Template-assisted fabrication of moon-shaped channels for protein breakthrough analysis

Cylindrical column with packed stationary phase is the workhorse of liquid chromatography systems. These stationary phases are commonly classified on the basis of different form factors namely, beads and monoliths for protein chromatography. Monolithic rods are one of the important geometries derived from polymers through complex polymerization schemes with additional requirements such as cross-linkers and specific reaction conditions. To address these practical difficulties and enable ease of fabrication at laboratory scale, acrylic copolymers are hypothesized to perform as a monolithic stationary phase suitable for protein chromatography. The present work proposes a rapid fabrication technique to obtain monolithic rods that could be reconditioned without any of the above additional steps. It is characterized with monolith diameter that could be controlled using acrylic copolymer concentration. Formation of the copolymeric stationary phase inside microchannel led to annular geometry and in turn, demonstrated fabrication of moon-shaped channels (MSCs) for the first time in literature. An online monitoring system facilitated tracer breakthrough analysis with MSCs to report sharp peak front and an estimate of channel void volume. Breakthrough curves with single protein validated the selection of blue dextran as tracer and indicated retention of proteins due to electrostatic interactions on the functional copolymer surface.