Absorbance-Corrected Synchronous Fluorescence with a Fiber-Optic-Based Fluorometer

Abstract

Synchronous fluorescence spectroscopy is frequently used to resolve multiple components in mixtures without separation. However, uncompensated inner-filter effects can lead to unexpected and/or large quantitative errors. An instrument and a method that correct for primary and secondary inner-filter effects in synchronous fluorescence spectroscopy have been designed and characterized. A bifurcated fiber-optic-based diode array fluorometer that simultaneously measures front-surface fluorescence and absorbance was constructed. A transfer function that mathematically describes the coupling efficiency between the excitation and emission legs of the fiber optics is defined and characterized. Once characterized, such a fluorometer is capable of calculating what the fluorescence measurement would be in the absence of inner-filter effects. Several experimental procedures for determining the instrument's transfer function are compared and discussed. Applications of the various procedures to experimental fluorescence inner-filter corrections to problematic samples are demonstrated.