In the Laboratory edited by
Cost-Effective Teacher
Harold H. Harris University of Missouri—St. Louis St. Louis, MO 63121
A Simple Device to Demonstrate the Principles of Fluorometry Néstor J. Delorenzi,* César Araujo, Gonzalo Palazzolo, and Carlos A. Gatti Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina; *
[email protected] Instrumentation has become integral to physicochemical measurements. However, the high cost of most instruments poses a restriction to the adoption of this approach in undergraduate physicochemical courses. Moreover, commercial instruments do not effectively teach the principles of the applied technique and the instruments generally appear to students as black boxes. To improve this situation, we propose a simple, low-cost device for the demonstration of fluorometry and its use in an experiment illustrating the fluorescence of quinine bisulfate and its quenching by chloride ion (1–3). Figure 1. Schematic diagram of the apparatus (top view).
A schematic diagram of the fluorometer is shown in Figure 1. The components of the fluorometer are described below.
To simplify data acquisition and the calculations associated with experimental work, we have used a digital multimeter provided with an RS232 interface. The data transferred to a personal computer were processed by software written in Basic.
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1. Excitation light source: Mini ultraviolet fluorescent lamp (a) with a 4-W Westinghouse Daylight F4T5/D lamp powered by 3–12-V (dc 500 mA max) power supply. 2. Cell holder (b): Black plastic housing (4.5 × 4.5 × 10 cm) to hold the cylindrical cell with two holes perpendicularly positioned (c, d), and a black plastic cap. 3. Cell (e): Cylindrical borosilicate glass tube (13 × 100 mm). 4. Detector (f ): A photoresistor (LDR) whose resistance, which depends on the intensity of radiation striking its surface, is measured by a digital multimeter (4).
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Design of the Apparatus
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Figure 2. Concentration and relative fluorescence intensity of QBS solutions vs LDR resistance.
The Experiments Quinine bisulfate (QBS) was dissolved in 1 M H2SO4. The maximum absorbance for QBS is about 350 nm, with emission at about 456 nm (2). The lamp emission peaks at 370 nm, overlapping the QBS absorption, making it suitable as an excitation source.
Calibration Curve QBS satisfies the Beer–Lambert law and its fluorescence intensity (FI) is directly proportional to concentration for dilute solutions (absorbance less than 0.5) (1). For that reason, dilutions of 50 µM QBS stock solution were prepared and the apparatus response for each of them was measured. Figure 2 shows the resistance as a function of QBS concentration. To convert the resistance data collected to FI measurements, relative FI values were assigned to the dilutions, demonstrating the linear relationship between them and the concentrations.
In Figure 2 a value of 1 was assigned to the quarter dilution of the stock solution. Note that by removing the cap during the measurement students can observe the following features of the fluorescence process: The wavelength shift between the emission and the excitation light. The increase of the emission intensity with increasing fluorophore concentration. The inner filter effect for highly concentrated QBS solutions, which appears as a more fluorescent zone near the incidence point of the excitation light.
Fluorescence Quenching Experiment To 6 mL of a suitable QBS dilution (a quarter dilution of the stock solution), small aliquots (20 µL) of a 1 M NaCl stock solution were sequentially added. The FI of the mixtures
JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Journal of Chemical Education
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In the Laboratory
was measured after each addition using the calibration curve shown in Figure 2. Final quencher concentrations ranged from 0 to .025 M. Fluorescence quenching can be interpreted quantitatively by the Stern–Volmer equation:
where FIo and FI represent the maximum fluorescence intensities of QBS in the absence and presence of NaCl, respectively, and KSV is the Stern–Volmer constant for the quenching process (5). The experimental Stern–Volmer plot of FIo/FI vs [NaCl] is a straight line, as shown in Figure 3. The value of the slope obtained (119 M{1) was in reasonable agreement with KSV determined for the same system in a Jasco FP 770 spectrofluorometer (108 M{1), with 350 nm and 456 nm as excitation and emission wavelength, respectively. From the K SV value it is possible to estimate k q , the bimolecular quenching constant, and to compare it with the value of the Debye–Smoluchowski diffusion-limited second-order rate constant (2, 3). Conclusion The apparatus involves low-cost components that are readily obtained from electronic shops, and it can be easily constructed by the students. This approach enhances the students’ understanding of physicochemical principles and their appreciation of the benefits of using the PC in data acquisition and processing.
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FI° = 1 + K SV [NaCl] FI
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Figure 3. Stern–Volmer plot for the quenching of QBS fluorescence by NaCl. m Data from the device; , data from the spectrofluorometer.
Literature Cited 1. O’Reilly, J. E. J. Chem. Educ. 1975, 52, 610–612. 2. Sacksteder, L. A.; Ballew, R. M.; Brown, E. A.; Demas, J. N.; Nesselrodt, D.; DeGraff, B. A. J. Chem. Educ. 1990, 67, 1065–1067. 3. Bigger, S. W.; Ghiggino, K. P.; Meilak, G. A.; Verity, B. J. Chem. Educ. 1992, 69, 675–677. 4. Andres, R. T.; Sevilla, F. III. J. Chem. Educ. 1993, 70, 514–517. 5. Coutinho, A.; Prieto, M. J. Chem. Educ. 1993, 70, 425–428.
Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu
