N-acetylcysteine-functionalised a multimodal HPLC stationary phase for broad-range separations

In this study, a new multimodal HPLC stationary phase derived from N-acetyl-l-cysteine was synthesized and comprehensively evaluated across a broad polarity spectrum. In chromatographic stationary phase design, two fundamental criteria are particularly important: a facile, low-cost synthesis and broad applicability across diverse analyte classes. The present stationary phase fulfils both requirements, as it is prepared from a commercially available starting material via a simple single-step immobilisation onto silica, providing a practical and economical synthetic route alongside wide analytical utility. The molecular architecture of the ligand, incorporating a pH-responsive carboxyl group, a hydrogen-bond-donating and -accepting amide moiety, a polarisation-sensitive sulfur atom, and hydrophobic methyl and methylene units, creates a versatile interaction environment capable of mediating hydrophobic, polar, dispersive, and weak cation-exchange processes. This multifunctional design enables the stationary phase to adapt its selectivity according to analyte structure and mobile-phase conditions, supporting both reversed-phase-like and HILIC-like retention behaviour within a single chromatographic platform. The chromatographic performance of the NAC-derived stationary phase was systematically evaluated using a wide range of chemically and biologically relevant analytes. Under HILIC conditions, the column successfully resolved 7 of the 9 highly polar nucleobases and nucleosides. In reversed-phase mode, compounds with high hydrophobicity-including 6 alkyl benzenes, 4 polycyclic aromatic hydrocarbons, 6 Sudan dyes, 8 anilines, and 7 benzoic acid derivatives-were efficiently separated. Under the same reversed-phase conditions, the stationary phase successfully resolved 10 analytes from a twelve-analyte plant growth regulator mixture and 9 sulphonamides. In addition, a 6 herbicide was also effectively separated, highlighting the multimodal selectivity of the stationary phase. Retention trends were interpreted using analyte logD and pKa values, clarifying the combined contributions of hydrophobic, polar, and weak ion-exchange interactions to overall selectivity. The N-acetyl-l-cysteine-based stationary phase provides a finely balanced interaction profile, enabling high separation performance for analytes spanning from extreme polarity to pronounced hydrophobicity and demonstrating broad analytical utility.