Liz Nunez A.P.

Biology Lab #4 Partners: Clea Howard, Madeline Smith, Vivian Cheng, Xindi Tan December 4, 2011 Plant Pigments and Photosynthesis Abstract: The process of Photosynthesis is the chemical exchange between carbon dioxide and water as they get converted into oxygen and glucose. Pigments, located in chloroplasts, are the molecules that help initiate the process. There can be various pigments in one single plant. Paper chromatography is a technique used to separate different pigments by sorting them according to their solvencies. Photosynthesis can also be measured quantitatively by use of a spectrometer, which calculates the percent of a specific light that is transmitted through a substance. By the use of indicator DPIP based on the production of NADP, the process of photosynthesis was measured and calculated. Plants with unboiled chloroplasts were the most sufficient in demostrating photosynthesis. Results: Exercise 4A: Plant Pigment Chromatography

Figure 4.1

Table 4.1 Distance Moved by Pigment Band Band Number 1. 2. 3. Distance (mm) 50 mm 13 mm 12 mm Band Color Lime green Emerald Yellow

Distance solvent front moved 75 millimeters. Observation: The farther away from the pigment origin, the darker the color was. Analysis of Results: Rf = distance pigment migrated (mm) distance solvent front migrated (mm)

Table 4.2 Relationship of the distance moved by the pigment to the distance moved by the solvent (Rf) 12/75 = 0.16 = Rf for Xanthophyll (yellow) __13/75 = 0. 17____ = Rf for Chlorophyll a (bright green to blue green) __50/75 = 0.66 ____ = Rf for Chlorophyll b (yellow green to olive green) Exercise 4B: Photosynthesis / The Light Reaction Table 4.3 The Photosynthesis Setup 1 blank (no DPIP) Phosphate Buffer Distilled H2O DPIP Unboiled Chloroplasts Boiled Chloroplasts 1 ml 4 ml ----3 drops -----2 unboiled chloroplasts Dark 1 ml 3 ml 1 ml 3 drops -----3 Unboiled Chloroplasts Light 1 ml 3 ml 1 ml 3 drops ----4 Boiled Chloroplasts Light 1 ml 3 ml 1 ml ----3 drops 5 No Chloroplasts Light 1 ml 3 ml + 3 drops 1 ml -----------

Figure 4.2 : Incubation Setup

Table 4.4 Transmittance Percent Expected Results Cuvette 0 5 2 Unboiled/ 31.3% 32.5% Dark 3 Unboiled/ 32.7% 54.5% Light 4 Boiled / 32.7% 32.9% Light 5 No 31.3% 31.3% Chloroplasts /

10 35.5% 63.7% 33.1% 31.3%

15 57.7% 60.1% 48.5% 60.5%

Light Observation: As chloroplasts absorb more light, NADPH production increases. DPIP is an indicator that measures the amount of photosynthesis occurring based on the production of NADPH. DPIP changes from blue to colorless as it is reduced, as a result of more NADPH being made and more photosynthesis occurring, since more H2O is being split and light is absorbed for the light reaction. Analysis of Results: Plot the percent transmittance from the four cuvettes on the graph below. a) The independent variable Time (minutes) b) Dependent variable Percent Transmittance Discussion Questions Plant Pigments Chromatography 1. What factors are involved in the separation of pigments? One of the main characteristic that defines the separation of pigments is solvency. Since different pigments have different solvencies, they will separate at different levels of the paper. 2. Would you expect the Rf value of a pigment to be the same if the solvent was different? No. Since the Rf value is a ratio, as long as the ratio is the same between the distance traveled and the front distance the value could be the same. However, if there is a different solvent, there will be a different solubility, and thus the distance traveled by a particular pigment and its proportion to the overall front distance should be different. 3. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments? The reaction center contains chlorophyll a, which helps convert the photons from light energy into chemical energy in the light reactions. The other pigments in the reaction center absorb photons from different light waves specific to their pigment and transfer the energy. 4. What was the purpose of DPIP in the experiment? DPIP was the indicator that measured, in a quantitative manner, the amount of photosynthesis occurring based on the production of NADPH. It was the electron acceptor in the reaction, and thus it was reduced as photosynthesis occurred. 5. What molecule found in chloroplasts does DPIP replace in this experiment? What is the source of the electrons that reduce DPIP? DPIP replaces NADP, which is the natural electron acceptor during photosynthesis. The free electrons that reduce DPIP come from water.

