Introduction / Purpose:
The technique of paper chromatography used for this section of the lab is used when trying to separate and identify plant pigments from the mixture of molecules found in plant cell extracts. Capillary action, the tendency of a liquid to rise or fall due to surface tension, is what makes the solvent move up the paper. Yet, this capillary action is caused by the adhesive properties of solvent molecules to each other, as well as the attraction of the solvent molecules to the paper. This then transfers to how the pigments are carried upward on the paper, since how far they are carried is dependent on how soluble each pigment is in the solvent and the degree to which the pigments molecules are attracted to the paper due to the formation of intermolecular bonds, including hydrogen bonds.
Since Beta carotene, the most abundant carotene in plants, is the most soluble in the solvent and will not be attracted to the paper because it does not form hydrogen bonds, it will be carried near the solvent front.
Unlike Beta carotene, the plant pigment xanthophyll contains oxygen molecules, which make it both less soluble in the solvent and more attracted the hydrogen bonds. For this reason, xanthophyll is found further from the solvent front.
Procedure:
1. Obtain a 50-mL graduated cylinder that has 1 cm of solvent in the bottom. The cylinder is tightly stoppered because this solvent is volatile, and you should be careful to keep the stopper on as much as possible.
2. Cut a piece of filter paper that will be long enough to reach the solvent. Cut one end of this filter paper into a point. Draw a pencil line 1.5 cm above the point.
3. Use a coin to extract the pigments from spinach leaf cells. Place a small section of leaf on top of the pencil line. Use the ribbed edge of the coin to crush the cells. Be sure that the pigment line is on top of the pencil line. You should repeat this procedure 8 to 10 times, being sure to use a portion of the leaf each time
4. Place the chromatography paper in the cylinder so that the pointed end is barely immersed in the solvent. Do not allow the pigment to be in the solvent.
5. Stopper the cylinder. When the solvent is about 1 cm from the top of the paper, remove the paper and immediately mark the location of the solvent front before it evaporates.
6. Mark the bottom of each pigment band. Measure the distance each pigment migrated from the bottom of the pigment origin to the bottom of the separated pigment band. In Table 4.1 record the distance that each front, including the solvent front, moved. Depending on the species of plant you used, you may be able to observe 4 or 5 pigment bands.
Discussion / Analysis:
Overall our experiment was set up correctly, however we were unable to obtain results. The error occurred because not enough pigment was rubbed off onto the chromatography paper before it was placed in the cylinder. This resulted in very minimal migration of the pigments. You can see that the pigments began to migrate upward on the chromatography paper, but stop very early because it ran out of pigment to carry. Luckily, we were able to obtain data from our helpful classmates and use these numbers for our calculations. (The first photo is our chromatography paper and the second photo is from another lab table who had successful migration)
The primary pigment found in plants is known as Chlorophyll a. While there also exists chlorophyll b, chlorophyll is known to contain both nitrogen and oxygen, and also bind more tightly to paper than other pigments. For this reason, it is plausible to believe that chlorophyll molecules will not be found near the solvent front. Chlorophyll a, b and carotenoid molecules are used to capture energy from light and transport it to chlorophyll a molecules located at the reaction center for photosynthesis.
Part 4B:
Introduction / Purpose:
Leaf pigments absorb light with the purpose of increasing each electron in the photosystems of plants up to a higher energy level. Once the electrons move to a higher energy level, this energy is then used for the reduction of NADP to NADPH, as well as to make ATP. ATP and NADPH will then be used during carbon fixation, when CO2 is incorporated to organic molecules.
For the experiment, the main purpose will be to investigate photosynthesis with the use of a dye-reduction technique. The experiment seeks to prove whether or not light and chloroplasts are instrumental for photosynthesis to occur. NADH, the electron acceptor, will be replaced with DPIP. As the electrons of DPIP become reduce the substance will change to colorless. As the DPIP is reduced it will then therefore increase the light transmission within the color spectrometer since DPIP is becoming clear.
Procedure:
1. Turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm by adjusting the wavelength control knob.
2. While the spectrophotometer is warming up, your teacher may demonstrate how to prepare a chloroplast suspension from spinach leaves.
3. Set up an incubation area that includes a light, water flask, and a test tube rack. The water in the flask acts as a heat sink by absorbing most of the light's infrared radiation while having little effect on the light's visible radiation.
