of 35°C. All data were processed with Origin 6.0 in order to properly graph and fit the van Deemter curves. Efficiencies (N/m or plates/m), and height theoretical plates (H), were not corrected for extra-column band broadening. Void volume was obtained by injecting an unretained marker (CCl4 and CHCl3

in NP in UHPSFC).

Figure 1. A) van Deemter curve analysis on both enantiomers of trans¬-stilbene oxide (Solid line for first enantiomer and dashed lines for the second one). B) Column efficiencies vs. Flow-rate on first enantiomer of trans¬-stilbene oxide. Columns: Regis-Whelk-O1 150x4.6 mm 5 µm (Black lines), Regis-Whelk-O1 150x4.6 mm 3.5 µm (Red lines) and UHPC-Whelk-O1 1.8 µm (Green lines). Eluent: Hex/EtOH 90:10 + 1% MeOH. T: 35°C. (For interpretation of the colours in this legend, please refer to the web version of this article)

The resolution (Rs), efficiencies and peak asymmetry values were extracted from Chromeleon 6.8 or Empower 3.0 softwares (according to European Pharmacopeia using peak width at half height (W0.5

)). Results and Discussion

Figure 2. A) Chromatographic traces of the separation of the enantiomers of trans-stilbene oxide on Regis-Whelk-O1 150x4.6 mm 5 µm (Black lines), Regis-Whelk-O1 150x4.6 mm 3.5 µm (Red lines) and UHPC- Whelk-O1 1.8 µm (Green lines) at their optimal flow-rates (0.6, 1.0 and 1.8 mL/min respectively). B) Same chromatograms reported replacing time on the x-axis with retention factor (k’). Eluent: Hex/EtOH 90:10 + 1% MeOH. T: 35°C. (For interpretation of the colours in this legend, please refer to the web version of this article)

compatible with temperatures up to 90°C, an UV detector equipped with an 8 mL flow– cell and a backpressure regulator (BPR). The injector/column inlet and column/detector connection tubes were 600 mm long and had an I.D. of 0.175 mm. The extra-column volume of this instrument was estimated to be 60 mL [29]. Data acquisition and control of the UHPSFC system was performed with the Empower 3.

Materials and chemicals

Solvents and reagents were from Sigma- Aldrich (St. Louis, Mo, USA) and used without further purification. HPLC grade solvents were filtered on 0.2 mm Omnipore filters (Merck Millipore, Darmstadt, Germany). Kromasil silica from Akzo Nobel (pore size 100 Å, particle size 1.8 mm and specific surface area 320 m2

g−1 ), Whelk-O1

selector and the two commercially available columns Whelk-O1 150x4.6 mm 3.5 and 5 mm were a gift from Regis Technologies Inc®

. 10 cm and 5 cm long empty stainless steel columns with an internal diameter of 4.6 mm were from IsoBar Systems by Idex (Wertheim-Mondfeld, Germany).

Synthesis of UHPLC Whelk-O1 CSP

The UHPC-Whelk-O1 chiral stationary phases (CSPs) were synthesised according to the procedure described by Pirkle in 1992 [11,25]. All UHPLC-Whelk-O1 columns were slurry packed with a pneumatically driven Haskel pump (Pmax: 1000 bar) into 100 x 4.6, 50 x 4.6 mm L.xI.D.


The kinetic performances of this new sub- 2µm CSP were evaluated under Normal Phase conditions (NP) and Super/sub-critical fluid chromatography (SFC). The mobile phases used for all tests were a mixture of Hexane/Ethanol 90:10 + 1% MeOH and CO2

/MeOH 80:20 in NP and SFC conditions

respectively. All injections were performed setting a Vinj of 0.1-0.5 mL in isocratic elution mode, repeated twice and the average values were used in the van Deemter construction. Kinetic evaluation were performed by the analysis of van Deemter curves starting from a minimum flow-rate of 0.1 mL/min up to 3.0 mL/min in NP and up to 4.0 mL/min in UHPSFC at the temperature

Kinetic performances of the new UHPC- Whelk-O1-1.8 columns (100 x 4.6 mm) were evaluated under UHPLC conditions through van Deemter curves analysis, correlating the efficiency, expressed as theoretical plate height, H (µm), with the flow-rate (mL/ min) at the temperature of 35°C. Plots were compared to those of the commercially available Whelk-O1 3.5 µm and 5 µm 150x4.6 mm (Figure 1A). Looking at the UHPC-Whelk-O1-1.8 curves, there is a clear advantage obtained in terms of efficiency. Efficiencies higher than 260,000 plates/meter were recorded on the first eluted enantiomer of trans-stilbene oxide versus the almost 140,000 plates/meter on the 3.5µm and the only 70,000 of the 5 µm. The same trend was observed for the second eluted enantiomer with a gain in efficiency of 75% going from the commercial 3.5 µm to UHPC- Whelk-O1-1.8. Taking into consideration the 5 µm Whelk-O1 in comparison with the sub- 2µm CSP, the efficiency was increased more than 3.5 times (68,000 versus more than 250,000 N/m) A large gain in optimal flow- rate range was also observed. Maximum efficiencies were recorded at 1.8, 1.1 and 0.6 mL/min, for the first enantiomer, on the 1.8 µm, 3.5 µm and 5 µm respectively. This permits the use of higher flow-rates with only a small loss of efficiency at the optimal flow-rate (more than 63% efficiency loss on the Whelk-O1 5 µm versus only 13% efficiency loss on the 1.8 µm for the first enantiomer at flow-rate: 3.0 mL/min). This trend can clearly be observed in Figure 1B, where the column efficiency was correlated to the flow-rate. Looking at this graph it is easy to understand the kinetic improvement of the 1.8 µm that yielded higher optimal flow-rates and better efficiencies with a 10 cm long column compared to 3.5 and the 5 µm commercial CSP packed into 15 cm columns. In Figure 2A are shown the chromatograms obtained injecting

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