Photosynthesis: Hill Reaction, A Hypertonic Analysis

The following paper was submitted in a Biology 101 Lab session at the University of North Carolina at Chapel Hill. It is not to be copied or taken from without reference and contacting the author.


Photosynthesis, represented in a chemical reaction as H20 + C02 –(light)–> (CH2O)n + 02, is one of the most important processes in plant life and chloroplast function plays a huge role in producing food for those plants. During photosynthesis, chloroplasts convert the water and carbon dioxide (using the energy from a light source) into food that the plant can use and gives off oxygen in the process.

The absorption of the light from the light source occurs in the thylakoid membrane of the chloroplasts. The formation of carbohydrates for use within the plant (also known as the synthesis reaction) takes place stroma. The stroma is a fluid substance in the chloroplasts that surrounds the thylakoids.

The Hill Reaction was focused on mainly for our experiment. Hill wanted to know if light was the only reason that photosynthesis took place or if it was another chemical phenomenon that was happening at the same time to further the production of oxygen and food for plants in the chloroplasts. He took a solution known as DPIP and used it in conjunction with the chloroplasts and placed them in a dark area. The solutions were blue as a result of the DPIP being added and turned out to be clear once the solutions were removed from the darkness. The colored DPIP was used instead of a hydrogen ion in photosynthesis, thus turning the solution clear after it had been processed. Hill concluded that the following equations were true as a result of his successful experiment: 2DPIP + 2H20 + Chloroplasts –(light)–> 2DPIPH2 + O2 and DPIP (blue) + e- –> DPIP (colorless).

In this experiment, we will be testing chloroplast function in various levels of hypertonic solutions. Saltwater concentration will be altered for the solutions and spinach will be the supplier of the chloroplasts in question. This experiment will be carried out to assess how well plants can function in environments in which they are receiving water from a hypertonic source. If chloroplasts are exposed to light in a hypertonic solution, then they will complete less photosynthesis than if they were in an isotonic solution. This trend will continue depending on the molarity of salt in the water, meaning that the higher the molarity, the less photosynthesis there will be taking place within the chloroplasts of the spinach.

Materials and Methods

To begin the experiment, we turned on the spectrophotometer by using the POWER switch on the back of the machine. We allowed the machine around 10 minutes to fully warm up, however, if the spectrophotometer was already on, we would not have waited. The wavelength of light used for this experiment was 605 nm since the blue solution (referenced later as DPIP) allowed the greatest amount of the orange light that 605 nm produced to pass through. We left the sample holder empty and the lid closed while proceeding through the experiment until it was needed so as not to skew the results by letting in outside light, which could have led to a miscalibration of the 605 nm wavelength selection. Four test tubes were numbered 1-4, respectively. All test tubes handled in the lab were handled with Kimwipes so as to not leave residue or anything else on the outside of the test tube that may block light and alter results of the transmittance test. Next, our TA prepared a chloroplast solution for use in the experiment as detailed in the next paragraph.

The unboiled chloroplast solution utilized chilled spinach leaves that had been kept in a refrigerator for more than a few hours. The spinach leaves were kept in this dark, cold location to minimize the amount of photosynthesis taking place within the leaves. Stems were removed from each spinach leaf since most chloroplasts are located within the leaf and not the stem and the leaves were placed under a bright light to ‘jump start’ photosynthesis in the chloroplasts. Photosynthesis was initiated at this point so that they would be most active in our solution and ultimately provided better results when their productivity was analyzed in our collected data. Our TA poured 0.5M chilled saltwater into a blender that had also been kept in the refrigerator with the spinach. The saltwater concentration was set at 0.5M, as this is isotonic to the cells in the spinach leaves. The chilled saltwater and cold blender worked towards the same goal of keeping photosynthesis as a result of heat at a minimum during the experiment. Too much heat at any time during the experiment may have caused the chloroplasts to become denatured and hinder the results that we received. Our TA blended the chloroplast solution for three short bursts of ten seconds so as not to heat the blender up too much, while pausing in between each burst. Once the chloroplasts were blended, our TA poured the chloroplast solution through a cheesecloth and into a cold beaker. The cheesecloth strained the particles and allowed only the liquid and chloroplasts to pass through. Again, the beaker was kept cold to avoid denaturing the chloroplasts while preparing the solution. The chloroplast solution was then poured into a light-blocking vial and placed on ice in a cooler. The light-blocking vial kept light from activating excessive photosynthesis within the chloroplasts so as to provide better results when the chloroplasts were tested for the first time in the spectrophotometer.

