Optimizing selectivity during reversed-phase high performance liquid chromatography method development: Prioritizing experimental conditions

•The influence of changing %B (or tG), °C, and solvent type on selectivity is reexamined.•These changes can be as effective as changing pH or column type when adjusting selectivity.•These changes can mimic changes in pH, reducing or eliminating the need to optimize pH.•Such continuous changes can be more effective than a step-wise change in column type

Several different conditions can be varied to improve selectivity for reversed-phase chromatography (RPC). A reexamination of literature data suggests that changes in selectivity due to a change of column or mobile phase pH are largely replicated by changes in temperature or mobile phase composition (concentrations of acetonitrile and/or methanol). This suggests a reconsideration of the role of mobile phase pH and the column during method development.