A Fluorometric Analysis of Quinine in Tonic Water

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1 A Fluorometric Analysis of Quinine in Tonic Water Introduction In this Laboratory Exercise, we will determine the amount of quinine in Tonic Water using a fluorometric analysis. Fluorescence Spectroscopy is an extremely sensitive technique and quinine is one of the most active fluorescers known. Thus, the determination of even small amounts of quinine is relatively easy. As is typical of spectrophotometric analyses, we will first prepare a calibration curve for the Fluorescence Intensity of quinine and then use this curve to determine the quinine concentration in a diluted sample of Tonic Water. Luminescence involves emission of photons from excited atoms or molecules. Fluorescence and Phosphorescence, both luminescent processes, involve emission of photons from systems that have been excited by absorption of photons. In molecular Fluorescence Spectroscopy, the analyte molecule first absorbs a photon (excitation, ΔE excite ) that leaves the analyte in an electronically and vibrationally excited state. At this point, the molecule rapidly loses excess vibrational energy by non-radiatively relaxing to the ground vibrational level of the excited electronic state. This occurs because energy is transfered to solvent molecules as the analyte jostles against them. This relaxation process is very efficient and very rapid. Finally, the molecule can fluoresce (ΔE relax ). Alternatively, the molecule can undergo a non-radiative transition (Internal Conversion) to the ground state. Molecules undergoing Internal Conversion transit to the Ground State without emitting radiation. This is an efficient relaxation process when higher vibrational states of the Ground Electronic State overlap with lower vibrational states of the Excited Electronic State.

2 Because of the non-radiative relaxation in the electronically excited state, the excitation energy is always larger than the relaxation energy. ΔE excite > ΔE relax (Eq. 1) Because the energies of the photons involved in these transitions are related to their wavelengths via: E photon = h c / λ (Eq. 2) (c is the speed of light and h is Planck s constant), the wavelength of an exciting photon is always shorter than that of a photon emitted during relaxation: λ excite < λ relax (Eq. 3) Quinine is an example of a molecule that undergoes both fluorescence and internal conversion. It has two possible excitation wavelengths; 250 and 350 nm. However, because internal conversion is very efficient between the two electronically excited states, only a single emission at 450 nm (blue) is observed. Fluorescence spectra can be measured using a Spectrofluorometer. Light from the source is dispersed and the excitation wavelength is selected using a monochrometer. The excitation radiation impinges upon the sample, which then begins to fluoresce. Fluorescent radiation is itself dispersed and the spectrum is measured using an appropriate detector.

3 In an actual spectrofluorometer, the dispersing element is usually a diffraction grating. In simpler Fluorometers, the dispersing elements are replaced with filters. The Flourescent Intensity (F) will be proportional to the radiant Power absorbed by the sample (P o -P): Inserting Beer s Law: F = K (P o P) (Eq. 4) P/P o = 10 -εbc (Eq. 5) and expanding the exponential term, gives us: F = K P o {2.3εbc (2.3εbc) 2 /2 -...} (Eq. 6) Provided the sample Absorbance is relatively low, we can truncate the expansion: F = 2.3 K P o εbc (Eq. 7) When P o is constant, we see the Fluorescent Intensity is proportional to the Concentration of the analyte: F = K c (Eq. 8)