6. What is measured by the spectrometer in this experiment? The spectrometer measures the percent of transmittance within the reaction occurring inside a cuvette. This is the percent of light that is transmitted, not absorbed by the NADP reduction taking place. 7. What is the effect of darkness on the DPIP reduction? If the reaction occurs in obsolete darkness, there will be no DPIP reduction. This is because photosynthesis takes place because photons from light waves are converted into chemical energy, and the reduction of NADP (in this case DPIP) is one of the products. If there is no light to initiate the reaction of photosynthesis, there will be no reduction of DPIP. 8. What is the effect of boiling chloroplasts on the reduction of DPIP? Chloroplasts, like many other proteins, denature in the exposure of extreme temperatures about the optimum. This means that their tertiary structure begins to unfold and thus the function and ability of the structure starts to disintegrate. 9. What reasons can you give for the difference in the percent transmittance between the live chloroplasts that were incubated in the light and those that were kept in the dark? The live chloroplasts incubated in light had a higher transmittance than the ones in darkness. This is because photosynthesis is rapidly taking place in the chloroplasts in light, and thus the amount of light photons being converted is higher than that of chloroplasts in darkness. 10. Identify the function of each cuvette: Cuvette 1 (Unboiled, dark) Control to the light reaction. Cuvette 2 (Unboiled, light) the expected experiment Cuvette 3 (Boiled, light) - denaturation Cuvette 4 (No chloroplasts, light) Control group. Error Analysis: Improper attention to the details of the materials, such as the effect of skin oils on the cuvettes inside the spectrometer can affect the overall success of the experiment. Also, malfunctioning equipment, such as an out-of-date spectrometer disallowed the proper correct results. Conclusion: Photosynthesis is a process where carbon dioxide and water are converted into oxygen and glucose. Mainly occurring in plants and photosynthesizing bacteria, this reaction begins when pigments located inside organelles called chloroplasts absorb photons from different light wave lengths and transfer the resulting energy to chlorophyll a pigment. Paper chromatography can be used to identify different pigments and their solvencies when placed in different solvents. The experiment showed that in alcohol, the pigments progress

to darker hues of color as they separate from the pigment origin. The second part of the experiment measured photosynthesis in a quantitative calculation. By using the transmittance percent of orange light passing through the cuvettes, one could observe that as time progressed, photosynthesis occurred in mainly the cuvette with the unboiled chloroplasts in light. Transmittance percent is directly influenced by the indicator DPIP, which turns colorless as more NADPH is produced. As this production increases as a result of photosynthesis, more light is being transmitted through the cuvettes, which results in a higher transmittance percent. In this way, the percent of light transmitted is a quantitative form of measuring the success and progress of photosynthesis. The experiment also showed the effects of exposure to optimum temperatures and the lack of sunlight over the entire photosynthesis process. If there is no light present, barely if not any photosynthesis will occur. Likewise, if the chloroplast is boiled, its tertiary structure would denature and unfold, disallowing it to fit its function. Thus, the most crucial elements for the continuation of photosynthesis are light, a normal environment, and carbon dioxide. Citation: http://www.scribd.com/doc/7847067/AP-Biology-Lab-Four-Plant-Pigments-andPhotosynthesis http://web.archive.org/web/20060412042050/http://sps.k12.ar.us/massengale/lab_4__a p_sample_2.htm