This experiment was done to test the hypothesis: Boiling and decrease in light will have negative affects on the rate of photosynthesis in chloroplasts.
In this experiment we were told that we would be measuring the rate of photosynthesis. The reduction of a dye called DPIP is a part of the measurement technique. The transfer of electrons during the light-dependent reactions of photosynthesis reduce DPIP, changing it from light blue to colorless.
The information on the lab sheet said that the light is a part of a continuum of radiation or energy. Wavelengths of energy have greater amounts of energy. Wavelengths of light with in the visible part of the light spectrum power photosynthesis. Electrons within each photosystem are excited to a higher energy level and this energy is used to produce ATP and reduce NADP to NADPH, when light is absorbed by the leaf pigments. Then ATP and NADPH are used to incorporate CO2 into organic molecules. This process is known as carbon fixation.
In this experiment we were to use a dye reduction technique to study photosynthesis. We used this method to see if light reactions were taking place. The DPIP is taking place of the electron acceptor NADP. When light strikes the chloroplasts the electrons will be excited to high energy levels and will reduce DPIP. The color will change from light blue to colorless. While this occurs there is an increase in light transmittance through the solution. This increase in transmittance of light is measured by using a spectrophotometer.
Materials and Method:
The teacher provided us with an outlined procedure of the lab. The teacher started the spectrophotometer and informed us on what it does and how it operates. Chloroplasts were prepared while the spectrophotometer got ready. There was an ice bin that contained the boiled and unboiled chloroplasts at all times, except when they were placed in cuvettes. They are of incubation, a light shining through a fish bowl onto a test tube rack, was set up. The cuvettes were labeled 1-5. Each was carefully cleaned and prepared as the lab instructed. Cuvette 1 received 1 mL of phosphate buffer and 4 mL of distilled water. Cuvettes 2, 3, and 4 received 1 mL of phosphate buffer, 3 mL of distilled water and 1 mL of DPIP. Cuvette 5 received the same as 2,3, and 4 but there was an extra 3 drops of distilled water added. Cuvette 2 was covered in tin foil so that no light could enter.
Once the spectrophotometer was ready, we adjusted the amplifier control until we had a 0% readout on transmittance. We then placed 3 drops of unboiled chloroplasts into cuvette 1 and covered the top with parafilm, and tipped it upside down to mix the contents and quickly placed it into the spectrophotometer. We adjusted it to 100% transmittance. This was used to adjust the machine between readings. Cuvette 1 was placed back on the rack for a period of 5 minutes.
Cuvette 2 was our next measurement. It was taken out of the foil and 3 drops of unboiled chloroplasts were added and the top was then covered with parafilm. It was also tipped upside down to mix the contents and was placed into the spectrophotometer.. We took a reading of the percent of light transmittance and recorded the data in the data table. It was then taken out and placed back in its foil and put on the rack.
Cuvette 3 was given the same treatment as cuvette 2.
Cuvette 4 was given the same treatment as cuvette 3 except that it received 3 drops in the spectrophotmeter.
These measurements were repeated every 5 minutes, the last reading was at 15 minute. The spectrophotometer was always recalibrate with cuvette 1. Cleaning the outsides of the cuvettes is a must by the way. After the last measurement everything was cleaned and the spectrophotometer was turned off.
This is a table that contains the results from each of the four groups that were performed in the experiment. The average has been calculated for each group of measurements.
Our experiment was designed to determine whether boiling and decrease in light will have negative effects on the rate of photosynthesis in chloroplasts. Our results proved the hypothesis true.
Our results confirm with our predictions because by looking at the cuvette that had boiled chloroplast you see that it did not have a lot of photosynthesis occurring neither did the chloroplast that received no light. However the ones that were unboiled and in the light did have a high rate of photosynthesis occurring.
From our results it is possible to see that in cuvette 2 there was a small increase in % of light transmittance, from 19.5% to 26.4%. In cuvette 3 there was a very significant climb in % of light transmittance up to the 10 minute mark, from 26.6% to 98.4%. In cuvette 4 there was a very small increase in % of light transmittance, from 21.2% to 23.3%. In cuvette 5 there was actually a decrease in % of light transmittance, from 29.0% to 25.6%. These were the averages of the classes results.
Basically the boiling of chloroplasts and the decrease in light did have negative effects on the rate of photosynthesis.
Some of the groups may have gathered their data in a different way and they might have made a mistake in the paths that they had chose to complete this experiment. If they had gathered false information they would have distorted everyone’s graphs. So the class average would have been way off.
Some problems that occurred was during the recalibraion of the spectrophotometer. It seemed almost impossible to get the dial on 0% light transmittance. This would have affected the data collected from the spectrophometer.
To get more reliable results it might have been better for everyone to do the experiment one day and then have them do the experiment again the following days. This would ensure that the procedure is followed one way and it would not change and the next day would be to do the experiment again and make sure it was done properly.
The chloroplasts that were boiled had little photosynthesis occurring and the chloroplast that was not exposed to light also had little photosynthesis occurring. The boiling chloroplasts and the decrease of light will have negative effects on the rate of photosynthesis.