Purpose: The purpose of this lab was to measure the different rates of photosynthesis in isolated chloroplasts using DPIP. We used a dye-reduction technique, which shows that light and chloroplasts are necessary for the light reactions to occur. The independent variable is boiled versus unboiled chloroplasts and the amount of time spent in the light. The dependent variable is the percent transmittance. The control group is the group with no DPIP and the group with no chloroplasts. We were trying to see if the amount of time that each group of chloroplasts was left in the light would effect the percent transmittance.
For the chromatography portion, we wanted to see the distance that pigments in spinach leaves would travel with a solvent.
Introduction: DPIP was the compound we used in place of the electron acceptor NADPH in photosynthesis. The dye-reduction technique was what showed us how many reactions took place in each group. Each time the DPIP accepted electrons, or was reduced, DPIP changed from blue to colorless. This means that the more color was lost, the more reactions were taking place. The percent transmittance shows how much light was absorbed and how much was allowed to pass through.
Methods: To set up the lab we had a flood light, in front of a heat sink, then eventually our five cuvettes would be lined up behind the heat sink. We first got our Labquest 2 set up and attached our colorimeter to it. We had five cuvettes that were all filled differently with our solutions. The 1st cuvette was the blank control and we were using that to collaborate our colorimeter. That had 1 mL of phosphate buffer, 4mL of distilled water, and 3 drops of unboiled chloroplast. The next was one that had unboiled chloroplasts dark which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of unboiled chloroplasts. Cuvette 2 was then covered in foil so that no light could get through to the solution. The 3rd cuvette was unboiled chloroplasts light, which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of unboiled chloroplasts. Cuvette 4 boiled chloroplasts light which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of boiled chloroplasts. The 5th cuvette was the no chlorplasts light, which was our negative control group. This had 1 mL of phosphate buffer, 3mL + 3 drops of distilled water and 1 mL of DPIP. We set the colorimeter to 1 by using the 1st. We weren't able to put the chlorplasts in until we were about to start the procedures. So we put all the chlorplasts in th colorimeter one at a time and at 0 min we measured the transmittance for all 5 cuvettes. Then we put all five in front of the heat sink where the light was shining through. Then at 5, 10, and 15 minutes, we took the cuvettes away from the light to measure the transmittance again and then put it back after each test.
|Colorimiter being calibrated before the cuvettes were tested for time 0|
|The cuvettes and flood lamp|
Below are pictures shown from the Chromatography Lab. Here, we used cells from spinach leaves on paper, with the end dipped in a solvent, to assess the pigment distribution.
For the chromatography lab, we have measurements of each band seen in the picture above.
This means that the Rf factor for the first band is 0.14, the second band is 0.27, the third is .35, the fourth is .51, and the final is the solvent front.
Then, we have our measurements from the Photosynthesis lab. In the first chart, we have our measurements from the first round of trials. Then, we have measurements from the second round of trials. The significant differences In results will be explained.
Graphs and Charts:
Using this key, two graphs are shown. The first graph is from the first round of results, and we know these are inaccurate since transmittance can not be greater than 100%. The second graph depicts results from the second round of trials.
In our first run through of the photosynthesis lab, we immediately knew something was wrong. In our first measurements at time 0, we had transmittance readings over 100%, which is not possible. For this reason, we increased the concentration of chloroplasts from our first round of trials. This way, the reactions would not be nearly finished taking place before we even took measurements in the colorimeter. After making this change, we saw an improvement in our results. Test tube 2, with unboiled dark chloroplasts, remained fairly constant. This makes sense due to the necessity of light for photosynthesis to take place and DPIP to be used. The transmittance of test tube 3, unboiled light chloroplasts, increased over time. This also makes sense due to the fact that the chloroplasts are functioning, and provided light to fuel their reactions. Test tube 4, boiled light chloroplasts, increased initially and then decreased. This is interesting because boiled chloroplasts become denatured, and therefore do not react. The graph should be relatively flat, and more trials could be taken to determine if a mistake was made. Finally, test tube 5 had no chloroplasts, and therefore no reactions taking place, which resulted in little or no change in the amount of transmittance. Excluding test tube 4, these results turned out the way we had hypothesized.
In the chromatography lab, we used the below formula to determine the Rf factor for each pigment. The results come out different for each pigment because they are not equally soluble in the solvent. Beta carotene traveled the furthest because it was the most soluble, forming no hydrogen bonds. Xanthophyll, on the other hand, is less soluble and forms hydrogen bonds with cellulose, causing it to be found further from the solvent front. We did not have extensive prior knowledge about pigments such as these, but the information provided supported our results.
Conclusion: Cuvette 1 stayed similar in it's transmittance rates because it didn't have DPIP, and acted as the NADP+, which is the electron carrier in photosynthesis. Cuvette 2 didn't have any light going through to it so therefore there were no electrons being produced,so the color didn't change. Then Cuvette 4, which had unboiled chlorplasts which didn't cause much electron production or color change since some proteins had probably been denatured through the process of boiling them. Then, Cuvette 5 was our negative control that didn't have either type of chlorplast, so therefore it didn't have anything to react with and it basically had very similar transmittance each time like Cuvette 2 had.