Exercise 10 PHOTOSYNTHESISStudent Learning Outcomes At the completion of this exercise you should: (1) Be able to describe, using both nanometers and color descriptions, the wavelengths of visible sunlight (2) Be able to describe how the “paper chromatography” technique can be used to separate out the different pigments present in plant leaves.(3) Be able to describe the two most important wavelength of sunlight used in photosynthesis.(4) Understand the experiment that was used to measure the relationship between light intensity and the rate of photosynthesis.(5) Be able to write out the summary equation for photosynthesis.undefined*Ignore the two video links included in the virtual images file. You will only use the virtual images to look at images. These parts of the lab are highlighted in purple.undefinedI. The Nature of Light Striking the Surface of a LeafSunlight is a relatively small but very important part of the vast electromagnetic spectrum of energy. Visible light, together with a small amount of invisible radiation at its boundaries, is the only part of this spectrum which is useful to life. Close examination of visible natural light reveals that it, too, contains a spectrum, which is revealed to our eyes in the colors of a rainbow. Two physical properties of light are of special interest to biologists: wavelength and energy.Procedure:1. Point the slit end of the Spectroscope at a white light source, such as a small light bulb. The prism within the spectroscope will separate the various wavelengths (colors) of which the white light is composed. Instead of looking through an actual spectroscope, you will refer to the chart of the visible light spectrum located on the bottom of page 2 of the virtual images. Zoom in so that you can see the wavelength values. Use this information to answer question #2. The units on the chart are in angstroms, but you need to use nm (nanometers) in your answers for question #2. I’ve completed the first two colors (violet and blue) for you, to show you how it should be completed. Table 1.Metric System: Length Measurement UnitsQuantityNumerical ValueEnglish EquivalentConverting to Metrickilometer (km)1,000 m1 km = 0.62 mile 1 mile = 1.609 kmmeter (m)100 cm1 m = 3.28 feet = 1.09 yard1 yard = 0.914 m1 foot = .305 mcentimeter (cm)0.01 m1cm = 0.394 inch1 foot = 30.5 cmmillimeter (mm)0.001 m 1 mm = 0.039 inch1 inch = 2.54 cmmicrometer (1 µm),also called a "micron (µ)"0.000001 m nanometer (nm),also called a"millimicron(mµ)"0.000000001 m angstrom (Å)0.0000000001 m*Note: In other sources, you will sometimes see another unit, the nanometer (nm), used to measure wavelength. Each nanometer is equal to ten angstroms: for example, 7000 Å=700 nm. Although both the angstrom and the nanometer are used to measure wavelength, the nanometer is currently the more frequently used unit.Question 2. The visible spectrum chart gives angstroms as the until of wavelength. Since one nanometer is equal to 10 angstroms, 3500 (Å) = 350 nm. Write down the wavelengths in nanometers, using this simple conversion. Nanometer is currently the more frequently used unit. Complete columns 2 and 3 of the table. In column 2, list the six visible light colors starting with the one with the shortest wavelength and ending with the color with the longest wavelength. I’ve completed the first two colors for you. The remaining colors are green, orange, red, and yellow. I’ve listed the remaining colors in alphabetical order, but you need to place them in order in terms of ascending wavelength. In column 3, indicate the approximate wavelength range of the colors.WavelengthObserved ColorApproximate Range of Wavelengths (nm)Relative EnergyShortest1. Violet 380-450nm Most energy2. Blue 450-500nm3. 4. 5. Longest6. Least energyQuestion 3. It is known that shorter wavelengths of light possess more energy than longer wavelengths. Which color from the table in Question #2 has the highest energy and which color has the lowest energy.undefinedReplace this text with your answer.undefinedII. The Photosynthetic Pigments Pigments are light absorbing substances. They are useful to humans in decoration because they absorb certain wavelengths and reflect others, thus coloring our environment. In this case, we appreciate the pigments for the energy they reflect to our eyes. Plants contain several pigments, (like chlorophyll), each pigment having its own light absorbing qualities. The colors which we see on plants represent the wavelengths reflected by their pigments. In this lab, it is the absorbed light that is more important to us, since this energy is