Optimization of the Preparation of Hydrophilic Poly(DHPMA-co-MBA) Monolithic Capillary Columns: A New Support for Affinity Chromatography

Abstract

In miniaturized affinity chromatography, the development of hydrophilic organic monoliths with reduced non-specific interactions and high-protein grafting capacity remains a hot topic. In this work, we propose the one-step synthesis of a diol organic monolith to replace the gold-standard epoxy-based organic monoliths (which require post-modification, namely hydrolysis, prior to use). The synthesis of this new monolith builds upon the use of N-N’-Methylenebis(acrylamide) (MBA), as a hydrophilic crosslinker, and 2,3-dihydroxypropyl methacrylate (DHPMA), a diol monomer that eliminates the time-consuming epoxy ring opening step and its associated side reactions. The optimization of one-step synthesis parameters led to a monolith with a satisfactory permeability ((4.8 ± 0.5) × 10−14 m2), high efficiency (117,600 plates/m at optimum flow velocity (uopt = 0.09 cm s−1)) and reduced non-specific interactions. It is exemplified by its separation ability in the HILIC mode (separation of nucleosides), and by the retention data set of 41 test solutes, which were used to evaluate the non-specific interactions. This new poly(DHPMA-co-MBA) monolith has not only hydrophilic surface properties, but also improved protein grafting capacity compared to the glycidyl-based monolith (13 ± 0.7 pmol cm−1). The potential of this monolith is illustrated in affinity chromatography, where the concanavalin ligands are ranked according to their Kd values.

Extending the Affinity Range of Weak Affinity Chromatography for the Identification of Weak Ligands Targeting Membrane Proteins

“…To reduce non-specific interaction, we envisioned the use of poly(DHPMA-co-MBA), a more hydrophilic monolith, synthesized with a diol acrylate monomer (2,3-Dihydroxypropyl methacrylate, DHMPA) and a more hydrophilic crosslinker (Methylene bis acrylamide, MBA). This hydrophilic monolith offers reduced preparation time (the DHMPA is a diol monomer that eliminates the epoxy ring opening step, and its thermal polymerization at 87 • C takes only 30 min) and increased hydrophilicity, i.e., reduced non-specific interactions due to the hydrophobic effect [18]. The immobilization of NDs was performed using the same strategy as for the poly(GMA-co-EDMA) monolith.…”

“…In the present study, we first show by a brief rationale that a higher density of grafted proteins per unit volume combined to reduce non-specific interactions is necessary to extend the affinity range to higher K d values. We then present a detailed characterization of existing affinity columns based on poly(GMA-co-EDMA) monoliths generating non-specific hydrophobic interactions in order to propose a more adapted alternative setup to achieve our goal, i.e., the use of a more hydrophilic poly(DHPMA-co-MBA) monolith previously developed for hydrophilic interaction chromatography [18] combined with a multilayer nanodisc grafting strategy.…”