Observing Plant Pigments, Photosynthesis, and Cellular Respiration in Plants and Peas
Statement of the Problem:
What are the most prominent pigment bands of a spinach leaf? What different kinds of bands and colors of bands are there? What will happen to the color of the chloroplasts when they are exposed to the light with added DPIP? Will these color changes help distinguish if the light reactions are taking place or not? What will happen when we submerge the germinating peas into the water? Will the movement of oxygen indicate cellular respiration is taking place?
Paper chromatography paper is used in science to separate mixtures of compounds. Leafs are used by rubbing the leaves compounds onto the chromatography paper with a coin or rough object. Then strong fumes are used to pull the pigments up and separate them onto the chromatography paper. Different molecules will travel up the chromatography paper at different rates due to their dissolving characteristics to the solvent. The separation and attachment to the paper by the compounds are usually caused by the hydrogen bonds.
During the light reactions of photosynthesis, light energy is absorbed to excite the electrons by chlorophyll. The electrons then travel to one of two chains: one chain converts ADP + P to ATP, and the other converts NADP + H to NADPH. When the isolated chloroplasts are exposed to light, their color will change. This will indicate the light reactions are taking place.
Aerobic respiration provides energy by the oxidation of glucose. This involves many reactions instigated and hosted by enzymes through the equation C6H12O6 + 6O2 = 6CO2 + 6H2O + energy.
If we put chromatography paper in a jar of petroleum and acetone, expose isolated chloroplasts to light, and submerged germinating peas and glass beads after reaching equilibrium, then we can separate different pigments in the spinach leaf, visually see light reactions taking place by color change of chloroplasts, and visually seeing oxygen moving toward the peas for cellular respiration.
AP Lab #4 Activity A: Leaf Pigment Chromatography
Chromatography Jar (Tightly Capped with Solvent)
First, we received an 8-cm square piece of chromatography paper and a leaf of spinach. We made two pencil marks 1.8 cm from one edge of the chromatography paper. We then laid the spinach on the chromatography paper near the marked edge and laid the ruler on top of the leaf. We made sure the edge of the ruler was on the paper at 1.5 cm from the edge of the chromatography paper. Using the ruler, we then rolled the coin over the leaf to drive the pigments into the chromatography paper in a line 1.5 cm from the edge.
After we could see a dark green line of the pigments of the leaf on the chromatography paper, we marked the bottom of the pigment line to use it as the origin. We formed a cylinder with the chromatography paper and stapled the paper together at each end. Then we placed the chromatography paper into the jar so the pigment is near the solvent, but would not touch the solvent. We then capped the jar and let the jar sit for several minutes. When the solvent was 1 cm from the top edge of the chromatography paper, we removed the paper from the jar and marked the location of the solvent before it evaporated. Then we marked the bottom of each pigment band and measured the distance each pigment band traveled by the solvent front at the bottom of the paper.
For the solvent and cellulose chromatography paper, each pigment band will move a different distance that is proportional to the distance the solvent moved. This was referenced as the Rf (Reference Front) value, and is a constant value. Rf = distance of pigment from origin/distance of solvent front from origin. We calculated the Rf values for each pigment band and recorded the values in Table 1.
Table 1 : Chromatography of Plant Pigments
AP Lab #4 Activity B: The Light Reactions of Photosynthesis
4 Dropping Pipets
Vial of DPIP Solution
Bucket of Ice
Test Tube Rack
First we labeled each cuvette with a number between 1 and 5 respectively. Using a clean, new dropper, we added 4 mL of distilled water to Cuvette 1. Using the same dropper, we added 3 mL of distilled water to Cuvettes 2 through 5. Again, using the same pipet, we added an additional 3 drops of distilled water to Cuvette 5. Once more, using the same pipet, we added 1 mL of phosphate buffer to each cuvette. Using a clean, new pipet, we add 1 mL of DPIP solution to Cuvettes 2 through 5. We covered Cuvette 2 with aluminum foil to prevent light from entering the cuvette.
We referred to Table 2 as to how we were to set up the cuvettes. We set up our work station so the water aquarium could absorb infrared radiation from the light so the chloroplasts won’t be damaged. We then labeled each of the cuvettes between 1 and 5. Using a clean dropper, we added 4 mL of distilled water to Cuvette 1. Using the same pipet, we then added 3 mL of distilled water to cuvettes 2 through 5. Using the same pipet again, we added three extra drops to Cuvette 5.