used to produce glucose through photosynthesis. As time passes, energy absorbed by plant pigments flows through the entire food web, providing energy for nearly all forms of life.Refer to page 3 of the virtual images and notice the color change in the test tube on the right when light is shined upon it.undefinedYou are viewing a phenomenon known as fluorescence, a process during which energy absorbed at one wavelength is emitted at another wavelength.Various wavelengths of light are being absorbed by the mixture of pigments. Chlorophyll a molecules would normally use this absorbed light to excite electrons in the light reactions. But, in this case, the excited electrons of chlorophyll a are forced to release their energy as lower energy light in the red range. Under normal circumstances, a leaf is not blended and dissolved in a solution of acetone. Normally, the chlorophyll pigments of a leaf are organized as photosystems so that the sunlight energy can be harnessed and used to generate ATP and NADH. Since the grass leaf cells have been disrupted and the acetone has dispersed all membranes, the normal electron acceptors and photosystems of photosynthesis are all jumbled up and not able to function properly. That is why the light shined upon the test tube of grass pigment extract is unable to harness the light energy, and emits it as red fluorescence. Note: This little activity is a bit odd to me. I’m not sure why a picture of a test tube with green food coloring is shown. If you find it confusing, don’t worry about it. undefinedQuestion 4. Describe the color change (from green to what color?) that occurs when light is shined upon on the blended pigment extract from grass leaves:Replace this text with your answer.undefinedB. Paper ChromatographyPaper chromatography is a technique used for separating components of complex mixtures quickly and cheaply. The process in this case, includes three separation phases: the paper, petroleum ether, and acetone. Separation occurs because of the differential affinities of the components of the pigment mixture for the three different phase substances. Those pigments favoring the solvent system (the ether and acetone) will race ahead with the solvent as it moves up the paper, while those favoring the chromatography paper will lag behind. In addition smaller (lighter) pigments will move up faster than larger ones.undefinedRead the following procedure and then watch the following video: Paper Chromatography being used to Separate out the Various Pigments within a Spinach LeafProcedure:1. Obtain a piece of chromatography paper. Pick it up by the straight cut end, not the angled cut end or the center of the paper. 2. Place the chromatography paper on a clean space of the lab bench. Obtain a bright green spinach leaf, and place it on top of the chromatography paper near the angled cut, as shown in Figure 1. Then, roll the coin firmly in a straight line over the spinach, so that the coin presses the spinach leaf’s pigments into the paper.3. Attach the stopper and paper clip to the straight end of the chromatography paper, as shown in Figure 1, and place on your group’s lab bench.4. Obtain a test tube rack with two large test tubes for your group. Carry the rack from the bottom. You only need one of the tubes but leave the other in the rack. At the side lab bench, pour the 90% petroleum ether: 10 % acetone solvent mixture to fill the tube to an approximate depth of 2 cm. Be sure not to breathe in this solvent mixture, as it is not good for you. Walk back to your group’s lab bench with your test tubes and rack. Place your test tube rack in the place you want to leave it in for the remainder of this exercise.5. Place the chromatography paper, paper clip, and stopper set-up inside the test tube, so that the solvent just touches and soaks the end of the chromatography paper, as shown in Figure 3. Adjust the paper clip so that the solvent does not soak the pigment line but continues to touch the tip of the paper. undefined6. Place the test tube in as vertical a position as possible. Do not move the test tube rack or test tube during the duration of the exercise, or the banding patterns will not be distinct. Start your timer.undefined7. When the fastest moving pigment approaches the top at around 15 – 20 minutes, remove the strip from the tube. Otherwise the pigments may crowd together at the top. Allow the paper to dry. Immediately, pour the solvent back into the original container, being careful not to breathe it in.8. When developed, the chromatogram should show 4-5 fairly distinct bands. The bands from