The distortion of gradient profiles in reversed-phase liquid chromatography

Severe distortions of the axial concentration profiles of modifiers in steep RPLC gradients were recently observed. These distortions are directly explained by the results of measurements of the excess adsorption isotherms of the strongest mobile phase component, the concentration of which is made to increase linearly with time at the column inlet. A front shock or a discontinuity of the organic modifier concentration may arise and grow along the column. The position where it forms is determined by the reciprocal of the second derivative of the excess adsorption isotherm with respect to the concentration of the strongest mobile phase component. It forms when two characteristic lines intersect for the first time. Gradient profiles are continuous and diffuse as long as characteristic lines do not intersect but diverge from each other. However, acetonitrile-water gradients are systematically distorted and deviate significantly from assumed ideal, linear, non-retained gradients. This challenges the validity of classical theories of gradient chromatography regarding the prediction of retention times, peak widths, and band compression factors when steep gradients are applied.