spectrometer a smaller solid angle than the acceptance angle of the spectrometer itself. Bgrresen's procedure does, however, suffer from another possible source of error. Since only the central area of the collimating mirror and prism, or grating, is used, a single measurement under these conditions does not give the spectral sensitivity curve of the spectrometer pertaining to fluorescencemeasurements in which the collimator is filled with light. At long wavelengths with a prism spectrometer the sensitivities for peripheral and central rays are not expected to differ greatly. At short wavelengths, where mirror reflectivities decrease and prisms start to absorb, considerable variations are possible. With grating instruments, variation in reflectivity over the grating surface will also introduce differences between the sensitivities for central and peripheral rays. It is recommended therefore that when Parker's method is used, the exciting light should be focussed with
WAVELENGTH nu^ 400
350
250
300
emission spectra to restrict the beam to the same central area of the collimator as that for which the calibration curve was determined. If, however, it is desired to utilize the full aperture of the fluorescence spectrometer, several calibration runs must be made with the narrow beam passing to various positions on the collimator so that a value may be derived for the mean spectral sensitivity of the spectrometer. LITERATURE CITED
WAVENUMBER Figure 1 .
JI-'
Values of 4An/(F, synthetic silica
-
B@rresen,H. C., Acta Chem. Scand. 19, 2089 (1965). (2) Parker, C. A., ANAL.CHEM.34, 502 (1962). (3) Parker, C. A., Nature 182,1002 (1958). (4) Parker, C. A., Rees, W. T., Analyst 85, 587 (1960). (1)
1 ) for
an achromatic lens or a concave mirror, If a silica lens has to be used, the correction factors indicated in Figure 1 must be applied to the results obtained. When Bgrresen's calibration procedure is employed, it is necessary in subsequent measurements of fluorescence
H. C. B@RRESEN C. A. PARKER'
Institute of Clinical Biochemistry Rikshospitalet, University of Oslo Oslo 1, Norway Admiralty Materials Laboratory, Holton Heath, Poole, Dorset, England.
Polarographic Reduction of the Pyridinium Ion in Pyrid i ne-W a ter Mixtures SIR: The polarographic reduction of pyridinium ion in pyridine solution proceeds by a direct attack on the pyridine ring, apparently producing a free radical intermediate, which then dimerizes (6) [ring reduction of pyridinium ion is also indicated (1, 2) by the occurrence of such a process in the electrochemical reduction of the analogous Lewis acid-base adduct formed by Al(II1) in pyridine solution]; in aqueous solution, containing small amounts of pyridine, the reduction proceeds by a catalytic evolution of hydrogen (6). Consequently, a fundamental change in mechanism must occur a t some intermediate pyridine-water ratio. Because the reduction of pyridinium ion in pyridine can be used for the determination of Lewis and Brgnsted acids, the effect of varying amounts of water present in the polarographic test solution on the half-wave potential, El,z, and diffusion current constant, I , should be known to ascertain its effect on the reproducibility of the analytical measurement. The latter consideration is of practical importance because pyridine is quite hygroscopic and acids may have to be determined in samples which are, a t least partially, aqueous. Consequently, the polarographic behavior was investigated of pyridine solutions, ImM in benzoic acid and 0.1M in lithium perchlorate, which contained from 0 to nearly 100% water by volume. 1074
ANALYTICAL CHEMISTRY
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