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.