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48 August / September 2016


Sub-2µm High Performance Enantiomeric Separations on Whelk-O1 chiral Stationary Phase


Omar H. Ismail1 , Gioacchino L. Losacco1, Ted Szczerba2 , Jelena Kocergin2 , Scott Anderson2


1. Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Roma, Italy 2. Regis Technologies, Inc, 8210 Austin Avenue, Morton Grove, IL 60053, USA *Corresponding author: Scott Anderson, E-mail address: sanderson@registech.com


Ultra High Performance Chromatography (UHPC) Whelk-O1 1.8 µm Chiral Stationary Phase (CSP) was evaluated from the kinetic point of view through van Deemter curves analysis on trans-stilbene oxide under Normal Phase conditions and compared to commercially available columns. The sub-2µm CSP exhibited more than 260,000 theoretical plates/m on the first eluted enantiomer at an optimal flow-rate of 1.8 mL/min (T: 35°C), obtaining efficiencies more than three times higher than the Regis HPLC- Whelk-O1 5 µm and, at the same time, maintained very high efficiencies at large linear velocities. High reproducibility in terms of thermodynamic performances (retention factor (k’) and selectivity (a)) was recorded between 1.8 µm CSP and the 3.5 and 5 µm columns. This chiral stationary phase was also employed in sub-critical fluid conditions. A van Deemter curves analysis was performed maintaining efficiencies of more than 250,000 theoretical plates per meter. This chiral selector was also used in the Ultra High Performance Super Criticalfluid Chromatography (UHPSFC) screening, in a single day, of a large library of 129 acidic, neutral and basic racemates employing a 9-min screening run using a 5 cm long column.


Introduction


Over the last ten years, technological advances have led to the development of achiral stationary phases bonded on ever smaller silica particles (sub-2µm) and instrumentation (UHPLC/UHPSFC) with a reduced extra-column volume able to reach very high pressures. These improvements allow the user to obtain high efficiencies and resolutions, and to reduce analysis time and eluent consumption. However, only in the last few years have chiral stationary phases started moving in this direction [1-2]. In enantio-chromatography, research has been focusing on the design and development of selectors with a very broad spectrum, with the aim to resolve the highest number of racemates. Since there is not yet a unique and universal Chiral Stationary Phase (CSP), many CSPs are needed [3-17]. Recently many CSPs have been covalently bonded on very small silica particles, Fully Porous Particles (FPP) and Superficially Porous Particles (SPP). The 3,5-dinitro- benzoyl derivative of 1,2- diaminocyclohexane (DACH-DNB) [18], β-cyclodextrin [19], Whelk-O1 [2-20-21], cyclofructan derivatives [22] and macrocyclic antibiotics [8,23] are all examples of covalently bonded phases


A new sub-2µm Ultra High Performance Chromatography (UHPC) chiral stationary phase was developed using Whelk-O1 as


a selector, bonded on 1.8 µm Kromasil silica, with the aim to obtain very high efficiencies, very high resolutions and fast separations [24]. Whelk-O1 is a Pirkle type selector, introduced firstly by Pirkle in 1992 [11,25], with a broad application field. Aryl propionic NSAIDs and all compounds that have aromatic systems with hydrogen bond acceptor sites near the chiral centre [26] are ideal targets for the Whelk-O1 CSP. Because of the fast interaction provided by the Whelk-O1 selector efficiencies of more than 260,000 theoretical plates/m were achieved on trans-stilbene oxide in Normal Phase – Ultra High Performance Liquid Chromatography (NP-UHPLC) analysis. UHPSFC is also a valid alternative analysis technique [27]. In fact, because of the low viscosity of the CO2


high flow-rates with low


backpressure can be achieved. SFC is an excellent choice to perform very fast analysis with high resolution values, allowing high throughput enantioselective screening [21] and reducing the consumption of organic solvents.


Experimental Instrumentation


The UHPLC system used for all tests was an UltiMate 3000 RS system from Thermo Fisher Dionex (Sunnyvale, California), consisting of a dual gradient RS pump (800


bar under normal phase conditions; flow rates up to 8.0 mL/min), an in-line split loop Well Plate Sampler, a thermostated RS Column Ventilated Compartment (temperature range 5-110°C) and a diode array detector (UV Vanquish detector) with a low dispersion 2.5 µL flow cell. The UV Vanquish detector was set at a filter time constant of 0.002 s, a data collection rate of 100 Hz and a response time of 0.04 s. Viper capillaries and fittings were used, with the two capillary Viper tubes (2 x 350 mm × 0.10 mm I.D.). Data acquisition and processing was performed with Chromeleon 6.8 software from Thermo Fisher. Detection of tested analytes was carried out at two different wavelengths (214 nm and 220 nm). The extra-column volume (obtained by injecting naphtalene) of this equipment was 12.7 µL (variance,s2


v,extra= 6.86 µL2 at flow-


rate 1.0 mL/min, calculated with second statistical moment, eluent: Hexane/EtOH 90:10 + 1% MeOH, T: 35°C) [28].


A Waters Acquity UPC2 (Ultra Performance Convergence Chromatography) was used to perform SFC analyses. The system was equipped with a binary solvent delivery pump compatible with mobile phase flow rates up to 4 mL/min and maximum system pressure of 414 bar. A 250 mL mixing chamber is present in the delivery system. The system also comprised an autosampler with a 10 mL loop, a column oven


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