Advancing Integrating-Sphere-Assisted Resonance Synchronous Spectroscopy for Quantitative Absorbance Analysis in...

Integrating-Sphere-Assisted Resonance Synchronous (ISARS) spectroscopy is a recently developed spectrofluorometer-based technique for quantifying ultraviolet-visible (UV-vis) absorbance in turbid solutions, where conventional UV-vis spectrophotometry fails due to scattering interference. Here, we advance ISARS spectroscopy capability by addressing three critical limitations: restricted spectral range, limited linear dynamic range (LDR), and susceptibility to sample volume variations. We (i) expanded the usable spectral range from 350-650 nm to 250-750 nm using a neutral-density-filter (NDF)-based "divide-and-conquer" strategy, (ii) extended the upper limit of the ISARS absorbance quantification via short-path cuvettes, (iii) mitigated susceptibility to sample volume variation by ensuring direct "first-strike" geometry, and (iv) increased the convenience of data analysis by developing a streamlined, segment-specific second-order polynomial models and accompanying Python GUI for rapid conversion of ISARS absorbance to conventional double-beam values. The enhanced method enabled the first reliable quantification of protein absorbance in undiluted commercial milks, where optical density exceeds 150 and protein absorbance accounts for <5% of the total UV-vis extinction at 280 nm, demonstrating ISARS's unique capability to determine absorbance in solutions with extremely high scattering activities. These advances establish ISARS as a powerful, accessible platform for quantitative absorbance analysis in optically complex media.