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7. Is the mathematical relationship between absorbance and concentration the same at the two different wavelengths? Explain.8. Determine the concentration of the “Unknown” Kool-Aid solution using one of your calibration plots. Explain which calibration curve you chose to determine the concentration.
9. The linear relationship between absorbance and concentration is described mathematically by Beer’s Law: A=εlCwhere Ais the absorbance, Cis concentration, lis the path length (the width of cuvette, here 1 cm exactly) and εis the molar absorptivity. Substances with large molar absorptivities absorb many photons (a lot of light) at that particular wavelength. Using your trend line equation fit and the information above, determine the value of the molar absorptivity of your Kool-Aid solution at the wavelength of maximum absorbance. Make sure that you report the molar absorptivity with the correct units.
Name: __________________________ Page 9 of 10 LAB 3:SPECTROPHOTOMETRY &BEER’S LAWPRE-LAB QUESTIONSDUE:WEDNESDAY 14SEPTEMBERA spectrophotometry simulation can be found at the following site: * Note: this site only seems to run on Internet Explorer on Windows…you may need to do the following to allow it to run: Go to “All Programs” (click on the Windows icon on the bottom left of your main screen) à“Java” à“Configure Java”. Under the Security Tab, click on “Edit site list”. Add . Now cross your fingers and reload the website.) Six topics are listed. Your objective is to complete the first two topics in the table: Spectrophotometry and Absorbance Spectrum. Access the site, read the relevant material, perform the simulations, and record your answers in the space provided on the work sheet below. 1. Spectrophotometry – Basic Principles Elapsed Time (s) Intensity ࠵?࠵?࠵?࠵?࠵?࠵?࠵?࠵?2. Absorbance Spectrum By running the Part 1 simulation, determine the percent transmittance and absorbance at the indicated wavelengths. l I0࠵?࠵?࠵?࠵?࠵?࠵?࠵?࠵?I ࠵?࠵?࠵?࠵?࠵?࠵?࠵?࠵?%T A 400 nm 500 nm 600 nm 700 nm
Page 10 of 10 3. Determination of Analyte Concentration Access the website below and complete the simulation: In this simulation, you need to determine the absorbance of four standard solutions and an unknown. After plotting a graph of Absorbance vs. Concentration for the standard solutions and determining the equation of the straight line produced, it will be possible to determine the concentration of the unknown solution. Blank, I0= ______________________ Sample I ࠵?࠵?࠵?࠵?࠵?࠵?࠵?࠵?%T (%) A 20.0 ࠵?࠵?࠵?࠵?࠵?40.0 ࠵?࠵?࠵?࠵?࠵?60.0 ࠵?࠵?࠵?࠵?࠵?80.0 ࠵?࠵?࠵?࠵?࠵?Unknown Slope: _________ y-intercept: _________ Equation of line*: _________________________ *Express in terms of Absorbance, A, and Concentration, c in standard “y = mx + b” formatConcentration (with units!) of unknown: Additional Questions 1. Why is the intensity of transmitted light less than the intensity of incoming light? 2. Explain why a certain paint sample (we’ll call it “Royal Versailles”) appears to be blue.