Gradient Scouting in Reversed-Phase HPLC Revisited

Gradient scouting is the best way to decide the most suitable elution mode in reversed-phase high-performance liquid chromatography (RP-HPLC). A simple rule for this decision involves the evaluation of the ratio δt/t G (where δt is the difference in the retention time between the last and the first peak and tG is the time of the gradient), which is equated to a "magic" reference number. In this article, we demonstrate that the reference number depends on the cosolvent selected. This is illustrated with an example, using methanol as cosolvent. For methanol/water mobile phases, when δt/tG < 0.15, an isocratic elution can be used, otherwise, gradient elution is advisable. In the example, a gradient elution was needed and a segmented gradient program was used to reduce the time of analysis. This article can serve as a reference and basis for laboratory experimentation in an upper-level analytical chemistry course. Copyright © 2010 The American Chemical Society and Division of Chemical Education, Inc.