Re-equilibration in HILIC Chromatography

Introduction

Interest in chromatography using hydrophilic interaction liquid chromatography (HILIC) has continued to build in recent years. Full adoption of the technique, however, has been slowed by experiences of poor reproducibility. Reproducibility issues generally stem from a lack of understanding of the controls one sets up in a chromatographic system. Re-equilibration times in HILIC, for example, have been reported as being exceptionally long as compared to reversed-phase chromatography. Is it possible, then, that some reproducibility issues are a result of improper re-equilibration settings?

In this study, re-equilibration times in HILIC, for both aqueous:organic gradients and buffer gradients are systematically explored. Repeatability of retention and selectivity was studied as a function of equilibration times following aqueous:organic gradients.

The impact of the use of buffer gradients and subsequent equilibration procedures on retention times were also investigated using several HILIC stationary phases and probes. The results not only promise to improve method development practices, but also provide valuable insight into HILIC retention mechanisms across a diverse set of polar stationary phases.