Fiber Optic Filter Fluorometer for Improved Analysis of Absorbing Solutions Douglas 0. Mitchell, John S. Garden," and Kenneth M. A ~ ~ Q U S New York State Department of Health, Division of Laboratories and Research, Albany, N. Y. 1220 1
Almost all fluorescence measurements are carried out on solutions that are transparent at both excitation and emission wavelengths. If the solution shows significant light absorption a t either wavelength, variations in absorption from sample to sample will result in significant, and perhaps gross, analytical error. For example, fluorometric determination of zinc protoporphyrin (ZPP) (1)in blood is subject to interference by hemoglobin, which absorbs strongly a t 424 nm, the excitation wavelength of ZPP (2). . This problem can be avoided by chemical separation of the absorbing species (e.g., by extraction of protoporphyrin into acetic acid-ethyl acetate and back extraction into hydrochloric acid (3-6), by sample dilution, or by using much smaller cuvettes. However, chemical separation procedures usually increase overall analysis time, while sample dilution and use of smaller cuvettesboth reduce sensitivity. Reduction in cuvette size reduces the ratio of fluorescence to reflected radiation signals and thus aggravates the problem of reflected radiation from the cuvette surface, which is particularly significant with disposable glassware. Extraneous material on the container wall can cause further error. An ideal fluorometer for routine analysis, particularly of absorbing solutions, would (a) have a short path length in solution, to minimize error due to variations in absorption; (b) observe a large solid angle of fluorescence, to maximize sensitivity; and (c) have no walls in the path of excitation radiation, to minimize blank signals that arise from light reflected to the detector. In this paper we describe a simple fiber optic fluorometer which approximates this ideal. Excitation radiation passes along one arm of a Y-branched fiber optic bundle (Figure 1) to the sample solution. Fluorescence radiation is collected by another group of fibers randomly distributed throughout the same bundle and is transmitted through a filter to a photomultiplier. Since the combined end of the fiber optic is dipped in the solution, neither excitation nor emission radiation passes through container walls. The performance of the fiber optic fluorometer has been evaluated by studying the fluorescence of ZPP in ethanolic and in aqueous solution.
EXPERIMENTAL
transmitted by them through a second interference filter (A, = 595 nm, half-bandwidth = 9.6 nm, transmission at 595 nm = 45%; Corion) to a photomultiplier tube (1P21, Hamamatsu Corp., Middlesex, N.J.). Electronic System. A regulated power supply (5 A, 12 V d.c.,
