The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.
本发明涉及聚酰胺键合亲
水相互作用色谱(HILIC)固定相和新型 HILIC 方法,用于表征用极性基团修饰的大
生物分子,本领域技术人员已知这些极性基团为聚糖。本发明特别提供了设计用于高效分离大型
生物分子的新型聚酰胺键合材料,例如具有较大孔隙比例的材料(即宽孔)。此外,本发明还有利地提供了新型 HILIC 方法,该方法可与本文所述的固定相材料结合使用,通过消除以前未解决的问题(如蛋白质样品的柱上聚集、糖分子的色谱检测灵敏度低以及由于孔扩散受限和颗粒内/颗粒间扩散距离长而导致的峰分辨率低)来有效分离蛋白质和肽的糖型。