Monolithic silica capillary columns having immobilized lectins and surface bound polar functionalities for lectin affinity and normal phase nano‐LC and CEC of glycoconjugates, respectively

Abstract

In this report, monolithic silica-based capillary columns were produced by the sol-gel process and subsequently silanized with gamma-glycidoxypropyltrimethoxysilane to form on the surface of the monolith a reactive gamma-glycidoxypropylsilyl sublayer to which an interactive top layer can be covalently attached. The interactive top layer consisted of either an immobilized lectin or polar cyano functions to perform lectin affinity chromatography (LAC) of glycoproteins or normal phase chromatography (NPC) of glycans, respectively. Two lectins were immobilized, namely Con A and wheat germ agglutinin (WGA) due to their utility in LAC of a wide range of glycoconjugates. On the other hand, 1H-imidazole-4,5-dicarbonitrile was covalently attached to the silica monolith to yield polar silica monolith referred to as 2CN-OH monolith. The two lectin monolithic columns were evaluated in Nano-LC with standard glycoproteins over a wide range of conditions and proved useful in capturing the glycoforms bearing the pertinent saccharidic motifs for interaction with the given lectin. The 2CN-OH monolithic columns were effective in the profiling of glycans derived from glycoproteins in both NP-CEC and NP-Nano-LC with NP-CEC offering enhanced separation when compared to NP-Nano-LC.