Methylene-tuned sulfonic acid-functionalized silica stationary phases for multi-mode chromatography

Traditional single-mode stationary phases often suffer from limited separation capability for complex samples containing both hydrophobic, hydrophilic, and ionizable components, requiring multiple column switches that reduce efficiency and increase method complexity. Mixed-mode stationary phases providing two or more retention mechanisms on a single column offer a promising solution. In this study, two novel stationary phases, phenylmethanesulfonic acid-modified silica (Sil-SMSa) and benzenesulfonic acid-modified silica (Sil-SSa), were designed and prepared via thiol-ene click chemistry, offering reversed-phase chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and ion exchange chromatography (IEC) modes, enabling effective separation of nucleobases and nucleosides, vitamins, flavonoids, sulfonamides, polycyclic aromatic hydrocarbons (PAHs), and rare earth elements (REEs). Compared with in-house C18 column and HILIC columns, both Sil-SMSa and Sil-SSa showed better selectivity, broader polarity coverage, and lower solvent consumption. The methylene spacer for Sil-SMSa improved sulfonic acid group (-SO₃^-) arrangement and hydrophilic layer order, enhancing the resolution of structurally similar compounds. In contrast, the sulfonic acid groups of Sil-SSa are directly attached to the benzene ring, the surface is more rigid and the hydrophilic layer is restricted, leading to stronger electrostatic interactions in HILIC and IEC. This comparison provides evidence for how surface structure regulates multi-mode separation performance and guides the rational design of advanced materials. This work demonstrates that subtle structural variations, such as the presence or absence of a methylene spacer, can significantly modulate separation selectivity and retention behavior, highlighting the importance of the spacer in the development of high-performance multi-mode stationary phases.