To test the effects a set wavelength and different variations of light intensities had on the rate of photosynthesis, isolated thylakoids from Spinach leaves (Spinacia oleracea) were placed under different light conditions. These conditions consisted of no light, white light, green light and red light. Under each condition, the change in transmittance of a mixture, containing thylakoids and 2, 6-dichlorophenol-indophenol (DPIP), was measured using a spectrophotometer, in order to track to rate of photosynthesis. Compared to the effects of no light on the rate of photosynthesis, the thylakoids that were exposed to a white light condition experienced an elevated rate of photosynthesis. While comparing the exposure to green and red-light conditions, the rate of photosynthesis was measured to be much more elevated in that of the red-light condition. This experiment has yielded that exposure to white light and red-light conditions hold a greater effect on the rate of photosynthesis in thylakoids than that of no-light and green-light conditions. This conclusion drawn from the experiment may be useful for individuals and/or systems that wish to speed up the rate of photosynthesis, by producing ATP and NADPH in excited states, in many different forms of plant life. Therefore, replacing the oxygen being used in processes like respiration and combustion.
To determine under which light condition, no light or white light and green light or red light, the rate of photosynthesis in thylakoids was the most elevated, this experiment took light conditions and exposed isolated thylakoids from Spinach leaves to the intensities. DPIP, a blue dye, was used to replace NADPH in the light reactions of this experiment. When reduced the dye turns from blue to colorless. Therefore, since DPIP replaces NADPH, it will turn from blue to colorless. The samples were exposed to the various light conditions and then measured for color change and transmittance using a spectrophotometer, thus, allowing the rate of photosynthesis to be tracked. Due to some wavelengths of light being absorbed at higher rates than others, the hypothesis for this experiment was that the effects of white light conditions on thylakoids will allow the rate of photosynthesis to occur at a faster rate than that of the rate of photosynthesis with a no light condition in thylakoids. Furthermore, it was hypothesized that the effects of red-light conditions will allow the rate of photosynthesis to be at a much faster rate than that of thylakoids exposed to a green-light condition. This is due to the absorption of light in the green portion of the visual spectrum.
Within this study, there were 5 cuvettes tested. Before beginning, the spectrophotometer was set to measure absorption at a wavelength of 610nm. This was due to the fact that DPIP is a deep blue color and should absorb red wavelengths. In order to calibrate the spectrophotometer, a cuvette was filled to the top with deionized water. Wiping any residue off the outside of the cuvette, the cuvette was placed in the holder with the arrow facing the light source. With a closed lid, the 0.00 button was pressed, and the screen flashed 100.0 when successfully calibrated.
When preparing the materials and reagents for the experiment, a small flask, labeled DPIP, was filled with 5mL of pH 7 buffer, 15mL of deionized water and 5mL of 0.01% DPIP stock, then mixed by swirling the contents. Cuvettes, labeled 1 through 5, were wiped of any residue and 5 1.2 cm X 1.2cm squares were cut out of the Parafilm strip. To continue the experiment, 3mL of the DPIP solution was added to cuvette 1, filling it to the top line. Then, a 1.2 cm X 1.2 cm square of Parafilm was pressed to the top of the cuvette to prevent spills. The cuvette was wiped of any residue and placed into the holder of the spectrophotometer to measure the transmittance (%T). This value was displayed on the screen and was recorded in the first row of Table 1 (Time 0) for all 5 treatments. This cuvette (1) contained all experimental components except thylakoids and represents %T value of the zero-time point for all samples and was placed aside in a safe place.
With a stopwatch ready, 10 drops of thylakoid suspension were added to the DPIP flask and mixed by swirling. Moving quickly, 3 mL of the DPIP/thylakoid mixture was pipetted into Cuvette 2, 3, 4 and 5, filling them to the top line. Squares of Parafilm were then pressed to the top of each. Each cuvette was then placed on a rack along with Cuvette 1 and applied the appropriate treatments. Cuvette 2 was covered with a black paper sleeve, Cuvette 3 was not covered at all, Cuvette 4 was covered with a green filter sleeve and Cuvette 5 was covered with a red filter sleeve. Then, quickly, the rack of cuvettes was placed 8-10 inches in front of a lamp that was placed behind a glass container full of water. This water acts as a barrier to disperse the light rays from the bulb, allowing the light to be spread about and not in intense rays that could harm the samples.
Starting the stopwatch, the transmittance was measured in each sample every 5 minutes for 30 minuets. This was done by wiping the cuvette of any residue and placing it inside the spectrophotometer holder and closing the lid. With each measurement, the %T was recorded in a table. Then, once finished, the contents of the test tubes were dumped into a hazardous waste jug labeled DPIP, then rinsed with deionized water in the sink and dried by placing upside down on towel. The lamp was cut off and the experiment was finished. Information collected for each light exposure was that of % transmittance along with the time in which it was measured in 5-minute intervals.