The Evolution of Chiral Stationary Phases from HPLC to UHPLC

There is a need for chiral stationary phases (CSPs) designed for high performance liquid chromatography (HPLC) to switch to enantioselective applications using ultrahigh-performance liquid chromatography (UHPLC). Although important goals have been achieved to rapidly separate achiral compounds, enantioselective LC remains solidly attached to 3-μm and 5-μm totally porous particles and pressure values in the HPLC domain. This article describes strategies aimed at immobilizing or coating well-established chiral selectors onto sub-2-μm silica particles, and aims to illustrate the potential of enantioselective UHPLC (eUHPLC) in terms of high speed, throughput and resolution.

Since the first commercially available ultrahigh-performance liquid chromatography (UHPLC) system was introduced in 2004, there has been a constant evolution of LC both in terms of instrumentation (ability to sustain high pressures and yield lower volume dispersion) and in terms of particle design, leading to a more universal use of UHPLC in routine and research applications. UHPLC offers new possibilities to the analytical chemist who can now choose between either ultrafast separations without (or with acceptable) efficiency loss or higher performance and resolution without speed gain in analysis time (1–3). Given the most recent advances in the field, it is now possible to reach efficiency values up to 300,000 N/m using columns packed with sub-2-μm particles, either totally porous or with a solid nucleus (4–5). The most commonly used reversed-phase and hydrophilic interaction chromatography (HILIC) chemistries are now available in the sub-2-μm format, allowing a scale-down of existing methods.

However, enantioselective LC is still performed on 3- and 5-μm totally porous particles, yielding pressure values in the high performance liquid chromatography (HPLC) domain. In fact, although HPLC and UHPLC C18 columns for reversed-phase separations have been extensively discussed and evaluated in literature in terms of kinetic and thermodynamic phenomena, little is known on the efficiency of enantioselective columns. Up to now, the main concern of both users and manufacturers has been a qualitative one: Analyses on a 45-min timescale or longer are considered typical in chiral separations. Is it really not possible to overcome this limitation?