胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

Preparation and evaluation of pepsin affinity organic polymer capillary monolithic column

以热引发原位聚合方法制备了聚(甲基丙烯酸缩水甘油酯(glycidyl methacrylate,GMA)-乙二醇二甲基丙烯酸酯(ethyleneglycol dimethacrylate,EDMA))毛细管整体柱,对整体柱的性能进行了表征。结果表明,柱内部结构均匀、渗透性好;整体柱能够实现苯等中性小分子化合物的分离,具有反相色谱特征,重现性和稳定性良好。利用整体柱环氧基团的活性,采用间接法,以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究,并对缓冲液pH值和运行电压等分离条件进行了考察。结果表明,亲和整体柱对4种碱性手性药物(奈福泮、氨氯地平、西酞普兰、扑尔敏)有拆分效果,奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。

The protein modified monolithic column in affinity capillary electrochromatography （CEC）has attracted considerable attention over the past decades because of its great enanti-oseparation ability. A porous polymethacrylate ester-based capillary monolithic column poly （glycidyl methacrylate-co-ethyleneglycol dimethacrylate ）（ poly（ GMA-co-EDMA ）） was pre-pared by in situ co-polymerization. The process was initiated thermally by azobisisobutyronitrile （AIBN）. The polymerization mixture contained GMA as the function monomer and EDMA as the crosslinking agent with 1,4-butanediol and 1-propanol as the binary porogen solvent. Under the optimized reaction conditions,including the proportion of monomer and porogens,reaction temperature etc,the column exhibited a uniform structure,sufficient permeability and excel-lent pressure resistance. The separation of alkyl benzenes on the column was mainly based on typical reversed-phase chromatographic retention mechanism. The reproducibility and stability were good with RSDs less than 9. 0% . A pepsin functionalized organic polymer monolith was prepared by covalently bonded pepsin to poly（GMA-co-EDMA）monolith with glutaraldehyde as a spacer based on the activity of epoxide group. The enantioseparation performance of the pep-sin affinity monolith for basic enantiomers has been investigated by CEC. Nefopam,amlodip-ine,citalopram and chlorpheniramine were resolved,and baseline separations of nefopam, amlodipine,citalopram were achieved. The influences of pH,operating voltage,temperature and sample quantity used on the chiral separation were studied. The chiral recognition mecha-nism of enantiomers on the monolithic column in CEC is discussed. This work developed a new method for the preparation and application of protein affinity monolith in CEC.