Separations by gradient elution: Why are steep gradient profiles distorted and what is their impact on resolution in reversed-phase liquid chromatography

• The excess isotherms of organic modifiers explain the distortion of steep gradient profiles.
• Distorted gradient profiles are expected below a critical value of the gradient time.
• The critical gradient time is inversely proportional to the applied flow rate.
• It is proportional to the initial value of the second order derivative of the excess isotherm.
• Distorted gradient profiles affect the resolution performance of steep gradients.

The formation of a concentration shock layer of either methanol or acetonitrile used as the modifier in steep RPLC aqueous/organic gradients was predictable from their excess adsorption isotherm and was directly observed from their elution profiles recorded in UV absorption. A discontinuity of the concentration profile of the organic modifier arises and grows along the column. Its effects on the peak shapes and the resolution levels achieved in steep gradient chromatography were measured in the recorded chromatograms of a sample mixture containing 14 different analytes uniformly distributed across the gradient retention window. The results showed severe peak shape deformation for some compounds and a significant degradation of the baseline resolution of weakly (when using acetonitrile) and moderately (when using methanol) retained analytes. Solutions to limit this loss of resolution may lie in changing the initial gradient conditions or in adjusting the curvature of the gradient profile at the column inlet.