III. Hypothesis, Materials, and Method
Exercise 4A: Plant Pigment Chromatography
Hypothesis: If crushed spinach leaves are placed on chromatography paper in solvent, then the chlorophyll a bands will migrate the largest distance.
Materials: 50 mL graduated cylinder with stopper, solvent, filter paper, spinach leaves, coin, and ruler.
Method: The first step in the experiment was to pour 1 cm of solvent into the 50 mL graduated cylinder. The next step was to cut one end of the filter paper into a point and to draw a line 1.5 cm above the tip. After, a spinach leave was placed above the line and crushed into the filter paper using a coin. The paper was then placed into the cylinder, with the point barely immersed in the solvent. When the solvent was 1 cm from the top of the paper, the paper was removed and the solvent line measured. The next step was to measure the distance migrated by each pigment band.
Exercise 4B: Photosynthesis and the Light Reaction
Hypothesis: If chloroplasts are placed under different conditions and percent of transmittance measured, the chloroplasts that are unboiled and subjected to light will have the greatest percent of transmittance.
Materials: Spectrophotometer, light, test tube rack, 5 cuvettes, aluminum foil, phosphate buffer, distilled water, DPIP, unboiled chloroplasts, and boiled chloroplasts.
Method: The first step of this experiment was to set the spectrophotometer to a wavelength of 605 nm and to label the cuvettes. Then, 1 mL of phosphate buffer was added to each cuvette. 4 mL of water was added to cuvette one. 3 mL of water and 1 mL of DPIP was added to cuvettes 2, 3, and 4. In cuvette 5, 3 Ml plus 3 drops of water and 1 mL of DPIP was added. The spectrophotometer was then brought to zero and cuvette one was placed inside. Cuvette one had unboiled chloroplasts and was used to calibrate the transmittance of the device. Unboiled chloroplasts were added to cuvettes two and three and each cuvette was measured for transmittance. Boiled chloroplasts were added to cuvettes three and four. No chloroplasts were added to cuvette five. Each cuvette was tested for fifteen minutes, with cuvette one used to calibrate the spectrophotometer.
IV. Variables
Exercise 4A:
The independent variable is the different types of pigment. The dependent variable is the distance migrated by these pigments.
Exercise 4B:
The independent variables being tested are the use of chloroplasts, boiled or unboiled chloroplasts, and light and darkness. Essentially, the experiment tests the effects of each of these conditions on the photosynthetic rate. The dependent variable is the percent of transmittance of light. The control group is cuvette one.
V. Data and Observations
Exercise 4A:
Observations: There was a slight variation between the colors of the bands. This made it difficult to tell the differences in pigment.
Table 4.1: Distance Moved by Pigment Bands (mm)
Distance Solvent moved: 190 mm
Exercise 4B
Table 4.4
Time (minutes)
VI. Analysis
Exercise 4A:
1. There are many factors that are involved in the separation of pigments. One factor is the amount of solvent. One of the ideas behind chromatography is that the pigments are attracted to the solvent. Thus, if there is more solvent, the solvent travels up the chromatography paper faster. Another factor involves the type of pigment used. Different pigments are attracted differently. The pigments are carried along at different rates because they are attracted differently to the solvent.
2. The Rf factor would be different if a different solvent was used. Each solvent has a different rate of attraction. Thus, the pigment bands might migrate greater or smaller distance depending on the type of solvent used.
3. The reaction center contains chlorophyll a. When light is absorbed, this type of chlorophyll donates one of its electrons to the primary electron acceptor. Other pigments include chlorophyll b and carotenoids. These pigments aid in absorbing photons for use in the reaction center.
Exercise 4B:
The independent variable: Time in minutes
The dependent variable: Percent of transmittance
1. The function of DPIP in this experiment is to act as an electron acceptor. DPIP is blue in the oxidized state. As light is absorbed, DPIP is reduced and becomes clearer in color. Thus, DPIP can also be used to determine the amount of light being absorbed by the solution.
2. DPIP replaces the NADP+ found in chloroplasts.
3. Light is the source of electrons that will reduce DPIP.
4. The spectrophotometer was used to determine the change in light transmittance at a wavelength of 605 nm. As more light was absorbed by the chloroplasts, there was a change in the amount of light that passed through the DPIP. This was measured by the change in transmittance.
