Light Reactions and Plant Pigments

The Effect of Light Reactions on Plant Pigmentation Alyssa Martinez AP Biology 4th pd E. Perkins Abstract In this lab, we were to separate pigments and calculate Rf  values using plant pigment chromatography, describe a technique to determine the photosynthetic rate, compare photosynthetic rates at different light intensities using controlled experiments and explain why rate  of photosynthesis varies under different environmental conditions. In the second part of the lab, we used chloroplasts extracted from spinach leaves and incubated then with DPIP and used the dye-reduction technique. When the DPIP is reduced and becomes  colorless, the resultant increase in light transmittance is measured over a  period of time using a spectrophotometer. If pigments are separated, then Rf values can be determined. Introduction Paper chromatography is a  useful technique for separating and identifying pigments and other molecules from cell extracts that contain a  complex mixture of molecules. As solvent moves up  the  paper, it carries along any  substances dissolved in it. The more soluble, the further  it travels and vice-versa. Beta carotene is  the most abundant carotene in plants and is  carried along near the solvent front since it is very soluble and  forms no hydrogen bonds with cellulose. Xanthophyll contains oxygen and is found further from the solvent front since it  is less soluble in the solvent and is  slowed down by hydrogen  bonding to cellulose. Chlorophyll a is  primary photosynthetic pigment in plants. Chlorophyll a, chlorophyll b, and carotenoids capture light energy and transfer it to  chlorophyll a at the reaction center. Light is  part of a continuum of radiation or energy waves. Shorter wavelengths of energy have greater amounts of energy. Wavelengths of light within the visible spectrum of  light power  photosynthesis. Light is absorbed by leaf  pigments while electrons within each photosystem are boosted to a higher energy level. This energy level is  used to produce ATP and reduce  NADP to NADPH. ATP and  NADPH are then used to  incorporate CO2 into organic molecules. In place of  the electron accepter, NADP, the compound DPIP  will be substituted. It changes chloroplasts from blue to colorless. Methodology Obtain a 50 ml graduated cylinder which has about 1 cm of solvent at the bottom. Cut a piece of  filter paper which will be long enough to reach the solvent. Draw a line about 1. 5 cm from the bottom of the paper. Use a quarter to extract the pigments from spinach leaf cells and place a small section of leaf on top of the pencil line. Use the ribbed edge of the coin to crush the leaf cells and be sure the pigment line is on top of the pencil line. Place  the chromatography  paper in the cylinder and cover the cylinder. When the solvent is about 1 cm from the top of the paper, remove the paper  and immediately mark the location of the solvent front before it evaporates. Mark the bottom of each pigment band and measure the distance each pigment migrated from the  bottom of the pigment origin to the bottom of the separated pigment band and record the distances. Then, turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm. Set up an incubation area that  includes a light, water flask, and test tube rack. Label the cuvettes 1, 2, 3, 4, and 5, respectively. Using lens tissue, wipe the outside walls of each cuvette. Using foil paper, cover the walls and bottom of cuvette 2. Light should not  be permitted inside cuvette 2 because it is a control for this experiment. Add 4 mL of distilled water to cuvette 1. To 2, 3, and 4, add 3 mL of distilled water and  1 mL of DPIP. To 5, add 3  mL plus 3 drops of distilled water and 1mL of DPIP. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts and cover the top of cuvette 1 with Parafilm and invert to mix. Insert cuvette 1 into  the sample holder and adjust the  instrument to 100% transmittance. Obtain the unboiled chloroplast suspension, stir to mix, and transfer 3 drops to cuvette 2. Immediately cover and mix cuvette 2. Then remove it from the foil sleeve and  insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it. Replace cuvette 2 into the foil sleeve,  and place it into the incubation test tube rack and turn on the flood light. Take and record additional readings at 5, 10, and 15  minutes. Mix the cuvette’s contents before each reading. Take the unboiled chloroplast suspension, mix, and transfer 3 drops to cuvette 3. Immediately cover and mix cuvette 3 and insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record. Replace cuvette 3 into the incubation test tube rack. Take and record additional readings at 5, 10, and  15 minutes. Mix the cuvette's contents just prior  to each readings. Obtain the boiled chloroplast suspension, mix, and transfer 3 drops to cuvette 4. Immediately cover and mix cuvette 4. Insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it. Replace cuvette 4 into the incubation test tube rack and take and record additional readings at 5, 10, and  15 minutes. Cover and mix the contents of cuvette 5 and insert it into the spectrophotometer's sample holder, read the percentage transmittance, and  record. Replace cuvette  5 into the incubation test tube rack and take and record additional readings at 5, 10, and 15 minutes. Results Table 4. 