In this experiment you will obtain the excitation and emission spectra of quinine and use this information to design a method for determination of quinine in tonic water.
Sawyer, Heineman, & Beebe, Chemistry Experiments for Instrumental Analysis, Wiley, 1984. Expt. 10-1.
Additional
Cuvette, fused silica,10-mm path, for fluorescence
Volumetric flasks: two 1000 mL; six 100 mL; one 25 mL
Volumetric transfer pipets: one 10 ml; one 5 mL; one 2 mL
Quinine stock solution, 100.0 mg/mL. Weigh to the nearest 0.1 mg or 100 mg quinine (or an equivalent amount of the acid salt) into a 1-L volumetric flask, add 50 mL 1 M sulfuric acid, and dilute to the mark with water. Prepare fresh daily and protect from light (wrap with aluminum foil).
Unknown: Quinine solutions in 0.05 M sulfuric acid.
1 M sulfuric acid (to prepare 0.05 M sulfuric acid for dilutions)
Fluorescence is luminescence stimulated by absorption of electromagnetic radiation. In molecular fluorescence, radiation from an intense UV source (e.g., a xenon discharge lamp) is typically used to excite the fluorescent molecule to an excited electronic state. Some of the energy thus absorbed by the molecule is typically dissipated by vibrational relaxation via collisions, followed by relaxation to the ground electronic and accompanying emission of a photon. Because the relaxation process can involve transitions between various vibrational levels within a given electronic state, the emitted radiation (fluorescence) from a fluorescent medium occurs over a range of frequencies, and the resulting emission spectrum appears as a broad band over a range of wavelengths.
Excitation and emission spectra can be obtained using a fluorescence spectrometer, or spectrofluorimeter. The major components of a spectrofluorimeter include a UV excitation source, excitation monochromator, sample compartment, emission monochromator, and detector. An excitation spectrum is obtained by varying the wavelegths of the exciting radiation and monitoring the emitted radiation at a fixed wavelength. Likewise, an emission spectrum is obtained by setting the exciting radiation at a fixed wavelenth and recording the emitted fluorescence by scanning with the emission monochromator.
For quantitative analysis using a spectrofluorimeter, readings are made at fixed wavelengths in order to minimize noise and maximize repeatability. Appropriate excitation and emission wavelengths are chosen based on the excitation and emission spectra. To obtain maximum sensitivity, the excitation and emission wavelengths corresponding the to the maximum fluorescence signals are typically used.
Ideally a plot of fluorescence versus concentration should yield a straight line. This is unfortunately almost never the case due to concentration quenching. The dependence of fluorescence on concentration is given by the relation
Prepare 1 liter of approximately 0.05 M sulfuric acid by diluting 50 mL of 1 M sulfuric acid to 1 liter with water. Careful measurement of the 50 mL volume with a graduated cylinder will provide adequate precision for this solution. Use this as the blank solution and as the diluting medium for solutions for the following procedures.
Prepare a 2.0-mg/mL stock solution of quinine by making by diluting 2.0 mL of the 100-mg /mL solution to 100 mL with 0.05 M sulfuric acid. Obtain the excitation and emission spectra of this solution from 200 nm to 800 nm. The following stepwise procedure applies to the Cary Eclipse spectrofluorimeter.
Prepare a series of quinine standards from the 100-mg /mL stock standard. Make sequential tenfold dilutions with 0.05 M sulfuric acid to yield the following concentrations (mg /mL): 10.0, 1.00, 0.100, 0.0100,…. Continue making dilutions until the lowest concentration used yields a fluorescence signal roughly equal to that of 0.05 M sulfuric acid. Avoid exposure of these solutions to direct sunlight or prolonged exposure to room light.
Construct a calibration curve and determine quinine in an unknown sample or samples (e.g., different brands of tonic water) using the Cary Eclipse spectrofluorimeter.
Plot the logarithm of the relative fluorescence intensity vs the logarithm of the quinine concentration (a log-log plot). Examine the plot for any deviations from linearity. This may occur especially at higher concentrations. If the fluorescence of your sample falls in a nonlinear region, you have two choices