Unit 10: Photosynthesis Abstract: Last week, you observed the way in which organisms and the cells within those organisms perform respiration as a means of creating energy. You will recall that in both aerobic and anaerobic respiration, glucose was one of the reactants needed to eventually produce ATP. Mammals are heterotrophs, which means that they cannot produce their own glucose, and must therefore consume other organisms to accrue the glucose needed for respiration. Plants, on the other hand are autotrophs, which means that they can produce their own glucose. Photosynthesis is the process by which plants acquire light energy and convert it into chemical energy. This process is carried out in two general processes: the light reactions and “dark reaction” (the dark reaction involves a cycle known as the Calvin cycle). Furthermore, the entire process of photosynthesis is carried out primarily in the chloroplasts of the leaf cells. In the light reactions, pigments in the leaves absorb different wavelengths of sunlight. You will recall from Unit 3 on spectrophotometer that light travels in waves of varying length depending upon the type of light. Additionally, you will also recall that the visible spectrum of light consists of different colored lights traveling at different wavelengths. Light on the red end of the visible spectrum will have a much larger wavelength, and therefore travel with much less energy, than light on the violet end of the visible spectrum. Pigments in plant leaves such as chlorophyll a and b and arytenoids absorb light of varying wavelengths to provide the plant with the energy that it needs to convert carbon dioxide and water into glucose and water as seen in the equation below. 6CO2 + 6H2O + light energy C6H12O6 + 6O2
10.1 Determining the absorption spectrum of photosynthetic pigments using a spectrophotometer Introduction: In this exercise you will observe the ability of differing pigments in vegetables to absorb and transmit different wavelengths of light. For this experiment, spinach leaves and carrots have been soaked in acetone to extract the pigments from each. Recall that the colors that we perceive with our eyes are the wavelengths of the visible spectrum that are transmitted rather than absorbed (example: a blue shirt is transmitting blue, but absorbing other colors of the spectrum). You will once again use the spectrophotometer to measure the ability of each pigment extract to absorb or transmit different wavelengths of light. You will now observe the ability of spinach extract and carrot extract to absorb or transmit light of different wavelengths (400 – 700 nm). The spinach extract in this experiment is green in color, while the carrot extract is orange in color.
Additionally, figure 1 denotes the approximate range of wavelengths for each color of the visible light spectrum. Color Wavelength Violet 380-450 nm Blue 450-500 nm Green 500-570 nm Yellow 570-590 nm Orange 590-620 nm Red 620-750 nm Figure 1. Colors of the visible light spectrum with their approximate corresponding wavelength range. Use the information given in figure 1 to write hypotheses about the wavelengths that you think spinach extract and carrot extract will absorb and transmit: Based on what I know about transmittance and absorbance of the visible light spectrum, spinach extract will _____________________________________________ ________________________________________________________________________ _______________________________________________________________________. Based on what I know about transmittance and absorbance of the visible light spectrum, carrot extract will _____________________________________________ ________________________________________________________________________ _______________________________________________________________________. Materials and Methods – Photosynthetic Pigment Absorption Spectrum Experiment: 1. Turn on the spectrophotometer using the power switch/zero control knob and allow it to warm up for five minutes. 2. With no sample tube in the holder and the lid closed, turn the power switch/zero transmittance control knob until the meter read 0% transmittance. 3. Adjust the wavelength control knob until the display reads 525 nm. 4. You have been provided with three test tubes: one labeled B, which contains your blank (in this case the blank is acetone), one labeled S, which contains spinach extract (the extract was made by placing spinach leaves in acetone), and one labeled C, which contains carrot extract (the extract was made by placing carrot shavings in acetone). 5. Insert the blank into the sample holder and close the lid. Using the 100% transmittance control knob, adjust the meter reading to 100% transmittance.
6.
7. 8. 9.
REMEMBER that as you go through this experiment you will be changing the wavelength. EVERYTIME you change the wavelength, you MUST again set the blank to 100% transmittance. Failing to do this may drastically affect your results. Remove the blank and insert the spinach extract tube. Measure the % transmittance, if it reads 90% transmittance record the reading in figure 2. IF THE SPECTROPHOTOMETER DOES NOT READ 90% TRANSMITTANCE FOR THIS SAMPLE, dilute the sample with acetone until it yields a reading of 90% transmittance. Press the mode button to obtain the absorbance of the spinach extract and record it in figure 3. Place the tube containing carrot extract into the spectrophotometer and repeat steps 6 and 7. After you have successfully obtained the transmittance and absorbance for both spinach and carrot extract at 525 nm, move the wavelength in 25 nm integrals both up and down the spectrum. REMEMBER to reset the blank to 100% transmittance at every step. Report the values you obtain in figure 2 and figure 3 accordingly.
Results: Wavelength (nm) T%
400
425
450
475
500
525
550
575
600
625
650
675
700
675
700
Spinach Carrot Figure 2. Transmittance of Spinach and Carrot Extract at varying wavelengths.
Wavelength (nm) A
400
425
450
475
500
525
550
575
600
625
Spinach Carrot Figure 3. Absorbance of Spinach and Carrot Extract at varying wavelengths.
650
Figure 4. Relative transmittance of spinach extract to each corresponding wavelength.
