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 hues and calculate Rfvalues using plant pigment chromatography, describe a technique to determine the photosynthetic rate, compare photosynthetic rates at different dizzy intensities using checkled experiments and explain wherefore rateof 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 trim and becomescolorless, the resultant increase in light transmittance is calculated over aperiod of time using a spectrophotometer. If pigments are separated, then Rf values can be determined. Introduction Paper chromatography is auseful technique for separating and chance uponing pigments and other molecules from cell extracts that contain acomplex mixture of molecules. As solv ent break downs upthepaper, it carries on anysubstances brush offd in it. The more dissoluble, the furtherit travels and vice-versa.Beta carotene isthe most abundant carotene in plants and iscarried along near the solvent front since it is very soluble andforms no hydrogen bonds with cellulose. Xanthophyll contains oxygen and is found further from the solvent front since itis less soluble in the solvent and isslowed down by hydrogenbonding to cellulose. Chlorophyll a isprimary photosynthetic pigment in plants. Chlorophyll a, chlorophyll b, and carotenoids father light energy and transfer it tochlorophyll a at the reaction center. Light ispart of a continuum of radiation or energy waves.Shorter wavelengths of energy commit greater amounts of energy. Wavelengths of light within the visible spectrum oflight powerphotosynthesis. Light is absorbed by leafpigments while electrons within apiece photosystem are boosted to a elevateder energy level. This energy level isused to produc e ATP and reduceNADP to NADPH. ATP andNADPH are then used toincorporate carbon dioxide into organic molecules. In place ofthe electron accepter, NADP, the compound DPIPwill be substituted. It changes chloroplasts from sick to colorless. MethodologyObtain a 50 ml graduated cylinder which has about 1 cm of solvent at the bottom. Cut a piece offilter paper which will be long enough to excrete 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. Placethe chromatographypaper in the cylinder and cover the cylinder.When the solvent is about 1 cm from the top of the paper, except the paperand immediately mark the location of the solvent front before it evaporates. Mark the bottom of from each one pigment band and measure the distance e ach pigment migrated from thebottom of the pigment origin to the bottom of the separated pigment band and account book the distances. Then, turn on the spectrophotometer to warm up the prick and dress out the wavelength to 605 nm. Set up an incubation area thatincludes a light, water flask, and test tube rack. Label the cuvettes 1, 2, 3, 4, and 5, respectively. utilize lens tissue, wipe the outside walls of each cuvette. Using foil paper, cover the walls and bottom of cuvette 2. Light should notbe 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, minimal brain dysfunction 3 mL of distilled water and1 mL of DPIP. To 5, add 3mL plus 3 drops of distilled water and 1mL of DPIP. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter takes 0% transmittance. Add 3 drops of unboiled chloroplasts and cover the top of cuvette 1 with Parafilm and invert to mix.Insert cuvette 1 intothe sample holder and adjust theinstrument 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 andinsert it into the spectrophotometers sample holder, read the fortune 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 15minutes. riffle the cuvettes 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 spectrophotometers 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, and15 minutes. Mix the cuvettes contents just priorto each readings. Obtain the boiled chloroplast suspen sion, mix, and transfer 3 drops to cuvette 4. Immediately cover and mix cuvette 4.Insert it into the spectrophotometers 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, and15 minutes. Cover and mix the contents of cuvette 5 and insert it into the spectrophotometers sample holder, read the percentage transmittance, andrecord. Replace cuvette5 into the incubation test tube rack and take and record additional readings at 5, 10, and 15 minutes. Results Tcapable 4. 1 Distance Moved by Pigment Band (millimeters)Band Number Distance (mm) Band Color Distance resultant 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/ blue-blooded 3 Unboiled/Light Boiled/Light 5 No Chloroplasts/ Light Analysis of Results Graph Discussion Chromatographyisatechniqueusedtoseparateand 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 surgery ofphotosynthesis. Photosynthesis is the process by which plants use light energy to produce chemicalenergy 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 suchpigment. These pigments along with many others are contained in organelles known as chloroplasts. One of the problems encountered during the descent 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 grouping 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 percentageof transmittance.This resulted in skewed data, which meant that the experiment had to be repeated once more. During the first part of thelab, the group made an error by allowing some part of the pigmentto 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 thepigmentsfrom thespinach plantsare the pigments solvability 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 charge in a hydrophobic solution, 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 m ore high energy electrons are absorbed by it. 2. What molecule found in chloroplast does DPIP 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 throttle 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 remainder 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 hi gher come 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