Photosynthesis Lab 11 PDF Fall 2024

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

Lab 11: Photosynthesis Lab Manual 6.1-6.4, Fall 2024 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 to Generate an absorption spectrum for separate molecules various photosynthetic pigments Roadmap of Today’s Activities 6.3: Separation of Pigments ○ Use chromatography paper to see individual pigments. ○ One chromatography paper per student 8:30- 6.2 is a demo. 6.1: Wavelengths of Light for Photosynthesis 10:30- 6.1 is a demo. ○ Observe photosynthesis in a leaf with several filters attached. 6.2: Pigments of Photosynthesis ○ Observe photosynthesis in a leaf containing several different colors. 6.4: Absorption Spectrum of Leaf Pigments ○ Use spectrophotometry to find the wavelengths best absorbed by your pigment. 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, 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 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 Also in leaves– visible in fall chlorophyll a and b Carotenoids (yellow, orange): present with chlorophyll in chloroplasts (and contribute Chlorophyll absorbs light at the ends to photosynthesis) of the visible spectrum, mainly blue Two groups: and red light, but reflects green light ○ carotene (yellow, orange, and red) ○ xanthophylls (yellow and brown) Pigments used in photosynthesis are Anthocyanins (dark red and violet): found in bound to proteins in the thylakoid membranes of chloroplasts the cell vacuole Also includes Chlorophyll b. 6.3: Paper chromatography 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- groups present for most each major polar/ slowest) pigment. Which is the most Chlorophyll ?? a polar? The least? Hypothesize how Chlorophyll ?? b these pigments would behave in a paper Beta ?? Carotene chromatography experiment. Xanthophyll ?? Predictions? 6.3: separation of substances by paper chromatography 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 forceps 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 paperclip (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 nonpolar 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. 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. 6.1: Wavelengths of light used in photosynthesis Light filters used, so only certain wavelengths reach the leaf (white light contains all wavelengths). Green filter above leaf 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 filters, in terms of photosynthetic products? 6.1 Predictions (and making your own!) 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 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? 6.1 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. Procedure and Predictions 6. Record your predictions for which wavelengths will be transmitted or absorbed. 7. 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 6.1 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 6.2: Coleus leaves (DEMO) 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 6.2: What are the pigments used in photosynthesis? 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, yes Anthocyanins Pink Anthocyanins no White None no 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. 6.4: absorption spectrum of various leaf pigments 6.4: absorption spectrum of various leaf pigments 1. Cut each pigment band from chromatography paper and Pigment Table distribute to assigned team (see table to the right). 2. Place your team’s assigned pigment bands into 20 ml Chlorophyll a 1 beaker w/10 ml ethanol, and swirl. Chlorophyll b 2 3. Label two cuvettes: “P” (pigment) and “B” (blank). 4. Pour 8 ml of beaker liquid into cuvette “P” and 8 mls of Xanthophyll 3 ethanol to cuvette “B”. 5. Turn on spectrophotometer, set wavelength to 400 nm. Carotene 4 6. Blank with sample “B”. Record the Absorbance of sample “P” from 400 nm to 720 All pigments 5/6 7. nm at 20-nm intervals. record readings in Table 6.3 from * Remember to recalibrate your lab manual. (blank) when you move to a new wavelength Predictions and results: absorption spectrum of various leaf pigments Absorption spectrum: Pigment Predicted wavelength Observed wavelength absorption pattern of a (nm) of peak absorption (nm) of peak absorption particular pigment across (lowest %T) (lowest %T) different wavelengths of light. Chlorophyll 400-500, 600-700 a Ex. 6.1: Which wavelengths support photosynthesis? Chlorophyll 400-500, 600-700 b Ex. 6.2: Which pigments support photosynthesis? Carotene 400-500 Hypothesize about Xanthophyll 400-500 absorption spectrum of the four pigments to the right: predict their peaks. Results: absorption spectrum of various leaf pigments  Lab Report Lab Report Notebook 1. You will write your lab report on this From 6.1: hypotheses, predictions (table ok), lab. and before and after drawings of the leaf From 6.2: hypotheses, predictions (table ok), 2. There will be two drafts- a first draft and and before and after drawings of the leaf a final draft. You are graded on both! From 6.3: Ranking of the four pigments in 3. Your first draft is due November 7th order of number of polar groups present. Hypotheses, predictions, chromatography 4. Please refer to the scientific writing lab paper sketch. when writing your report and make sure From 6.4: Hypotheses, predictions, and you include all components of a Tables 6.2 and 6.3 from the lab manual. scientific paper. 5. The final draft will be due TBD