Hanover College



Photosynthesis Biology 185, Winter 2020Objective of laboratory: Determine the absorbance spectrum for total pigment and isolated pigments in spinachVisualize different spinach pigments and examine relative solubility using paper chromatographyMeasure O2 production in Elodea when exposed to varying concentrations of CO2Overview:During the previous lab, the conversion of sugars into carbon dioxide was examined. In this lab, we explore the process that produces the sugars needed by many organisms. Most energy trapped in organic molecules used to power metabolism originates from the sun. In essence, the process of photosynthesis converts visible light produced by the sun into chemical energy. While most organisms oxidize glucose into simpler waste products such as carbon dioxide, plants are able to do the opposite by converting carbon dioxide into sugars. The two processes (respiration verses photosynthesis) can be visualized with the following reactions: Respiration: C6H12O6 + 6 O2 6 CO2 + 6 H2O + ATP energy (glucose) Photosynthesis: 6 CO2 + 6 H2O + solar energy C6H12O6 + 6 O2 (glucose) Absorption Spectra of Total Spinach Pigments Similar to the reactions that extract energy from glucose, photosynthesis is a two-step process that involves trapping radiant energy from the sun to make ATP. Light is “trapped” by pigments, which absorb light energy of specific wavelengths while reflecting or ignoring the energy of others. The most prominent is chlorophyll a, a compound that absorbs energy corresponding roughly to blue and red light while reflecting green light, giving plants their green color. Auxiliary pigments absorb and transfer light energy that chlorophyll a misses (primarily those in the blue color—and therefore more energetic—wavelengths of light, resulting in their primarily yellow and red colors). These pigments include chlorophyll b, xanthophylls, and carotenoids. The xanthophylls and carotenoids produce the colors observed in the fall after the chlorophylls have decomposed in the dying leaf.The light-trapping ability of pigments can be studied by isolating pigments and examining the wavelength of light absorbed. Through the use of a pigment mixture (chlorophyll, xanthophyll, and carotenoid) isolated from leaves, a graph of the absorbance at different light wavelengths provides the absorption spectrum and is a good approximation of the light wavelengths most effectively utilized by plants during photosynthesis. Obtain the following supplies from the back benches for each group:1 Speciman jar2 Spectophotometer tubesSmall chromatography paperGlass graduated cylinderSpinach leafProcedure:Isolate total spinach pigment as follows: Obtain a spinach leaf, the smallest sheet of chromatography paper, and a coin. Place the spinach leaf on top of the chromatography paperUsing the coin to “run” over the leaf, creating a thin stripe on the chromatography paper.Repeat this process, without moving the spinach leaf, until you have a strong band of green.Cut the band of green from the chromatography paper and soak the strip in a vial of 5mL prepared 2:1 acetone:ethanol mixture. Incubate for 5min.Note: acetone is an organic liquid that disrupts membranes and dissolves hydrophobic molecules, namely the pigmentsTurn on the spectrophotometer to warm-up, set to 380nm. Zero spectrophotometer with the blank (a 2:1 acetone:ethanol mix).Transfer dissolved pigments into a second spectrophotometer tubeRecord absorbance values for total isolated pigment at 380nm, repeating the reading in 20nm intervals to 700nm (Table 1).Record the values in the Data collection: Absorption spectrum pigments (Total pigment) spreadsheet posted on Moodle Table 1: Spinach Leaf Extract Absorbance ValuesWave length (nm)AbsorbanceWave length (nm)AbsorbanceWave length (nm)AbsorbanceWave length (nm)Absorbance380480580680400500600700420520620440540640460560660Isolation of Chlorophyll a and Chlorophyll bAbsorption spectra can be determined for individual pigments in a leaf extract, but first each must be isolated from the mix. This can be done by cutting dried paper chromatograms into strips containing only one pigment. Soaking these strips in an organic solvent dissolves the pigment and the liquefied purified molecule may be used to determine the absorption spectrum of individual pigments. The absorption spectrum of the individual pigments can then be compared with the pigment mixture. Several techniques effectively separate pigments, and paper chromatography is simple, inexpensive, and effective. In this technique, a small amount of the extract is applied to and dried on a special paper. The paper is placed in a container with the edge near the sample touching a solvent. The paper acts as a wick, and when the advancing solvent encounters the sample, the liquid attempts to dissolve the pigments away from the paper. To develop the most effective separation system for any group of pigments, solvents are chosen that dissolve some pigments better than others while at the same time papers that bind pigment molecules with different efficiencies are also evaluated. The best combination of paper and solvent allows some pigments to dissolve into the solvent well and bind the paper poorly, so they tend to travel with or near the solvent front, the leading edge of the solvent as it moves along the paper. Other pigments may bind the paper well and dissolve poorly and tend not to move far from the spot on which they were applied (called the origin). Finally, most molecules fall somewhere in between and move only partially up the paper. Thus, pigments can be