Understanding and managing peak shape for basic solutes in reversed-phase high performance liquid chromatography

Abstract

While other separation mechanisms can challenge the dominance of reversed phase (for example in the separation of native proteins), reversed phase liquid chromatography is the method of choice for the analysis of a wide variety of samples. However, basic solutes (including small molecules, peptides and proteins) can give broad peaks, ofteh with severe peak tailing, which negatively affects peak identification and quantitation. In this feature article, the causes of low efficiency and peak asymmetry are discussed, including the choices of stationary and mobile phases that can minimise these detrimental effects. The contributory effects of column overloading to peak asymmetry are also considered although the exact causes of these effects remain of considerable debate.