PHOTOSYNTHEIS – LAB 10 Photosynthesis Photosynthesis is one of the most important series of chemical reactions that occurs on earth as virtually all life within earth’s biosphere depends on photosynthesis either directly or indirectly. Photosynthesis is a complex chemical process by which radiant solar energy is converted to chemical energy. The following equation summarizes photosynthesis. 6 CO2 + 12 H2O → Light/Chlorophyll → C6H12O6 + 6 H2O + 6O2 Carbon dioxide + Water → Light/Chlorophyll → Glucose + Water + Oxygen Paper Chromatography of Photosynthetic Pigments Light must be absorbed before its energy can be used. A substance that absorbs light is a pigment. The primary photosynthetic pigments that absorb light for photosynthesis are chlorophylls a and b. However, chlorophylls are not the only photosynthetic pigment; accessory pigments such as carotenes and xanthophyll also absorb light and transfer energy to chlorophyll a. Paper chromatography is a technique for separating dissolved compounds such as chlorophyll, carotene, and xanthophyll. When a solution of these pigments is applied to strips of paper, the pigments adsorb onto the fibers of the paper. When the tip of the paper is immersed in a solvent, the solvent is absorbed and moves up through the paper. As the solvent moves through the spot of applied pigments, the pigments dissolve in the moving solvent. However, the pigments do not always keep up with the moving solvent- some pigments move almost as fast as the solvent, 1 PHOTOSYNTHEIS – LAB 10 whereas others move more slowly. This differential movement of pigments results from each pigment's solubility and characteristic tendency to stick (i.e., be adsorbed) to the cellulose fibers of the paper. A pigment's molecular size, polarity, and solubility determine the strength of this tendency; pigments adsorbed strongly move slowly, whereas those adsorbed weakly move fastest. Thus, each pigment has a characteristic rate of movement, and the pigments can be separated from each other. In procedure 1, four bands of color will appear on the strip-a yellow band of xanthophyll, a yellow-orange band of carotenes, a blue-green band of chlorophyll a, and a yellow-green band of chlorophyll b. The relationship of the distance moved by a pigment to the distance moved by the solvent front is specific for a given set of conditions. We call this relationship the Rf value and define it as follows: Rf = Distance Moved by Pigment/ Distance From Pigment Origin to Solvent Front Thus, paper chromatography can be used to identify each pigment by its characteristic Rf value. This Rf is constant for a given pigment in a particular solvent-matrix system. Exercise 1 Separate Plant Pigments by Paper Chromatography 1. Observe the contents of the provided container labeled "Plant Extract." 2. Obtain a strip of chromatography paper from your lab instructor. Handle the paper by its edges so that oil on your fingers does not contaminate the paper. 3. Use a Pasteur pipet to apply a stripe of plant extract approximately 2 cm from the tip of the paper. Be sure to mix “Plant Extract” before each use as it may settle. Blow the stripe dry and repeat this application at least 15 times. For this separation to work well, you must start with an extremely concentrated application of extract on the paper. 4. Place the chromatography strip in a test tube 2 mL of chromatography solvent (9 parts petroleum ether: 1 part acetone). Position the chromatography strip so that the tip of the strip (but not the stripe of plant extract) is submerged in the solvent. You can do this by hooking the strip of paper with a pin inserted in the tube's stopper. 5. Place the tube in a test-tube rack and watch as the solvent moves up the paper. Keep the tubes capped and undisturbed during solvent movement. 2 PHOTOSYNTHEIS – LAB 10 6. Remove the chromatography strip before the solvent front reaches the top of the strip (i.e., after 2-3 min). Mark the position of the solvent front with a pencil (ink will bleed) and set the strip aside to dry. Observe the bands of color, and then draw your results. Use your textbook or other materials in lab to identify the different bands of pigments according to their position and color. For example, xanthophyll appears yellow. 7. Use a ruler to measure the distance from the pigment origin to the solvent front and from the origin to each pigment band. Calculate the Rf value for each pigment; record your data in table 1. Table 1: Rf Values for Plant Pigments Pigment Rf Value Carotene 13.3/13.3 Xanthophyll 11/13.3 Chlorophyll a 7.3/13.3 Chlorophyll b 10/13.3 Exercise 2 Uptake of Carbon Dioxide During Photosynthesis Phenol Red (phenol-sulfonphthalein) is a pH indicator that turns yellow in an acidic solution and becomes red in neutral or basic solutions. In this experiment, you will use the pH indicator phenol red to detect the uptake of CO2 by a photosynthesizing aquatic plant, Elodea. To detect CO2 uptake you will put a plant into an environment that you have made slightly acidic with your breath. Carbon dioxide in your breath will dissolve in water to form carbonic acid; which will lower the pH of the solution. H2O + CO2 /→ H2CO3 /→ HCO3 + H+ Water + Carbon dioxide /→ Carbonic Acid /→ Bicarbonate ion + Hydrogen ion 3 PHOTOSYNTHEIS – LAB 10 Observe the Uptake of CO2 During Photosynthesis 1. Fill two test tubes half full with a dilute solution of Phenol Red (2%). 2. Using a straw, gently blow into the phenol red solution. Stop blowing as soon as the solution turns yellow. Continuing to blow will result in an excess of carbonic acid in the solution and will significantly lengthen the experiment. 3. Add 10cm pieces of healthy Elodea (or green algae) to one of the test tubes. Pour out any excess solution that is above the Elodea. The second tube will not contain Elodea, as it is the control. 4. Cover the tops of both tubes with parafilm to prevent gas from entering into the tubes. 5. Place both tubes approximately 0.5 meters in front of a 100-watt bulb for 30-60 minutes. 