Preparation of a novel C18-sulfobetaine stationary phase based on silica monolith particles for multimodal high performance liquid chromatography

Mixed-mode stationary phases enhance separation flexibility for complex samples by integrating multiple retention mechanisms in high performance liquid chromatography (HPLC). In this study, a novel mixed-mode stationary phase was prepared by functionalizing sub-1 µm porous silica monolith particles (pore size ∼ 35 nm) with 4-vinyl-1,3-sulfopropyl pyridinium betaine (VSPB) and octadecyl silane (C18). The stationary phase (SMP-C18-SB) was characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy, transmission electron microscopy, solid-state 13C NMR spectroscopy, Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) analysis. The resultant stationary phase was packed into stainless-steel column (150×4.6 mm) by slurry packing method with sequential pressure of 80-100 MPA. The SMP-C18-SB column demonstrated excellent separation performance for alkylbenzenes, nucleosides, zwitterionic compounds, peptides, and proteins, facilitated by hydrophobic, hydrogen bonding, and hydrophilic interactions from C18 and SB groups. SMP-C18-SB column exhibited superior separation performance in both reversed-phase and hydrophilic interaction modes. Moreover, the column back pressure was quite low owing to the irregular shape of SMPs. The SMP-C18-SB column also exhibited excellent repeatability, with intra-day relative standard deviations (RSDs) of <0.11% for retention time and <1.02% for peak area (n = 10 each) after nearly 1000 consecutive injection. Owing to its versatile mixed-mode capability, the SMP-C18-SB column could be used for the separation of complex mixtures containing polar, non-polar and zwitteionic compounds.