Exercise 4A
Purpose
In this lab, our experiment was to use chromatography paper to separate plant pigments and to measure the rate of photosynthesis in isolated chloroplasts with the use of a dye. Capillary action helps move the solvent up the chromatography in order to show us the different rates in which chlorophyll's pigments travel and are thus separated.
Introduction
Chromatography is the separation of a mixture by passing it in a solution through a medium, such as paper, in order to reveal the different rates in which the components travel at. Pigments on a chromatography paper may travel at different rates due to their solubility in the solvent. In spinach leaves, pigments that are found inside the plant are chlorophyll, xanthophyll, and carotene. While chlorophyll is the most abundant pigment in spinach as well as all plants, all of these pigments will still show up on chromatography paper.
Methods
We first crushed up a leaf, and then smeared the green die on chromatography paper. We then placed a small amount of water in a graduated cylinder and placed the paper in it, making sure to keep it suspended.
By means of capillary action, water was absorbed through the chromatography paper, spreading out the green die.
Data
This is a picture of the chromatography paper from our lab. Although we had a gaping hole at the bottom, the results still came out fine. Using the ruler, we marked off the bottom of each color.
This table shows all the measurement of each color band in millimeters and the total distance the water moved up the chromatography paper.
Discussion
In chromatography, water will be absorbed vertically by means of capillary action. As noted in the provided pictures, not all of the plant pigments were carried the same distance as the water. This phenomenon occurs because not all of the pigments are equally soluble, and will therefore stop at various points on the paper. While chlorophyll a and b are really the only pigments necessary to carry out photosynthesis, carotenoids are important to absorb various wavelengths of light that will otherwise cause damage to plant cells. To focus specifically on the distance traveled by all pigments, scientists use an Rf value, which is a ratio that compares the distance migrated by the pigment to the distance traveled by the entire solvent. The Rf value could change substantially if a solvent were used that cannot be absorbed into the paper as well as water. The pigments will not change their own properties, yet they will only travel as far as the solvent will let them. As a result, it is important to use a very polar liquid (like water) for this experiment in order to see the pigments separate as much as possible.
Conclusion
From this lab we discovered that carotenoids are the most water soluble, and chlorophyll b the least soluble in water. From this, we can learn why leaves change color in the fall. After the tree cuts off water supply to the leaves, the green chlorophyll pigments break down before the yellow pigments, leaving a yellow leaf. In time, the yellow pigments break down as well.
References
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromintro.html
Exercise 4B
Purpose
In the second part of our experiment, our purpose was to study photosynthesis by measuring the transmittance of light over a certain period of time with a spectrophotometer and a dye reduction technique. Since we know wavelengths of light power photosynthesis and cause the production of ATP, we wanted to find out the different levels of transmittance during the photosynthesis process within a chloroplast (in our case, spinach) as well as prove that in order for light reactions to occur, chloroplasts and light need to be present.
Introduction
A spectrophotometer is a device that consists of two instruments: a spectrometer which produces light of any selected color/wavelength, and a photometer which measures the intensity of light. Since light is part of a continuum of radiation or energy waves, the energy that is given off from light is used to power the process of photosynthesis in chloroplasts. Dye reduction is a technique where a dye, sometimes DPIP which is a redox dye, is used to look at the change of color of the solutions or mediums in an experiment. In plants, carbon fixation is when light is absorbed by leaf pigments atmospheric carbon dioxide is converted to carbon compounds which can provide energy to produce ATP.
Methods
We first created different environments using various chemicals, some being favorable to chloroplasts, some not. In addition, some environments were given boiled chloroplasts, some un-boiled chloroplasts, and some no chloroplasts at all. In the photo above, Judd is creating these environments.
We then shined an incandescent light bulb on the different test tube environments (sparing one which used to calibrate the spectrometer.) The large jug of water was used as a heat sink, as 95% of an incandescent's energy is given off as heat. After 5 minutes, we removed the test tubes from the light as recorded their transmittance. We did the same at 10 and 15 minutes and recorded our results.
Data
This graph shows the transmittance percentage of each cuvette from our lab. We got this data from the LoggerPro and recorded the measurement at different times.
Graphs
This graph shows the results of our data In the key, the colors correspond to the numbers and the number represent the different cuvettes. The numbers and the type of cuvette can be determined from the table above. As we can see, the cuvette with no chloroplast had no change while the unboiled/light had a great change. Unboiled/dark and boiled/light both decreased slightly.
Discussion
An important part of this lab was the use of DPIP, an electron carrier that served to replace NADP throughout the experiment. As our results will show, the presence of an electron carrier is essential in order for photosynthesis to take place at all. Five cuvettes were used in this experiment to test what ingredients are needed for plants to make sugar and survive with sunlight, water, and good soil. In the first cuvette, we added our chloroplasts but did not supply an electron carrier. Without electrons, we cannot create ATP molecules to power the Calvin Cycle, where our sugar is actually created via carbon fixation. In addition to this, the second cuvette was wrapped in film to keep out light, proving that light energy must be absorbed by the plant pigments in order to excite electrons. Our third cuvette was truly the only specimine that should have shown a high level of transmittance, because the environment had all of the necessary components to photosynthesize, reduce DPIP and essentially turn the color of the cuvette clear. In doing so, light would pass through it in the colorimeter and yield a low absorbance level when compared to the other four cuvettes. As for the fourth cuvette, we were able to prove that chloroplasts must be alive for DPIP to be reduced. Our fifth cuvette acted as another control when no chloroplasts were added at all, and this creates a serious problem for the plant if there are no reactants to power the dark reactions. With five cuvettes to test five different combinations, we were successfully able to determine a powerful recipe for plants to use when making their own food.
Conclusion
Our data was complex, but in the end, it was the tubes with the chloroplasts that absorbed the most light especially before the possible errors in calibration. This makes sense, as the chloroplasts role in a plant is to absorb light and donate electrons so the processes of photosynthesis can carry out.
References
http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html
http://en.wikipedia.org/wiki/Dichlorophenolindophenol
Glowing Spinach
We were all confused when Mr. Filipek told us to go an make spinach glow without any direction. After playing around with the different solutions and equipments, we finally figured it out. We ripped up the spinach into tiny pieces and placed them in a funnel. We placed the funnel in a beaker and then added isopropyl alcohol into the funnel to drip through. At this point, Judd decided to get his hand dirty and rubbed the spinach and alcohol. Afterwards, we poured a little bit of the liquid into a graduated cylinder and shined a backlight through. And voila! Spinach glowed red!
When running the alcohol through the spinach leaves, we were able to extract chlorophyll into the liquid.The chlorophyll absorbs photons and takes it from its ground state and into an excited state. This raises the energy level and releases heat and fluorescence. This fluorescence is what causes the liquid to glow red with the help of a black light.
References
References
http://c-lab.co.uk/default.aspx?id=9&projectid=58
Biology Book
Biology Book
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