Biology Unit 2, Structure of Life, Lab Activity 2-2

Transcription

1 Biology Unit 2, Structure of Life, Lab Activity 2-2 Photosynthesis is the process by which energy used by living systems is converted from electromagnetic radiation from the sun to chemical energy. This chemical energy is stored in organic molecules. The entire process takes place in the chloroplasts found in plants. There are a variety of photoactive pigments contained in the chloroplasts. Each pigment is reactive to different wavelengths of light energy. Those wavelengths not utilized by the pigment are reflected and account for the color we see. We see green leaves because chlorophyll a and b do are not active in that portion of the visible spectrum. While chlorophyll is the primary pigment active during photosynthesis, there are other 'accessory' pigments found throughout the plant. These 'accessory' pigments have a wide variety of uses. Carotenoids, accessory pigments produced in chromoplasts, are associated with many colors observed in vegetation. There are hundreds of different types of carotenoids. Carrots get their color, which is often orange but is not restricted to orange, from carotene. And carotene is not so much a specific compound as a family name for several compounds that also go by the name terpene. Another type of carotenoid phyto-pigment is called anthocyanin. The purplish color of a red cabbage and the rusty red of the flesh of a blood orange are a result of the presence of anthocynanins, which also have the curious property of changing color with changes in ph. Anthocyanins absorb UV light, which is used by plants to perform two important functions: to attract insects, which augment pollination, and as a sunscreen to protect the other parts of the plant cells such as DNA from harmful UV radiation. In this experiment you will extract pigments from spinach and carrots and measure their visible absorbance spectra with a spectrophotometer. The spectrophotometer measures both the amount and wavelength of a light passed through a sample. While you wait for the extracts to develop, you will measure the absorbance of blue and yellow food-colored water samples, which will provide an analogy to the absorbance of the plant pigment extracts. Research Questions 1) What wavelengths of light are captured by plants for photosynthesis? 2) Why have plants evolved to utilize those frequencies of light? Write your answers to the research questions AFTER you have completed all other parts of the Lab. 1) 2)

2 Protocol Materials computer carrot slices or shavings Logger Pro 70% isopropanol (IPA) spectrophotometer petroleum ether cuvettes yellow and blue food colored solutions 10 ml graduated cylinder distilled water funnel plastic Beral pipets filter paper balance, ± 0.1 g accuracy three small flasks mortar and pestle mm glass test tube, or paper cups glass cuvette fresh spinach Procedure Data collection for this lab is to be completed as a lab table group. Data reporting and questions are to be completed individually. 1.Obtain and wear goggles. 2. Connect a spectrophotometer to the USB port of your computer. 3. Start Logger Pro. If it is already running, choose New from the File menu. 4. Calibrate the Spectrometer. The calibration sets the spectrophotometer to baseline 0 for the solvent being used. In Part 2 water is the solvent. In Part 3 the solvents are alcohol and ether. a. Prepare a blank by filling an empty cuvette 3/4 full with distilled water. b. Choose Calibrate Spectrometer from the Experiment menu. c. When the warmup period is complete, 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. d. Click Finish Calibration, and then click. Answer Stop question #1-2 before continuing Part I Prepare the Plant Pigment Extracts 5. Measure out 0.5 g of fresh spinach. Add 20 ml of 70% isopropanol (IPA) to a clean mortar. Tear the spinach into tiny pieces and grind them with the pestle. We are just extracting the pigments so killing the plant cells doesn t affect the experiment. Continue to grind until solvent turns color. Transfer the mixture to a small beaker. Allow the mixture to sit. 6. Measure out 0.5 g of carrot slices (or shavings). Add 20 ml of petroleum ether to a clean mortar. Grind the carrot with the pestle. Continue to grind until solvent turns color. CAUTION: Handle the petroleum ether, with care. The fumes may irritate your nasal passages. Set the two beakers of extracted pigment aside for Part 3. Answer Stop question #3-4 before continuing Part II The Absorbance of Food Coloring Portions adapted from Advanced Biology with Vernier by permission Page 2 of 9

3 7. Conduct a full spectrum analysis of the blue food coloring. a. Empty the blank cuvette and rinse it twice with small amounts of the blue food coloring mixture. Fill the cuvette 3/4 full with the blue liquid and place it in the spectrophotometer. Align the cuvette so the clear sides are facing the light source of the spectrophotometer. b. Click. A full spectrum graph of the blue food coloring sample will be displayed. c. Click. Examine the graph, noting the peak or peaks of very high absorbance or other distinguishing features. 8. Choose Store Latest Run from the Experiment menu to store your data. 9. Repeat Steps 7 and 8 with the yellow food coloring sample. Remember to store the data. 10. Mix equal amounts of the blue and yellow solutions in a paper cup or small beaker. Repeat Steps 7 and 8 with the mixture. 11. Display a graph of all three plots. a. Choose Graph Options from the Options menu. b. In the Graph Options dialog box, select the Axes Options tab. c. Select Absorbance for all of the successful runs so that all three color analysis are displayed on the same graph. Cick. 12. Save your experiment file. Print your graph (it will show all three pigments on the same graph). Make a copy for each lab member. Paste printed graph in Data Graph 1.Write down your observations of the graph in Table 4. Answer Stop question #5-6 before continuing Part III Measure the Absorbance of the Plant Pigment Samples 13. Prepare a purified sample of your spinach extract. Use a funnel and filter to slowly pour the alcohol/spinach extract into a clean flask. 14. Calibrate the spectrophotometer. You will calibrate the spectrophotometer with isopropanol because your solvent in the spinach extract is isopropanol, not water. a. Choose New from the File menu. b. Prepare a blank by filling an empty cuvette 3/4 full with clear isopropanol. c. Choose Calibrate Spectrometer from the Experiment menu. d. When the warm-up period is complete, 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. e. Click Finish Calibration, and then click. 15. Measure the absorbance spectrum of the spinach extract. a. Pour out the isopropanol from the cuvette, rinse, and fill it 3/4 full with the spinach filtrate. b. Place the cuvette in the spectrophotometer. c. Click to see a plot of the absorbance spectrum for the spinach extract. d. Click. 16. Examine the graph, noting the absorbance peak ranges for chlorophyll described in the introductory remarks. If any of the peak absorbance values are greater than 1.5 or off the top of your graph, dilute your sample to bring the peaks down to a more reasonable level. Repeat data collection. 17. Save your experiment file. Print out the graph and paste in Data Graph 2.Write down Portions adapted from Advanced Biology with Vernier by permission Page 3 of 9

