Derived Absorbance Spectral Parameter as a Tool for Sensitive Fluorescence Measurements of Optically Dense Systems

The present study proposes an approach to use the derived absorbance spectral parameter (D_abs) as a tool for sensitive fluorescence measurement of optically dense multifluorophoric systems. Mishra and group have demonstrated the use of D_abs in obtaining optimum offset wavelength for sensitive fluorescence measurement in synchronous fluorescence spectra (SFS) (Anal. Chim. Acta 2016, 940, 113-119; Anal. Chim. Acta 2008, 630 (1), 47-56). The D_abs is obtained with the knowledge of UV-vis absorbance spectral data of the system under study and fluorescence observation filed (FOF) parameters of the instrument. D_abs is proportional to the observed fluorescence intensity of the system. Excitation of an optically dense sample at D_abs maximum leads to maximum fluorescence intensity obtainable for that system even in the presence of considerable inner filter effect (IFE). The earlier studies were applicable to SFS at right-angle fluorescence measurement geometry and for samples showing only primary IFE. In the present study, the work has been expanded to general fluorescence measurements, even when both primary and secondary IFE is present and for different fluorescence measurement geometries (right-angle, 45° and front-face). The procedure for obtaining D_abs is simple and can be applied to other analyte-specific/unconventional fluorescence measurement strategies reported in the literature.