Putman’s Biol 160 Lab 7: Photosynthesis

LAB 7: Photosynthesis Introduction Photosynthesis is the process of taking carbon dioxide, CO2, and water, H2O, and “gluing” them together using light energy to produce sugars, (CH2O)n with oxygen, O2, as a waste product. We also call this process carbon fixation because carbons are “fixed” together into larger, higher-energy compounds. Organisms that possess the biochemical machinery for photosynthesis are termed photoautotrophs. Photoautotrophs are capable of taking light energy and transforming it into usable chemical energy (sugars). Photosynthetic pigments are relatively large molecules capable of capturing light and using the energy absorbed to elevate electrons to higher energy levels. They then pass this energy to energy carriers such as ATP and NADPH which, in turn, pass the energy off to the molecules that “glue” CO2 and hydrogens from water into (CH2O)n, the basic class of food molecules for all herbivores, organisms that eat plants. Evidence supports the endosymbiotic theory of Dr. Lynn Margulis. This theory suggests that, in the early evolution of life, the ancestors of chloroplasts were free-living, photosynthetic bacteria. These presumptive chloroplasts were ingested by larger cells and incorporated within vacuoles into their cytoplasm without being digested. These newly-formed eukaryotes then evolved into eukaryotic algae and true plants. Other organelles that probably originated in this way mitochondria, nuclei and basal bodies/centrosomes. Chloroplasts contain three major classes of photosynthetic pigments. These classes are the chlorophylls, which are green, the xanthophylls, which are yellow, and the carotenoids, which are orange. Photosynthetic pigments are colored by the wavelength of light they do not absorb as this unabsorbed wavelength is reflected back, unused, from the pigment and interpreted by our brain as color. As such, chlorophylls are green because they absorb, to a greater or lesser extent, all other wavelengths of light; the same principle holds for xanthophylls and carotenoids. The process of photosynthesis is very important to life on earth. One estimate suggests that some 550 billion tons of CO2 are removed from the atmosphere and 400 billion tons of O2 are released into the atmosphere by photoautotrophs each year, up to 90% of this taking place within the photic zone of the World Sea. Is Light Necessary For Photosynthesis Well, of course light is necessary for photosynthesis! Everyone knows that! But, how do you know? How do you collect evidence consistent with that theory? We know that a qualitative overall statement of photosynthesis is: CO2 + H2O  (CH2O)n + O2 So, in a system where photosynthesis is occurring, without appreciable respiration (the reverse equation), the system will be increasing in oxygen and decreasing in carbon dioxide. Although we can measure CO2 and O2 levels in terrestrial systems, using a land plant as our experimental system, it’s really easy to qualitatively check this in aquatic systems.

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Putman’s Biol 160 Lab 7: Photosynthesis Recall from chemistry that carbon dioxide plus water yields carbonic acid: CO2 + H2O  H2CO3 So, if photosynthesis occurs in an aquatic system, the amount of carbon dioxide will decrease, causing the amount of acid to decrease, causing the pH to increase! (Think about this for a moment—it’s an important concept!) A good aquatic plant to use for this exercise is Egeria densa (anacharis, Brazilian Elodea), and a good qualitative pH indicator to use is phenol red. Phenol red turns pink at a pH of 8.1 and above (basic solution), yellow at a pH of 6.5 and below (acidic solution), and a mixture between yellow and pink from a pH of 6.6 to 8.0. Wavelengths of Light Light exists as waves with particular wavelengths (). (We are ignoring the quantum theory of light here as it is not pertinent to our discussion.) Visible light is a narrow subset of the entire electromagnetic spectrum. When light within the visible spectrum hits the cones of your eyes, your cones send an impulse to your brain. Relatively low-energy wavelengths of 720 nm to about 600 nm range from deep red to pink; beyond 720 nm is infrared light, which is invisible to the human eye (some animals can detect it). From about 600 nm to about 575 nm is yellow. Green is about 525 nm to 575 nm. Blue to deep blue extends from about 525 nm to 400 nm or so. Beyond 400 nm is ultraviolet, which is not visible to the naked eye. (See your textbook for a further discussion on wavelengths of light.) Wavelengths in the blue region have the most energy; this is why blue light penetrates the deepest in the ocean. Recall that 1 m = 109 nm? Extraction of Photosynthetic Pigments Chemicals that can dissolve in a particular solvent are said to be soluble in that solvent. For instance, salt and sugar both dissolve in water, so are both water soluble. This property can be used for the extraction of various compounds. For instance, anthocyanins are water-soluble pigments found in plants, so we can extract anthocyanins from plant sources such as purple cabbage by heating them in water. Photosynthetic pigments are soluble in acetone. By grinding plant sources rich in photosynthetic pigments in acetone, we extract the pigments. The other major pigments found in plants, anthocyanins, are not acetone-soluble, so we get mostly photosynthetic pigments if we use acetone to extract them. Then by filtering the crude extract through cheesecloth, we obtain a workable collection of photosynthetic pigments. Paper Chromatography If you spilled a drop of ink on a paper towel then placed the paper towel in a liquid, what does the liquid do? It absorbs into and creeps up the paper towel then, when it reaches the ink, it smears the ink as it continues on up the towel. This is, in crude form, the principle of chromatography. In chromatography, you need a substrate (the paper towel) and a running medium (the liquid). You put the mixture of compounds you wish to separate on or in the substrate and let the

