Ultra-high-pressure liquid chromatography (UHPLC) in method development

In recent years, ultra-high-pressure liquid chromatography (UHPLC) became the modern standard HPLC platform. UHPLC, with its shorter analysis time and quicker column equilibration, is ideally suited to rapid method development. This article provides a critical review of the current status, the benefits and the limitations of UHPLC in method development. We use case studies to describe best practices and recent advances. Examples include conversion of existing HPLC methods to faster analysis, rapid column/mobile-phase screening, and automated method optimization. While we focus on the development of reversed-phase methods for assay and impurity analysis of small-molecule pharmaceuticals, our insights and conclusions can be extended to other applications and sample types. Besides generating faster analysis (when used with short, small-particle columns), the higher pressure limits of UHPLC also allow the effective use of longer columns for superior routine analysis of complex samples.