The influence of stationary phase on pressure-induced retention, selectivity and resolution changes in RP-LC

This paper reports the influence of a diverse range of stationary phases and differing mobile phase modifiers on pressure-induced retention changes in reversed-phase liquid chromatography (RP-LC). The practical implications of these effects in the Tanaka column characterization using ultra high-performance liquid chromatography (UHPLC) conditions, and implications for HPLC to UHPLC translations in order to increase productivity and resolution are investigated. The stationary and mobile phase combinations responded to a similar degree to elevated pressure; hence, the authors believe that reliable column characterization parameters should be obtainable when UHPLC format columns are evaluated using the Tanaka approach. Analytes exhibited differing pressure-induced retention changes even for only modest increases in pressure (i.e. ΔP(total) 85 bar as shown when one transfers from a 3- to 2-μm particle). The degree of pressure-induced retention changes correlated with the analyte's molar volume and refractivity. The hydrophobicity of the analytes, as measured by logD, only exhibited a weak correlation. Hence, translating a RP-LC methodology from large to smaller particle size material of the same type may result in an increased or decreased selectivity and hence resolution between two analytes depending on their differing response to the pressure-induced retention changes. This potentially has a major impact on LC method development/optimization strategies and LC method translations.