ABSORBENCY-CORRECTED SYNCHRONOUS FLUORESCENCE WITH A FIBER-OPTIC-BASED FLUOROMETER

Synchronous fluorescence spectroscopy is frequently used to resolve mu ltiple components in mixtures without separation. However, uncompensat ed inner-filter effects can lead to unexpected and/or large quantitati ve errors. An instrument and a method that correct for primary and sec ondary inner-filter effects in synchronous fluorescence spectroscopy h ave been designed and characterized. A bifurcated fiber-optic-based di ode array fluorometer that simultaneously measures front-surface fluor escence and absorbance was constructed. A transfer function that mathe matically 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 f luorescence measurement would be in the absence of inner-filter effect s. Several experimental procedures for determining the instrument's tr ansfer function are compared and discussed. Applications of the variou s procedures to experimental fluorescence inner-filter corrections to problematic samples are demonstrated.