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Putman’s Biol 160 Lab 7: Photosynthesis running medium “run” past the sample. The compounds in the mixture (sample) will separate out depending on their weight, interaction with the substrate, and ability to dissolve in the running medium. Molecules that are heavy, strongly attracted to the substrate or less soluble in the running medium will move quite a bit slower up the substrate than molecules that are light, weakly attracted to the substrate or highly soluble in the running medium. Types of substrates can include paper, glass beads coated with various chemicals, and other substances. Running media can include various solvents (water, alcohol, acetone, and others) or various gases. Figure 7.1. Schematic of paper chromatography. chromatography paper separating substances

sample

running solvent

In paper chromatography, the medium is a finely-made (and expensive) paper called chromatography paper. The running medium is called running solvent. In this type of chromatography, a drop or line of the chemical mixture you wish to separate is drawn on the chromatography paper and allowed to dry. The paper is then placed in the running solvent so that the sample is just above the level of the running solvent. (See Fig. 7.1.) As the solvent “runs” up the chromatography paper, it carries with it the various chemicals found in the mixture at the rates indicated above. When you remove the chromatography paper from the running solvent, the chemicals stop moving. All of those of a particular size, affinity for the paper or solubility in the running solvent will be found at a certain level. This results in bands of separated chemicals. These bands can then be literally cut out of the paper with a pair of scissors and soaked in solvent, yielding separated and purified compounds. A 10% acetone-90% petroleum ether running solvent has been found to work well in the separation of photosynthetic pigments using paper chromatography.

Laboratory Objectives After mastery of this laboratory, including doing the assigned readings and required laboratory work, the student should be able to: 1. Describe and demonstrate an experiment using phenol red and pH change that provides evidence that light is necessary for photosynthesis.

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Putman’s Biol 160 Lab 7: Photosynthesis 2. State which wavelength of light has the most energy. 3. Describe and demonstrate the theory and technique for the extraction and maximum absorption wavelength determination of photosynthetic pigments using acetone/ether extraction, paper chromatography and a spectrophotometer. 4. Determine the relative molecular weights of carotenoids, xanthophylls, chlorophylls a and b from a chromatogram.

Materials and Methods Is Light Necessary for Photosynthesis? 

The hypothesis we are testing in this experiment is that light is necessary for the process of photosynthesis.

1. Obtain two test tubes, enough aluminum foil to completely cover one test tube, and two pieces of Parafilm large enough to seal the ends of the test tubes. Also obtain two sprigs of Elodea of approximately equal length, a light source, a dropper bottle of phenol red, a coffeestirrer straw and a 250 mL beaker. 2. Put approximately 100 mL of tap water in the 250 mL beaker. Using your straw, blow bubbles into it vigorously for about 15 seconds. 3. Insert an Egeria sprig into each test tube. Completely fill each test tube with water from the 250 mL beaker. Make sure the lip and outside of each test tube is dry; if it is not, use a bit of paper towel to dry it. 4. Stretch Parafilm over each test tube to seal. Place test tubes in test tube rack. 5. Without crinkling it, wrap aluminum foil smoothly around one of the test tubes so that light cannot get in and so that the test tube can fit into the test tube rack. If you do happen to crinkle the foil so that the test tube does not fit into the test tube rack, empty the water from your 250 mL beaker and put the foil-covered test tube in that. 6. Place the test tube rack (with test tubes) a few inches from a light source. Turn the light source on and allow the experiment to run for at 30 to 60 minutes. While you’re waiting, go on to the next exercise. 7. Approximately 30 to 60 minutes later (you should see bubbles forming from the Egeria leaves in the uncovered test tube), REMOVE the Egeria from each test tube and return the sprigs undamaged to their holding container. After you have removed the Egeria from the test tubes, place 3 drops of phenol red into each test tube, agitate, and note the color of each. Note the results in your lab report. Also, analyze the color the phenol red indicator turned, and note the approximate pH as “acid,” “base” or “neutral.”

