The Hill Reaction and Light Wattage
Wabash College| The Effects of Light Wattage on the Rate of the Hill Reaction| | | Mark Stoops| 11/29/2012| | Introduction: In the Hill Reaction lab we will be measuring the rate of photosynthesis in light dependent reactions. The goal is to measure the change of absorbance of 2,6-dichlorophenolindophenol (DCIP) and examine the rate of the photosynthetic reactions using this data. The Hill Reaction can be used to study photosynthesis because we can directly measure the rate of the reaction of photosynthesis using DCIP.
The Hill Reaction is defined as the photo reduction of an electron acceptor by the hydrogen ions from water, which then produce oxygen. In naturally occurring reactions NADP+ is the final electron acceptor. In the Hill Reaction we will be using 2,6-dichlorophenolindophenol (DCIP) as an electron acceptor instead of NADP+. DCIP is blue in its oxidized state and is colorless in its reduced form. This change in color can be utilized. As the photosynthetic reaction proceeds the DCIP will become increasing transparent. This reduction in blue color leads to change in absorbance and can be measured by the spectrophotometer in lab.
Using the Hill Reaction, we hypothesized that the amount of light,(change in wattage) affects the rate of change of absorbance of DCIP in solution. In order to test our hypothesis we set up the experiment with three different strengths of light (15W, 60W, 120W), as well as a light free, negative control. Each run was conducted for ten minutes under similar conditions with a difference in wattage being the only variable. The negative control was conducted with no light to see how the reaction would proceed with no external influences. Having a control allows us to have a baseline of comparison for our three lighted runs.
Due to the fact that light dependent reactions use light, we can predict that an increase in the amount of light will increase the rate of reaction of photosynthesis, thus lowering the absorbance. We can also predict that our control will have no change in absorption after a ten minute period without light. Method: To begin the Hill Reaction we first isolated the chloroplasts. This was done by placing the pieces of leaves into a mortar with 15ml of ice cold NaCl-buffer. The mixture was then ground for two minutes. After grinding the leaves we filtered the solution through 8 layers of cheesecloth.
The juice was rung out and the solution put into a 15ml centrifuge tube. The solution was than centrifuged for one minute at 400xg. Then we decanted the supernatant into another clean, chilled centrifuge tube and spun it at 1000xg for 5 minutes. After the centrifuge process, we decanted the supernatant and suspended the pellet in 7ml of ice cold Nacl. This solution was kept on ice the entire time of experiment. To begin our runs we made a warm water bath for our solutions, then prepared the solutions shown in Figure 1 below. | NaCl buffer| DCIP| DI H2O| Chloroplats (on ice)| Blank| 3. 5 ml| -| 1. 0ml| 0. 5 ml|
Control | 3. 5 ml| 0. 5ml| 0. 5ml| 0. 5 ml| Reaction 15W| 3. 5 ml| 0. 5ml| 0. 5ml| 0. 5 ml| Reaction 60W| 3. 5 ml| 0. 5ml| 0. 5ml| 0. 5 ml| Reaction 120W| 3. 5 ml| 0. 5ml| 0. 5ml| 0. 5 ml| Figure 1. Experimental solutions to be prepared in lab. The blank solution was used to zero our spectrophotometer. To zero our spectrophotometer, we used the instructions provided at the spectrophotometer. To prepare the control, we added all solutions shown above and then wrapped it in two layers of aluminum foil to completely block any sources of light. After 10 minutes the control absorbance was tested to provide a negative control.
We prepared the 15W, 60W, and 120W reaction tubes immediately before each respective run to avoid light pollution. The procedure we used to test each solution was to prepare the tube and place it 25cm from the source of light. Then, turn on the light and leave it on for a minute. Then at the minute mark the light was turned off and the clock stopped. The tube was placed in the spectrophotometer and a reading was taken. Then the tube was returned to the water bath, the light turned on, and the clock started. We followed this procedure for ten times for a total of 10 minutes for each solution.
