Scattering and absorption differ drastically in their inner filter effects on fluorescence, resonance synchronous,...

The sample inner filter effect (IFE) induces spectral distortion and affects the linearity between intensity and analyte concentration in fluorescence, Raman, surface enhanced Raman, and Rayleigh light scattering measurements. Existing spectrofluorometric-based measurements treat light scattering and absorption identically in their sample IFEs. Reported herein is the finding that photon scattering and absorption differ drastically in inducing the sample IFE in Stokes-shifted fluorescence (SSF) spectrum, resonance synchronous spectrum (RS2), and the polarized resonance synchronous spectrum (PRS2) measurements. Absorption with an absorption extinction as small as 0.05 imposes significant IFE on SSF, RS2, and PRS2 measurements. However, no significant IFE occurs even when the scattering extinction is as high as 0.9. For samples that both absorb and scatter light, one should decompose their UV-vis extinction spectra into absorption and scattering extinction component spectra before correcting the sample IFE. An iteration PRS2 method was introduced for the experimental decoupling of the photon absorption and scattering contribution. The methodology presented in this work can be easily implemented by researchers with access to one conventional UV-vis spectrophotometer and one spectrofluorometer equipped with a pair of excitation and detection polarizers. This work should be of broad significance in chemical research given the popularity of fluorescence spectroscopy in material characterization applications.