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Photosynthesis is the conversion of light energy to chemical energy which is stored in glucose and other organic compounds, occurring in all plants. One way to study photosynthesis is through paper chromatography. The pigments within a plant can be studied and analyzed by applying a method known as paper chromatography. In applying this method, molecules can be broken apart from one another by analyzing their characteristics. By applying such means, these divided molecules can be easier to identify. Chromatography includes various parts such as a solute. A solute is a solution in which a substance becomes dissolved in, which carries a variety of pigments which is carried upward on a piece of paper. This rising of the solute is performed through capillary action. Capillary action occurs due to the attractions among the paper and the solvent molecules. As the solvent moves upward the paper, it carries the pigment along. Yet, these pigments travel at varying speeds, since each pigment has varying solubility and attraction to fibers contained within the paper. While some pigments form stronger intermolecular bonds, others form weaker intermolecular bonds. These bonds are mostly hydrogen bonds. In addition, the pigments that are more soluble usually move higher up on the paper, as the solute allows the pigment to stay existing for a longer period of time. In contrast, pigments with lower solute levels usually travel a shorter distance upward on the paper.
Chlorophyll a is the crucial pigment that forms approximately seventy five percent of the pigmentation in all living plants. Chlorophyll b contains approximately twenty five percent of the pigmentation in all living plants. In addition, carotenes and xanthophylls are known as accessory pigments, which makes up the rest of all pigmentation. Finally, the pigments chlorophylls a and b reach the highest point on the paper, since such chlorophylls bind tightly to the paper. Because of this, these pigments to travel slowly.
Beta carotene, the most abundant carotene found in all plants, is the most soluble of all pigments. Because it also forms no hydrogen bonds with cellulose this allows it to be capable of being carried the furthest by the solvent. The paper displays a spectrum of the different pigments contained inside the spinach leaves. Using the formula with constant Rf, the relationship between the distance of the solvent traveled to the distance the pigment traveled can be calculated.
Another pigment, Xanthophyll is different from carotene because it has no oxygen. Xanthophyll is found further from the solvent because it is not as soluble in the solvent and has been slowed down by hydrogen bonding to the cellulose.
By using unboiled and boiled chloroplasts, soaked in phosphate buffer, distilled water, and DPIP, and then carefully added into small cuvettes. The control cuvette was covered with aluminum foil, as a barrier, so that no light could pass into the cuvette. Light absorbance was analyzed and noted every five seconds for an entire duration of twenty minutes, while the cuvettes were placed in front of a flood lamp. In this experiment, the DPIP (2, 6-dichlorophenol-indophenol), replaced NADP+ as an electron acceptor.). To be reduced means that the electrons got added, and to be oxidized, means the electrons got taken away. When this DPIP becomes reduced, the solution turns colorless, and as a result, was not able to soak in as much light. In contrast, when the DPIP becomes oxidized, the solution transforms into a shade of blue, which allows a high percent absorbance. A colorimeter was very used throughout the experiment to measure how much light was absorbed by each solution.
I hypothesize that as time passes by, the pigments will spread out on the filter paper. I hypothesize that the rate of photosynthesis in plant cells is controlled by the light it attains and the temperature it is in. I hypothesize that the beta carotene will go highest up on the filter paper, while cholorophylls a and b will be last, with xanthophylls in the middle. I hypothesize that the dark cuvette (D) and the cuvette with the boiled chloroplasts in the light (B) will have minimal changes in the reduction of DPIP; while, the cuvette with chloroplasts that are unboiled in the light (U) will have the highest reduction of DPIP.
To being the experiment, we turned on the provided flood lamp, and placed a 600mL beaker of water in front of it to assure that the chloroplasts do not get warmed up from the flood lamp. A vial with approximately one centimeter of solvent in the bottom was obtained and the lid was kept as on as much as possible due to the volatility of the provided solvent. One end of a piece of filter paper was cut into a point and the top of the filter paper was cut off so that it would fit into the provided vial. Then, using a pencil, a line was drawn one and a half centimeters above the point. With a provided coin, pigments were taken from a spinach leaf cells by rubbing the coin onto the spinach leaf. The pigment line was made so it was on top of the pencil line. This was done approximately eight to ten times to reassure that the pigment retained; caution was taken so as to use a new portion of the leaf each time the coin was rubbed on the leaf. The chromatography paper was carefully placed in the cylinder so that the pointed end of the paper is minimally immersed inside the solvent making sure that the pigment was not placed in the solvent. The cap was then carefully placed on the vial. When the solvent was approximately one centimeter from the top of the paper, the paper was removed; immediately after removal of paper, the mark was made of the location of the solvent front before evaporation. The bottom of each pigment band was marked. The distance that each pigment traveled from the bottom of the pigment origin to the bottom of the separated pigment band was measured using a given ruler and the distance that each front, with inclusion of the solvent from, traveled was recorded. After this portion of the lab was performed, we proceeded to perform the second portion of the lab experiment.