The only difference between runs was the changing of bulb wattage. Results: How does the amount of light affect the rate of reaction of photosynthesis and therefore absorbance of the DCIP solution? The data shown in Figure 2 is the results of three reaction runs and a negative control run. The time in minutes is shown on the left and the percent absorbance of the 15W, 60W, 120W, and negative control run are provided in the subsequent columns. Time (minutes)| 15W %A| 60W %A| 120W %A| Negative control| 0| 1. 1| 0. 99| 0. 89| | 1| 1. 09| 0. 945| 0. 716| | 2| 1. 08| 0. 9| 0. 55| | 3| 1. 8| 0. 815| 0. 422| | 4| 1. 07| 0. 772| 0. 322| | 5| 1. 06| 0. 702| 0. 237| | 6| 1. 07| 0. 638| 0. 176| | 7| 1. 055| 0. 578| 0. 125| | 8| 1. 05| 0. 53| 0. 088| | 9| 1. 035| 0. 464| 0. 064| | 10| 1. 025| 0. 408| 0. 032| 1. 11| Figure 2. Data values for absorbance of DCIP solutions on a one minute basis. Also shown is the negative control with absorbance taken at 10 minutes. Figure 3 shows us a visual of the data in Figure 2. Figure 3. Time in minutes versus % absorbance of 15W, 60W, 120W, and negative control runs. Figure 4. The effect of bulb wattage on rate of absorbance. Discussion:
Our results for our data runs show a common theme which is, the amount of light does have an effect on the rate of photosynthetic reaction. We can see by looking at the data in Figure 2 and depicted in Figure 3 that the amount of light has a direct influence on the rate of absorbance. The 15W run has a very small decline ending with a change in absorbance of only 7. 5%. The 60W bulb shows a change in absorbance of 58. 2%, and the 120W shows a change of 85. 8%, with a final absorption of almost 0. As shown in Figure 4, the rates of change of the 15W, 60W, and 120W runs are 0. 75%, 5. 8%, and 13. 06 % absorbance/minute respectively.
These results show that the higher the wattage, the faster DCIP turns clear, and the faster photosynthesis proceeds. Although the total change and rate of change of the 120W bulb are greatest, the reaction slows down towards the end of the run, as shown in Figure 3. This slowing of the reaction means that the amount of DCIP in its reduced state is very high, and can no longer accept electrons. This corresponds to Figure 3 because the absorbance is 3. 2% at the end. Which show a very low level of DCIP in the oxidized state. If the DCIP is no longer oxidized it can’t accept electrons which is a vital step in the light dependent reaction.
Therefore we expect to observe a slowing of the reaction, and this is seen in Figure 3. The positive and negative controls give us a reference to compare our results to. In our case the 60W run is our positive control and is used in our data runs as a part of our data. It shows a linear decline in absorbance providing a solid point of reference for a normally functioning system. The negative control provides a reference to a non-functioning Hill Reaction. The negative control shows a system without light and shows that the system will not react without sunlight. It also provides a base for 100% absorbance for each run.
In conclusion our data does support our hypothesis and our prediction. As shown in the results, a change in the amount of light will produce a change in rate of the photosynthetic reaction. We predicted that a higher wattage will increase the rate. This was indeed shown in figure 2, with the 120W bulb showing the highest rate of reaction, and the 15W bulb with the slowest rate of synthetic reaction. Also we predicted that the negative control would show no reaction without light. This was supported as shown in Figure 2 with no change in absorption over the 10 min period.
To test if the slowing of the reaction is due to a shortage of oxidized DCIP in solution, and not from high wattage, I would run each experiment again for a longer period of time. By doing this it would allow each run to reach a lower absorption. At this low absorption we would expect the rate to slow down due to the lack of oxidized DCIP. If this were true, each wattage would show the same slowing effect at low absorption. If the slowing of the reaction is not observed, the change would be due to a different reason such as a high wattage reducing DCIP’s functionality over time. References: Biology 111 Lab Manual. 2012