4 your observations of the graph in Table Calibrate the spectrophotometer with a different solvent for testing the carrot extract. a. Choose New from the File menu. b. Choose Calibrate Spectrometer from the Experiment menu. c. Filter your carrot extract in the same way you filtered the spinach extract. d. Obtain a mm glass test tube(these solvents will melt plastic. The spectrophotometer would be permanently damaged). Mark the test tube so that you can align it in the spectrophotometer the same way each time you use it. Fill the test tube ~1/2 full with the clear petroleum ether that you used with the carrot slices. This test tube of solvent will serve as your blank. e. When the warm-up period is complete, place the test tube in the spectrophotometer, being careful to line it up with the mark on the tube so the same sides are always facing the light source of the spectrometer. f. Click Finish Calibration, and then click. Answer Stop question #7 before continuing 19. Measure the absorbance spectrum of the carrot extract. a. Pour out the solvent from the test tube, rinse, and fill it ~1/2 full with the carrot extract. b. Place the test tube in the spectrophotometer, being careful to line it up with the mark. c. Click to see a plot of the absorbance spectrum for the carrot extract. d. Click. 20. Examine the graph. If any of the peak absorbance values do not show on the graph use Autoscale to show the range of the data on your graph. Repeat data collection. Answer Stop question #8 before continuing 21. Save your experiment file. Print a copy of the graph for each lab member and paste in Data Graph 3.Write down your observations of the graph in Table 4. EXTRA CREDIT OPTION Extract red cabbage or blood orange pigments using steps Extract red bell pepper pigments using steps (remember to use glass with acetone). Prepare graphs for extra credit as you did for the other parts of this lab. Now that you have read these procedures construct a flow chart on the following page. Portions adapted from Advanced Biology with Vernier by permission Page 4 of 9

5 Flow Chart Lab 2-2 Photosynthesis Portions adapted from Advanced Biology with Vernier by permission Page 5 of 9

6 Data Graph 1 Food Color Spectrum Prediction Graph 2 Chlorophyll Spectrum Prediction Portions adapted from Advanced Biology with Vernier by permission Page 6 of 9

7 Graph 3 Carotene Spectrum Prediction Table 4 Graph Observations Trial Sample Wavelengths of peaks and other unique features of the spectrum graph (multiple peaks etc.) 1 Blue 2 Yellow 3 Mixture 4 Spinach Extract 5 Carrot Extract Portions adapted from Advanced Biology with Vernier by permission Page 7 of 9

8 Stop Questions 1) What does a spectrophotometer measure? 2) Why is it important to calibrate all measuring devices? 3) What are the solvents in this step used for? 4) Why aren't we concerned that the grinding and solvents will kill the plants? 5) Why do the food coloring samples peak where they do? 6) Why do the mixed food colors look the way they do? (use the term absorbance and/or reflectance in your answer) 7) Why is it important to use a glass test tube in this step? 8) What pigment accounts for the orange color of carrots? Analysis Questions 1.Describe, in detail, the absorbance spectrum of each food coloring sample. With the mixture of blue and yellow food coloring, can you clearly distinguish the characteristics of each coloring? Blue Yellow Mixture Explain in terms of wavelengths of light 2. Consult a reliable resource (Wikipedia is good) to identify the major absorbance peaks (give wavelength) of chlorophyll a nm and chlorophyll b nm Examine the absorbance vs. wavelength graph for your spinach extract. You will see peaks for both Chlorophyll a and b. Are there other peaks on your graph that are not characteristic of chlorophyll? Speculate why there might be even if you did not see extra peaks. 3. Have carrots always been orange? What are some other colors carrots have? (try the carrot museum) 4. Why are leaves green in the spring and summer, then orange or yellow in the fall? Refer to what you have learned in this unit to answer. Portions adapted from Advanced Biology with Vernier by permission Page 8 of 9

9 Conclusion. Write a one paragraph (three sentence minimum) answer to the following question. You may use internet sources as reference (not copied) but they must be cited. You may share sources with classmates. Why have different pigments evolved in the plant kingdom? Plants and animals have co-evolved. You have probably heard that carrots are good for your vision. Write a one paragraph (three sentence minimum) answer to the following question. You may use internet sources as reference (not copied) but they must be cited. You may share sources with classmates. How does the pigment beta carotene from carrots work in helping your vision? Portions adapted from Advanced Biology with Vernier by permission Page 9 of 9