Neutral diol methacrylate-based monoliths were developed for normal phase chromatography (NPC) and NP-CEC of polar compounds including N-glycans. Four different diol methacrylate-based monoliths were synthesized via the copolymerization of a functional monomer using either glyceryl monomethacrylate (GMM) or glycidyl methacrylate (GMA) and a crosslinker either ethylene dimethacrylate (EDMA) or trimethylolpropane trimethacrylate (TRIM). While the GMM-based monoliths yield in one reaction step polar diol methacrylate monoliths that are ready for use in NPC or NP-CEC, the GMA-based monoliths required a postmodification with hot sulfuric acid to convert the epoxy functions into diols before use in NPC or NP-CEC. All the four monoliths are neutral and void of fixed charges on their surfaces but yet exhibited relatively strong EOF in NP-CEC. The EOF is attributed to the adsorption of ions from the mobile phase thus forming the electric double layer necessary for producing a bulk mobile phase flow. Under the same in situ copolymerization conditions of GMM or GMA with either EDMA or TRIM, the GMM-EDMA monolith was the best choice in terms of retention, separation efficiency, EOF velocity in CEC and linear flow velocity in Nano-LC.
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.
Two polar ligands, namely 3-hydroxypropionitrile and 1H-imidazole-4,5-dicarbonitrile (IDCN) were covalently attached to epoxy-activated silica-based monolithic capillary columns via an epoxide ring-opening reaction to yield CN-OH-Monolith and 2CN-OH-Monolith, respectively. The silica monolith was prepared by a sol-gel process, and the resulting "rod-like" stationary phase was subjected to pore tailoring with an alkaline solution to convert small pore domains to mesopore domains, thus yielding a monolith with bimodal pore structure consisting of flow through pores (i.e., flow channels for mobile-phase flow) and mesopores that provide most of the adsorption capacity of the monolith toward the separated solutes. The two polar monoliths, CN-OH-Monolith and 2CN-OH-Monolith, were evaluated in normal-phase CEC with organic-rich mobile phases less polar than the stationary phase. The 2CN-OH-Monolith bearing more polar functions than the CN-OH-Monolith exhibited more retention and improved selectivity toward model polar solutes.
In this report, a novel polar monolithic capillary column is described for normal phase CEC (NP-CEC) of representative polar compounds including mono- and oligosaccharides, peptides, and basic drugs. The polar monolithic column, which was described in detail in the preceding paper, consisted of silica-based monolith bonded with 1H-imidazole-4,5-dicarbonitrile (IDCN) and is denoted as 2CN-OH-Monolith. Various retention parameters for neutral polar solutes (e.g., mono- and oligosaccharides) and charged polar solutes (e.g., peptides and basic drugs) were evaluated over a wide-range of elution conditions. These retention parameters yielded quantitative assessment for the polar interactions between the model solutes and the stationary phase under investigation as well as the effect of electromigration of charged solutes on their overall migration in NP-CEC. Furthermore, this investigation demonstrated that despite the possibility of achieving isocratic separation in NP-CEC for widely differing polar species, multistep-gradient elution in NP-CEC is preferred to bring about the rapid separation of a large number of polar species in a single run.
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