To begin the second portion of the experiment, we connected the provided Colorimeter with the computer interface. Following that, we proceeded to obtain four cuvettes and lids for the cuvettes. We labeled one of the cuvettes (BL) for blank, one of the cuvettes (U) for unboiled, one of the cuvettes (D) for dark and one of the cuvettes (B) for boiled. The blank cuvette was used as the control for the experiment, and therefore, no DPIP was added. The cuvette labeled (U) was used to examine the absorbance of light in unboiled chloroplasts. The cuvette labeled(B) was to examine the absorbance of light in boiled chloroplasts. The cuvette labeled (D) is for unboiled chloroplasts in an lightless environment. All four sides and bottoms of the cuvette labeled with (D) were carefully covered with aluminum foil. Then phosphate buffer, distilled H2O, and DPIP was added to each of the four cuvettes. One milliliters of phosphate buffer, four milliliters of distilled water, and three drops of unboiled chloroplasts was dropped in the (BL) cuvette. 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP and 3 drops of unboiled chloroplasts was added to the (U) cuvette. 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP and 3 drops of unboiled chloroplasts was added into the dark cuvette. Lastly, 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP and 3 drops of boiled chloroplasts was added into the (B) cuvette. Following the colorimeter was calibrated. We cleaned the outside of the cuvettes carefully, and made sure it was void of particles and fingerprints. Following that, we carefully opened the lid of the colorimeter and then placed the (BL) cuvette inside. (The calibrate portion was performed by the instructor). Immediately after we had calibrated the colorimeter, we placed all three of the cuvettes in front of the beaker and light. After waiting for approximately five minutes the cuvettes were removed from the light and the (U) cuvette was placed inside the colorimeter. After closing the lid of the colorimeter, we waited for approximately ten seconds and noted the absorbance value. Following that, we placed the cuvette in front of the light and beaker. Then we did the same thing with the (D) cuvette and (B) cuvette right after. Then we waited for another five minutes, we performed the same activity and put each cuvette in the colorimeter. After recording the data we placed them in front of the light again. Following another five minutes cuvette was removed from the light and took turns placing them in the colorimeter. When the data was noted we placed them in front of the light for the final trial. When time was over we placed them into the colorimeter and recorded the results. When we finished the lab, we cleaned up and poured the cuvettes containing DPIP into a special beaker and poured the ones without down the drain. We then switched off the computer interface and proceeded to clean our area.
Conclusion and Error Analysis
My conclusion was found to support my initial hypothesis. I hypothesized that the rate of photosynthesis in plant cells is controlled by the light it attains and the temperature it is in; this was confirmed throughout the experiment. In addition, I also predicted that the dark cuvette and the cuvette with the boiled chloroplasts in the light will have slight changes in DPIP and that the cuvette with the unboiled chloroplasts will reduce more DPIP; this was confirmed with the performed experiment. Lastly, I observed that the cholorophylls a and b was at the very bottom of the filter paper, with xanthophyll above cholorphylls a and b, in the middle, and with the pigment beta carotene located just below the line representing the solvent front.
We realized that the various pigments contained within chloroplasts are all involved in gathering energy from sunlight. The spectrum of colors shown on the filter paper displayed the solubility and pigments of each.
The spectrophotometer calculated the light transmittance through the different cuvettes and the chloroplast solutions found in each. The purpose for this was to examine the DPIP change from a blue color into a clear color. This showed that photosynthesis was happening and at what rate it was happening at. The cuvette with the unboiled chloroplasts which had been exposed to light showed the largest change in percent transmittance, which indicates that the amount of light available has a significant effect on the rate in which the light reactions of photosynthesis happen.
For photosynthesis to occur, light energy must be present; this is relevant for the dark reactions as well dark reactions as both need the products of light reactions to occur. In the dark cuvette, during the experiment, although the chloroplasts were unboiled, the cuvette was completely covered with aluminum foil. The foil sets a barrier between the chloroplasts and the light source, disabling the ability for chloroplasts to absorb light. The boiled chloroplasts did not decrease significant amounts of DPIP because when the chloroplasts were boiled, the chloroplasts become denatured along with the enzymes within them disabling the ability for them to function properly. DPIP is reduced only when light reaches the chloroplasts, allowing the enzymes to be boosted to a higher level of energy. Yet, because the chloroplasts and the enzymes are already disabled, it will no longer perform photosynthesis even with the presence of a light source. The cuvette with the unboiled chloroplasts in the light held the highest reduction of DPIP because this cuvette has the proper conditions for photosynthesis to carry out. Differentiating from the other cuvettes, this cuvette had light and the chloroplasts inside the cuvette were not denatured from boiling. When the light reached inside the chloroplasts the enzymes jumped to a higher level of energy. As increased amounts of electrons were boosted, the DPIP was slowly reduced along with its change in color.
Various sources of errors could have occurred throughout the experiment but one of them was that the cuvettes may not have been probably handled prior to insertion into the colorimeter. My peers and I did not handle the cuvettes by the top edge of the ribbed sides only, but we also accidentally touched the four sides of the cuvette. This very likely left plentiful fingerprints that may have formed a barrier for the light to enter the chloroplasts at its potential, which in turn, would have caused the colorimeter to miscalculate the amount of DPIP reduced. When placing the three cuvettes in front of the light we did not remember the positions were placed them in. After each absorbance reading for the cuvettes, the cuvettes were placed in a different spot in front of the light. This could have caused more light to enter one part and less to enter another causing our data and results to not be a sufficient as they could have been. A source of error that occurred during the first part of the lab was we did not rub enough spinach pigments onto the filter paper so we did not have that many pigment bands.
I feel like we did not carry enough trials for our experiment to have accurate results. If we had carried out another trial and found the average of the two trials, it would have provided more accurate results. To make the experiment more scientifically sound, one could avoid careless errors that we did like leaving fingerprints and placing them in different spots of the light with wearing gloves and marking where the cuvettes were placed each time. Some further experiments could be done to expand my knowledge could be to see how photosynthesis occurs in photoautotroph or algae. Another experiment that could be performed would to be to, instead of using spinach cells, to use cells from lettuce, or other plant cells to see which cell type contains highest levels of photosynthesis. This would be interesting because instead of seeing how photosynthesis works in chloroplasts we could see how photosynthesis works in animals that can provide its own food.