Comparative analysis of fluorescent nanoparticles in beers using size-exclusion HPLC and Brix-normalized fluorescence...

Carbon nanodots (CNDs) formed during brewing may contribute to the fluorescence properties of fermented beverages, yet their presence and behavior in commercial beers remain poorly characterized. This study investigated CNDs-related fluorescence in commercial beers using steady-state fluorescence spectroscopy and size-exclusion HPLC with fluorescence detection (HPLC-SEC-FLD).

Five commercially produced beers - representing dark stouts, abbey ales, dark lagers, and pale lagers - were analyzed for CNDs-related fluorescence. Glycine-derived CNDs were used as a calibration reference to obtain CNDs-equivalent concentrations. Method performance was evaluated for linearity, limit of detection (LOD), and limit of quantification (LOQ). ATR-FTIR spectroscopy was additionally used to characterize the chemical structure of the fluorescent species.

The SEC-FLD method showed good linearity over the range of 6.25-100 mg kg^-1 Gly-CND equivalents (R2 > 0.99), with an LOD of 0.05 mg kg^-1 and an LOQ of 0.15 mg kg^-1. Dominant fluorescent fractions eluted at 6.57-6.90 min, corresponding to apparent molar masses of 260,000-330,000 Da, consistent with high-molecular-weight nanocarbon-like assemblies. Dark and semi-dark beers exhibited higher CNDs-related fluorescence than pale lager, while Brix-normalized values revealed that brewing technology and malt roasting intensity, rather than total soluble solids content, are the primary determinants of CNDs-related fluorescence. ATR-FTIR analysis further confirmed the presence of thermally derived, highly oxygenated organic structures consistent with Maillard-derived carbonaceous assemblies.

These findings indicate that CNDs-related fluorescent species in beer originate primarily from Maillard-driven thermal processing during brewing rather than from raw material solids content. The combined fluorometric and chromatographic framework developed here provides a reproducible approach for assessing CNDs in fermented beverages, with potential applications in quality control and process monitoring across beverage types.