50 August / September 2016
trans-stilbene oxide on the three columns at the respective optimal flow-rates. The separation provided by the UHPC-Whelk-O1 1.8 µm (100 x 4.6 mm ) was completed in just 1.65 minutes, with a gain of more than four times in speed compared to the 5 µm commercial column (150 x 4.6 mm). One of the most important advantages, due to the kinetic profile described previously,
is
the resolution (Rs), which was 17.74 on the 100x4.6 mm 1.8 µm Whelk-O1, almost two times larger than the 150x4.6 mm 5 µm column. The thermodynamic profile was characterised in Figure 2B, where the same chromatographic traces of Figure 2A were reported with the k’ on the x-axis, in order to evaluate the performances of these columns independently of the column geometries. Extremely reproducibility k’, and selectivity, was achieved between the 1.8 µm Whelk-O1 and the two commercially available Whelk-O1 columns. In fact, an enantioselectivity factor of 2.42-2.49 was recorded on all columns. This reproducibility is extremely important in the transition from enantioselective ‘e’ High Performance Liquid Chromatography (eHPLC) to ‘e’ Ultra High Performance Liquid Chromatography (eUHPLC). Using 1.8 µm columns instead of the 3.5 µm (or 5 µm) without any change in the analytical methodology, maintaining the same conditions (i.e. mobile phase composition), it is possible to improve the separation of two enantiomers with better efficiencies and resolutions thus improving the speed of analysis and reducing the analysis time.
Figure 3 shows two different separations, 1-acenapthenol (Figure 3A) and benzoin (Figure 3B), on the UHPC-Whelk-O1 1.8 µm 100x4.6 mm and on the two Regis Whelko-O1 150x4.6 mm 3.5 and 5 µm. In both cases the sub-2µm, Whelk-O1 reached efficiencies two and four times greater in comparison with the 3.5 and the 5µm HPLC Whelk-O1 CSPs. All injections were performed at the optimal flow-rate of each column, increasing the speed of analysis by 5 times and enhancing the resolution values more than 30%. With the very high reproducibility obtained on the UHPC- Whelk-O1, the retention factors and the enantioselectivity values were almost the same, when using the same mobile phase, on all columns and on both samples.
This phase was also used in Sub/super-critical fluid chromatography with a mobile phase consisting of 80% CO2
and 20% methanol.
van Deemter curves analysis was performed also in this case (Figure 4). Efficiencies up to 250,000 were recorded on the sub-2µm
Figure 3. Examples of separations of 1-Acenaphthenol (A) and Benzoin (B) 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). 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).
Figure 4. van Deemter curve analysis in HPSFC/UHPSFC on both enantiomers of trans¬-stilbene oxide (Solid line for first enantiomer and dashed lines for the second one). 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: CO2
/MeOH 80:20. ABPR: 1800 psi. T: 35°C. (For interpretation of the colours in this legend, please refer to the web version of this article)
Whelk-O1 with a gain of more than three times in comparison to the 5µm column. Also the optimal flow-rate was more than twice that of the commercial Whelk-O1. Another interesting aspect is the optimal linear velocity, which can’t be achieved with the current instrumentation (UPC2
has an instrumental
limit at 4 mL/min and 6000 psi). The curves of the two enantiomers are not able to reach the minima, and those can only be estimated at an optimal flow-rate of 5-6 mL/min [27] and consequently it was not possible to record maxima efficiencies of the column.
Figure 5 shows the chromatographic traces recorded on trans-stilbene oxide on the three columns in SFC conditions at their optimal flow-rates. Under these conditions, an analysis time gain of more than three times was observed and the separation
of the two enantiomers was completed in only 1.08 minutes at the flow-rate of 3.7 mL/min (instrumental pressure limit) in comparison to the 5 µm. Also the resolution was extremely improved going from 13.5 to 19 and up to 21.5 on the 5, 3.5 and 1.8 µm Whelk-O1 columns respectively.
In order to demonstrate the potential of this CSP a large library screening was performed by Gasparrini’s group [21]. A previous version of the Whelk-O1 sub-2 µm was used, where the selector was bonded on 1.7 µm Thermo Syncronis silica and packed into a 50x4.6 mm. column (Figure 6). 129 chiral compounds of pharmaceutical interest where randomly collected (acidic, neutral and basic) and analysed in one working day: 81 out of 129 racemates were resolved under identical eluting conditions using a 9 min methanol in
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