Photosynthesis lab BIO 101

BIO 101 Lab: Photosynthesis Objectives:________________________________________________________________ • Determine the absorption spectrum of leaf pigments • appreciate the impact of light on photosynthesis • recognize that photosynthesis is occurring • identify the various pigments in plant leaves Background:_______________________________________________________________ Sunlight provides the majority of energy for organisms living in most ecosystems, however only a subset of organisms are capable of harvesting this energy. The process by which most autotrophs construct carbohydrates, is called photosynthesis . Chloroplasts in leaves are used to absorb the energy from sunlight. This energy is then stored in the covalent bonds of glucose, a monosaccharide. Animals ( heterotrophs ) can eat plants to obtain glucose, and then produce energy through a process called cellular respiration. Photosynthesis is made up of two phases, or sets of reactions: the light reactions and the Calvin cycle reactions. In the first phase, the light reaction , chlorophyll absorbs light energy which is used to split apart a molecule of water. The O atom from the water molecule is combined with an O from a second water molecule, producing O 2 , a gas, which is given off from the plant. The H from water is used in a proton gradient that diffuses through an ATP synthase to make ATP. In the Calvin cycle reaction, H from the light reaction is used to reduce CO 2 to glucose (C 6 H 12 O 6 ). The overall reaction for photosynthesis is represented by the chemical equation: 6 CO 2 + 6 H 2 O + sunlight → C 6 H 12 O 6 + 6 O 2 From the equation above the three key elements for photosynthesis to occur are carbon dioxide (CO 2 ), water (H 2 O), and light. If any of the three are missing from the system then photosynthesis will not occur, and glucose production in the plant will be negatively affected. Photosynthesis occurs in the green parts of plants, mainly in the leaves. The cells in the leaf tissues are loosely packed, and there is a large amount of space between the cells. This space is filled with gas, some of which is CO 2 from the air which the plant uses in photosynthesis and some of which is O 2 produced by photosynthesis. MEASURING PHOTOSYNTHESIS IN SPINACH LEAVES When small circular disks cut from leaves are placed in water they will float because the gasses in the leaf make it buoyant and lighter than the surrounding liquid. The gas can be pulled out of the leaves using a vacuum and the spaces will fill with liquid from the surrounding solution. Since fluids are heavier than gas, replacing the gas in the leaf with liquid causes the leaf disks to sink to the bottom of the solution. During the process of photosynthesis, as more O 2 is produced it will diffuse into the intercellular spaces and replace the liquid with gas. When enough O 2 has accumulated, the leaf disk will become buoyant again and turn on its edge or float to the surface of the solution. This technique, that is, measuring the % of leaf disks that float after a given time period, can be used as a method of measuring photosynthesis. Sodium bicarbonate (NaHCO 3 ) will be used as the solution to replace the gas in the leaves. It breaks down to provide the CO 2 which plants need for photosynthesis. Materials:__________________________________________________________________ 1

● spinach leaves, 4 dishes, 1 larger dish ● flask with side arm and stopper ● cork borer, rod or dissection probe, plastic forceps ● distilled water, vacuum pump ● NaHCO 3 solution, plant stand Procedure:________________________________________________________________ 1. Get 4 dishes and label them 1, 2, 3, and 4. Fill dish 1 about 2/3 full with distilled water. Fill the other 3 dishes about 2/3 full with 0.2% NaHCO 3 solution. Set these aside for later. 2. Pour about 100 ml of 0.2% NaHCO 3 solution into a flask with a side arm. 3. Get three or four spinach leaves and cut 40–50 disks with a cork borer. Do not include the large veins in the disks that you make. Stack the leaves so you can cut through them all and cut 3 or 4 disks at once. Use a glass rod or a probe to push the disks out of the cork borer into the flask containing NaHCO 3 solution. 4. Take the disks in the flask to your instructor, who will use a vacuum pump to draw the air out of the disks. As the flask is evacuated, you will see the water bubble and you may see bubbles coming out of the edges of the disks. Evacuate for about 15 seconds, then stop to see if the disks sink to the bottom of the flask. If they do not sink, continue to apply the vacuum. It is not necessary to sink all of the disks. The tissue will be damaged if it is over-aspirated. 5. Take the flask of disks back to your bench and pour its contents into the large oval dish. Discard any disks that are still floating (in the trash, not the sink). With forceps, gently transfer 10 disks to each of the 4 dishes. Spread the disks apart so that they are not resting on each other. Label them with your name and place as follows: Dish 1. under the plant light stand as close as possible to the bright light (bright light - low CO 2 ) Dish 2. next to dish 1 under the plant light stand (bright light - high CO 2 ) Dish 3. on your lab table under normal room light (low light - high CO 2 ) Dish 4. inside a cabinet or drawer in complete darkness (no light - high CO 2 ) 6. Wait at least 45 minutes for photosynthesis to occur. Write a hypothesis for this experiment here: 7. Bring all four of the dishes back to your lab table and count the number of disks that are either floating or turned on their edge in each dish. 8. Record your results in the Spinach Leaf Photosynthesis Data Table. 9. Clean up your materials and discard used disks in the trash, not in the sinks. 2