5. Without light, the DPIP was not reduced. This can be seen in the example of cuvette two. Because there was no light, the DPIP did not receive any electrons. Light is source of electrons that reduce the DPIP. Thus, there was not a change in the transmittance. In cuvette three, there was light. This accounted for the change in transmittance because electrons were being accepted by the DPIP.
6. Boiling chloroplast destroys vital enzymes and damages them. Because the chloroplasts are damaged, they are not able to absorb light energy. As seen in cuvette four, boiled chloroplasts were not able to absorb the light. This led to DPIP not being reduced. If the chloroplasts had not been boiled, then the DPIP would have been reduced.
7. In the dark, the DPIP was not reduced. This can be seen in the example of cuvette two. Because there was no light, the DPIP did not receive any electrons. Light is source of electrons that reduce the DPIP. Thus, there was not a change in the transmittance. In cuvette three, there was light which allowed the chloroplasts to photosynthesize. . This accounted for the change in transmittance because electrons were being accepted by the DPIP. As the DPIP is reduced, the percent of transmittance changed.
8. Cuvette 1: This cuvette was used as a “blank” to calibrate the spectrophotometer.
Cuvette 2: This cuvette was used as to show that light was responsible for photosynthesis.
Cuvette 3: This cuvette was used to show the conditions needed for photosynthesis to take place: undamaged chloroplasts and light.
Cuvette 4: This cuvette was used to show that heat destroyed the enzymes with in chloroplasts. This cuvette also shows that chloroplasts are needed.
Cuvette 5: This cuvette also shows that chloroplasts are the molecules that photosynthesize.
VII. Discussion
In this lab, chromatography was performed and the rate of photosynthesis was calculated. Chromatography is useful in separating pigments. The solvent carries pigment bands up the chromatography paper. However, the solvent carries the different pigments at varying degrees of speed. This results in the creation of the pigment bands seen on the chromatography paper. Rf value is one way to compare the distance moved by the pigment in relation to the distance moved by the solvent front. After analyzing the results of this experiment, it was determined that beta carotene traveled the farthest distance. Chlorophyll b migrated the shortest distance. There seemed to be relatively few errors in this part of the experiment. The results of the experiment could be made more accurate by using a longer piece of chromatography paper. This would allow the pigments to be more separated.
One way to measure photosynthetic rates was to measure the amount of dye that changed from blue to clear. This was done by using the electron acceptor DPIP. In this experiment, DPIP “replaced” NADP+. As the chloroplasts absorbed more light for photosynthesis, the DPIP accepted more electrons. By reducing DPIP, the color of the solution changed from blue to clear. In cuvettes where photosynthesis was taking place, the color of the solution changed. To get accurate results, a spectrophotometer was used to measure the percent of light transmittance. As DPIP was reduced, the percent of transmittance increased as more light was able to go through the solution.
Photosynthetic rates can change depending on light intensity and wavelengths of light. Shorter wavelengths of light have greater amounts of energy. Thus, these short wavelengths provide more energy for the light reaction. As there is more energy, the photosynthetic rate increases. However, chlorophyll only absorbs light of certain wavelengths. Therefore, some wavelengths of light may have no impact on the photosynthetic rate. Another factor that influences photosynthetic rate is light intensity. When the light intensity is low, there is a lower rate of photosynthesis. This is because there is not enough light to maintain the conditions for light reactions to take place. As the light intensity increases, the chlorophyll is able to absorb more light and thus, more energy.
Environmental factors play a huge role in determining the photosynthetic rate. One factor that changes is light intensity. When there is more sunlight, the photosynthetic rate increases. This is because light provides the energy source for the light reactions. Without light, photosynthesis can not take place. Another factor is heat. According to the results of the experiment, excessive heat can damage the chloroplasts. If the chloroplasts are damaged, then photosynthesis cannot occur. Other factors include the availability of carbon dioxide and water. These compounds are key in the photosynthetic process.
Overall, this experiment was important in learning about the process of chromatography and the rate of photosynthesis.
Bibliography:
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Campbell, Neil A. and Jane B. Reece. Biology: 6th Edition. San Francisco: Benjamin Cummings, 2002.
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Information from Lab Four: Plant Pigments and Photosynthesis
- Lecture / Class Notes
