Plant Pigments and Photosynthesis

4A: Plant Pigment Chromatography

In this experiment we used paper chromatography to measure the movement of pigment from plants. The mixture of solvent and pigment moves up the paper due to the attraction of solvent molecules to one another. In plants Beta carotene is the most commonly found carotene found in plants and attracted near the solvent because it has no hydrogen bonds with cellulose. The chlorophylls in plants are filled with oxygen and nitrogen and bind much tighter to the paper then the other pigments

In this experiment we wanted to use chromatography to separate plant pigments and isolate chloroplasts by using dye DPIP and then measure the rate of photosynthesis

First we got a 50mL graduated cylinder that had one cm of solvent and got a piece of filter paper that would be long enough to reach to solution. We then smashed a spinach leaf on top of the piece of filter paper with a coin to extract the pigment out. Once the pigment was on the paper we stuck it in the tube so the pigment was just above the solvent. Then we let the solvent be absorbed into the filter paper until it was about a cm from the top. Each time we noticed a pigment change me marked it and measured how far the pigment migrated until the next strand of pigment.

We resulted with a paper that had different colors at different distances from the base line.  If the pigments were farther from the line, then their color was lighter.  Each color represented a different pigment.  These pigments were Carotene, Xanthophyll, and Chlorophyll.   Our paper showed different pigments because of the bonding taking place between them and the paper.  Carotene was the farthest from the starting line because it is the most soluble and does not bond with the paper thus spreading along the paper the most.  Xanthophyll was next because it's bonds with the paper.  As a result, the distance was less than carotene since it had more resistance.  Chlorophyll bonds tightly to the paper resulting in even less distance from the starting point.  It also depended on their solubility.  If they were more soluble, they would travel up the paper faster.   Therefore, if another solvent was used, the Rf value would be different because of its solubility.  Finally, the reaction center would contain chlorophyll a.  All of the other pigments trap the light energy and send it to the reaction center.  

Distances from the base line.

1:Carotene, 2:Xanthophyll, 3:Chlorophyll a, 4:Chlorophyll b

Pigments climbing up the chromatography paper.  

4B: Photosynthesis, the Light Reactions

In this experiment we were trying to see if photosynthesis needs light and chloroplasts in order to occur. The chloroplasts were taken from spinach leaves and mixed the DPIP solution and placed in front of a light. Photosynthesis will become apparent when the color in the liquid begins to disappear due to when the light hits chloroplasts and boost high energy levels which then reduce DPIP.

In this experiment we were test if light and chloroplasts are both needed for photosynthesis to occur.

First we received two beakers with boiled chloroplasts and un-boiled chloroplasts. We set the spectrophotometer to 0% transmittance. Cuvette 2 was covered so no light could enter because it was the control group. Then we added three drops of un-boiled chloroplasts, 1mL of phosphate buffer and 4 mL of distilled H2O to cuvette 1.  Then to the remaining cuvettes 2, 3 and 4 we added 3 mL of distilled H2O and 1mL of DPIP. Then finally to cuvette 5 we added 3 mL and 3 drops of distilled water and 1 mL of DPIP Each cuvette was then placed in front of the light for 5, 10 then 15 minutes. Then we inserted cuvette 1 into the sample holder and set transmittance to 100%. We then measured how much light was transmitted through each of the other tubes. After that we put 3 drop of un-boiled chloroplasts into cuvette 2 and covered it with foil, but then removed the foil and put it in the spectrophotometer and measured the % of transmittance. We repeated this step for cuvette three and measured the % of transmittance as well. Then for cuvette 4 and 5 we added the un-boiled chloroplasts and measured the transmittance. Finally we compared the different % transmittance difference between boiled and un-boiled chloroplasts.

In this experiment we used DPIP to act as an electron acceptor which replaced NADP molecules. Our data shows that overall the dark cuvette had less activity than the others suggesting that the darkness resulted made it difficult to absorb the light. There was clearly an error with the no chloroplast cuvette because ideally, there would have been 100% transmittance or close to that for each trial because there were no chloroplasts to absorb the light. As the data shows, the unboiled chloroplasts/light and boiled chloroplasts/light had the most transmittance after 15 minutes which would suggest that they stopped absorbing light.  We could have run into errors when our logger pro machine froze midway through the experiment. It might not have given us a completely accurate reading. Also, the amount of time it took us to take our readings. Some cuvettes might have been exposed to the light for more time which would have an effect on the transmittance of light.

Percent Transmittance of Light through the Cuvettes

Cuvettes being exposed to light