Protocol to compare column performance applied to hydrophilic interaction liquid chromatography

Abstract

The lack of retention of highly polar solutes in liquid chromatography with reversed phase columns (RPLC) can be solved through the use of the hydrophilic interaction liquid chromatographic (HILIC) mode. Due to the complexity of the separation mechanisms in HILIC and the different factors that may have significant influence, the selection of the appropriate stationary phase plays a fundamental role in the development of analytical procedures to obtain good performance. In this work, the chromatographic performance of bare silica and six polar stationary phases with different functionalized groups (with neutral, cationic, anionic and zwitterionic character) are investigated. The behaviour is compared in terms of retention, selectivity, peak shape, and resolution, using acetonitrile-water mobile phases buffered at pH 3 and some correlation tools previously developed for RPLC. Six nucleosides and uracil were used as probe compounds. The results revealed significant differences among the columns regarding all inspected parameters. Some insight on the interactions established between solutes, and both stationary and mobile phases, is also given.