Cool Photosynthesis Lab

The main idea with chromotography paper is to be able to see the separation of pigments. In this case we are seeing the variety of pigments found in the chloroplast to see how it helps with photosynthesis. As seen below with the paper strip, we can see the wide variety of pigments being separated and then the pencil mark is there the solvent ends. The very bottom of the paper is a dark green color and as it progresses to the top it begins to get lighter and lighter. The dark green color can be concluded to be chloroplast-a which is the main pigment used to absorb every light except for green. That has a low Rf value compared to Carotene which has an Rf value of 1 because it flows along with the solvent. What factors can play a role in the distance travels can be based on the polarity of the pigment or even the intermolecular forces of the pigment. The more complex and stronger it in hydrogen bonds, then the harder it is to move along the strip. This concept is great to think about in term of thinking about why a plant needs off of these complex pigments in the chloroplast and how Rf and absorbance and relatively be related. It can be concluded that the lower the Rf, the higher the absorbance. The rest is to be seen in the following data and lab procedure. 

The relationship between absorbency and transmittance is important to understand photosynthesis. In order to calibrate the colorimeter we put regular tap water in a cuvette and zeroed the absorbance and transmittance values. This served as a baseline value for the rest of the solutions.  

This is a picture of the colorimeter that we used.

In order to find this relationship we put a 100% blue dye solution in cuvette and put it in a colorimeter to test the amount of light that absorbed and transmitted. 

This is a picture of the 100% blue dye solution for trial one. 

We then followed these same steps but instead of a 100% blue dye solution, we diluted it to 50%, 25%, 12.5%, 6.25%, 3.125% and again measured the absorbable and transmittance values. 

This is a data table of the values we have obtained. 

From this data table we can conclude that the values between absorbency and transmittance have an inverse relationship.

In the next part of the lab, we conducted trials with either boiled or unboiled chloroplasts (dead or living) and we controlled whether or not the solution was exposed to light or not. This was done in order to observe the effects of how changing these factors would effect the rate of photosynthesis. 

First we made five different test tubes with the following contents:

Test tube one with 1mL phosphate buffer, 4mL distilled water, and 3 drops of unboiled (alive) chloroplasts

Test tube two with 1 mL phosphate buffer, 3 mL distilled water, 1 mL DPIP, and 3 drops of unboiled (alive) chloroplasts

Test tube three with 1mL phosphate buffer, 3 mL distilled water, 1 mL DPIP, and 3 drops of unboiled (alive) chloroplasts 

Test tube four with 1 mL phosphate buffer, 3 mL distilled water, 1 mL DPIP, and 3 drops of boiled (dead) chloroplasts 

Test tube five with 1 mL phosphate buffer, 3 mL+3 drops of distilled water, and 1 mL DPIP

Then we poured the solutions into cuvettes for testing in ur colorimeter to test the change in their transmittance over time. 

Going from left to right, these are cuvettes  1 to 5 

The first cuvette was done in order to calibrate the colorimeter and set a baseline for comparison for the rest of the cuvettes. 

We exposed all the cuvettes to light using the 2 liter Erlenmeyer flask filled with water in front of a heat lamp so that heat wouldn't affect the chloroplasts, but only the light. To simulate dark reactions for cuvette two we wrapped the cuvette in aluminum foil. This prevented light from entering the cuvette. 

This is a picture of our heat lamp and Erlenmeyer flask set up. 

This is a data table of the values we have obtained. 

Unfortunately due to an error with the lab equipment, some of the data was not stored correctly and we could not retrieve it. 

However, in a later discussion of what our results should have been we found that all the cuvettes except the third one should have had steady transmittance. Cuvette three was the only one that should have increased transmittance since it had all the requirements for photosynthesis to occur. As photosynthesis progressed, DPIP would change from blue to colorless because the chloroplasts would reduce the DPIP. As DPIP changes from blue to colorless, the colorimeter perceives it as an increase in the solution's transmission of light. 

The function of DPIP in this experiment is to act as a primary electron acceptor. DPIP replaced P680 as an electron acceptor. The source of electrons for DPIP was the distilled water. 

The colorimeter measured the absorbence and transmittance of the solution in each cuvette.

The darkness doesn't allow for the light to excite the electrons so DPIP cannot be reduced and in turn cannot gain electrons. 

Boiling the chloroplasts kills them causing photosynthesis to not occur. Which means that DPIP cannot occur because there is nothing for it to accept. 

Live chloroplasts kept in the light will have photosynthesis occurring which means they give off electrons for DPIP to accept. As DPIP accepts electrons it's reduced and turns from blue to colorless, so it's transmittance increases. If there's no light, photosynthesis can't occur so there's no electrons for DPIP to accept so DPIP can't reduce which results in the color not changing, and so the transmittance doesn't change. 

The purpose of cuvette one was to serve as a calibration of the colorimeter and to tell the colorimeter what 100% transmittance looks like. 

Cuvette two deprived the chloroplasts of light. This shows us that deprivation of light hinders the rate of photosynthesis. 

Cuvette three contained all the necessary requirements for photosynthesis to occur, living chloroplasts and light. 

Cuvette four contained all the necessary requirements for photosynthesis to occur except living chloroplasts. This tells is that death of chloroplasts hinders photosynthesis. 

Cuvette five contained no chloroplasts which tells us that having no chloroplasts hinders photosynthesis.

Cuvette five specifically behaved as a double negative control to show that DPIP does not reduce on it's own. If it did reduce on its own, that that would reduce the reduction that photosynthesis contributed to DPIP. 

This labs shows us that photosynthesis occurs only when chloroplasts are alive and are exposed to light and transmittance and photosynthesis have a direct relationship.