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Putman’s Biol 160 Lab 7: Photosynthesis Photosynthetic Pigment Extraction Before you proceed, read and understand the lab safety advisory below! Lab safety advisory: Biohazard! Acetone is a carcinogen. Do not get it on your skin or breathe in excess fumes. 1. Obtain spinach and remove the stem and large veins. 2. Cut or tear the spinach into small pieces and place in a mortar. 3. Add just enough acetone to moisten; grind with a pestle for several minutes until you have obtained at least 3 mL of dark-green fluid. 4. Filter the solution through 3 layers of cheesecloth into a 100 or 150 mL beaker. You now have a crude extract of photosynthetic pigments! Set your extract aside. Pigment Separation Using Paper Chromatography Lab safety advisory: Biohazard! Do not breathe in the fumes of petroleum ether as they may cause unconsciousness. 1. Squirt a small amount of detergent onto your work station and clean it well; make sure you rinse it well with water and ray it well. DO NOT TOUCH THIS WORK AREA! The work area needs to be oil free! 2. Using latex or nitril gloves, if available, obtain a piece of chromatography paper and lay it down on your newly-cleaned work station. Skin oils will interfere with the chromatogram, so handle the chromatography paper by the edges only! 3. With a black lead pencil and ruler, lightly draw a line about 12 mm from the bottom edge of the chromatography paper. 4. With a fine paint-brush or capillary pipette, apply a narrow line of your photosynthetic pigment extract over the pencil line and allow it to dry. Repeat this 10 to 20 times. 5. Once the final application of pigment extract is dry, and again without touching the chromatography paper with your fingers, roll the paper into a cylinder so that the edges overlap minimally, and paperclip together securely. 6. Your instructor will have already prepared chromatography jars containing 5 mL of running solvent (10% acetone, 90% petroleum ether). Carefully place your undeveloped chromatogram in your running jar and cap the jar. Don’t agitate the jar! 7. Allow your chromatogram to run until the solvent is about 1 cm from the top; watch this carefully—if the pigments run over the top, your chromatogram will be ruined!

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Putman’s Biol 160 Lab 7: Photosynthesis 8. Under a running fume hood, using forceps, remove the chromatogram from the chromatography jar and stand the chromatogram on a paper towel to dry. Immediately cap the chromatography jar so that the running solvent doesn’t evaporate! 9. Once your chromatogram has dried, take it back to your work station. You should see four bands of color, with carotenoids at the top, followed by xanthophylls, chlorophyll a, then chlorophyll b. Mark them with a pencil (NOT pen!) AND sketch your chromatogram in your lab report. 10. We will decide in class who will do the extraction of each of the four pigments from the filter paper. Once those four people are “volunteered,” they need to each obtain a 100 mL beaker with 15 mL acetone. Beakers should already be labeled with “carotenoids,” “xanthophylls,” “chlorophyll a” and “chlorophyll b.” 11. Using the scissors provided, cut the filter paper stained with each photosynthetic pigment out and, using your forceps, deliver it to the person extracting that pigment from the paper. 12. The students doing the extractions from the filter paper should take each strip delivered to them and, with forceps, agitate it up and down in the acetone. In this manner, we will eventually have a high-concentration, chromatographically-purified solution of each photosynthetic pigment to analyze. The “washed” filter paper strips may be thrown away in the trash. Pigment Absorption Spectra Determination 1. Your instructor will demonstrate for you the proper use of a spectrophotometer. Do not attempt to operate this machine by yourself; it is very, very expensive and easy to break! 2. The class should divide into four groups, each group with the student who did a particular photosynthetic pigment extraction. 3. Each group will need to obtain two cuvettes. DO NOT DROP the cuvettes! They are made of optically-ground glass and are very, very expensive. Obtain a beaker to hold your two cuvettes. DO NOT WRITE OR MARK ON THE CUVETTES! 4. Each group should fill a cuvette with the solution to be tested. 5. Each group should fill a second cuvette with acetone to serve as the acetone blank. 6. Set the initial wavelength at 360 nm, insert the acetone blank, the set T = 100% and set A = 0. T means “transmission” and A means “absorbance.” By setting T at 100% and A at 0%, you’re telling the machine to ignore acetone. 7. Remove the acetone blank, insert the cuvette with the photosynthetic pigment concentrate, read and record the absorption in your lab report. 8. Now set the wavelength to 380 nm, insert the acetone blank, set T = 100% and A = 0%.

