Enantioselective ultra-high and high performance liquid chromatography: A comparative study of columns based on the Whelk-O1 selector

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150 mm, 100 mm, 75 mm and 50 mm columns, either 4.6 or 3.0 mm ID. A 100 mm × 4.6 mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250 mm × 4.6 mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated Hmin values of 3.53 μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μinter) of 5.07 mm/s, and Hmin of 4.26 and 4.17 μm for the two enantiomers of acenaphthenol, at μinter of 4.85 mm/s and 4.24 mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150 mm, 100 mm, 75 mm and 50 mm, either 4.6 or 3.0 mm ID), with respect to the standard HPLC column (250 mm × 4.6 mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10 s, while a 50 mm × 3.0 mm ID column was used as a compromise between reduced mobile phase consumption (less than 1 mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600 bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods.