School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
Mikrochim Acta. 2023 Sep 20;190(10):404. doi: 10.1007/s00604-023-05986-7.
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 30 nm, 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-C 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.
建立了一种简化过程的硬模板法，通过两次碱热处理和煅烧路线合成具有均匀结构的单分散大孔硅胶微球。通过水解缩聚法合成了多孔乙烯基功能化聚倍半硅氧烷微球（V-PMSQ），并用作模板。通过调节两次碱热反应的 pH 值和反应时间，获得了具有大孔径和高孔体积的模板颗粒。煅烧后，制备出具有平均孔径为 30nm、均匀孔结构和窄粒径分布的单分散二氧化硅微球，无需分类即可直接用作色谱基质。之后，通过“硫醇-烯”点击反应将 1-乙烯基-3-辛基-咪唑配体键合到上述硅胶微球上，制备了一种新的反相/强阴离子交换（RP/SAX）混合模式固定相 Sil-S-VOIM。通过比较单一咪唑修饰的 Sil-S-VIM 柱和辛基修饰的 Sil-C 柱，评估了 Sil-S-VOIM 柱的性能。选用了一种酸性蛋白（转铁蛋白）和两种碱性蛋白（溶菌酶、α-糜蛋白酶）进行评估。由于静电排斥和疏水相互作用的协同效应，仅在 Sil-S-VOIM 柱上观察到上述蛋白质的基线分离，溶菌酶和转铁蛋白之间以及转铁蛋白和α-糜蛋白酶之间的分辨率分别为 2.55 和 2.01，表明与单模式固定相相比具有良好的选择性和分离能力。该方法成功应用于蛋清样品的分离，通过 SDS-PAGE 和 MALDI-TOF-MS 鉴定了峰。结果表明，成功实现了卵白蛋白和卵转铁蛋白的选择性保留和分离，产率分别为 78.8%和 67.2%。本工作中描述的方案更简单、更快，且具有更高的蛋白质回收率。总体而言，这种新的混合模式固定相为完整蛋白质的分离和测定提供了有前景的潜力。
