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

Abstract

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.

Insights into Spectral Distortion and Nonlinearity in UV–Vis and Fluorescence Spectroscopy of Molecular Fluorophore Solutions: Effects of Cascading Optical Processes (Part IV)

“…Second, the model assumes that fluorescence involves only two cascading optical processes: photon absorption followed by fluorescence emission with no possibility of light absorption of the emitted photons. However, conventional spectrofluorometers detect only a portion of the fluorescence emission produced within the effective sampling volume defined by the pinhole effect of the instrument. ,,− Therefore, any absorption of excitation photons before they reach the effective sampling volume reduces the intensity of the excitation light within that volume, consequently decreasing the fluorescence intensity. This reduction in fluorescence caused by absorption of excitation photons is commonly referred to as the primary IFE in the literature. ,,, We refer herein to the IFE due to absorption as (aIFE) to distinguish it from the IFE caused by light scattering, as the scattering IFE behaves drastically differently than the aIFE …”