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Putman’s Biol 160 Lab 7: Photosynthesis 9. Remove the acetone blank, insert the cuvette with the photosynthetic pigment concentrate, read and record the absorption in the Results table. 10. Repeat this procedure for each 20 nm increment up to 700 nm and record the data in the Results table. 11. Using a graphics program, if one is available, or a piece of graph paper, graph each of the four absorption spectra and determine the maximal absorption for each pigment. 12. When finished with the cuvettes, rinse well with DW and return to your instructor!

Make sure you cleanup your work station, clean all equipment you used and put it back, and help in general to keep the lab clean and in order!

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Putman’s Biol 160 Lab 7: Photosynthesis

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Putman’s Biol 160 Lab 7: Photosynthesis

Biol 160 Lab 7: Photosynthesis Prelab (5 points)

Name: ___________________________________ Date: ________________ Lab Section: ________

~Complete this prelab before coming to lab; it is due at the beginning of lab! 1. In the first experiment (Is Light Necessary for Photosynthesis?), why does the protocol ask you to blow bubbles into the 250 mL beaker in the first experiment? What will you be adding to the water? What will this help you to visualize?

2. Which wavelength ( of light has the most energy?

3. Why do we use acetone and not water to extract photosynthetic pigments from plants?

4. What are the two safety precautions in this lab? a)

b) 5. Regarding the spectrophotometer, what is the function of the acetone blank we’ll be using?

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Putman’s Biol 160 Lab 7: Photosynthesis

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Putman’s Biol 160 Lab 7: Photosynthesis

Biol 160 Lab 7: Photosynthesis Lab Report (20 points)

Name: ___________________________________ Date: ________________ Lab Section: ________

Is Light Necessary for Photosynthesis?

Results and Analysis 1. Color of water treated with phenol red in which Egeria sprigs were incubated for 30 to 60 minutes, with and without light, along with pH interpretation of the results.

Color of water treated with phenol red

pH (acid, base or neutral)

With light Without light

Discussion 6. Is light necessary for photosynthesis? Explain fully the evidence you collected that supports or disproves our starting hypothesis.

7. Why did we blow bubbles into the 250 mL beaker in the first experiment? What were we adding? What did this help us to see?

8. In the first experiment, what were the bubbles forming off the leaves of the Egeria?

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Putman’s Biol 160 Lab 7: Photosynthesis 9. In the first experiment, what was the independent variable?

10. In the first experiment, what was the dependent variable?

Pigment Separation Using Paper Chromatography

Results 11. Sketch of chromatogram WITH the four photosynthetic pigments labeled. Top

12. How can you make a crude extract of photosynthetic pigments from a natural source?

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Putman’s Biol 160 Lab 7: Photosynthesis 13. Explain fully how paper chromatography works to separate substances.

14. What three factors work to separate mixtures of chemicals in paper chromatography?

15. How can you isolate and concentrate photosynthetic pigments (or any other chemicals, for that matter) found on a chromatogram?

16. Which photosynthetic pigment was the largest/heaviest? How do you know? Rank the photosynthetic pigments from heaviest to lightest, based on your data!

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Putman’s Biol 160 Lab 7: Photosynthesis Pigment Absorption Spectra Determination

Results 17. Absorption of Photosynthetic Pigments. Wavelength,  (nm) 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

Absorption of photosynthetic Pigments % carotenoid xanthophyll chlorophyll a chlorophyll b

Analysis 18. Include a graph of the above data with your report! Graph paper is included on the next page of this lab. (Hint: Turn the graph paper sideways and put the % absorption on the y-axis and the wavelength, , on the x-axis.) If a computer is available with a graphics program, use that to generate your graph.

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Putman’s Biol 160 Lab 7: Photosynthesis

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Putman’s Biol 160 Lab 7: Photosynthesis Discussion

19. From our results, what was the peak absorption for each of the pigments? a) Chlorophyll a __________ nm b) Chlorophyll b __________ nm c) Carotenoids __________ nm d) Xanthophylls __________ nm

20. Which photosynthetic pigment absorbed light over the broadest wavelengths? How is this an advantage for a plant?

21. What is the advantage for a plant to have multiple photosynthetic pigments?

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