Serial coupling of reversed-phase and hydrophilic interaction liquid chromatography to broaden the elution window for the analysis of pharmaceutical compounds

It is presently a common practice in drug discovery to analyse samples by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). To increase throughput, HILIC was connected in series to RPLC by means of a T-piece with make-up flow. The first column is a 2 mm I.D. column having an optimal flow between 0.1 and 0.2 mL/min. Via the T-piece, the flow for the second dimension column with an I.D. of 4.6 mm is adjusted to 1.5-2.0 mL/min with a high acetonitrile content (i.e. ≥80%) mobile phase. Therefore, even in gradient RPLC analysis starting with a mobile phase with high water content, the HILIC column is always operated at high acetonitrile concentration which is required to obtain retention on the HILIC column. The performance of the hyphenated RPLC/HILIC set-up is illustrated with the analysis of two model samples of pharmaceutical interest. Optimization of the conditions in the HILIC dimension is discussed.