top to bottom are: the orange-yellow carotene; the yellowish bands are xanthophylls; the bluish green band is chlorophyll a, and the olive green band is chlorophyll b. As the strip dries and is exposed to light and oxygen, some of the pigments will fade.undefinedThe point of this experiment is to show you the various types of pigments located within a leaf that are involved in absorbing sunlight for photosynthesis.Question 5. Refer to the finished paper chromatogram at the end of the video or the paper chromatogram that I’ve included (to the right). Draw the paper chromatogram: Color and label the 4-5 different pigment bands.undefinedMake your drawing by hand, take a picture and paste it in place of this text. From the menu above, choose Insert > Image.undefinedC. Determining the Absorption Spectrum of the Leaf PigmentsAs light strikes a pigment, certain wavelengths will be reflected, and some will be absorbed. Refer to the absorption spectrums on page 5 of the virtual images to answer Questions #7 and 8.undefinedQuestion 7. Generally speaking, which wavelengths and their corresponding colors are most strongly absorbed by the following pigments? The peaks on the spectrums should be your answers. Be sure to include both peaks for each pigment. Refer to your answers from Question 2 for the corresponding colors for these wavelengths. I’ve completed one of the absorption peaks for chlorophyll a to show you how it should be completed.undefinedPigment:Wavelengths:Colors:Chlorophyll a 430 nm VioletChlorophyll bundefinedQuestion 8. Which wavelength ranges and corresponding colors are least absorbed? Refer to your answer from Question 2 for the corresponding colors for the wavelength ranges. The colors that are least absorbed, are the colors that are reflected.undefinedPigmentRanges:Colors:Chlorophyll a 450-625 nm Bluish green, green, yellow, and orange light. Primarily green and yellow.Chlorophyll bundefinedIntroduction to SpectrophotometersIn today’s lab you will be using the Spectronic 200, a type of spectrophotometer, to perform an analysis. Spectrophotometers measure light transmission and absorption. For your lab activity, you will concentrate on the light absorption measurements.undefinedA spectrophotometer works by using a light source that emits light of various wavelengths (see Figure 4). An adjustable filter removes all but a single wavelength of light (chosen by the experimenter). This wavelength of light passes through the sample tube, and an analog scale in the spectrophotometer measures the percent transmittance. This is the percentage of that wavelength of light, that is not absorbed by the solution in the sample test tube–meaning that it passes straight through the solution.undefinedFigure 4. Basic function of the spectrophotometer measuring transmittance of lightundefinedThe amount of light that a substance transmits is called the % Transmittance or %T for short. A substance that transmits no visible light is opaque (very murky) and has a 0% T. A substance that is completely transparent, transmits all visible light, has a 100% T. We will use acetone as a calibration or blanking solution; it is completely transparent and therefore will have a 100% T. You will shine various wavelengths of light through a sample test tube containing leaf pigments that have been prepared as a solution. Since these are photosynthetic pigments, they will absorb some of the light and transmit/reflect other wavelengths of light. This experiment will allow us to experimentally determine the specific wavelengths of light that are primarily absorbed by the major photosynthetic pigments within a leaf, to power photosynthesis. If this experiment is done accurately, the results should mirror the absorption spectrum on page 5 of the virtual images. Realize that the absorption spectrum in the virtual images includes separate absorption spectra for chlorophyll a and b, but the following experimental results will look at all pigments at the same time–meaning the graph will have less defined peaks and be a little “blobbier” (for lack of a better term). undefinedProcedure:undefined*Your instructor has done the following steps prior to lab.1. Basic calibration and Dark Zeroing of Spectrophotometer2. Set the Spectrophotometer to read % Transmittance (%T)3. Set the initial wavelength for today’s experiment4. Prepared samples for today’s experimentundefinedWatch the following video: Determining the Absorption Spectrum of Leaf Pigments to obtain the % Transmittance values for Table 1 (below). I’ve recorded the %T for the first wavelength (400nm) to show you