Some students did an experiment to determine if different wavelengths (colors) of light are equally effective at producing photosynthesis. Their results are shown in the Effect of Light data table below. Table: Effect of Light Color on Photosynthesis. Color of Light Number of disks floating % of disks floating none 1/30 3 white 25/30 83 blue 24/30 80 yellow 3/30 10 green 2/30 7 red 24/30 80 9. What conclusions can be drawn from the results in the table above? white light produces the most oxygen and red and blue can also produce oxygen ABSORPTION SPECTRUM OF CHLOROPHYLL IN SPINACH LEAVES Light energy is a small part of the electromagnetic spectrum that is visible to the human eye. The wavelength of visible light lies between 380 nm and 760 nm. In order to obtain the energy from light, plants must absorb light energy using pigments, namely chlorophyll a, chlorophyll b, carotene, and xanthophyll. These pigments show characteristic colors because they do not absorb all light equally. By measuring the absorbance at different wavelengths, the absorption spectrum of the leaf pigments can be obtained. 4

The color of leaves is determined by wavelengths of light that their pigments absorb and reflect. White light is made of all the colors or wavelengths in the visible electromagnetic spectrum, however, only those wavelengths of light that are absorbed are used in photosynthesis. The other wavelengths are reflected, and we interpret these as colors of the leaves. The mixture of chlorophyll molecules found in green leafy plants like spinach absorbs wavelengths of visible light with distinct absorbance peaks in the blue range (400–500 nm) and in the yellow-red range (600–700 nm). The wavelengths that are reflected tend to be in the wavelengths in the middle of these two that we interpret as green or shades of green. The specific wavelengths that a pigment absorbs can be determined by using a spectrophotometer. Light of specific wavelengths is passed through a solution of plant pigments and the light absorbed can be determined. The graph created is referred to as an absorption spectrum and the peaks on the graph represent the light the solution absorbs while the valleys on the graph represent the light that is reflected. In this experiment you will measure the visible absorbance spectrum of spinach pigments with a Vernier Spectro V Plus spectrophotometer. You will first measure the absorbance of blue and yellow food colored water samples which will provide an analogy to the absorbance of the chlorophyll extract. Materials:__________________________________________________________________ • Vernier Spectro Vis Plus • LabQuest • LabQuest App • One cuvette filled with distilled water • One cuvette filled with Yellow food coloring • One cuvette filled with Blue food coloring • One cuvette filled with Yellow and Blue food coloring mixed • One cuvette filled with isopropanol • One cuvette with Spinach extract Procedure:________________________________________________________________ 1. Connect the SpectroVis Plus spectrophotometer to the USB port of LabQuest. 2. Calibrate the spectrophotometer by A) using a blank cuvette filled with ¾ distilled water. B) choose calibrate from the Sensors menu of LabQuest. C) Place the blank in the spectrophotometer; make sure to align the cuvette so that the clear sides are facing the light source of the spectrophotometer. When the warmup period is complete, select Finish Calibration. Select OK. PART I. THE ABSORBANCE OF FOOD COLORING. 3. Conduct a full spectrum analysis of the blue food coloring. A) Use a cuvette filled with ¾ blue food coloring and place it in the spectrophotometer. B) Start data collection. A full spectrum graph of the blue food coloring sample will be displayed. C) Stop data collection by hitting the “stop” button. Examine the graph, noting the peak or peaks of the very high absorbance or other distinguishing features. 4. Store the run by tapping the File Cabinet icon in LabQuest. 5. Repeat steps 3 and 4 with the yellow food color sample. 6. Use a cuvette filled with equal amounts of blue and yellow food coloring (Mixed) and repeat steps 3 and 4. 5