separated by relative ability to move up the paper in the presence of a specific solvent. Of course, even identical samples run for different lengths of time on different papers will not travel the same distance, making comparisons between anything other than samples run on the same paper at the same time difficult. To solve this problem, scientists compare not the absolute distance traveled by each sample, but rather the distance relative to the solvent front, a value called Rf. The ratio of the distance each pigment travels to the maximum possible distance, the solvent front, is the same for all samples, hence the need for Rf values to compare data from different runs.Obtain the following supplies from the back benches for each group:46863002727960Figure 1. Application of pigment to chromatography paper00Figure 1. Application of pigment to chromatography paper468630022860001” x 8” Whatman #1 chromatography paper1 spinach leafProcedure:Touching on the long edges (grease from fingers can disturb test), Lay one 1” x 8” Whatman #1 chromatography paper strip on the lab bench. Cut one end to a point. Use a coin to roll a stripe of concentrated pigment extract from spinach immediately above the pointed tip of the paper. Best results are obtained when the stripe is applied in a thin band. Let the sample dry, repeat application until the entire stripe is a dark green. In the fume hood, hook the broad end of one paper onto a clip embedded in a cork, insert this into a large test tube containing 9:1 petroleum ether so that the tip of the point just touches the solvent in the bottom of the tube (Figure 1). Safety note: Petroleum ether is highly flammable and intoxicating, hence the need for proper ventilation in the fume hood. While working in the fume hood, Pull the chromatogram out of the tube when the solvent is near the top (approximately 1/4cm). Mark the position of the solvent front before solvent dries completelyDry the chromatogram in fume hood.Identify chlorophylls a (blue-green), b (yellow-green) and carotenoids (orange-ish), and xanthophylls (yellow). Mark the middle of each band.Measure the distances from the origin (location where samples were applied) to the solvent front and then to each of the pigments (Table 2). Determine the Rf values for each pigment with the formula (Table 2):Rf = distance to sample spot / distance to solvent frontRecord the values in the Data collection: Pigment distance spreadsheet posted on Moodle Table 2. Pigment distance (Rf)PigmentDistance to sample (cm)Distance to solvent front (cm)Rf (cm)Chlorophyll a (blue-green)Chlorophyll b (yellow-green)Carotenoids (orange)Xanthophylls (yellow)The next part of the procedure is to isolate specific chlorophyll a and b pigments from chromatography paper.Obtain the following supplies from the back benches for each group:2 Speciman jar2 Spectophotometer tubesGlass graduated cylinderProcedure:Using the chromatography paper strip form the previous step, identify chlorophyll a and chlorophyll b bands by circling the yellow-green (chlorophyll b) and blue-green (chlorophyll a) band(s) of pigment with a pencil. If needed, confirm identification with the UV-light test and adjust the pencil markings:The blue-green pigment (chlorophyll a) glows red when exposed to UV light. Cut out two strips: one containing only chlorophyll a and another containing only chlorophyll b.Place strips in separate vials with 5mL of a 2:1 acetone:ethanol mixture. Incubate for 5 min.Place strips in separate vials with 5mL 2:1 acetone:ethanol mix. Soak 5min.Turn on the spectrophotometer to warm-up, set to 380nm. Zero spectrophotometer with the blank (a 2:1 acetone:ethanol mix).Pour the dissolved pure pigment into separate spectrophotometer tubes.Record absorbance values for total isolated pigment at 380nm, repeating reading in 20nm intervals to 700nm (Tables 3 and 4).Record the values in the Data collection: Absorption spectrum pigments (Chlorophyll a or chlorophyll b) spreadsheet posted on Moodle Table 3: Chlorophyll a from SpinachWave length (nm)AbsorbanceWave length (nm)AbsorbanceWave length (nm)AbsorbanceWave length (nm)Absorbance380480580680400500600700420520620440540640460560660Table 4. Chlorophyll b from SpinachWave length (nm)AbsorbanceWave length (nm)AbsorbanceWave length (nm)AbsorbanceWave length (nm)Absorbance380480580680400500600700420520620440540640460560660Production of Oxygen by ElodeaCarbon and energy are fixed at the same rate oxygen is produced during photosynthesis thus the rate of oxygen production illustrates the effect of varying CO2 concentrations and light intensities on the rate of photosynthesis. The amount of oxygen generated is easily determined. Oxygen is a gas that does not dissolve well in water, thus changes in the amount of gas in a closed tube with a plant conducting photosynthesis can be attributed to oxygen production. This is easily done in an apparatus similar to that used in the previous lab to follow CO2 production in yeast, though this time an aquatic plant (Elodea) will be submerged in water containing varying concentrations of dissolved CO2 and exposed to different amounts of light.Obtain the following supplies from the back benches for each group:4 test tubes4 1mL pipet in rubber stopped4 Elodea200mL beaker with diH203% bicarbonate2 pieces aluminum foilProcedure:Label 4 test tubes, which will be set-up with the following conditions in the following steps (Table 5).Table 5: Sample preparationTube #OrganismSolutionLight1ElodeadiH2OLight: No foil 2ElodeadiH2ODark: Foil3Elodea3% bicarbonateLight: No foil4Elodea3% bicarbonateDark: Foil*The