how it should be completed. The spectrophotometer is first blanked/calibrated with a test tube of acetone for each wavelength.undefinedUse a Spectronic 200 spectrophotometer to determine the absorption spectrum of your grass leaf pigment extract. Record %T in your Table 1.Table 1.Percent TRANSMITTANCE at Wavelengths 400 nm - 700 nm400425450475500525550575600625650675700 60.7Subtract the above values from 100%, since % T + % A = 100%. Write the results in the Table 2 blanks below:Table 2.Percent ABSORPTION at Wavelengths 400 nm - 700 nm40042545047550052555057560062565067570039.3 undefinedUsing the % absorption values from Table 2, to create a leaf pigment absorption spectrum graph. I’ve included the link to a Google Spreadsheet here. You will need to insert the % absorption data and then copy and paste the resulting graph into the answer box belowundefinedPaste the graph from Google Spreadsheets undefinedQuestion 9. On the graph that you pasted above: Describe the two wavelength ranges where you see the "peaks" of light absorption? What colors of light are found at these two peaks?Replace this text with your answer.Question 10.a. On the graph that you pasted above: Describe the wavelength range where absorption is low?Replace this text with your answer.undefinedb. What colors of light are found in this range?Replace this text with your answer.undefinedQuestion 11.a. What happens to the light that is not absorbed by a solution? How does this relate to the fact that leaves appear green?undefinedReplace this text with your answer.undefinedIII. Leaf Anatomy: Model of a Leaf Cross-SectionProcedure: undefinedRefer to pages 6 and 7 of the virtual images to answer Questions #12-14undefinedPage 6 shows the bottom of a leaf: the lower epidermal cells, guard cells, and stomatal pores; Page 7 shows the cross section of a leaf. This is just like the cross section of a leaf that we cover in Module 8 and is good practice for the Module 8 quiz. undefinedQuestion 12.Although a plant’s leaf is its primary organ for photosynthesis, not all the cells in a leaf are photosynthetic. undefineda. Observe the image of the bottom of a leaf (page 6 of the virtual images). What is the name of the cells on the bottom surface of the leaf that have chloroplasts? Hint: They control the opening and closing of stomatal pores.Replace this text with your answer.undefinedb. Why do you think these cells (your answer for part a) need chloroplasts for undergoing photosynthesis. Hint: Think about the job of these cells and the amount of energy that it would demand.Replace this text with your answer.undefinedc. Now observe the diagram leaf cross section (page 7 of the virtual images).Which two cell types have chloroplasts in this part of the leaf? Hint: These are the two primary sites of photosynthesis in a leaf.undefinedReplace this text with your answer.Question 13. Observe the openings in the leaf’s epidermis (page 6 of the virtual images). Each opening is between a pair of guard cells. What are the openings (pores) called? undefinedReplace this text with your answer.undefinedThese pores are the site of gas exchange in a plant. State the gas that enters these leaves (it’s a reactant of photosynthesis). State the two gases that exit these pores (one of them is a product of photosynthesis)Replace this text with your answer.undefinedQuestion 14a. Observe the leaf cross section (page 7 of the virtual images). Is the cellular material inside the leaf arranged so that the leaf is a solid mass, or are there areas with spaces inside? What are the names of the cells that have air spaces around them? Refer to Module 8 if you are unsure.Replace this text with your answer.undefinedQuestion 14b. Refer to the diagram of the cross section of a leaf (above). Make your own sketch of the diagram (above). Label and point to the: cuticle, lower epidermis, upper epidermis, spongy mesophyll, air/empty space within the leaf, palisade mesophyll, vascular tissue (xylem + phloem), guard cells, and a stomatal pore. Refer to Module 8 and the cross-section of an actual Lilac leaf below for reference.undefinedMake your drawing by hand, take a picture and paste it in place of this text. From the menu above, choose Insert > Image.undefinedundefinedShown above is a Syringa (lilac) leaf cross section:undefinedBelow the upper epidermis is a region of palisade mesophyll tissue, consisting of one or more layers of elongated cells with the long axis of the cell perpendicular to the surface of the leaf. Palisade cells contain numerous chloroplasts and are the primary site of photosynthesis. Beneath the