Absorption spectrum of leaf extract
To submit this assignment, students will complete the Lab Worksheet on pages 7-10, then upload their completed document as a DOC or PDF file in Canvas
BIO 101 Lab 08: Photosynthesis
Notification: If you have a disability that makes it difficult to complete this lab, please contact your instructor. Please provide your instructor a copy of the Memorandum of Accommodation (MOA) from NVCC Disability Support Services.
· Determine the absorption spectrum of leaf pigments
Sunlight provides the majority of energy for organisms living in most ecosystems, however only a subset of organisms are capable of harvesting this energy. Plants use their chloroplasts to absorb the energy from sunlight. This energy is then stored in the covalent bonds of glucose, a simple sugar, and can be used by the plant for structural purposes (cellulose), as usable energy (ATP generation), or for energy storage (starch). Animals can eat plants to obtain glucose and produce energy through a process called cellular respiration.
The overall reaction for photosynthesis is represented by the chemical equation:
6 CO2 + 6 H2O + sunlight → C6H12O6 + 6 O2
The entire process is complex and involves many enzymatic reactions. You may notice that the photosynthesis reaction is nearly the exact reverse of cellular respiration. From the equation above the three key elements for photosynthesis to occur are carbon dioxide (CO2), water (H2O), 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.
There are two phases of photosynthesis:
1) In the light-dependent phase chlorophyll molecules located in the thylakoid membrane stacks of the chloroplasts absorb the energy from light resulting in the production of ATP and NADPH.
2) In the light-independent reactions (the Calvin Cycle) the energy stored in ATP and NADPH is used to ultimately convert carbon dioxide to sugar. The process of taking carbon dioxide from the air to build carbohydrates is called carbon fixation.
In solution, CO2 can be converted to carbonic acid (H2CO3) when dissolved in water. The carbonic acid will then release hydrogen ions (H+), causing the pH of the solution to decrease.
CO2 + H2O H2CO3 HCO3- + H+
Bromothymol blue is a pH indicator that changes color based on the pH of a solution. Bromothymol blue turns yellow at lower pH and blue as the pH is increased. This indicator can be used to track respiration (turns yellow as CO2 is added to the system) or photosynthesis (turns blue as CO2 is consumed).
Different colors of Bromothymol blue at the indicated pH conditions
Light energy is a small part of the electromagnetic spectrum which is visible to the 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.
Follow all standard laboratory safety procedures.
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. Watch the following video about setting up the photosynthesis lab from time 0:00 until 5:46.
Ignore the use of a second plant in the video, we only use Elodea in this laboratory activity. In the video, Tubes 1 and 2 contain Elodea plant cuttings and Tube 1 is wrapped in foil instead of green film.
In our photosynthesis lab experiment, we wrap Tube 1 in green film, which only allows green-colored light to reach the Elodea plant cutting. The green film we use ends up having the same effect as the aluminum foil used in the video.
6. In Table 1 in the Lab Worksheet, record the color of the solution before blowing into it in the “before exhaling” column.
7. In Table 1 in the Lab Worksheet, record the color of the solution after blowing into is in the “after exhaling column.
8. Answer question 1 in the Lab Worksheet
9. 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.
10. Record the color of the solution in each test tube in Table 2 of the Lab Worksheet
11. 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.
12. Watch the following video showing the results of photosynthesis lab from time 0:00 until 2:41.
In the video, Vial A corresponds to Tube 1 in our photosynthesis lab experiment. Ignore Vial B from the video. Vial C in the video corresponds to Tube 2 in our photosynthesis lab experiment and Vial D in the video corresponds to Tube 3 (the control).
Note that in our photosynthesis lab experiment, we wrap Tube 2 in green film, which only allows green-colored light to reach the Elodea plant cutting. The green film we use ends up having the same effect as the aluminum foil used in the video.
13. Using the results diagrammed below, fill in Table 2 in the Lab Worksheet
14. Record the color you observe for each test tube in Table 2 in the “Observed color” column.
15. Answer questions 2 – 3 in the Lab Worksheet.
Experiment 2. Absorption spectrum of leaf extract (work in groups)
1. In the laboratory, you would first turn on the spectrophotometer and allow it to warm up for 15 min.
2. You would then insert the blank cuvette (which contains alcohol) in sample holder marked “B” and the cuvette containing leaf extract in sample holder “1”, making sure that the orientation of the cuvettes is correct.
3. Then, you would select a wavelength to measure and calibrate the spectrophotometer by measuring the absorbance of the blank cuvette at this wavelength. You would then see the absorbance reading set to 0.
4. Now, you would measure the absorbance of the cuvette containing the leaf extract and record the absorbance in Table 3 in the position corresponding to the wavelength you selected.
5. You would repeat steps 3 – 4, changing to a different wavelength each time, until all the wavelengths in Table 3 had been measured. Record absorbance readings in Table 3.
6. Watch the following video about using a spectrophotometer from time 0:00 until 4:43.
Note that the video uses a tube containing a red liquid, while in our photosynthesis lab, we would use a tube containing leaf extract (which would appear green) as our sample tube and a tube containing alcohol (which would be transparent) as our blank tube.
7. Table 3 has been partially filled in for you in the Lab Worksheet. Complete Table 3 by filling in the color of light that corresponds to the grouped wavelengths.
8. Answer question 4 by making a Line Graph of the data in Table 3.
9. Answer questions 5 and 6 based on your Line Graph
10. Answer question 7 based on the results of both Experiment 1 and Experiment 2.
BIO 101 Lab 08: Photosynthesis Worksheet
Name: __________________________ Section: ______________________
Data Analysis and Synthesis Questions:
Table 1. pH-indicating dye color
|Color before exhaling into beaker||Color after exhaling into beaker|
1. Why did the solution in the beaker change color after you exhaled?
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|
(Elodea + green film)
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:.
Table 3. Absorbance of Different Wavelengths of Light
|Color of Light||Wavelength (nm)||Absorbance||Color of Light||Wavelength (nm)||Absorbance|
4. Make a Line Graph of the absorbance readings versus the wavelength of light data from Table 3. Be sure to include x-axis and y-axis labels and a chart title.
5. Which wavelength(s) and colors of light are most effectively absorbed by leaf pigments (where are the peaks in your Line Graph)?
6. Which wavelength(s) and colors of light are poorly absorbed (where are the valleys in your Line Graph)?
7. Draw a general conclusion about which color(s) of light are best for plant growth based on your data from both the measurement of absorbance of light in leaf extracts and the observations of CO2 use by Elodea cuttings in different light conditions.
|BIO 101 Lab 08: Photosynthesis||10|