Versatile Spectrophotofluorometer-Polarization Fluorometer

Abstract

A highly versatile spectrophotofluorometer with unique optical as well as electronic features is described. In addition to corrected excitation, emission, and polarization spectra, absorption and transmission spectra can also be produced. Spectra may be recorded as a function of wavelength or wavenumber. The sample area is designed to permit temperature and p_H control and use of an automatic titration assembly. A three quartz plate beam splitter produces two identical beams (within 0.5%) over a range of 280 to 600 mμ, regardless of the polarization of the incident beam, thus allowing the measurement of accurate corrected excitation spectra. Total stray light present at exciting wavelengths from 210 to 240 mμ was measured: it amounted to 7% at 240 mμ. Polarization spectra (excitation as well as emission) can be measured continuously without altering the apparatus; a discriminator was built which separates two normally polarized components. Degree of polarization can be obtained within 1%, and this figure can be further improved by correcting for imperfect discrimination of the two polarized components. Corrections for light source fluctuations and nonlinearities as a function of wavelength and continuous polarization measurements are effected by a digital ratio system which allows unlimited time integration and has an infinite dynamic range. The long range drift introduced by this system and the photomultiplier current preamplifiers is below 0.03%. The limit on overall stability is determined by the photomultipliers, and amounts to 0.5% over the long range (due to gain variations).