4 This, then, provides a method for quantifying the amount of analyte in a system based on fluorescence measurements. A few words of caution: if the concentration of the analyte is high enough, higher order expansion terms become important and the relationship between F and c is no longer linear. And, if the concentration becomes very high, the system begins to absorb its own emitted radiation, causing a decrease in fluorescence intensity and severe non-linearities set in. Fluorescence spectroscopy is much more sensitive than corresponding absorbance spectroscopic techniques. This is because light emitted against a dark background (fluorescence) is much easier to detect than a slight dimming of intensity against a light background (absorbance). Imagine the difference between observing a photographers flash going off in a dark sports stadium (fluorescence) versus trying to detect the same flash on a bright sunny day (absorbance). However, fluorescence techniques are severely limited by the number of analytes that actually fluoresce. Most systems shed their excitation energy via radiationless pathways. Structurally, molecules that possess unsubstituted aromatic rings or other structurally rigid elements have a propensity for fluorescing. Fused-ring heterocycles also fluoresce nicely. Finally, other species in solution can act as quenching agents (Q); they absorb radiation nonradiatively from the excited analyte (S*) causing a decrease in the fluorescence intensity. S + hν excite S* (excitation) S* S + hν relax (fluorescence) or S* + Q S + Q (quenching) It can be shown the ratio of unquneched-to-quenched fluorescent intensities (F o /F) is related to the quenching agent concentration ([Q]) via (Stern-Volmer Relation): (F o /F - 1) ~ [Q] The proportionality is related to the fluorescence lifetime and the rate constant for the quenching process. In many cases, Oxygen (O 2 ) acts as a very effective quenching agent for those systems that do fluoresce. Fortunately for our experiment, O 2 does not quench quinine fluorescence. But, chloride ions (Cl - ) do act as a quenching agent for quinine. Thus, it is imperative to keep the Chloride Ion concentration low throughout. In particular, we should not use the quinine hydrochloride, a common salt of quinine, to prepare our standard solutions.

5 Quinine, as already mentioned, is a very efficient fluorescer with excitation wavelengths at 250 and 350nm. Emission occurs at 450nm. Quinine, originally derived from the bark of the Cinchona tree, is an effective cure for malaria. Traditionally, because of its bitter taste, quinine was mixed with gin, giving rise to the Gin and Tonic cocktail. Quinine is used as a flavoring agent in Tonic Water, Bitter Lemon and Vermouth. This is limited by the Food and Drug Administration to 83 ppm; with most commercial preparations at ppm. We will measure the amount present in a commercial product. Pre-lab Exercises 1. Assuming the tonic water used in the experiment contains 50 ppm quinine, what will be the final diluted concentration after following the dilution in the procedure? 2. Based on the answer to number one, determine a good range of quinine concentrations to analyze, and develop a plan for dilution to create 5-6 standards within that range. A few things to keep in mind: you will be given a 1 L stock solution of ppm quinine in 0.05 M H 2SO 4 as well as a separate 1 L of 0.05 M H 2SO 4. All standards will be made using these two components. You will use 100 ml volumetric flasks, and can use 1, 5, 10 and 20 ml volumetric pipettes. Procedure Preparation of Quinine Standards 1. A ppm stock solution will be prepared by the TA, from which several standard dilutions will be made. The stock solution is prepared by accurately weighing mg of quinine sulfate

6 dihydrate, transferring to a 1L volumetric flask, adding 50 ml of 1M H 2SO 4 and diluting to the mark with Distilled Water. This stock solution should be prepared daily and be protected from the light. 2. A series of 10X dilutions of the quinine stock should be prepared with 0.05M H 2SO 4. After each dilution, measure the Fluorescence. Continue with the dilutions until the Fluorescence intensity is approximately that of the blank. 3. Prepare a graph of log(rel. Fluor. Intensity) vs log(concentration). This is your calibration curve. Discuss any deviations from linearity with your instructor. Do a Linear Least Squares Analysis of the linear portion of the curve only. Preparation of the Tonic Water 1. Pipet 5.00 ml of Tonic Water into a 250 ml Volumetric flask. Dilute to the mark with 0.05M H 2SO 4 and mix thoroughly. 2. Pipet 5.00 ml of the above solution into a 25 ml Volumetric flask. Dilute to the mark with 0.05M H 2SO 4 and mix thoroughly. 3. Record the Fluorescence intensity of this solution. 4. Determine the quinine concentration using your prepared calibration curve. Collection of Fluorescence Data 1. Your laboratory instructor will demonstrate the use of the Fluorometer. Quinine has two excitation wavelengths; 250 and 350nm. Either can be used. The wavelength of maximum fluorescence for quinine is 450nm.

A Fluorometric Analysis of Quinine in Tonic Water

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