Photosynthesis Lab

Part A

The purpose of this part of the lab is to separate and identify the pigments in chloroplast using chromatography paper, and to measure the rate of photosynthesis in separate chloroplasts.

Introduction: Chromatography paper is used for identifying and separating pigments. A solvent moves up the chromatography paper via capillary actions. This is due to the attraction of the molecules to the paper and the attraction of the pigments to each other. Paper chromatography is a way to view different pigments of plant cells. When the liquid with the pigments travels up the paper, it uses capillary action. The pigments travel at different rates up the paper, and they travel different distances as well. This is because they are not equally as soluble and have varying attractions to the to the paper through intermolecular bonds like hydrogen bonds. Beta carotene is very soluble pigment  in the solvent and doesn't form hydrogen bonds.  The pigment xanthophyll is less soluble and does form hydrogen bonds to cellulose. The pigment chlorophyll a is the primary pigment in chlorophyll.







 





This is when we took a piece of filter paper and cut it to be around 20cm long. At the end, we cut the edges of the paper off so that it made a point. We measured 1.5 cm from the tip of the point and drew a pencil line at that spot.



 

We took a leaf of spinach, and with a coin rubbed the spinach onto the pencil line drawn on the piece of paper. This causes the pigments of the spinach to be transferred on to the filter paper.



Then, we put 1mL of solvent into the graduated cylinder and inserted the filter paper so that the pigment was not touching the liquid. After we did this, the solvent started moving up the paper.

We watched the solvent travel up the paper until it was 1 cm from the top. Then, we took the filter paper out and immediately drew a pencil line to mark where the solvent was. We also marked the bottom of each pigment band that had formed.

 

 

 

This shows the measurements of  the distance of each pigment that was found.

 



This table displays the color that travelled the furthest which would reflect directly with the rate of photosynthesis.

Discussion part a:

The carotene travelled the furthest up the paper, and it had the highest Rf value. Xanthophyll had the second highest Rf factor . While chlorophyll a and b had lower Rf values. The reaction center contains chlorophyll a pigments. The other pigments capture light energy to protect against damaging effects of ultraviolet rays. Pigments are separated through the attraction of the solvent molecules to the paper. This would lead to the pigments not travelling the same distance. Our data is able to represent that photosynthesis occurs when light is absorbed. We can see that yellow travelled the furthest (112mm) and green travelled the least distance (25mm) and from this we are able to tell that green light is reflected and not absorbed. When light is reflected, photosynthesis does not occur as completely as when light is absorbed. The further the distance that a pigment travelled, the higher the rate of photosynthesis. Our data is valid because it shows the green band travelling the least distance. If we were to do this lab again and if a different solvent was used the Rf values for each would change. Since the new solvent could be attracted to each of the pigments differently which would lead to different values, since the pigments would travel up the page at different rates and distances.

Conclusion part a:

In conclusion, from our data you could see that the dark green pigment travelled the least distance (25mm) and the yellow pigment travelled the furthest (112mm). This shows that since the plant is green it will reflect green light and not absorb it. Since the plant is green it uses other colors of light (such as yellow in this case) to make photosynthesis occur. This is shown by the distance that the yellow pigment travelled.

Part b

The purpose of this part of the experiment was to see why light and chloroplasts are required in order for light reactions to occur.

Introduction: Light consists of waves of energy. Each wave of light has a different wavelengths. Wavelengths that are shorter have higher energy, while longer wavelengths have less energy. The light is taken in by the plant through their photosystems, which ultimately produces ATP while also reducing NADP to NADPH. Carbon is fixed when ATP and NADPH are used with carbon dioxide.Chloroplast used will be from spinach leaves. The electron acceptor will be DPIP. Light striking the chloroplast should boost the electrons to higher energy levels reducing DPIP. DPIP becomes colorless as it reduces or gains electrons. This results in an increase of light transmittance which will be measured via a spectrophotometer.

This photo depicts us preparing the chloroplast solution by adding DPIP and water (and buffer to the first) to the chloroplast.

This photo shows the spectrophotometer where we placed the cuvettes in to measure the amount of transmittance for each cuvette.

Our graph shows that the unboiled chloroplasts had the highest percentage of transmittance even though the unboiled dark percent transmittance was close.

 

Discussion part b:

    In each of the cuvettes, the percent transmitted was effected by the amount of time that they were exposed to the light. Before we exposed the solutions to light, the chloroplasts that were boiled and exposed to light were the ones which had the highest percent transmittance. After all the cuvettes were exposed to the light for 5 minutes, all of their percent transmittance increased. However, the amount that they increased varies significantly. The cuvettes with “unboiled/dark” and “unboiled/ light” increased the most at a change of 17.65% and 19.53%. The remaining two, “boiled/light”, and “no chloroplasts/light” only increased by 1.96% and 1.31%. After being left in the light for an additional 5 minutes after this reading, the percent transmittance increased, but not as drastically as before. The highest percent transmittance was 5.18%, which was for the “unboiled/dark” chloroplasts. For the final time, we left the cuvettes in the light for 5 minutes, and this time not all of the chloroplasts increased their percent transmittance. “Unboiled/dark” chloroplasts decreased percent transmittance by 1.35%. “Unboiled/light” chloroplasts also decreased percent transmittance by 3.21%. “Boiled/light” chloroplasts also decreased at 1.05% “no chloroplasts/light” was the only cuvettes increased their percent transmittance, but only at     .33%.

   Although our graph depicts that the unboiled light chloroplasts had the highest rate of transmittance, the unboiled dark were fairly close, which isn't accurate. Changing of the foil for the “unboiled/dark” chloroplasts could have skewed our data, resulting in a higher transmittance. When taking the foil on and off before and after testing, some light could have gotten in. Since light effects the amount transmitted, this could have caused more transmittance than actually exists. If we were to do this lab again, we would have to be sure to take the foil on and off with as little light exposure as possible. We would also have to make sure that the entire solution is covered by the foil, to prevent the light from entering. This may have also been a problem, especially if the tinfoil did not completely cover it while it was exposed to the light.

Conclusion:    In conclusion, the unboiled light chloroplasts should have the highest percentage of transmittance because we have not denatured the chloroplasts and it's in a more natural environment. Overall, the unboiled dark chloroplasts should have had the smallest change in transmittance because light would not be able to get through which would allow photosynthesis to occur. The boiled light chloroplasts and no chloroplasts have little change, as our graph shows, since photosynthesis cannot occur when the chloroplast is denatured or there are no chloroplasts.