6. During this waiting period, predict what you think will happen to both tubes and explain why. 7. Observe the tubes every 10 minutes and take notes on the condition of each tube. 4 PHOTOSYNTHEIS – LAB 10 Apply the Knowledge 1. What does a small Rf value tell you about the characteristics of the moving molecules? 2. Which are more soluble in the chromatography solvent, xanthophyll, or chlorophyll a? How do you know this? 3. Would you expect the Rf value of a pigment to change if you altered the composition of the solvent? Why or why not? 4. If yellow xanthophylls were present in the extract, why did the extract appear green? 5. Is it possible to have an Rf number greater than 1? Why or why not? 6. What happens to the color of indicator in exercise 2 during the course of the experiment? 7. What is the reason for the color change in exercise 2, if any? 8. Did the solution in the control tube change color in exercise 2? Why or why not? 9. What is the basis for the color change you observe in exercise 2? Consult the equation for photosynthesis as a guide. 5 A Separate title page consisting of the following: Name Course Title Instructor Date Experiment Completed Completed Lab Format Purpose: Short explanation of what is being tested. Give the reader the main idea in as little as one sentence or as many as you need to reference all the procedures being completed. Background: Paragraph format. This section states what is important about the experiment. What is the reason you are doing this experiment, why is relevant? Where do we see this experiment applicable in the everyday world, where could it be useful? Use the lab manual to make this section nice and dense with information. Read the information before the procedures and you can make a great background. If you feel it is necessary, feel free to use an outside source as long as it is sited. Do not copy any material. This will result in a zero and is plagiarism. Use your own words!!! For example, if we are analyzing color pigments associated with photosynthesis in leafs why would we be using spinach? What is the purpose of homogenizing the spinach leafs in chromatography solvent (a non-polar solution)? What is chromatography, how does the process work? What does it mean when the colors separate, which are the heaviest and lightest molecules and what are the molecules? Explain what the retardation factor Rf value is and how it is calculated. Describe any chemical equations if necessary. What results are we looking for to make a conclusion? The previous paragraph is an example of some information you would want to have in your background for a pre lab. This ensures that any questions you may have regarding the methods or materials can be answered. You cannot go wrong if you are too detailed. I DO NOT WANT TO SEE BACKGROUNDS THAT TELL ME NOTHING, IF YOUR GRANDMOTHER PICKED UP THIS PRE LAB SHE SHOULD BE ABLE TO GET THE IDEA OF WHAT IS GOING ON AND WHY. Materials: • A bulleted list is • a perfect way to • neatly show all • of the materials • needed to conduct • the experiment. • • • • • • This is like the recipe to make bread. If you forget yeast it does not rise, and the bread is bad. I like to use tables. Procedures: Procedure 5.1 – Number and name the individual procedures 1. Numbered directions that tell step-by-step what is done. 2. Model it after the procedures in the book. DO NOT COPY, PUT IN YOUR OWN WORDS IF POSSIBLE. I understand there is not many ways to say, “Add 3 drops of water to a test tube.” Use your best judgment. 3. Do not use the words: I, Me, We, Us, Our. 4. …… 5. …… 6. …etc. Procedure 5.2 – Number and name the individual procedures 1. Numbered directions that tell step-by-step what is done. 2. Model it after the procedures in the book. DO NOT COPY, PUT IN YOUR OWN WORDS IF POSSIBLE. I understand there is not many ways to say, “Add 3 drops of water to a test tube.” Use your best judgment. 3. Do not use the words: I, Me, We, Us, Our. 4. …… 5. …… 6. …etc. Procedure 5.3 – Number and name the individual procedures 1. Numbered directions that tell step-by-step what is done. 2. Model it after the procedures in the book. DO NOT COPY, PUT IN YOUR OWN WORDS IF POSSIBLE. I understand there is not many ways to say, “Add 3 drops of water to a test tube.” Use your best judgment. 3. Do not use the words: I, Me, We, Us, Our. 4. …… 5. …… 6. …etc. Data Table(s) / Results: Table 5.1 – Name of table Total Volume mL of Base of Organism, to Reach mL End Point Beaker with Organism (gold fish) Control Beaker, Distilled Water Relative Respiration Rate of the Organism Respiration Rate per mL of the Organism Table 5.2 – Name of table Total Volume mL of Base of Organism, to Reach mL End Point Relative Respiration Rate of the Organism Respiration Rate per mL of the Organism Beaker with Organism (gold fish) Control Beaker, Distilled Water Note: Add pictures in the results section if there are pictures to help with the lab. Discussion: A discussion should discuss the results you found in the lab. Explain the results you got and if they are what you expected why or why not. What could have happened to make the results not what you expected? Was it human error? Instrumental error? Were all of the steps completed how they should be? Use your discussion to talk about the results. The discussion can also talk about what could be changed if the experiment were to be repeated. Anything you feel is necessary to talk about, add it in. Note: The discussion is usually the longest portion of the lab report. A good thorough discussion can span a half page to multiple pages long. Conclusion: A short summary of what was done and found or not found while doing the lab. A conclusion could be as little as a few sentences, but no bigger than a paragraph. Post Lab Questions: 1. At the end of each chapter in the lab manual is a set of short questions asking specific questions about the lab. 2. In your final lab report, answer those questions. 3. These questions can be incorporated into the discussion. Reference(s): MLA format citing the lab manual and the pages used to do the lab, along with any other references you may have used.
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