Using the same pipet, we then added 1 mL of phosphate buffer to each cuvette. Using a new and cleaned pipet, we added 1 mL of DPIP to cuvettes 2 through 5. We then covered Cuvette 2 with aluminum foil to prevent light from entering. We then took samples of unboiled and boiled chloroplast suspension, which needed to be placed on ice during the activity. We mixed the unboiled suspension of chloroplasts by inverting the vial it was in. Using a new and clean pipet, we added 3 drops of the unboiled chloroplast suspension to Cuvette 1.
Table 2: Contents of the Cuvettes
By referring to Table 2, we made each Cuvette and mixed the contents. Since the spectrometer needed for this experiment was not working, we recorded the color change of the mixture of the chloroplast suspension. Cuvettes 3 and 4 were to be exposed to the lamp we set up while Cuvettes 2 and 5 were to be exposed to the dark. We recorded our results in Table 3.
Table 3: Color Change of Chloroplast Suspensions
*Cuvette 2 and Cuvette 5 are overlapping
When creating a hypothesis for this experiment, it would be “If unboiled and boiled chloroplast suspensions were exposed to the light or darkness for different time periods, then the color of the chloroplast suspension mixture will change to indicate light reactions”. Cuvette 1 is the control of this experiment because the cuvette contained distilled water, phosphate buffer, and unboiled chloroplasts, but was blank during the experiment. This makes cuvette 1 the control.
During the experiment, the rates of color are tested against time. Depending on the cuvette, the different color rates correspond to the different times each cuvette was tested. The results in Table 3 and the graph both show the color of Cuvette 3 drops the most while Cuvette 2 and 5 stayed the same and Cuvette 4 was between Cuvettes 2 and 5 and Cuvette 3. DPIP was not added to Cuvette 1 because Cuvette 1 was the control Cuvette and if DPIP was added, then the color of Cuvette 1 would have changed.
The purpose of adding 3 drops of chloroplast suspension to Cuvette 1 was to observe the initial chloroplast suspension color as a control so the other cuvette colors could be referred and compared to the control chloroplast suspension color. Three drops of water were added to Cuvette 5 because the cuvette did not contain any chloroplast suspension. The effect of boiling on the chloroplast suspension was it made the chloroplast suspension color become lighter, but at a slower pace than unboiled chloroplast suspensions because of the increase in temperature.
AP Lab #5: Cellular Respiration
Room Temperature Water Bath
Cold Water Bath
Container of Ice
Absorbent Cotton Balls
15% Potassium Hydroxide (KOH) Solution
Setting up Water Baths:
To set up the water baths, we first obtained two water bath trays and taped lined paper to the bottom of them so we could read the respirators easily without making an error in the experiment. After we taped the paper to the bottom of the trays, we filled one tray with water at room temperature and another tray with cold water at about 10oC. We placed ice cubes into the cold water tray to help lower the water temperature to 10oC. After we set up the water baths, we began the set up for the respirometers.
Preparing Peas and Glass Beads:
For Respirometer 1, we put 25 mL of water into a 50 mL graduated plastic tube and then added 25 germinating peas. We determined the volume of water that was displaced, which is also the volume of the peas. After we recorded the volume of the germinating peas, we removed the peas and placed them on a paper towel
For Respirometer 2, we refilled the graduated tube with 25 mL of water and added 25 non-germinating peas to the graduated cylinder. Then we added enough glass beads to the graduated cylinder so the volume is equal to the volume of the germinating peas. We then removed the non-germinating peas and beads and placed them on a paper towel.
For Respirometer 3, we refilled the graduated tube with 25 mL of water and added enough glass beads to equal the volume of the germinating peas. We then removed the beads and placed them on a paper towel.
Assembling the Respirometers:
For this lab, we needed a total of 6 respirometers (3 for the room temperature bath and 3 for the cold bath). When we assembled the respirometers, we placed an absorbent cotton ball in the bottom of each respirometer. Using a dropping pipet, we saturated the cotton with 2 mL of 15% KOH. Then we placed a wad of dry, non-absorbent cotton on top of the KOH-soaked cotton ball. This will prevent the KOH solution from coming in contact with the peas. For vial 1, we placed 25 germinating peas, for vial 2, we placed 25 dry peas and beads, and for vial 3, we placed beads only. After preparing 2 sets of the 3 vials, we inserted a stopper with a calibrated pipet into each respirometer.