Figure 5. Relative transmittance of carrot extract to each corresponding wavelength.
Conclusions:____________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________.
10.2 Pigment Chromatography Introduction: As mentioned previously, spinach leaves contain a myriad of different pigments. These pigments each absorb different wavelengths of light, and in doing so increase the total amount of energy the spinach leaf can obtain from the visible spectrum. Chromatography is a laboratory technique used to separate the components of a mixture. This technique can be performed in a variety of ways. In today’s lab you will use chromatography paper and solvent to separate the different pigments of the spinach leaf from one another. Furthermore, this technique separates molecules based on either a chemical or physical property. The chromatography paper will separate the pigments based upon their relative sizes, so larger molecules will travel through the paper slower while smaller molecules travel much faster. The relative sizes from smallest pigment to largest pigment are: carotene (red-orange-yellow), xanthophyll (yellow), chlorophyll a (blue-green), and chlorophyll b (green-yellow). Use the information given in introduction to write hypotheses about where the different pigments will run through the chromatography paper. Remember that the chromatography solvent is running from the bottom of the paper to the top of the paper. Based on what I know about chromatography and the relative pigment sizes ______ ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________. Materials and Methods - Pigment Chromatography Experiment: 1. Holding only the sides of the paper, draw a line with a pencil (DO NOT USE A PEN) approximately 2 cm from the bottom of the chromatography paper. 2. Use a coin to grind a spinach leaf on the line that you just drew. 3. Bring the large test tube and stopper to the instructor’s bench. Pour about 1 cm of chromatography solvent into the bottom of the tube.
4. Place the paper in the test tube and fix it in place with the stopper, making sure that the paper is just in the solvent without the solvent actually touching the pigment spot. 5. Place the stopper so that it closes the tube, and traps the top most part of the chromatography paper between itself and the glass wall of the test tube. 6. Holding the test tube carefully upright, return to your lab bench and place the test tube in the test tube rack. 7. Wait approximately 15-20 minutes, or until the separated pigments can be seen clearly. 8. Remove the paper from the tube and allow it to dry on a tissue on the bench. 9. Color what you see in figure 6. Results:
Figure 6. Representation of photosynthetic pigment separation on chromatography paper. Make sure that you know the names of the pigments and the order in which they travelled on the chromatography paper. Based on how chromatography works, which pigment do you predict is comprised of the largest molecules?______________________________ Which pigment is comprised of the smallest molecules? __________________________
Conclusions:____________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________.
10.3 Starch localization in leaves Introduction: While plants are autotrophs, they are still dependent upon sunlight as a source of the light energy that they will eventually turn into chemical energy. On cloudier days, or at night, plants continue to grow and therefore still require glucose for respiration. If there were no other means of acquiring glucose, this could be a problem, but plants have developed a means of storing the macromolecule starch so that it can be broken down into glucose. The following experiment is a demonstration that was performed by your instructor. In the experiment two coleus leaves were grown in the presence of light. One leaf was covered in aluminum foil, and therefore could not obtain any energy from the light, while the other was uncovered and could obtain energy from the light. At the end of the experiment the leaves were soaked in iodine to detect the presence of starch. Use the information given in introduction to write hypotheses about where you expect to see starch in either leaf. Based on what I know about starch storage in the presence of light energy, _______ ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________. Materials and Methods – Starch localization in leaves: 1. Coleus leaves were incubated in the presence of light for seven days. Some of the leaves were wrapped in aluminum foil, while others were not. 2. Your instructor took the leaves off of the stem at the end of the seven day period and placed them in a 400 ml beaker containing 95% ethanol. 3. You instructor then placed the 400 ml beaker in an 800 ml beaker containing water, and placed it on a hot plate at 100 C. 4. Once the water was boiling, you instructor allowed the leaves to boil for three minutes.
5. After the three minute time point, the leaves were removed from ethanol, and placed in tap water at room temperature for three minutes. This rehydrates the leaves so that they are not brittle and frail. 6. After three minutes, the leaves were placed in an empty petri dish, covered in iodine, and allowed to sit at room temperature for five minutes. 7. After the five minute incubation, the leaves were rinsed with tap water, and placed in a clean petri dish containing distilled water. 8. Record the results that you observe for each step of the procedure in figure 7. Results:
Coleus Leaf
Boiled
Placed in water, then placed in iodine:
Covered Leaf
Uncovered Leaf
Figure 7. Observation of Coleus Leaves at various stages of iodine staining for starch detection. Conclusions:____________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________. Points for Discussion: 1. Were the results that you obtained in the spectrophotometer experiment what you expected for spinach extract? Were there any surprising results? Please elaborate.
2. Were the results that you obtained in the spectrophotometer experiment what you expected for carrot extract? Were there any surprising results? Please elaborate.
3. A carrot is a root, which means that it spends most of its life underground. Knowing this, can you think of any reason as to why a root plant, such as carrots, beets, and radishes would have light absorbing pigments in it?
4. Today you separated the pigments of spinach leaf using chromatography. What physical property governed the separation of the pigments? Have you exploited this particular trait of molecules before in a previous lab? If so, which lab?
5. Were the results for the starch test in the leaves expected? Why or why not?
6. How do you humans store additional nutrients and carbohydrates in their bodies?