palisade tissue is a region of rounded, parenchymatous cells, which constitute the spongy mesophyll tissue. The palisade tissue and spongy parenchyma are collectively called leaf mesophyll. Note the presence of numerous intercellular air spaces in the mesophyll. Locate the stomatal pore surrounded by a pair of guard cells.undefinedQuestion 15. Write the summary equation for photosynthesis:undefinedReplace this text with your answer.undefinedQuestion 16. If we could somehow remove the gases from a leaf without killing all the cells and then expose that leaf to sunlight, water and CO2 for a given amount of time, what gas would be produced that would refill the spaces within the leaf?undefinedReplace this text with your answer.undefinedIV. Photosynthesis and Light IntensityMany questions may come to your mind when you first study photosynthesis. Perhaps you have never really thought about plants respiring and using oxygen. Just how much do they use in a given period of time? Is it about the same amount as they produce during photosynthesis? Is the photosynthetic rate the same in a plant on a cloudy day as it is on a sunny day? The questions are endless. Doubtless there are many that you may be pondering right now. Some of these questions may require considerable time and sophisticated pieces of equipment to answer, but many may be studied in our lab, with minimum equipment and a little thought.The question that you are trying to answer today is: “Does the intensity of the light striking a plant affect the rate at which plants undergo photosynthesis?The Effect of Light Intensity on Photosynthesis: ExperimentIt is quite likely that the experiment you suggested would be suitable to test your hypothesis, but in order to give some uniformity to today's laboratory experiment, it will be set up for you.Leaf material, placed in a buffered water solution, will be subjected to a vacuum, removing gases from within the spongy mesophyll layer of the leaf. As the gas is drawn out of the spongy mesophyll layer, the leaf will become denser than the water, and it will sink. Light of two different intensities will then be shined upon the leaf, and due to the gas generated by photosynthesis, the leaf will eventually regain its buoyancy and rise to the surface. The time taken for the leaf to come to the surface will be used as a rough estimate of the photosynthetic rate. The gas that is produced during photosynthesis that causes the leaf to rise, is oxygen. The rate will be compared for different light intensities.undefinedProcedure:1. Preparation of Leaf Discs:undefinedRead steps 1a-c of the following procedure and then watch the following video: The Effect of Photosynthesis on Light Intensity (Part 1) undefineda. Your experimental material will be the leaves from freshly a cut plant b. Obtain a 50 ml beaker and pour in a small amount of buffer solution.c. Using a #5 cork borer (punch) and cutting board, punch/cut out six or seven leaf discs of the same size. In cutting your discs avoid the major veins and make them as evenly-sized as possible. As you cut the discs, place them into the buffer solution so that they do not dry out. The buffer solution resists changes in the acidity or the alkalinity of the water, thereby ensuring that the pH variable will not interfere with the experiment.undefinedQuestion 17. Describe what the professor was punching/cutting out using the cork borer.undefinedReplace this text with your answer.undefinedQuestion 18. Why is it important for all of the leaf discs to be uniform in size? Hint: Think about the basics of proper experimental design.undefinedReplace this text with your answer.undefinedRead steps 1d-h of the following procedure and then watch the following video: The Effect of Photosynthesis on Light Intensity (Part 2). Use the video to answer question #21 undefinedd. Your instructor will prepare a large vacuum flask containing a buffer solution. Drop your leaf discs into the flask, along with those of the rest of the class. Be sure the discs are down in the buffer solution. Question 19. In preparation for the experiment: Observe the leaf discs before the vacuum is placed on them. Are they floating, or are they on the bottom of the flask? Explain why they are either floating or at the bottom of the flask.undefinedReplace this text with your answer.undefinedIn preparation for the experiment: Observe the leaf discs after the vacuum is placed on them. Are they floating, or are they on the bottom of the flask? Explain why they are either floating or at the bottom of the flask. I think it’s difficult to see the