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Over the duration of this lab our group successfully learned how to use and analyze samples using the Spec 20. These instruments are intuitive to use, however there are also some common errors that can easily be overlooked. Once the Spec 20 has been given time to warm up, simply select the desired measurement (absorbance or percent transmittance) along with the wavelength that you will be measuring at 3. Once the appropriate parameters are selected, a blank should be placed in the sample chamber to properly zero the machine by using the auto zero function 3. It is imperative to use an appropriate solution as a blank depending on the samples that will be measured. For example, when measuring food coloring and water solutions, water should be used as a blank. Once the machine is properly zeroed, individual samples can be measured for their absorbance and transmittance. While operation is fairly straightforward, it is important to avoid simple mistakes.
First, if transmittance is found to be out of a 15%-85% range, a different wavelength should be used. Data measured outside of this range may not accurately display the sample’s properties. Other common issues involve the cuvettes. If a cuvette is not placed in the proper orientation, readings could be incorrect. To avoid this, always be sure the triangle on the cuvette faces the light source. Cuvettes should also be cleaned with chemical wipes to ensure there are no contaminants such as finger prints or oils on the clear plastic, this will ensure accurate readings. Using data collected from various samples that were analyzed with the Spec 20, we were able to determine relationships between wavelength, transmittance and absorbance. Transmittance refers to the percent of light that passes through a sample from the source and is received at the detector compared to a blank, which has a transmittance of 100%. Absorbance refers to the amount of light that is absorbed by a sample compared to a blank, which has an absorbance of 0. After taking measurements of red and green food dye samples at various wavelengths, we were able to plot both transmittance and absorbance on a graph. These graphs showed that absorbance was at a maximum when transmittance was at a minimum and vice versa, giving them an inverse relationship. The red and green samples also had inverse relationships with each other. This relationship can be explained by looking at the color wheel. The color that we see is the color’s wavelength that a sample transmits the best. The complimentary color, which is on the opposite side of the color wheel gets absorbed best by the sample. For example, red and green are opposites on the color wheel. Red has the greatest absorbance reading when analyzed at a green wavelength (~500 nm) and has the greatest transmittance at red wavelengths (~625 nm). Green has the opposite data values. The observed color of KMnO4 is purple, whose compliment is yellow. As expected, we see that KMnO4 has it’s greatest absorbance in the yellow wavelength (~550 nm).
Another important attribute of absorbance is that it determines the analytical wavelength of a sample. This is found when absorbance is at a maximum, which occurs when light of complimentary wavelength is used, and will provide the best wavelength for measuring samples. We repeated these methods to find the analytical wavelength and create a calibration curve for phosphate. However, unlike the previous samples phosphate does not absorb a measurable amount of light on its own. Due to this property, our phosphate solution had to be mixed with AVM in a 2:1 solution in order to provide readable absorbance values. This also meant changing the blank from pure water to a 2:1 solution of water to AVM. After performing several dilutions of our phosphate solution and mixing with AVM, we collected absorbance values at the analytical wavelength of the solution (375 nm), which was determined by testing absorbance at different wavelengths. These values along with those collected from KMnO4 were then used to create a method for finding concentration. From absorbance, we can determine a sample’s concentration. To do this, a calibration curve must be created using absorbance readings from various dilutions of a solution, which have known concentrations, at the analytical wavelength. The concentration is plotted on the x-axis and absorbance on the y-axis. By creating a calibration curve, we can determine the concentration of an unknown substance from its absorbance value. The absorbance can then be substituted into the y value of the calibration curve equation and we can solve for concentration, or x.
Absorbance values are shown on the y axis and concentration on the x. Using these values, we can create a line of best fit for the calibration curve. With the measured absorbance (y values) of each cola solution, we can solve for their concentration (x values) by using the equation of our calibration curve. Each cola was first boiled to remove CO2, which could affect readings, then measured for absorbance at 375nm at a 1:50 dilution. Using the method described for finding concentration, it was determined that Sam’s Cola had the highest phosphate content, followed by Pepsi and Cheerwine as seen in Figure 10. It is important to note that we use absorbance to determine concentration instead of transmittance, because absorbance and concentration share a linear relationship. This can be explained using Beer’s Law. Beer’s Law states that a material’s absorbance is proportional to its concentration and can be written as Absorbance = eLc, where e is the molar absorptivity constant, L is the path length and c is the concentration 1. Beer’s Law also states that the ratio of concentrations is proportional to the ratio of absorbances. This can be written mathematically as Concentration 1/Concentration 2 = Absorbance 1/Absorbance 2 4. Using this formula, with one known concentration and absorbance value, we can determine an unknown concentration by measuring the solution’s absorbance.
We can also use the calibration curve formula method discussed above. While potential errors with the Spec 20 have already been discussed, there are other potential pitfalls when completing this experiment. The most difficult part of this laboratory experiment was accurately measuring the concentrations of each sample. Dilution errors can be avoided by carefully checking molarity calculations before performing a dilution. M1V1=M2V2 should be used when finding molarity and double dilution should be used as needed when performing large dilutions. In addition, you should take care to get proper and exact measurements of each part of the new solution. For example, when reading the markings on the burettes be sure to use the meniscus of the liquid. In addition, when adding a small amount of a substance to a solution, it is best to use a smaller burette to gauge a more accurate reading. It is also important to make sure your burette is clean and dry. This prevents both contamination of your sample, as well as incorrect volume readings.
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