4. Your teacher will provide you with two beakers, on containing a solution of boiled chloroplasts and the other containing unboiled chloroplasts. Be sure to keep both beakers on ice at all times.
5. At the top rim, label the cuvettes 1, 2, 3, 4, and 5 respectively. Make sure each cuvette is wiped and cleaned throughly. Cover the walls and bottom of cuvette 2 with aluminum foil. Light should not be permitted inside cuvette 2 because it acts as the control for this experiment.
6. Refer to Table 4.3 and prepare each cuvette accordingly.
7. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts to cuvette 1. Insert cuvette 1 into the sample holder and adjust the instrument to 100% transmittance. Cuvette 1 is the blank to be used to recalibrate the instrument between readings.
8. Add 3 drops of unboiled chloroplasts to cuvette 2. Then remove the cuvette from the foil sleeve and insert it into the spectrophotometer's sample holder, read the % transmittance, and record it's the time "0" reading in Table 4.4. Place cuvette 2 back in it's foil sleeve and return it to the incubation rack with the light on. Take and record additional readings at 5, 10, and 15 minutes.
9. Add 3 drops of unboiled chloroplasts to cuvette 3. Then insert it into the spectrophotometer's sample holder, read the % transmittance, and record it's the time "0" reading in Table 4.4. Next return cuvette 3 to the incubation rack with the light on. Take and record additional readings at 5, 10, and 15 minutes.
10. Add 3 drops of boiled chloroplasts to cuvette 4. Then insert it into the spectrophotometer's sample holder, read the % transmittance, and record it's the time "0" reading in Table 4.4. Next return cuvette 4 to the incubation rack with the light on. Take and record additional readings at 5, 10, and 15 minutes.
11. Cover and mix the contents of cuvette 5 (no chloroplasts are added). Then insert it into the spectrophotometer's sample holder, read the % transmittance, and record it's the time "0" reading in Table 4.4. Next return cuvette 5 to the incubation rack with the light on. Take and record additional readings at 5, 10, and 15 minutes.
Discussion / Analysis:
The purpose of this experiment was to test whether or not chloroplasts and light are necessary for photosynthesis to occur. Since NADP reduction and carbon fixation are major steps of photosynthesis in plants, it is plausible to believe that higher levels of NADP being reduced is indicative of more photosynthesis occurring. In the case of the experiment, NADP was replaced by DPIP. This means that DPIP will become reduced instead of NADP in the experiment. Therefore since DPIP becomes colorless as it is reduced, more light will be able to pass through the cuvette inside of the spectrometer. A higher percentage of light passing through the cuvette will cause the spectrometer to measure higher levels of transmittance. Therefore, higher measurements of transmittance will be indicative of more photosynthesis occurring.
Our data demonstrates that the cuvette with no chloroplasts and no light had the lowest measurement of transmittance, suggesting that for this cuvette, the least amount of DPIP was reduced, which therefore also indicates that the lowest amount of photosynthesis occurred in this cuvette. This may demonstrate how chloroplasts and light are indeed necessary for photosynthesis to occur. However, our data also indicated that the cuvette with 2 unboiled chloroplasts and no exposure to light had higher levels of transmittance than the cuvette with 3 unboiled chloroplasts and exposure to light.
In an ideal experiment, the cuvette with exposure to light and higher number of chloroplasts should have had a higher measurement of transmittance. Our data may have yielded these results due to several reasons. The cuvette with exposure to light may have had too much DPIP causing the measurement of transmittance to be lower than expected, while the cuvette with no exposure to light may have had too little DPIP which caused the higher than expected measurements of transmittance. Also, it is possible that the cuvette with exposure to light was not exposed to the light enough time. For future experiments measurements of DPIP should be taken more accurately and carefully. Time should also be monitored more closely.
Also in order to get more accurate results, it is important to measure the amount of transmittance for the cuvette having zero minutes in the light. Unfortunately, data was not collected at the zero minute mark for our experiment. This could have been prevented with a better preparation of how to use the spectrometer and record our data from the device. It is important to record data at the zero minute mark since it will act as a basis for comparison to how much the photosynthesis rate will increase in the cuvette once exposed to light.
