In the Laboratory JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Jo urna l o f Chemic a l Educ a tio n 1265 Cost-Effective Teacher edited by Harold H. Harris University o f Misso uri— St. Lo uis 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 Q uímica-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina; * mpires@fbioyf.unr.edu.ar Instrumentation has become integral to physicochemical measurements. However, the high cost of most instruments poses a restriction to the adoption of this approach in un- dergraduate 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 bisul- fate and its quenching by chloride ion (1–3). Design of the Apparatus A schematic diagram of the fluorometer is shown in Fig- ure 1. T he components of the fluorometer are described below. 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 per- pendicularly 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 ). To simplify data acquisition and the calculations associ- ated with experimental work, we have used a digital multi- meter provided with an RS232 interface. T he data transferred to a personal computer were processed by software written in Basic. The Experiments Quinine bisulfate (QBS) was dissolved in 1 M H 2 SO 4 . The maximum absorbance for QBS is about 350 nm, with emission at about 456 nm (2). T he lamp emission peaks at 370 nm, overlapping the QBS absorption, making it suit- able as an excitation source. Calibration Curve QBS satisfies the Beer–Lambert law and its fluorescence intensity (FI) is directly proportional to concentration for di- lute 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: T he wavelength shift between the emission and the ex- citation light. The increase of the emission intensity with increasing fluorophore concentration. T he inner filter effect for highly concentrated QBS solu- tions, 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. T he FI of the mixtures 0 400 800 1200 1600 30 20 10 0 3 2 1 0 FI R / kOhm [Q BS] / μM Figure 2. Concentration and relative fluorescence intensity of Q BS so lutio ns vs LDR resistance. Figure 1. Schematic diagram of the apparatus (top view).