Lab 11: Photosynthesis (Fall 2024) PDF

Lab 11: Photosynthesis Lab Manual 6.1-6.4, Fall 2024 (MK) Goals of this lab Concepts Output Understand the photosynthetic activity Separate pigments using paper of various wavelengths of light chromatography Understand the role of pigments...

Lab 11: Photosynthesis Lab Manual 6.1-6.4, Fall 2024 (MK) Goals of this lab Concepts Output Understand the photosynthetic activity Separate pigments using paper of various wavelengths of light chromatography Understand the role of pigments in Use a starch test to evaluate photosynthesis photosynthetic activity Understand how polarity can be used Generate an absorption spectrum for to separate molecules various photosynthetic pigments Lab Safety Today we will be working with both toxic/flammable substances and heat plates. It is very important that we all keep the following safety information in mind: Make sure you are using gloves for everything today, and using forceps for all items that will touch flammable materials (leaves, chromatography paper). Beakers with ethanol should never be placed directly on the heat plate. They should be nested in a larger beaker with water in it. Do not inhale solvents used for 6.3 and 6.4: close jar as quickly as possible to minimize potential inhalation. If any solutions spill, alert instructor immediately, and do not attempt to clean yourself. All chemical waste should go into provided glass waste containers, not the sink. Photosynthesis Plants absorb sunlight and use it to generate glucose, starch, and eventually: all other organic molecules Equation: 6H2O + 6CO2 6O2 + C6H12O6 + H20 It takes place in three stages: ○ capturing energy from sunlight ○ using the captured energy to produce ATP and NADPH ○ using the ATP and NADPH to make carbohydrates from CO2 in the atmosphere Photosynthesis Electromagnetic Spectrum Light is comprised of packets of energy called photons. Sunlight has photons of varying energy levels; these varying levels are represented by an electromagnetic spectrum. Human eyes only perceive photons of intermediate energy levels. This range of the spectrum is visible light. Pigments Pigments Accessory pigments Molecules that absorb light energy Absorb light levels that chlorophyll doesn’t Give color to flowers, fruits, vegetables The main pigments in plants are Present in leaves too: masked by chlorophyll a and b chlorophyll until fall when chlorophyll breaks down Chlorophyll absorbs light at the Carotenoids (yellow, orange): present ends of the visible spectrum, mainly with chlorophyll in chloroplasts (and blue and red light, but reflects green contribute to photosynthesis) light Two groups: ○ carotene (yellow, orange, and red) Pigments used in photosynthesis ○ xanthophylls (yellow and brown) are bound to proteins in the Anthocyanins (dark red and violet): thylakoid membranes of found in the cell vacuole chloroplasts Coleus leaves Contain multiple pigments that are visible: Areas that are green contain chlorophyll Areas that are pink contain anthocyanins Areas that are dark purple contain both chlorophyll and anthocyanin pigments Areas that are white and light yellow contain no pigments Experiment: What are the pigments used in photosynthesis? (LM 6.2) 1. Detach a multicolored leaf containing green, pink, purple and white regions from a Coleus plant. 2. Draw your leaf, mapping the outline of the regions containing the different colors. 3. Put the leaf in a beaker w/80% ethanol. Place the beaker with the leaves and the ethanol inside another beaker with boiling water on a hot plate. 4. After the leaf has lost its color, remove it from the beaker and cover with water in a petri dish. 5. Add I2KI until the water is amber. Let the leaf sit in solution for 5 minutes. 6. Observe/record the results comparing the sketch you drew of the leaf’s different pigments with the regions of the leaf that have starch present. Note which regions of the leaves had the most amount of starch, medium amounts of starch and little starch. Predictions and Results: What are the pigments used in photosynthesis? Table 2: Predictions and results of starch test for exercise 6.2 Color Pigments Starch Present? Starch Present? (Predicted) (Actual) Green Chlorophyll a, b yes Purple Chlorophylls, some Anthocyanins Pink Anthocyanins no White None no Others? 6.1: Wavelengths of light used in photosynthesis- Demo Light filters used, so only particular wavelengths reach the leaf (white light contains all Green filter above leaf wavelengths). Color you see is wavelengths not absorbed, i.e.: reflected and transmitted. Why are most plants green? The image to the right shows our hypothesis with a green filter. What do you predict for the other (red, blue and black) filters, in terms of photosynthetic products? 6.1 Predictions 1) Leaves w/ green filter papers will have no starch present, because this paper will absorb red and blue light and transmit green light 2) Leaves w/ blue filter papers will have some starch present, because this paper will absorb red and green light and transmit blue light 3) Leaves w/red filter papers will have some starch present, because this paper will absorb blue and green light and transmit red light 4) Leaves w/black construction paper do not get any light so no starch will be produced Note: Even though plants make glucose in the leaf mesophyll tissue during photosynthesis, they store it in the form of starch. As such, how can we test these predictions? Demo Procedure 1. Remove leaf containing filters and construction paper from geranium plant. 2. Draw/take a picture of the leaf in your notebook, noting where each filter/paper is located (need to ID it later). 3. Remove the filters/paper and paper clips. 4. Leaves placed in beaker w/80% ethanol, w/in a larger beaker of boiling water. 5. After leaf has lost color, it will be moved to a Petri dish and covered with water and iodine until amber. Let sit in this solution for five minutes. Demo Procedure and Predictions 7. Record your predictions for which wavelengths will be transmitted or absorbed. 8. Record your observations/results noting which of the leaves had the most amount of starch, medium amounts of starch and little starch Table 1: predictions and results of exercise 6.1 Green Filter Blue Filter Red Filter Black Paper Uncovered Green transmitted absorbed absorbed absorbed transmitted Wavelengths Blue Wavelengths absorbed transmitted absorbed absorbed absorbed Red Wavelengths absorbed absorbed transmitted absorbed absorbed Starch Present? no some some no yes Iodine test for starch If results support the hypothesis: photosynthesis will only occur where there are chloroplasts. Glucose is a product of photosynthesis. Excess glucose is converted into starch (food storage for plants). Areas of the leaf that contain chlorophyll should have starch present. Areas of the leaf that contain only anthocyanins (or no pigments) should have no starch present. Demo Results Table 1: starch test results of exercise 6.1 Green Filter Blue Filter Red Filter Black Paper Uncovered Green Light Transmitted Absorbed Absorbed Absorbed Transmitted Blue Light Absorbed Transmitted Absorbed Absorbed Absorbed Red Light Absorbed Absorbed Transmitte Absorbed Absorbed d White Light Absorbed Absorbed Absorbed Absorbed Absorbed Starch Present? Negative Some Positive Negative Positive Paper chromatography (LM 6.3) A technique used to separate different components in a mixture There is a stationary phase (paper, polar) and a mobile phase (solvent, less polar). A mixture is applied to the paper and placed in the solvent. The solvent moves up the paper carrying different components of the mixture. Depending on their chemical structure, molecules will want to interact differently with the paper and solvent (move slower or faster). Polarity Polar molecules bind to the paper (stationary phase), less polar molecules dissolve in solvent (mobile phase). More polar components: move slower and stay closer to where you application site. Non-polar components: get carried with solvent up the paper, moves faster and further. Estimate polarity by counting oxygen groups in the chemical structure of a compound. Estimating polarity Count the number Pigment Order of Polarity of polar Oxygen (1st- most groups present for polar/ each major slowest) pigment. Chlorophyll Which is the most a(5) polar? The least? Chlorophyll Hypothesize how b(6) these pigments would behave in a Beta paper Carotene(0) chromatography experiment. Xanthophyll (2) Predictions? Estimating polarity Count the number Pigment Order of Polarity of polar Oxygen (1st- most groups present for polar/ each major slowest) pigment. Chlorophyll 2nd Which is the most a polar? The least? Chlorophyll 1st Hypothesize how b these pigments would behave in a Beta 4th paper Carotene chromatography experiment. Xanthophyll 3rd Predictions? Experiment: separation of substances by paper chromatography (LM 6.3) Objective: Use paper chromatography to separate leaf pigments by polarity 1. Mark a line in pencil 1.5 cm from the edge of the chromatography paper. Use gloves and foreceps when handling. 2. Stick a capillary pipette into the spinach leaf extract solution and streak it on top of the pencil line. Re-apply a couple of times letting it dry in between applications. 3. Let it dry. Roll the paper up into a cylinder and secure it with a paper-clip (top edge). 4. Place the paper in the solvent. The pencil line should be above the solvent. 5. As the solvent starts to move up the paper, there will be a leading edge of the solvent called the front. 6. When the front approaches the top of the paper (within 3cm), end the separation process. Results: separation of substances by paper chromatography Carotene most non-polar pigment Xanthophyll Chlorophyll a Chlorophyll b most polar pigment The most non-polar will dissolve in the (non-polar) solvent first. The most polar will be attracted to the (polar) chromatography paper (cellulose), and move last. Experiment: absorption spectrum of various leaf pigments (LM 6.4) 1. Cut each pigment band from chromatography paper Pigment Table and distribute to assigned team (see table to the right). 2. Place your team’s assigned pigment bands into 20ml Chlorophyll a 1,2,5 beaker w/10ml acetone, and swirl. Chlorophyll b 1,2,6 3. Label two cuvettes: “P” (pigment) and “B” (blank). 4. Pour 8mls beaker liquid into cuvette “P” and 8 mls of Xanthophyll 3,4 pure acetone to cuvette “B”. 5. Turn on spectrophotometer, set wavelength to 400nm. Carotene 3,4 6. Blank with sample “B”. All pigments 5,6 7. Record the % T of sample “P” from 400 nm to 720 nm at 20-nm intervals. record readings in Table 6.3 from * Remember to your lab manual. recalibrate (blank) 8. Convert to Absorbance in your lab notebook using when you move to a provided conversion table. new wavelength Predictions and results: absorption spectrum of various leaf pigments Absorption spectrum: Pigment Predicted wavelength Observed wavelength (nm) of peak absorption (nm) of peak absorption absorption pattern of a (lowest %T) (lowest %T) particular pigment across different wavelengths of Chlorophyll 400-500, 600-700 light. a Ex. 6.1: Which wavelengths Chlorophyll 400-500, 600-700 support photosynthesis? b Ex. 6.2: Which pigments Carotene 400-500 support photosynthesis? Xanthophyll 400-500 Hypothesize about absorption spectrum of the four pigments to the right: predict their peaks. Results: absorption spectrum of various leaf pigments To do list Pigments in Photosynthesis (LM 6.1 – 6.2) Paper Chromatograph (LM 6.3) Absorption Spectrum (LM 6.4)