A Home-Built Spectrofluorometer Fred J. Hadley and Ali Mahloudji Rockford College, Rockford, IL 61108 We have returned into service two Beckman DU s ~ e c t r o photometers as the heart of a modest spectrofluorometer. Beckman DU's have excellent optics, and we were fortunate that one of the two had an attached photomultiplier system with attendant power supply. I t seemed a simple matter to put the two together to form the excitation and emission halves of a fluorometer and have something we could use to illustrate the basic principles of fluorescence, both qualitative and quantitative. The new instrument now supports one of our experiments in instrumental analysis. Putting the components together turned out to he an excellent summer project for one of our students (Mahloudji), combining work in the machine shop with some electronics, computer programming, and data acquisition.
students now use it to determine the quinine content in tonic water', typical results of which are shown in Figure 2. The content of fresh, commerrial tonic water is ahout 7 8 ppm. althoueh [his droos uuon standine in lieht. We diluted sam6 results 4 . on two ples b;a factor oi 20 kith 0.05 ~ ~ 2 ~ Our Canada Drv samnles were 64 and 66 ovm. Our ontimum concentratibn range for work with quinine is higher than one ordinarily uses on a commercial instrument (about 0.1-0.5 ppm), but the calibration curve is sufficiently linear that it eives eood results. We have been unable to work much below 6.5 p& with the quinine. If one knows the response of the detector as well as the output curve for the lamp, excitation and emission spectra can be obtained2. Sample spectra are shown in Figures 3a and 3h. In the emission spectrum, one canclearlj see the scattered light at 367 nm as well as the fluorescence maxi-
A l~lockdiagram of the arrangement of the components is shown in Figure 1. The Heckmans were mounted on a solid plywood baie and set side t ~ yside, with the second staggered and elevated with respect to the first so that the fluorescenre from the sample cell passed directly into the second instrument. The exit hole behind the sample cell wnv sealed shut, and a-~ hole was cur in the side uf the cell holder so that the beam could pass to the second DU. All passages and joints were made lieht tieht with black felt. The sample cuvette was a Wilmad near-UV quartz cell; the lamp source was the 200-W H e source of an Oriel arc lamn. The signal from the photomultiplier tube (PMT) and high-voltage power supply varied between 30and 34 V, so we floated a Tetronix PS 503A constant-voltage power supply between the output and a Keithley 197 digital voltmeter, thereby dropping the signal down to 0-4 V. It was then a simple matter to adjust the Tetronix power supply in order to zero solution blanks. The signal also led from the Keithley DVM to a Commodore P E T comouter where we could mint the data and draw graphs. This part of the project is still in progress-normally, the student reads the signal directly off of the DVM. ~
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Exoerlmenia A number of well-known experiments can he performed on the instrument. In our instrumental analysis course the
' O'Reilly, J . E . J. Chem. Educ. 1975. 52, 6610-612.
O'Reilly, J. E. J. Chem. Educ. 1976, 53, 191-193.
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Figure 1. Block diagram of me specbofluorometer, consisting ot a mercury lamp source, two Beckman DU specbophatometers(Mc = monochromator),a high-voltage power supply (HVPS) that runs a photomultiplier on the second DU, a Tetranix PS503A constant-voltage power supply, and a Keithley 197 digital voltmeter (DVM). The computer is a Commodore PET.
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[~uinin.] , ppm Figure 2. A calibration curve for quinine in 0.05 M H,S04. The dolled line showsthe result for a sample of tonic waterlhat wasdiluted by a factor of 20 in 0.05 M sulfuric acid.
mum at 450 nm. The students can also vary the two slit widths readily and see how this affects the quality of the spectra. One of the best aspects of the instrument, however, is that they can take the cover off the sample cell and look down into the cuvette while manually scanning the excitation wavelength (the students wear UV-protective goggles while o~eratinnthe instrument). They can see how quickly the flu&escence appears and then dibappears as one scans throunh - the hand. We hope to extend this instrument's use to include experiments for our hiochemistrv and physical chemistry labs as . . well (fur example, with experiments on ligand-protein hinding'and the emission spectrum ot molecular iodine'.)
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Wavelength, n m Figure 3. Excitation a d emiJsion spectra of quinlne in 0.05 M sulfuric acid. A. Excitation spectrum with the emission monitored at 450 nm. B. Emission Spectrum with excitation at 367 nm. in each, the monoChromators were menually rotated and the signal read directly off the DVM.
Marty, A.: Boiret, M.; Deumie, M. J. Chem. Edoc. 1986, 63.365366.
Shoemaker, D. P.: Garland. C. W.: Nibler. J. W. Experiments in Physical Chemistry, 5th ed.; McGraw-Hill; New York, 1989; pp 497507.
Acknowledgment The authors would like to thank Tom Herrinton and Debbie Wiegand for their help in the early stages of the design.
Volume 67
Number 9
September 1990
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