The Hill Reaction and Light Wattage

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Wabash College|
The Effects of Light Wattage on the Rate of the Hill Reaction| |
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Mark Stoops|
11/29/2012|

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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...
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