Capillary electrochromatography with monolithic stationary phases. 4. Preparation of neutral stearyl - acrylate monoliths and their evaluation in capillary electrochromatography of neutral and charged small species as well as peptides and proteins

Abstract

A neutral, nonpolar monolithic capillary column having a relatively strong electroosmotic flow (EOF) yet free of electrostatic interactions with charged solutes was developed for the reversed-phase capillary electrochromatography (RP-CEC) of neutral and charged species including peptides and proteins. The neutral nonpolar monolith is based on the in situ polymerization of pentaerythritol diacrylate monostearate (PEDAS) in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. PEDAS plays the role of both the cross-linker and the ligand provider, generating a macroporous nonpolar monolith having C17 chains as the chromatographic ligands. Despite the fact that the neutral PEDAS monolith is devoid of fixed charges, the monolithic capillary columns exhibited a relatively strong EOF due to the ability of PEDAS to adsorb sufficient amounts of electrolyte ions from the mobile phase. The adsorbed ions imparted the neutral PEDAS monolith the zeta potential necessary to support the EOF required for mass transport across the monolithic column. The absence of fixed charges on the surface of the neutral PEDAS monolith and in turn the adsorption sites for electrostatic attraction of charged solutes allowed the rapid and efficient separations of proteins and peptides at pH 7.0, with an average plate number of 255,000 and 121,000 plates/m, respectively. To the best of our knowledge, this constitutes the first report on the separation of proteins at neutral pH by RP-CEC using a neutral monolithic column.

Optimization of the porous structure and polarity of polymethacrylate‐based monolithic capillary columns for the LC‐MS separation of enzymatic digests

“…Silica-based monoliths emerged a few years later and their performance was demonstrated with fast separations of small molecules [12][13][14]. Polymer-based monoliths are typically prepared in-situ from a liquid precursor [15,16], a feature that makes the monolithic stationary phases very attractive for their use in narrow bore capillaries and microfluidic devices. Both the chemistry and the porous properties of the monoliths, which have a direct effect on their chromatographic performance, are readily controlled by the composition of the polymerization mixture and conditions used for the in situ polymerization [17][18][19][20].…”

Recent developments in CE and CEC of peptides

“…A neutral, nonpolar stearyl-acrylate monolithic column providing a relatively strong EOF but being free of electrostatic interactions with charged solutes was developed for the RP-CEC of neutral and charged species including peptides and proteins [141]. Another mixed-mode n-alkyl methacrylate-based monolith has been used for separation of therapeutic peptides [142].…”

Monolithic media in microfluidic devices for proteomics

“…Separation performance for proteins was compared with C18 and C8 stationary phase columns, and better resolution was achieved on C18 stationary phase. A one-step process for the preparation of RP chromatographic media has also been used by copolymerization of hydrophobic monomers, such as butyl-, hexyl-, octyl-, lauryl-, and stearyl acrylates or methacrylates [22,23,[36][37][38][39][40][41][42][43][44].…”