Acidification‐driven molecular fractionation of dissolved organic matter during direct analysis of freshwater using <scp>UPLC‐Orbitrap MS</scp>

Abstract While acidification is widely recognized as a critical step influencing the molecular formula distribution of dissolved organic matter (DOM), pH‐dependent molecular fractionation remains largely unexplored. Here, we conducted a comprehensive molecular characterization of DOM by direct analysis of original water samples across a pH gradient (non‐acidified, 5, 3, 2, and 1) using online ultrahigh‐performance liquid chromatography coupled with Orbitrap mass spectrometry. The results demonstrate that acidification‐induced molecules experience a compositional shift from saturated compounds to more oxidized polyphenolic and condensed aromatic species as pH decreases from 5 to 2. However, molecules with higher double‐bond equivalents and nitrogen‐containing compounds are largely detected when the pH is lowered to 1. This is primarily attributed to the enhanced protonation of DOM molecules under strongly acidic conditions, which reduce their ionization, further improves their retention in reversed‐phase systems and facilitates their effective separation from inorganic salts, indicating pH‐dependent fractionation of ionizable species. Compared with direct analysis of original water under optimized acidification (pH 1), terrestrially derived and microbial‐altered components are largely underestimated in DOM from solid‐phase extraction. Given that DOM sources and secondary alterations are key processes governing the fate of DOM, the method developed in this study provides new insights into the composition and transformation of DOM in the original water and offers a novel perspective for exploring its biogeochemical implications in aquatic systems. Future studies should systematically analyze samples from diverse environments to constrain the driving factors of DOM compositional heterogeneity and its environmental implications.