Respirometer Placement in the Water Baths:
We then placed the respirometers (1 of each viral in both baths) with their pipets resting on the edge of the water bath. We then waited 5 minutes for the respirometers to reach thermal equilibrium with the water. After the 5 minute wait period, we submerged all respirometers in the water bath. We positioned the respirometers so we were able to read the pipet scales clearly. When taking the readings, we allowed another 5 minutes for the respirometers to equilibrate. Then we observed the initial volume reading on the scale and recorded the data in Table 1 for Time 0. We repeated the observations and recorded them every 5 minutes for 20 minutes for each water bath.
Table 1: Respiration of Peas at Room Temperature
▲V = V at Time 0 – V at time of current reading
Corrected ▲V = ▲V(for Respirometer 1 or Respirometer 2) - ▲V of Respirometer 3
Table 2: Respiration of Peas at a Colder Temperature
For this experiment, one hypothesis would be “ If we submerge the 3 different vials into the room temperature water, then the germinating pea’s respiration would be a higher rate over the dry peas and beads with a medium respiration rate and the beads only with no respiration rate”, and “If we submerge the 3 different vials into the colder temperature water, then the germinating pea’s respiration rate would be a higher rate over the dry peas and beads with a medium respiration rate and the beads only with a lower respiration rate which drops down and rises back up”.
The formula for volume in this experiment would be V=nRT/p, where V=volume, n=kmoles of gas, R=universal gas constant (8314 joules/kmole x K), T=temperature of gas in K, and p=pressure of gas. For this experiment, the variables which had to be controlled for the data to be valid were n, R, T, and p. Each of these variables had to be controlled so the volume of the peas could be determined. Respirator 3 serves as a negative control because there are no germinating peas or non-germinating peas inside the respirometer but only glass beads. This makes Respirator 3 a negative control. A change to pressure in the experiment led to an observed change in volume because as the pressure changed, the oxygen in the pipet would move toward the peas.
During the experiment, between vials 1 and 2, vial 1 had the most respiration rate in both room temperature and cold water baths over vial 2. The graph and data table shows during the experiment in the colder water bath, the rate increased and then decreased for both vials.
For both of the activities for Lab 4 and Lab 5, the hypothesis accepts all the results and is supported by all of the data collected in the lab. For Lab 4 activity A, the hypothesis stated, “If we put chromatography paper in a jar of petroleum and acetone, then we can separate different pigments in the spinach leaf”. In this experiment, we followed the procedure and we were able to obtain 3 pigment bands from the spinach leaf. Each band was a different length and a different color, which indicated the separation of the pigments onto the chromatography paper.
For Lab 4 activity B, the hypothesis stated, “If we expose isolated chloroplasts to light, then we will visually see light reactions taking place by color change of chloroplasts”. During this experiment, when we exposed the suspended chloroplasts to the light or darkness, their color changed to indicate whether or not the light reactions were taking place. For our experiment, the colors of the chloroplasts changed and indicated the light reactions were occurring.
For Lab 5, the hypothesis stated, “If we submerged germinating peas and glass beads after reaching equilibrium, then we will visually seeing oxygen moving toward the peas for cellular respiration”. During the experiment, we were to indicate oxygen bubbles in the pipets of the vials and watch them move closer to the peas in a period of 20 minutes. During this experiment, the air bubbles moved down the pipets toward the vials with the peas inside of them indicating cellular respiration was occurring.
During these activities and experiments, I learned more about plant pigments, photosynthesis, and cellular respiration. When learning about plant pigments, I learned how to separate the pigments onto chromatography paper and I was able to visually see the different pigment types and the different colors of the pigments. The photosynthesis activity showed me when light reactions were occurring because of the change in color of the suspended chloroplasts and whether or not they were changing in the light or dark. The cellular respiration lab showed me how the peas were sucking in the oxygen bubbles in the pipets of the vial over a time period, and as the time increased, the volume of the vial decreased and the oxygen bubble would move closer to the peas. This indicated cellular respiration taking place. During this experiment, there was technological error with machinery, human error by not measuring properly and not fully understanding directions. Also, there were time errors in timing the cellular respiration lab.