A highly stable C18-modified polystyrene-divinylbenzene stationary phase on porous silica for HPLC

In order to further increase the chemical stability of polystyrene-divinylbenzene (PS/DVB) stationary phase on porous silica and to mitigate its irreversible π-π interaction with aromatic compounds, the cross-linked PS/DVB on porous silica (SiL-PSD-50%) was prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) and derivatized by C4, C8, and C18 (SiL-PSD-C4, SiL-PSD-C8, SiL-PSD-C18) by Friedel-Crafts alkylation, and then used for the separation of aromatic compounds and proteins by high-performance liquid chromatography (HPLC). The successful grafting of PS/DVB and alkyl chains was first confirmed by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and nitrogen adsorption-desorption. The cross-linking degree of PS/DVB was also determined as 52.8%, which was consistent with the initial feed ratio of 50%. It was found that SiL-PSD-C18 could resist corrosion in 0.1 M NaOH for at least 48 h and maintain its initial morphology, whereas bare silica, commercial C18, and SiL-PSD were corroded to some extent. For the separation of small aromatic compounds except alkylbenzenes, SiL-PSD-C18 significantly mitigated peak tailing and improved peak symmetry. During protein separation, SiL-PSD-C18 sharply decreased the mobile phase equilibrium time during gradient elution cycle. The relative standard deviation (RSD) of the retention times of six proteins on SiL-PSD-C18 was only 0.14%-0.34% within 90 h. Under accelerated aging conditions, SiL-PSD-C18 retained >98% of its initial column efficiency after being flushed with 16,000 column volumes of mobile phase. Among all tested columns, SiL-PSD-C18 exhibited the lowest column bleeding, which was about one-tenth of commercial Kromasil C18 column and one-third of SiL-PSD-50% at pH 11.