UV-Vis Data Analysis

A.  Open your data file in Excel. Make one well-labeled plot (A vs λ) showing the absorbance spectrum of your soft drink sample, a caffeine standard, and a benzoic acid standard.  (Make sure your legend describes which is which.) The purpose is to show that the soft drink spectrum is a combination of the caffeine and benzoic acid spectra.

B.  Make a second similar plot, this time with the spectrum from the unknown sample substituted for the soft drink.

C.  Choose four separate wavelengths for the analysis. Two should be the wavelengths of maximum absorbance, λmax, for both caffeine (~270 nm) and benzoic acid (~230 nm).  The other two should be wavelengths between these two limits, and both compounds should show fairly significant non-zero absorbance at each and the Beer's Law plot for each compound at each of the four wavelengths should be highly linear.

D.  Determination of molar absorptivities (ε).

• 1. 1. Calculate the concentration of caffeine (in mol/L) for your caffeine stock solution and then in all four of your caffeine standard solutions.

     2. For each of your caffeine standard spectra, find the absorbance value at all four of your chosen wavelengths.

     3. Determine the slope, intercept, and R-squared values for the Beer's law plots at each of your four wavelengths. (The slope is equal to your εb value at that wavelength.) You can do this by actually making plots, or by using Excel as shown in the figure below. In the highlighted cells you'll enter the concentration of each standard and the absorbance values at each wavelength. Entering the SLOPE, INTERCEPT, and RSQ equations as shown will calculate each automatically. Notice that the dollar signs ($) in the formula will allow you to fill these cells across to the right locking the concentrations, but incrementing the absorbance values.

4.  Repeat steps 1-3 for benzoic acid.

E.  Determine the value of the absorbance at each of your four wavelengths for both your soft drink sample and your unknown solution.

F.  Starting with your soft drink sample, you should be able to set up four linear equations, one at each of the four wavelengths, that have the form:

Atot,λ = εcaff,λbccaff + εba,λbcba

Set up your spreadsheet like the image below, and fill in the highlighted cells with your εb values from above. The Asample values are the absorbances of your soft drink sample. Cells F17 through F20 are the four linear equations, and cells G17 through G20 calculate the difference between the calculated and measured absorbances at each wavelength. (We square the difference to get rid of the sign.) 

Enter initial values of 0.001 M for the caffeine and benzoic acid concentrations, then use the Solver function in Excel to find the optimal values for the concentrations that minimize the sum of the square differences in column G. These values should be the concentrations of caffeine and benzoic acid in your measured solutions.

G.  Repeat step F using the absorbances from the unknown sample in place of the soft drink absorbances.

H.  Remembering to consider the initial dilutions of your samples, calculate the concentration of caffeine and benzoic acid in both your undiluted soft drink and in your undiluted unknown solution in units of mg/L.

I.  Upload all of your data analysis to LabArchives (making sure any spreadsheets are well labeled), then complete the Report Form.