Absorption-corrected chemiluminescence measurements with a dual-pathlength spectrometer

Abstract

typically 5-15 min. Light scattering and absorption by the support can affect and may distort the apparent intensities of the peaks in the spectrum, so care must be taken when attempting to do quantitative work.A mathematical model of secondary inner-fitter effects allows the derivation of a correction factor based on a determination of the ratio of chemiluminescence intensities from two pathlengths. A microprocessor-controlled instrument equipped with a unlque dual-path cell acquires intenstty measurements from a collimated, monochromatic optical system. Lumlnol chemiluminescence was observed from solutions containing the interfering chromophores picrate and ferroin. The effectiveness of the model and the characteristics and limitations of the instrument are discussed. For the case of negiiglbie reemission, absorption-free signals are accurately calculated for matrices wlth absorbances ranging to more than 0.75. An emission spectrum demonstrating ferroin activation Is recovered from an apparently quenched reaction.displacements of the through-cell paths of the excitation and emission beams (5-8). For a well-defined optical geometry, the fluorescence attenuation changes with the pathlength according to Beer's law. Intensity vs. pathlength information is used to determine the absorbance values, and a correction factor is calculated. No similar remedies have been described for correcting CL measurements for inner-filter effects, although an expression describing CL in terms of absorbance and pathlength has been reported by Stieg and Nieman (9). This expression was applied as a criterion for the efficient design of CL flow cells but not for developing a method to correct the inner-filter effects.In this paper we describe a procedure for correcting CL measurements that is based on a determination of the ratio of CL intensities from two different cell pathlengths. A microprocessor-controlled instrument with a unique dual Dathleneth cell is described. The instrument automaticallv Y produces absorption-corrected CL intensities a t a single wavelength or absorption-corrected CL spectra. Results of studies with a luminal cL reaction are presented that demonstrate the effectiveness of the correction procedure and the characteristics of the instrument. THEORYMany studies Of (cL) and bioluminescence reactions have suggested ambiguities or interferences attributable to inner-filter effects (attenuation of emitted radiant power due to absorption by the solution matrix) (1-3). Methods for correcting molecular fluorescence measurements for inner-filter effects have been reviewed (4, 5). These correction procedures used expressions that describe the fluorescence intensity as a function of the absorbance a t the excitation and/or the emission wavelength. The "cellshift" method for correction of inner-filter effects depends upon fluorescence measurements determined with several Consider a thin slice of solution of infinitesimal thickness dx, parallel to the observation window. The slice radiates monochromatic light of unattenuated ...