High pressure liquid chromatography (HPLC) packing materials have always been pursued to combine high mechanical strength with stable surface chemistry, ensuring reproducible and reliable chromatographic results even under ever-increasing operational backpressures [[1], [2], [3]]. Silica has been widely adopted as an ideal substrate material in practice, due to its remarkable mechanical and thermal stability, controllable pore structure and surface area, as well as flexible surface chemistry [[4], [5], [6]]. Conversely, silica itself and its stationary phases are prone to hydrolysis and detachment, especially in acidic or alkaline solutions [[7], [8], [9], [10]].

Various strategies have been proposed to alleviate silica hydrolysis and to shield metal sites and silanols on the surface. An endcapped silica column was prepared for use at pH 11 with organic buffers at temperatures below 40 °C [7]. Bridged ethylene hybrid silica (BEH) exhibited similar selectivity to the silica-based column, symmetrical peak shape, and significantly improved chemical stability compared to the C18 bonded silica phase [11]. Bidentate C18 coating showed significant higher stability at pH 11.5, and was used to separate tricyclic antidepressant drugs at pH 10.8. ​Silsesquioxane​s coated and C18/C8 derivatized stationary phases effectively separated pyridine and hinokitiol, and exhibited a high durability under an alkaline mobile phase (50 °C, pH 10) [12].

Alternatively, some acid- and alkali-resistant materials were also developed as HPLC packing substrates, including titanium dioxide [13], zirconium oxide [[14], [15], [16]], and polymer microspheres [17,18]. Among these, crosslinked polystyrene-divinylbenzene (PS-DVB) polymer microspheres had been widely studied due to their well developed preparation method, excellent chemical stability across the pH range of 0-14, and higher separation efficiency [19,20]. To date, porous PS-DVB HPLC microbeads have been commercialized on a large scale and are widely used worldwide for a variety of compounds, including peptides and proteins in RPLC mode, oligosaccharides in ion-exchange HPLC mode, and DNA fragments in ion-pair RPLC mode [[21], [22], [23]]. However, the application of PS-DVB microbeads is still hindered by two critical challenges: their susceptibility to swelling and shrinking during solvent exchange processes, and their insufficient mechanical strength under ultra-high pressure conditions [[24], [25], [26]].

Therefore, the development of HPLC stationary phases that combine the high mechanical strength of silica substrates and the surface stability of PS-DVB is of great significance. In 1989, styrene was polymerized and bonded to silica by using peroxide initiation and then crosslinked by gamma radiation, and was used for separation of alkylarylketones [24]. However, the PS coating amount and crosslinking ratio ​were difficult to control and the agglomeration between silica particles and the mesopore clogging within silica particles were largely inevitable. Recently, oxidized nanodiamonds hybridized PS-DVB and the corresponding surface quaternized microbeads were prepared for separation of benzene homologues and inorganic ions, respectively [25]. Generally, the mechanical strength and the organic solvent resistance of these porous PS-DVB microbeads incorporated with nanodiamonds still were limited. Hyper-crosslinked styrene heptamer stationary phase was also prepared on silica, and hydrophobic C8, ionizable –COOH and –SO3H, and or polar –OH could be further derivatized for subsequent different HPLC separations, respectively [4]. It was demonstrated that the hyper-crosslinked PS and subsequent various derivatized stationary phases could offer great acidic and thermal stability, excellent kinetic performance, and distinctive selectivity.

Atom transfer radical polymerization (ATRP), as an emerging polymerization technique, offers notable advantages, including a broad range of applicable monomers and excellent controllability [27,28]. Nowadays, ATRP had been widely applied in the preparation of stationary phases [29,30]. Among various polymerization methods, surface-initiated ATRP possesses unique advantages. The ATRP polymerization rate and the coating amount could be finely tuned via temperature, catalyst concentration, and monomer feed ratio. Moreover, the polymer stationary phases are completely grafted from and constrained on the surface, so the mesopore structure could be well preserved, and the particle agglomeration and the mesopore clogging are fully avoided. In previous HPLC stationary phases by ATRP [29,30], the bonding amount, crosslinking effect, and chemical stability had not been investigated systematically.

In this study, stable cross-linked PS-DVB stationary phases were prepared on silica with pore sizes of 100 Å and 300 Å (SiL-PSD-10, SiL-PSD-30) by using the surface-initiated ATRP method. The PS-DVB coating and the microscopic morphology of the microbeads were characterized by Fourier transform infrared (FT-IR), scanning electron microscope (SEM), thermogravimetric analyses (TGA), and nitrogen adsorption–desorption isotherms, and the crosslinking ratio was monitored by RPLC. With alkylbenzenes, fullerenes, and proteins as probes, the chromatographic performance of SiL-PSD was evaluated, respectively. Additionally, the chemical resistance to 0.1 M NaOH, column bleeding at pH 3 and pH 11, and the reproducibility of SiL-PSD were systematically assessed, and compared with bare silica and commercial C18 silica columns.