bicarbonate provides CO2 through the equilibrium reaction:HCO3- + H+ H2CO3 H2O + CO2 (bicarbonate) (carbonic acid)Select four healthy Elodea plants that have similar appearance and size. Plants should be about as long as the test tube (between 10 and 15cm each). Try to shake as much water as possible off the plants, roll them gently in a dry paper towel to remove as much free water as possible prior to weighing.Place a single plant into tubes #1-4. Pour the correct contents into each tube (diH2O or 3% bicarbonate); fill to about 1 inch from the top.Seal all 4 tubes with a rubber stopper pierced with a calibrated 1mL pipette, and ensure liquid is visible above the stopper. This device should resemble the apparatus constructed for the fermentation experiment. Wrap tubes numbered 2 and 4 with aluminum foil, leaving the pipette exposed. The goal is to prevent light from getting to the plants in these tubes.Set the tubes in a rack behind a water-filled beaker and place a 150watt lamp 75cm away from the tubes. The water is a heat sink and prevents the flood light from boiling the plants.Turn on lamp, wait 10min and record the initial level of the water in each pipette (Table 6). Note: The pipette measures 1mL, thus each major line represents 0.1mL.Wait 10 more min, record the water level in the pipettes again (after 20min since start) (Table 6). Subtract the initial reading from this value. Enter the change in volume (milliliters of O2 produced in 10min) (Table 6).Note: Pipettes measures 1mL, and each major line on it measures 0.1mL.Move the tubes to 25cm from the lamp, wait 10 min, record the water level (Table 6).Wait another 10 min, record again, and enter the difference (Table 6).Record the values in the Data collection: Photosynthesis spreadsheet posted on Moodle Table 6. O2 production by ElodeaTubeConditionmL O2 dissolved @75cmmL O2 dissolved @ 25cm10 min20 minDifference10 min20 minDifference1ElodeadiH2OLight2ElodeadiH2ODark3Elodea3% bicarb Light4Elodea3% bicarb DarkGlossaryAuxiliary pigments: absorb and transfer light energy missed by chlorophyll a Carotenoids: auxiliary pigment; can be split into two classes: xanthophylls and carotenesChlorophyll a: compound that absorbs energy corresponding to blue and red light, while reflecting green light; gives plants green colorChlorophyll b: auxiliary pigment; absorbs blue light; gives plants yellow colorPaper chromatography: Analytical method used to separate, isolate, or identify pigmentsPhotosynthesis: conversion of light energy into chemical energyPigments: molecules that absorb certain wavelengths of visible lightSolvent front: the leading edge of the solvent as it moves along the paperXanthophyll: auxillary pigment; type of carotenoid with contains oxygen; yellow pigmentLab write-upThe full scientific write-up will include:Goal: What was the reason the experiment(s) was/were performed (what was discovered/studied/identified)?Checklist:Is it brief? (Typically one sentence per unique experiment)Is it descriptive? (Does the goal mention the tested variables?)Results: What were the major findings of the experiments performed (include means, SD, and t-test values when appropriate)? What information do the figures present (figures must be cited in the text)? If t-tests were performed, what is indicated, does a significant difference exist between the treatments?ChecklistFindings in paragraph form, includingDescriptive statistics (Mean, SD) for each treatment are included?If a t-test was performed, do results include whether the means are significantly different or not?Do the results avoid interpretation or analysis?Does the results cite the figure (s) (at the end of sentences)?Pertinent figures from the Excel spreadsheet, with captionsDiscussion: How do the results help meet the goal(s)? How should the results be interpreted? Are trends present? If t-test was performed, what do the significance and non-significance mean?The questions below should be addressed in the Discussion section:?Absorption Spectra of Total Spinach Pigments Describe the absorption spectrum for the total pigment of spinach. Where are the peak absorbance, and what does this mean? What about the low absorbance areas between the peaks? Isolation of Chlorophyll a and Chlorophyll bLook up paper chromatography. You may use an online source, but please cite it. Discuss why certain pigments traveled farther up the paper than others, tying this in to the results you found. Describe the absorption spectrum for chlorophyll a. How many peaks does it have? What do these peaks indicate? What about the low absorbance areas between the peaks? Describe the absorption spectrum for chlorophyll b. How many peaks does it have? What do these peaks indicate? What about the low absorbance areas between the peaks? Production of Oxygen by ElodeaWhat are the effects of the distance from the light source on oxygen production? Why do you think you are observing this effect? What are the effects of varying bicarbonate concentrations on production of oxygen? Why do you think you are observing this effect? What are the effects of light vs. dark conditions on the production of oxygen? Why do you think you are observing this effect? Final Tips and Suggestions:Read over the report at least twice before submitting to ensure major points are addressed following correct grammatical rules. Use proper units throughout (mL, μL, g, nm, etc.).Do not do the report at the last minute; give yourself a few days to come up with an outline. Come see me if you need assistance. ................
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