The influence of grafted polymer architecture and fluid hydrodynamics on protein separation by entropic interaction chromatography

Abstract

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 +/- 0.005 chains/nm(2)) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities.

A New Stationary Phase Prepared from Ground Silica Monolith Particles by Reversible Addition-Fragmentation Chain Transfer Polymerization

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Various silica materials including porous silica particles, 1,4,[6][7][11][12][13][14][15][16] silica beads, 2,5,10 silica monolith, 9 and silica capillary 3,8 have been used for such purposes. Atom transfer radical polymerization (ATRP) was most frequently used in such studies [1][2][3][4][5][6][7][8][9][10]16 while reversible addition-fragmentation chain transfer (RAFT) polymerization was also sometimes utilized. [11][12][14][15] It is interesting to note that an initiator was attached to organic monolith particles instead of silica, and a polymer layer with ion exchange capability was added by ATRP in a specific study.…”

Use of chain transfer agent attached to silica particles in preparation of polystyrene‐based stationary phases

“…The concept of surface‐attached initiator has been extensively adopted in controlled polymerization of organic monomers on porous or nonporous inorganic surface, and the resultant organic‐inorganic combined phases have been used as stationary phases in chromatography. Porous silica particles, silica beads, silica monolith, and silica capillary have been employed to prepare such phases 1–17. Atom transfer radical polymerization (ATRP) 1–10, 16, reversible addition‐fragmentation chain transfer (RAFT) polymerization 11, 12, 14, 15, and conventional polymerization with a surface‐attached diazo type initiator 13, 14, 17 were used for such studies.…”

Synthesis, characterization and cleavage of linear polymers attached to silica nanoparticles formed using thiol‐acrylate conjugate addition reactions

“…7,10-13 Another popular technique is the incorporation of a chemically cleavable group, such as an ester group, in the anchoring molecule or the initiation of polymer chains from the surface, followed by its cleavage using acid or base hydrolysis. [14][15][16] In this work, polymer films grafted using thiol-acrylate conjugate addition reactions were investigated. Traditionally, thiol-ene step growth polymerizations are conducted using a radical-mediated, photoinitiated mechanism.…”