Expanding the potential of chiral chromatography for high-throughput screening of large compound libraries by means of sub-2µm Whelk-O 1 stationary phase in supercritical fluid conditions.

With the aim of exploring the potential of ultra-fast chiral chromatography for high-throughput analysis, the new sub-2 micron Whelk-O 1 chiral stationary phase (CSP) has been employed in supercritical fluid conditions to screen 129 racemates, mainly of pharmaceutical interest. By using a 5-cm long column (0.46cm internal diameter), a single co-solvent (MeOH) and a 7-min gradient elution, 85% of acidic and neutral analytes considered in this work have been successfully resolved, with resolution (Rs) larger than 2 in more than 65% of cases. Moreover, almost a half of basic samples that, for their own characteristics, are known to be difficult to separate on Whelk-O 1 CSP, have shown Rs greater than 0.3. The screening of the entire library could be accomplished in less than 24h (single run) with 63% of positive score. For well-resolved enantiomers (Rs roughly included between 1 and 3), we show that method transfer from gradient to isocratic conditions is straightforward. In many cases, isocratic ultra-fast separations (with analysis time smaller than 60s) have been achieved by simply employing, as isocratic mobile phase, the eluent composition at which the second enantiomer was eluted in gradient mode. By considering the extension and variety of the library in terms of chemico-physical and structural properties of compounds and numerousness, we believe that this work demonstrates the real potential of the technique for high-throughput enantioselective screening.

Screening of Pirkle-type chiral stationary phases for HPLC enantioseparations.

The majority of enantiomeric separations for purity analysis and quality control continue to be performed by normal-phase liquid chromatography and supercritical fluid chromatography. In this chapter, representative chromatographic screening procedures for the enantioseparations using Pirkle-type stationary phases are presented. As Pirkle-type phases are commonly applied to the preparative chromatographic isolation of enantiomers, volatile modifiers are used in this screen in order to be subsequently compatible with the techniques used to recover analytes from preparative scale isolations. The Stage 1 screen presented here is used initially for screening chiral entities. The gradients use cyclohexane and ethanol both with and without chromatographic modifiers. The Stage 2 screen is used for more challenging to resolve compounds that do not exhibit resolution using the Stage 1 screening procedure.

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, 100mm, 75 mm and 50mm columns, either 4.6 or 3.0mm 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×4.6mm ID, based on 5-µm porous silica particles. On the eUHPLC column, van Deemter plots generated H(min) values of 3.53 µm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (µ(inter)) of 5.07 mm/s, and H(min) of 4.26 and 4.17 µm for the two enantiomers of acenaphthenol, at µ(inter) 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, 100mm, 75 mm and 50mm, either 4.6 or 3.0mm 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 10s, 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.

Introducing enantioselective ultrahigh-pressure liquid chromatography (eUHPLC): theoretical inspections and ultrafast separations on a new sub-2-µm Whelk-O1 stationary phase.

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-µm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-µm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-µm particles, showed H(min) (µm) and µ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-µm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-µm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-µm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-µm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.