Chlorophyll A peaks more at blue and Chlorophyll B peaks more at purple. They both make green at the end. There aren’t any undefined peaks, it follows the example chart and shows the absorbance peaks 3. If distilled water had been used as the calibration blank for the chlorophyll test, would it have affected the absorbance measurements? Because its not on the spectrum CONCLUSIONS: 1. What physiological processes does a plant continue to perform even when it is in the dark? Respiration 2. A respiring plant produces CO 2 . What is the fate of that CO 2 ? The CO2 goes in to made into glucose 3. In order for photosynthesis to occur, the following must be present (Hint: Review the photosynthesis equation): a. Light as the energy source. b.CO2 is the carbon source. c. H2O as the electron donor. d. Photosynthesis for the absorption of light energy. 4. Explain why grow lights for house plants are never green. Because green is reflected not absorbed 5. Explain why plants that are kept in the dark for a week are still able to conduct cellular respiration. because light is not needed for cellular respiration 6. There are more pigments present in leaves than you can see. Propose an explanation for why leaves only appear green in the growing season, but turn a bright color, such as orange, in the autumn when the weather turns cold. There’s no green pigment so it’s just reflecting other colors that are already there 7. One hypothesis for the extinction of the dinosaurs is that a large meteor struck the earth, sending up large clouds of dust into the atmosphere. This would negatively affect all life on earth in at least two ways. One relates to photosynthesis and the other does not. Speculate what the two negative effects could be. 7

the dust cloud would block the sunlight, plants cannot get energy meaning they would die, and we would not get food and it would be really cold because there's no sun PHOTOSYNTHESIS AND pH EXperiment Experiment 1. Observing photosynthesis (work in groups) 1. In the laboratory, you would add about 60 ml of tap water into a beaker and then add about 1 mL of bromothymol blue to the water in the beaker. 2. Using a clean straw, you would then gently blow into the solution until the color of the solution turns yellow. 3. You would next fill three test tubes 2/3 full with this yellow-colored solution. 4. Then, you would place a 3-inch cutting of Elodea into Tube 1 and another 3-inch cutting of Elodea into Tube 2, making sure that the cuttings were completely immersed in the solution. After this, you would wrap Tube 1 with green film. Tube 3 will be left alone and will not contain a plant cutting. 5. In Table 1 in the Lab Worksheet, record the color of the solution before blowing into it in the “before exhaling” column. 6. In Table 1 in the Lab Worksheet, record the color of the solution after blowing into it in the “after exhaling column. 7. Answer question 1 in the Lab Worksheet 8. In the laboratory, you would then place the test tubes in front of a light source such as a fluorescent lamp. You would allow the tubes to be exposed to the light for 1 hour. 9. Record the color of the solution in each test tube in Table 2 of the Lab Worksheet 10. Make a hypothesis about how the color of bromothymol blue solution in each test tube will change and record the color you expect for each tube in Table 2 in the “Expected Color after 1 hour of light exposure” column. When making a hypothesis, consider why the pH-indicating solution turned color in the first place and what might happen if the substance that caused the color change was removed by a plant performing photosynthesis. 8

Table 2. Observing Photosynthesis Tube Color before 1 hour of light exposure Expected Color after 1 hour of light exposure Observed color after 1 hour of light exposure 1 ( Elodea + green film) 2 ( Elodea ) 3 (no plant) 2. In this experiment , what is the purpose of the tube without the plant? 3. Explain the color change or lack of color change in the three experimental tubes: a. Tube 1: b. Tube 2: c. Tube 3:. 10

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