leaf discs at all towards the end of the video (once they have been placed under a vacuum). Check out the start of the Part 3 video (below) and observe the leaf discs in the small beaker on the left, and the answer will be clear.undefinedReplace this text with your answer.undefinede. Connect the vacuum flask to the lyophilizer as demonstrated by your instructor. Turn on the vacuum.f. After applying the vacuum for 30-60 seconds (consult your instructor), turn off the vacuum and disconnect the flask. Generally, you need just enough vacuum time so that most of the discs sink to the bottom after you disconnect the flask. g. If most of the discs do not sink to the bottom, repeat steps e and f. In order to avoid overdoing it, apply vacuum for 5 second periods until most discs sink.h. Your instructor will pour the buffer with the discs into a large stacking dish. undefinedProcedure steps 1i-j, 2a-c, and 3a are not shown in the videos.undefinedi. Retrieve your 50 ml beaker, and using a graduated cylinder to measure, add 30 mL of buffer into it. j. Use forceps to retrieve 6 sunken discs of equal sizes and without leaf veins. Be sure to handle the disks carefully without squashing or folding the disks with the forceps. Place the discs in the buffer solution into your 50 ml beaker.2. Equipment Set-upa. Obtain a ring stand with a flood lamp with a ring attached and a timer.b. Place a stacking dish half full of cold water on the ring. This will serve as a heat trap. The position of the heat trap should be one inch above the beaker.c. Measure the distance from the top surface of the ring stand to the painted edge of the light bulb where the light shines from. One group at your table should use the 15 cm distance, and the other should use the 60 cm distance. These are the two different light intensities. Light intensity is the experimental variable. 3. Data Collectiona. Using forceps or a dissecting needle (not your finger), move the leaf discs around on the bottom of the beaker so that they are not resting on one another.undefinedRead steps 3b-e of the following procedure and then watch the following video: The Effect of Photosynthesis on Light Intensity (Part 3). Here you will observe the experimental group that has the leaf discs that are placed 15 cm away from the light source. You must use the video to record (the time in seconds) that it takes the first 3 leaf discs to first touch the surface of the buffered solution. Use this data to fill out Table 3. I’ve done the first leaf for you to show you how this should be completed. It touched the surface at 3 minutes and 5 seconds, which was converted into 185 seconds.undefinedRead steps 3b-e of the following procedure and then watch the following video: The Effect of Photosynthesis on Light Intensity (Part 4). Here you will observe the experimental group that has the leaf discs that are placed 60 cm away from the light source. You must use the video to record (the time in seconds) that it takes the first 3 leaf discs to first touch the surface of the buffered solution. Use this data to fill out Table 3.undefinedb. Add three drops of saturated sodium bicarbonate (NaHCO3 = baking soda) solution to the beaker (not the stacking dish). This dissociates in water to form CO2.c. Immediately place the beaker on the ring stand base, move it into position and begin timing.d. As gas is produced during photosynthesis, it will fill the spaces in the mesophyll and buoy up the discs in the solution. You will measure the rate of photosynthesis by determining the number of seconds that it takes each leaf disc to rise to the surface. (Note: A disc does not need to be flat on the surface, as long as its edge reaches the surface.)e. Obtain times (in seconds) for the first 3 leaf discs to rise. Enter the data in Table 3 below. Make sure to convert the data into seconds.Table 3. Leaf Disc DataDistanceTeamTime (sec)TeamNumberDisc 1Disc 2Disc 3Averages15 cm1185 sec. 60 cm1C. Photosynthesis Data AnalysisundefinedQuestion 20. What is the independent variable (the variable that was intentionally varied in the experiment)?Replace this text with your answer.undefinedQuestion 21. What is the dependent variable (the variable that was measured to see if the independent variable has an effect)?Replace this text with your answer.undefinedQuestion 22. According to your data (the data acquired in the previous two videos), explain the relationship between the intensity of light and the rate of photosynthesis. Explain how the rate of photosynthesis was specifically quantified in the experiment. Replace this text with your answer.