Biochem 300 Lab
Fall 2012 Lab #1
Theory of Photometry
In this lab we are conducting a series of test to verify the theory of photometry. A spectrophotometer is used to measure the absorbance of light of a sample. The absorption of light mechanism consist of a light bulb, a compartment to place the test tube, a wavelength selector, a slit a light detecting photocell and a meter to show the measurement of the sample. The compound can absorb a photon of light, if the energy of the photon, is equal to the amount of energy needed to emit electrons of the sample to a higher energy orbital. The mechanism works by turning a light bulb, which permits light to go through a prism that is set in a specific wavelength, then this light is deliver across a narrow slit. Once it passes the slit it then allows absorbance of light of the compound. Once the correct light goes via the test tube it lets the detector know the amount of absorbance in the sample. Absorbance is defined as the change in log between the intensity of light that enters the solution and the intensity of light that leaves the solution. Absorbance is determined on the number of molecules that beam of light hits as it passes the solution; this is based on the molecule path length and concentration. We used the Beer-Lambert law to review the data collected from the experiment. The law states that absorbance is equal to the extinction coefficient, which is the absorbance per concentration length, the concentration and the path length. The law also states that if we double the amount of our PNP then the absorbance should also double. If the results fit the BeerLambert law we know that would note that absorbance is linearly relative to the concentration of the compound. This relationship will aid in the construction of a standard curve that can be used to figure out the concentration or the absorbance of a unknown of the same compound. During the experiment we conduct a PNP assay. We know that PNP has a pH of 7.08, hence any pH above this number would be deprotonated and any pH below the known pH would be protonated. PNP does not absorb visible light when its protonated, this is why we add Na carbonate to the compound to increase the pH and deprotonated, which will allow absorption of light. The 4 aims of this experiment are to deprotonate our PNP in order to obtain a sample that is able to absorb light, determine the concentration of an unknown using a standard curve, to grasp a better understanding of Beer-Lambert Law by building an absorbance spectrum and to comprehend wavelength function. The experiment was conducted by first taking samples from a known PNP concentration and adding the buffer (pH 4) and then the Na Carbonate (pH 11). The Na Carbonate was added to the solution because its known that at pH 4 light will not absorb and needed the pH 11 that Na Carbonate for the light to be absorbed. Different volumes of the PNP and buffer were added to the test tubes, and it was noted that the solution remained clear, and then we added a constant amount of Na Carbonate. Once the Na Carbonate was added the solution turned a light yellow. This change in color can be accounted for the sample to have become deprotonated. We used a spectrophotometry to get absorbance rate on our sample. Once we retrieve the absorbance data and review it to verify the Beer-Lambert Law. The results obtain from the experiment did fit to an extent the Beer-Lambert Law, there where two point that where close to the doubling effect that is described by the Beer-Lambert Law but not quite. This can be accounted to a possible pipetting error. Once all of the liquids that are to be use are in the test tube you will vortex each tube before placing the in the spectrophotometry, making note of the color. Since the vortex aid the sample to actually mix completely we take note of any color change that might show more deprotonating of the sample. During the second part of the experiment when we took a sample to...
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