Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Feb 1;878(3-4):403-8. doi: 10.1016/j.jchromb.2009.12.013. Epub 2009 Dec 16.
Separation by hydrophilic interaction chromatography (HILIC) with fluorescence detection utilizing a sub-2 microm glycan column for the separation of 2-aminobenzamide (2-AB) labeled N-linked glycans is described. The HILIC column packed with a 1.7 microm amide sorbent improves the peak capacity compared to a 3.0 microm HILIC column by a similar degree as observed in reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results indicated that the optimal peak capacity was achieved at flow rate 0.2-0.5 mL/min. HILIC method transfer guidelines were shown to further enhance the resolution of glycans by changing initial gradient conditions, flow rate, column temperature, and different column lengths. Additionally, excellent resolution can be achieved in the separation of 2-AB labeled glycans released from fetuin, RNase B, and human IgG with a rapid analysis time.
本文介绍了利用亲水作用色谱（HILIC）分离 2-氨基苯甲酰胺（2-AB）标记的 N-连接糖肽，结合荧光检测，使用亚 2 微米聚糖柱进行分离。与 3.0 微米 HILIC 柱相比，填充有 1.7 微米酰胺固定相的 HILIC 柱通过类似的程度提高了峰容量，如在反相超高效液相色谱（RP-UPLC）中观察到的那样。结果表明，在流速为 0.2-0.5mL/min 时达到最佳峰容量。HILIC 方法转移指南通过改变初始梯度条件、流速、柱温以及不同柱长进一步提高了糖肽的分辨率。此外，还可以在快速分析时间内实现从胎球蛋白、核糖核酸酶 B 和人 IgG 释放的 2-AB 标记糖肽的出色分离。