1: Distance Moved by Pigment Band (millimeters) Band Number| Distance (mm)| Band Color| | | | | | | | | | | | | | | | Distance Solvent Front Moved ____ (mm) Table 4. 2: Analysis of Results __ = Rf for Carotene (yellow to yellow orange) __ = Rf for Xanthophyll (yellow) __ = Rf for Chlorophyll a (bring green to blue green) __ = Rf for chlorophyll b (yellow green to olive green) Table 4. 4: Transmittance (%) Time (minutes) Cuvette| 0| 5| 10| 15| 2 Unboiled/Dark| | | | | 3 Unboiled/Light| | | | | Boiled/Light| | | | | 5 No Chloroplasts/ Light| | | | | Analysis of Results Graph Discussion Chromatography  is  a  technique  used  to  separate  and identify pigments and other molecules from cell extracts that contain a complex mixture of molecules. This can be used to identify the pigments that are used in the  process of  photosynthesis. Photosynthesis is the process by which plants use light energy to produce chemical  energy in the form of food. This is where plant pigments come into play because they are the reason why the plant is able to absorb light. Chlorophyll a is one such  pigment. These pigments along with many others are contained in organelles known as chloroplasts. One of the problems encountered during the course of this lab included human error when using the spectrophotometer. The student made slight errors when setting the transmittance to the required levels. On a few occasions, the group accidentally introduced light into a cuvette where the variable being tested was the absence of light. This might have caused some error when taking measurements of the percentage  of transmittance. This resulted in skewed data, which meant that the experiment had to be repeated once more. During the first part of the  lab, the group made an error by allowing some part of the pigment  to be in the solvent. This did alter our results in the end. Topics for Discussion 4A: Plant Pigment Chromatography 1. What factors are involved in the separation of the pigments? The factors involved in the separation of the  pigments  from the  spinach plants  are the pigments’ solubility in the solution, how much they bind to the paper based on their chemical structure, and the size of the pigment particles. . Would you expect the Rf value of a pigment to be the same if a different solvent were used? Explain. No I would not expect the Rf values to be different because the pigments will dissolve differently in different types of solvents. For example, chlorophyll b is very soluble in hydrophobic solutions, so if the crushed spinach cells on the paper were put in a hydrophobic s olution, the chlorophyll b would move the highest and probably be right on the solution front, while the other pigments will move much less. 3. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments? Chlorophyll a is in the reaction center, and the other pigments are able to absorb light from the other wavelengths that chlorophyll a cannot absorb light from, and then they transfer the energy harvested from the other wavelengths to the chlorophyll a, providing more energy to be used in photosynthesis. 4B: Photosynthesis/The Light Reaction 1. What is the function of DPIP in this experiment? DPIP is the electron acceptor in this experiment (instead of NADP which is what is normally used in plants). The electrons boosted to high energy levels will reduce the DPIP, which will change its color from blue to clear as more high energy electrons are absorbed by it. 2. What molecule found in chloroplast does DPIP â€Å"replace† in this experiment? It replaces NADP molecules that are found in chloroplasts. 3. What is the source of the electrons that will reduce DPIP? The electrons come from the photolysis of water. 4. What was measured with the spectrophotometer in this experiment? The light transmittance was measured, which really was the measure of how much the chloroplasts reduced the DPIP 5. What is the effect of darkness on the reduction of DPIP? Explain. Darkness will restrict any reaction to occur. 6. What is the effect of boiling the chloroplasts on the subsequent reduction of DPIP? Explain. By boiling chloroplasts, we denature the protein molecules, ending the reduction of DPIP. 7. 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 percent transmittance grew to steadily higher numbers as the experiment progressed because the light reaction was able to occur. However, the dark cuvettes had stable levels of transmittance because light is necessary to excite electrons, which, in turn, reduces the DPIP. 8. Identify the function of each of the cuvettes. Cuvette 1: Used as the control Cuvette 2: Used to observe the rate of photosynthesis without light Cuvette 3: Used to observe the rate of photosynthesis with light Cuvette 4: Used to observe the rate of photosynthesis in boiled chloroplasts Cuvette 5: Used to observe the rate of photosynthesis