Questions from Old Exams on Ultraviolet-Visible Spectrophotometry

  1. List the essential parts of a spectrophotometer.

  2. A solution has Absorbance = 0.235 at 630 nm. What is the % Transmittance of the solution at this wavelength?

  3. At 425 nm a 1.2 x 10-3 M solution of compound Q has Absorbance = 0.879. Another solution of Q is prepared by diluting 25.00 mL of the 1.2 x 10-3 M Q to a total volume of 100.00 mL. What is the Absorbance of the new solution at 425 nm?

  4. The table below lists Absorbance readings at 500 nm for solutions of Z at various concentrations.

    [Z], MolarAbsorbance at 500 nm
    1.00 x 10-40.136
    2.50 x 10-40.338
    4.00 x 10-40.549
    1. Do these solutions of Z obey Beer's Law? Explain why or why not briefly.

    2. What is the molar absorptivity for compound Z at 500 nm?

    3. You are given a sample of Z of unknown concentration. A 5.00-mL aliquot of the unknown is diluted to 500.00 mL total volume. At 500 nm this dilute solution of Z has Absorbance = 0.375. What is the concentration of Z in the original unknown sample?


  5. Salicylic acid is found in the bark of willow trees.

    A 100.00-g sample of willow bark is boiled with water, the mixture filtered, and then cooled. The resultant solution is a complex mixture. The salicylic acid content of the aqueous extract was analyzed spectrophotometrically by the method of standard addition.

    Solution #1 was prepared by mixing 25.00 mL willow bark extract, 10 mL aqueous Fe(NO3)3, and sufficient water to equal a total volume of 100.00 mL. [The iron nitrate forms a complex with salicylic acid which absorbs in the visible region of the spectrum.] At the wavelength used for the analysis, Solution #1 has Absorbance = 0.075.

    Solution # 2 was prepared by mixing 25.00 mL willow bark extract, 5.00 mL of 1.00 x 10-3 M salicylic acid, 10 mL aqueous Fe(NO3)3, and sufficient water to equal a total volume of 100.00 mL. At the analysis wavelength, Solution #2 has Absorbance = 0.215

    1. What is the concentration of salicylic acid in the 25.00-mL aliquot of willow bark extract?

    2. A total of 1000.00 mL willow extract was prepared from the 100.0 g of bark. Based on your answer in part a, how many grams salicylic acid were obtained from the bark? FW of salicylic acid = 138

  6. A spectrophotometric analysis for Compound QXZ was carried out at 475 nm. A series of solutions of known concentration in QXZ were prepared and the absorbance of each determined at 475 nm. The relative uncertainty in the concentrations was 2 ppt, and the relative uncertainty in the absorbance readings was also 2 ppt. A linear regression analysis was performed on the absorbance versus concentration data so obtained. The results were as follows: (correlation coefficient = 0.998) slope = 1.536456 x 10+4 and intercept = 1.8 x 10-3.
    1. What is the formula relating absorbance to concentration of QXZ based upon this data?

    2. A solution containing QXZ of unknown concentration had an absorbance too large to be read directly at 475 nm. A 10.00-mL aliquot of this solution was diluted to 50.00 mL and the absorbance measured to be 0.346. What was the concentration of QXZ in the undiluted sample?
ANSWERS

  1. Light source, wavelength selector, sample holder, detector, read-out device.

  2. Absorbance = -log(Transmittance). So, 0.235 = -log(T). Please note, the specific wavelength is irrelevant for solving the problem.
    -0.235 = log(T)
    10-0.235 = T
    . 0.582 = T, or T x 100% = 58.2%.

  3. At every wavelength where the sample absorbs light, Absorbance = absorptivity x path length x concentration, or A = abc. The absorptivity is constant at a specific wavelength. We must assume that the same path length was used for both measurements. This means A/c = ab. This also means for different solutions of the same material at the same wavelength, A1/c1 = A2/c2, or A1 x c2 /c1 = A2. In this specific case:


  4. Answers:
    1. Beer's Law is "obeyed" if the Absorbance increases linearly with concentration. In going from the first to second set of data points, the concentration increases 2.5 times. The corresponding Absorbance change is 0.338/0.136 = 2.48. You can compare the data for points 2, 3 and 1,3 and see that the same holds true. Hence, the three data points lie on the same line.

    2. The molar absorptivity is the slope of a plot of Absorbance versus molar concentration. Slope = (change in absorbance)/(change in concentration). Any two sets of data points can be used to compute the slope. Slope = (0.549 - 0.136)/(4.00 x 10-4) - (1.00 x 10-4) = 1.38 x 103 M-1cm-1 for an assumed 1-cm path length.

    3. A = abc. From the previous step we know molar absorptivity = 1.38 x 103 M-1cm-1. So, A/ab = concentration in sample taken for the absorbance measurement. 0.375/(1.38 x 103) = 2.72 x 10-4 M. The concentration in the original sample must be 500.0/5.00 times larger, since all the moles Z in the sample for the absorbance measurement had come from the original 5.00 mL aliquot:
      2.72 x 10-4 M x 500.0 mL/5.00 mL = 2.72 x 10-2 M.

  5. In the method of standard additions, two identical samples are treated the same way, except one of these has a known amount of analyte added to it. When the absorabnces of the two solutions are measured, the increase in absorbance must be due only to the added concentration of analyte. Since the amount added is known, the molar absorptivity for the analyte can be calculated. The sampe that was not mixed with extra analyte can then be used to determine the concentration of the analyte in the unknown.

    For part b:


  6. for part a: Absorbance = molar absorptivity x path length x concnetration + constant. For error at rate of 2 ppt, we have Absorbance = 1.536 x 104 x b x [QXZ] + 0.002.

    For part b: 0.346 = 1.536 x 104 x 1 cm x [QXZ] + 0.002
    (0.346 - 0.002) / (1.536 x104) = 2.240 x 10-5 M in the sample analyzed.

    The original sample which was too concentrated was diluted 5-fold in order to do the analysis. The concentration of the unknown was thus
    (50.00 mL/10.00 mL) x 2.240 x 10-5 M = 1.120 x 10-4 M.
    copyright 2000, Larry McGahey, The College of St. Scholastica, Duluth MN.
    All rights reserved.