Imidazole-octyl mixed-mode stationary phase based on macroporous silica for the purification of ovomucoid and ovotransferrin.

Abstract

A process-simplified hard template approach was established to synthesize the monodisperse macroporous silica microspheres with homogeneous structures by twice alkali-thermal treatment and calcination routes. Porous vinyl-functionalized polysesquioxane microspheres (V-PMSQ) were synthesized through a hydrolyzation-polycondensation method and used as templates. The template particles with large aperture and high pore volume were obtained by adjusting the pH value and reaction time of the twice alkali-thermal reaction. After calcination, monodisperse silica microspheres with an average pore size of 30nm, homogeneous pore structures, and narrow particle size distribution were fabricated, which can be directly used as chromatographic matrices without classification. After that, a new reversed-phase/strong anion-exchange (RP/SAX) mixed-mode stationary phase Sil-S-VOIM was prepared by bonding the 1-vinyl-3-octyl-imidazole ligands to the above silica microspheres through a "thiol-ene" click reaction. The performance of the Sil-S-VOIM column was evaluated by one acidic protein (transferrin) and two basic proteins (lysozyme, α-chymotrypsin) and compared to a single imidazole-modified Sil-S-VIM column and an octyl-modified Sil-C8 column, respectively. Due to the synergistic effect of electrostatic repulsion and hydrophobic interactions, baseline separations of the above proteins were observed only on the Sil-S-VOIM column, with resolutions of 2.55 and 2.01 between lysozyme and transferrin, and between transferrin and α-chymotrypsin, respectively, indicating good selectivity and separation ability compared with single-mode stationary phases. It was applied to the isolation of egg white samples with peaks identified by SDS-PAGE and MALDI-TOF-MS. The results showed that the selective retention and isolation of ovomucoid and ovotransferrin were successfully achieved, with yields of 78.8% and 67.2%, respectively. The protocol described in this work is simpler, faster, and has higher protein recovery. Overall, this new mixed-mode stationary phase provided a promising potential for the separation and determination of intact proteins.