Modulator Sorption in Gradient Elution Chromatography

Abstract

Gradient elution, invented by Tiselius and his co-workers [1], employs a mobile phase whose composition at the column inlet varies with time. This is in contrast to isocratic elution, where the mobile phase composition remains constant at every point in the column throughout the separation. In fact, one of the reasons gradient elution was introduced was to hasten isocratic separations, by reducing tailing and by moving widely separated compounds closer together. It is also well-suited to the separation of macromolecules, whose retention tends to vary strongly with mobile phase composition [2], and is consequently a widely used technique in biotechnology.

Gradient elution chromatography is widely used to separate both small molecules and macromole- cules. The mobile phase additive (modulator) used to modify adsorbate retention is usually considered to be either unretained or linearly retained. In both cases, the shape of the gradient does not change as it moves down the column. However, the high mobile phase concentrations at which such a modulator is commonly used makes it likely to adsorb according to its nonlinear sorption isotherm. Here the quantitative consequences of such nonlinear modulator sorption are reviewed. Nonlinear sorption deforms the shape of the gradient during its passage through the column; ultimately a shock (or shock layer) could be formed. The condition for shock formation is discussed, and numerical simulations using representative parameters illustrate the magnitude of gradient deformation and the consequences for separation.