Next, we added a phosphorous-based buffer, various molarities of saltwater solutions, and DPIP to the appropriate test tubes. See Table 1 in the appendix for the amount of each substance and solution added to each test tube. DPIP, as stated in the introduction, turned our solutions a dark blue. We used this substance to take the place of hydrogen ions in the photosynthesis that took place within the spinach chloroplasts to measure their efficiency and effectiveness in different molarities of saltwater. After the three substances were added to their respective test tubes, we also added 3 drops of cooled chloroplasts from the icebox to each of the test tubes.

We added only 0.5M saltwater, chloroplasts, and the buffer to another tube to allow it to be our calibration tube. This tube had no DPIP so as to give us a result of what 100% transmittance would be without it hindering the light passing through. The calibration tube was covered with Parafilm and flipped so as to not lose any of the solution inside while still allowing us to mix it well. Once flipped back, the Parafilm was removed and the calibration tube was placed into the spectrophotometer. To provide a standard to compare the other tubes against, the calibration tube acted as a balance for the device to give a base reading. We pressed the balance/tare button and the machine set itself to 100% transmittance given the solution inside. We left this measurement of 100% transmittance the same for each trial before recalibrating the spectrophotometer. The test tube was placed on a test tube rack in the path of a desk lamp to activate photosynthesis but set behind a beaker of water so excess heat from the lamp would not reach the test tube. Immediately after we added the chloroplasts to the other three test tubes, a piece of Parafilm was stretched over the top of each tube to keep the contents secured while we flipped them over to mix all of the solutions inside. The Parafilm was removed and each test tube was placed into the spectrophotometer one by one. The reading displayed on the screen was recorded for the transmittance value of each test tube at the time 0 slot in our data table. After testing each tube, they were placed beside the calibration tube in the light of the test tube rack to undergo photosynthesis. All steps that involved calibration of the spectrophotometer and transmittance measurements were repeated after the test tubes had been sitting in the light for five, ten, and fifteen minutes as well to gather data for how effective the chloroplasts were at photosynthesis over time in their respective saltwater solutions.


After completing the experiment, we gathered our data into a table and graphed it as well to show the correlation of percent transmittance over time that the chloroplasts were exposed to light and utilizing the blue DPIP in their photosynthesis procedure. In Figure 1, located in the appendix, there are three lines representing the three test tubes that underwent consideration for support or rejection of our hypothesis. A line graph was chosen over a bar graph since we were tracking changes over time and not comparing different groups of data at a single time. Test tube 2 (the control tube) started at just over 20 percent transmittance and rose to 21.8 percent transmittance before slightly falling again at the fifteen-minute mark of our experiment. Test tube 3 (holding 2M saltwater) started at 30.9 percent transmittance and rose to an astonishing 59.9 percent transmittance after fifteen minutes had passed. Test tube 4 (holding 4M saltwater) began at 42.8 percent transmittance and rose before flattening off and becoming stagnant at 46.9 percent transmittance (all data in Table 2).

Using a formula, I determined the rate of increase of photosynthesis over the fifteen minutes that we were observing the chloroplast-infused substances. The rate was determined by the formula: Rate = change in %T / change in time. Test tube 2 was calculated at a rate of 0.06 %T per minute, test tube 3 was calculated at a rate of 1.93 %T per minute, and test tube 4 was calculated at a rate of 0.27 %T per minute.


Transmittance of the solutions was measured to calculate the activity of the chloroplasts over the fifteen-minute period that data was being collected. This means that, the less the transmittance change was during the experiment, the less productive the chloroplasts were in the solution. These rates show that, the order of photosynthesis productivity followed the order of test tube 2 chloroplasts having the most activity, test tube 3 chloroplasts having the second most activity, and test tube 1 chloroplasts having the least activity of the samples tested.

Unfortunately, our hypothesis was rejected by this experiment based on the results stated above. For our hypothesis to have been supported, the higher molarity tubes of saltwater containing chloroplasts should have had a less %T per minute than the lower molarity test tubes, signifying that the more hypertonic a solution is, the less photosynthesis takes place. However, tube 3 with 2M saltwater had the most %T per minute while tube 2 with 0.5M saltwater had the least %T per minute. This is the opposite of what our hypothesis states and therefore I must reject our thoughts.

An error that may have resulted in the outcome of this experiment could have originated from the fact that we failed to successfully create a control in the experiment and used data from a previous experiment with the same configuration of test tube. Another source of error may have been the amount of time that the test tubes were exposed to light before the testing actually began. If one test tube was exposed to outside light and room light longer than another test tube before the experiment of transmittance values began, this may have dramatically adjusted the results.

The control tube in our experiment contained a 0.5M saltwater solution that was isotonic to the chloroplast cells in the spinach leaves used in our experiment. This control was utilized to provide something to compare the two other solutions to, in order to determine if our hypothesis involving hypertonic solutions and their effect on photosynthesis proved to be correct. The control tube should have produced the greatest rate of photosynthesis and thus provided a base to compare what percent of potential effectiveness at which the other solutions were operating.


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