Correction of Polychromatic Luminescence Signals for Inner-Filter Effects

Abstract

Fluorescence (FL) and absorbance (ABS) data are acquired simultaneously with a multiple detector spectrometer. The measured ABS is utilized in mathematical equations for automatic correction of fluorescence signals for attenuation (inner-filter effects) caused by significant absorption of the excitation or emission radiation by the fluorophore or other species. Correction equations are presented which apply to situations in which the excitation or emission beam cannot be considered monochromatic, for collection geometries involving optical fibers, and for chemiluminescence measurements. The correction scheme was tested with quinine sulfate (QS) as the analyte in the absence and presence of other chromophores (gentisic acid and methyl red). Even when conditions are such that the FL signal is attenuated by a factor of 5, the correction scheme is accurate to 2% or better. These corrections extend the linear range of the QS calibration curve by a factor of over 100, and they can improve the accuracy up to a factor of ten in cases for which polychromatic effects are important.