Department of Chemistry University of Kentucky CHE 226 – Analytical Chemistry Laboratory 34 Quinine Fluorescence EXPERIMENT 6 Molecular Fluorescence Spectroscopy: Quinine Assay UNKNOWN Submit a clean, labeled 500-mL volumetric flask to the instructor so that your unknown quinine solution may be issued. Your name, section number, and your locker number should be written legibly on this flask. The flask does not need to be dry on the inside, but needs to have been rinsed with distilled water after it has been cleaned. The flask must be turned in at least 1 lab period before you plan to do the experiment so that the Teaching Assistants will have time to prepare the unknown. Each student will have his or her own unknown to analyze even if you are working in pairs. BACKGROUND Quinine (C20H24N2O2, 324.43 g/mol) is an alkaloid extracted from the bark of the cinchona tree. It has been used for many years as an antimalarial agent. Although it does not cure malaria, it is effective in alleviating the symptoms of malarial attacks. The usual medicinal form is quinine dihydrochloride or quinine sulfate dihydrate, (C20H24N2O2)2zH2SO4z2H2O, 782.97 g/mol. Quinine is a very strongly fluorescing compound, especially in dilute acid solution, and thus can be detected in very trace amounts. In 0.05 M H2SO4, quinine has two analytically useful excitation wavelengths: λex = 250 and 350 nm. Regardless of which excitation wavelength is used, the wavelength of maximum fluorescence emission intensity, λem or λfl, is 450 nm. The basis for quantitation is that the intensity of fluorescence emission in very dilute solutions is directly proportional to the concentration of quinine – if the intensity of the excitation source and other experimental factors are kept constant. Because the absolute emission intensity can vary considerably with small differences in experimental conditions, a calibration curve is prepared by measuring the fluorescence-emission intensity of accurately known quinine standard solutions. INSTRUMENTATION Turner Quantech Digital Filter Fluorometer, Model FM 109525 The instrument used in this experiment uses glass filters to select wavelength ranges appropriate for quinine, rather than some type of expensive grating or prism monochromator to isolate narrow excitation and fluorescence wavelengths. The latter type may typically have a bandpass of 0.1 or 1 nm. A bandpass is defined as the width at half-height of the maximum intensity of light that passes through the filter. It is also called the bandwidth.Department of Chemistry University of Kentucky CHE 226 – Analytical Chemistry Laboratory 35 Quinine Fluorescence A monochromator-based instrument is much better for obtaining spectra, particularly those with fine structure, but a filter-based unit will often be better for routine quantitative analysis. The much larger bandpasses permit greater fractions of the excitation and fluorescent light to excite the sample and to reach the detector, respectively. This greatly increases the sensitivity of the instrument and can thus usually lower typical detection by an order of magnitude or more. Filter fluorometers are also much less expensive. Excitation Source: A 5-watt quartz-halogen lamp, which emits intense broadband radiation from 340 nm to 750 nm. Excitation Wavelength Filter: A narrow-band 360-nm filter with a bandpass of 40 nm. Emission Wavelength Filter: A sharp cut-in, long-wavelength-pass filter which transmits essentially all light with λ > 415 nm. Detector: Photomultiplier tube, model 931B PMT. Detection Limit. Stated in the manufacturer’s literature as 30 ppt quinine sulfate, which is 30 ng/L or about 9 x 10-11 M. Turn the instrument on at least 15 minutes before using to allow it to warm up and stabilize. The ON-OFF switch is on the back panel near the power cord. When turned on, the instrument runs a countdown timer during which it undergoes self tests. PREPARATION OF SOLUTIONS Preparation of Stock Sulfuric Acid Solutions The two stock H2SO4 solutions should have already been prepared for you by the Teaching Assistants. However, if you use up all of the 0.05 M solution while doing your experiment, prepare another 2-L batch for those that follow. It is very straightforward. 1 M H2SO4. Slowly and carefully add 56 mL conc. H2SO4 to about 500 mL distilled water in a 1-L beaker with stirring. This solution should have been already prepared for your use. 0.05 M H2SO4. With a graduated cylinder, add 100 mL of 1 M H2SO4 with a graduated cylinder to about 500 mL of distilled water in the screw-capped acid reagent jug labeled for this solution. Mix and dilute to the 2.0-L mark on the bottle and mix thoroughly. This solution should already have been prepared for your use. If you use all of or most of what is there, prepare a new batch for the students who follow you. Preparation of Quinine Stock Solution, 1000 ppm 1. Carefully weigh exactly 0.1207 g of quinine sulfate dihydrate onto a folded glassine weighing paper or into a small plastic weighing boat, and transfer this quantitatively into a 100-mL volumetric flask. A few squirts of distilled water from a wash bottle should help to wash the solid material from the weighing boat and the neck of the flask.Department of Chemistry University of Kentucky CHE 226 – Analytical Chemistry Laboratory 36 Quinine Fluorescence 2. Pipet 5.00 mL of 1 M H2SO4 (located in hood #2) into the flask. Carefully dissolve all the quinine in this sulfuric acid solution by swirling before diluting to volume. This is critically important. 3. Carefully dilute to volume with distilled water and mix thoroughly. Preparation of Intermediate Quinine Stock Solution, 10.0 ppm 1. Pipet 5.00 mL of the 1000-ppm solution into a 500-mL volumetric flask. 2. Add 25.0 mL of 1 M H2SO4, dilute carefully to volume with distilled water, and mix thoroughly. Preparation of Quinine Standard Solutions 1. Using volumetric transfer pipets and/or a 10-mL graduated pipet, add 1.00, 3.00, 5.00, 7.50, and 10.00 mL of the 10-ppm intermediate stock solution into five properly labeled 100-mL volumetric flasks. This will result in standard solutions of 0.1, 0.3, 0.5, 0.75, and 1.0 ppm. 2. Carefully dilute to volume with 0.05 M H2SO4. 3. 0.05 M H2SO4 is used as the “blank.” Preparation of Quinine Unknown 1. Your unknown solution is obtained from the teaching assistants
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