 got darker and we did not reset the spectrophotometer each time, all the points on the graph would shift down. The darker tube 1 tells us that the plant pigments in tube 2, 3, and 4 are also getting darker. The darkness impairs the transmission of light; thus the percent transmission will be reduced. Regardless, the differences between the samples would still be conspicuous because the samples act as mutual controls.