Coupling Isotachophoresis with Affinity Chromatographyfor Rapid and Selective Purification with High Column UtilizationPart 1: Theory

摘要

We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensionalparameters. The analysis yields simple analytical relations for capturelength and capture time in relevant ITP-AC regimes, and it demonstrateshow ITP greatly reduces assay time and improves column utilization.In the second part of this two-part series, we will present experimentalvalidation of our model and demonstrate ITP-AC separation of the targetfrom 10,000-fold more-abundant contaminants.