Text Box: THE THEORY AND PRACTICE OF UHPLC AND UHPLC-MS DAVY GUILLARME AND JEAN-LUC VEUTHEY SCHOOL OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF GENEVA, UNIVERSITY OF LAUSANNE, BOULEVARD D’YVOY 20, 1211 GENEVA 4, SWITZERLAND Introduction Reversed-phase liquid chromatography (RPLC) is nowadays one of the most widely used separation techniques. Indeed, despite a lower chromatographic efficiency in comparison with capillary GC, the interaction of the analytes with both the stationary phase and the mobile phase provides an important selectivity, and thus RPLC can be applied to solve numerous analytical problems. During the last few years, some substantial improvements, such as innovative supports and instrumentation, helped to achieve high throughput analyses and highly efficient separations (Guillarme et al., 2007b; Novakova et al., 2006). Such advances were mainly driven by the need to handle either a growing number of analyses or more complex samples. Regarding high throughput separations, there is a growing demand in numerous fields, including toxicology, doping, forensic, clinical chemistry and environmental analyses, where the delivery time response must be reduced as much as possible. The pharmaceutical field, with its need for enhanced productivity and reduced costs, is the main driving force for faster separations (Wren & Tchelitcheff, 2006). Due to the high number of analyses required for common pharmaceutical applications, such as purity assays, pharmacokinetic studies, and quality control, rapid analytical procedures (less than 5 minutes including equilibration time) are often mandatory (Al-Sayah et al., 2008). Highly efficient separations are also necessary for many applications, including genomics, proteomics and metabolomics, which all deal with very complex samples, such as biological samples, tryptic digests, or natural plant extracts (Grata et al., 2008; Petricoin et al., 2004). With such difficult samples, conventional HPLC systems present some obvious limitations, thus demanding analytical procedures to yield high resolution within an acceptable analysis time, even when a large number of compounds need to be separated.

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