Fluorescence determinations with the universal spectrophotometerJ. E. Purkyne University, Brno
Abstract: A modification of the Carl Zeiss VSU-1 universal spectrophotometer for fluorescence spectrophotometry in the visible and uv region is described. By adding a linear recorder the fluorescence spectrum can be recorded directly. The arrangement is also suitable for the fluorimetric standardization of fluorescent substances with a high degree of sensitivity. It has the advantage that an excitation wavelength can be selected conveniently during the fluorimetric detn. with a 2nd monochromator. In the optical arrangement a light ray from the source (high-pressure Hg lamp, 120 v., 250 w., backed by a highly polished Al parabolic reflector) passes through an aperture, a condenser, an iris aperture, an interference filter and enters a rectangular cuvette in front of the entrance slit of the monochromator. A 2nd monochromator can be substituted for the interference filter. The fluorescent emission leaves the window of the cuvette at right angles to the entering excitation beam, passes through the slit and optical system of the monochromator with a glass or a quartz prism for visible or uv spectra, resp. The light ray leaves through the exit slit of the monochromator to be intercepted by the cathode of the photomultiplier. The disadvantages of the rectangular method are avoided by the off-center paths of the excitation and emission beams being confined tightly into one corner of the cuvette to minimize the depth of liquid in the light path. The cuvette and the sample can be very small. The transmitted portion of the excitation beam is absorbed in a light trap consisting of a tube curved into a 90° circular arc. The use of a 2nd monochromator was adopted later in this work and offers the advantage of a continuum of choice in the excitation spectrum to obtain max. emission intensity, as compared with the more laborious alternative with exchangeable interference filters at various wavelengths. Increased sensitivity of fluorometric detns. in anal. chemistry can thus be achieved. Fluorescence spectra are plotted for 10-4M quinine sulfate in 0.1M H2SO4 and 10-5M Acridine Orange in pH 6 buffer at excitation wave lengths of 366 and 436 mμ, resp., also on a common graph, 6 addnl. emission spectrograms of Acridine Orange soln. concns.: 1 × 10-5, 8 × 10-5, 6 × 10-6, 4 × 10-6, 2 × 10-6, and 1 × 10-6M at an excitation wavelength of 436 mμ. The results agree with published wavelength data on fluorescence spectra for max. excitation. A suitable source of 850 v. d.c. with a stability of 2.3 × 10-2 was not available and had to be built along with an electronic voltmeter for the photoelec. measurements. The wiring diagram is shown. Full paper in Portable Document Format: 204a299.pdf (in Czech)
Chemical Papers 20 (4) 299–306 (1966) |
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