Interest in chromatography using hydrophilic interaction liquid chromatography (HILIC) has continued to build in recent years. In previous studies, repeatability of retention and selectivity was studied as a function of equilibration times following aqueous:organic gradients. Surprisingly, retention and selectivity were repeatable even after short equilibration times, however, continued to change as a result of extended re-equilibration. The aim of this continuing study was to further enhance our knowledge of mechanisms of retention through fundamental investigations of factors contributing to robustness and repeatability.

Although HILIC chromatography is known to provide valuable retention and selectivity of polar compounds and provide highly compatible conditions for coupling with mass spectrometry, it often is avoided due to issues surrounding robustness and repeatability. In this study, systematic investigations of possible contributions to repeatability are reported. The impact of the use of buffer gradients and subsequent equilibration procedures on retention times were investigated using several HILIC stationary phases. 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.