40 August / September 2016

The difluoromethyl substitution phase (CCO- DiF), on the other hand, exhibited significant instability to solvent percentages above ~5% v/v, such that subsequent injections of the trans-stilbene oxide marker resulted in loss of selectivity (Figure 8a). In addition, the m/z found in the mass spectral trace (8b) indicates the presence of significant bleed of the benzylic moiety (8c).

This was observed across several columns and numerous modifier combinations, and use was only determined to be acceptable if the solvent percentage was kept very low (<5% v/v). This effect was not observed on any of the other phases tested (data not shown). Further studies with this column were halted pending assessment of the stability issues.

Figure 8. Preliminary results for the CCO-DiF column indicating significant instability of the phase. a) SFC/ UV chromatograms of replicate injections of trans-stilbene oxide using 15% isopropanol as a modifier; b) Mass spectra of the column effluent indicating a possible identity of the noise: c) Total ion chromatograms of the CCO-DiF column taken shortly after first installation (Blue) and after the 5th

injection (Red).

More specifically, improvements in chiral recognition can be achieved by adding substituents to the 3- or 4-position [9,11- 12]. Following this logic, we chose to incorporate readily available trifluoromethyl and fluorine on the 3-position of the benzylic ring, utilising the corresponding benzoyl chlorides to create the second generation phases: cellulose tris (4-fluoro- 3-trifluoromethylphenylbenzoate), or CCO-F4-CF3; and cellulose tris(3,4-difluoro- methylphenylbenzoate) or CCO-DiF.

Although we chose to use the benzoate linker to reduce costs, there is precedence that the chiral discrimination between the resulting benzoates and carbamates is consistently preserved [31]. Figure 7 demonstrates that the prescribed changes did not diminish the selectivity of miconazole or verapamil enantiomers even with the change in linker and the addition of the trifluoromethyl substituent. The preliminary data suggests that the chiral discrimination of the CCO-F4 and CCO-F4-CF3 columns are similar.

Figure 10. The overall trend in selectivity (a) and resolution (R) for pharmaceutical compounds supports effectiveness of positional substitution and dipole potential on the stationary phase.

Figure 9. Example chromatograms of (2,2-difluorocyclopropyl) methyl benzoate on a) CC4; b) CCO-F4-CF3; and c) CCO-F4 phases demonstrating the possible effect of stationary phase dipole moments on each separation. Each column is 4.6mm I.D. x 250mm length containing 5-µm particles maintained at 10°C. The mobile phase consists of 100% CO2

moment calculations reflect the magnitude differences in an ideal system and were not measured for each specific phase. Adapted from Crab [36]. R and a were calculated using Agilent Chemstation software.

delivered at a flow rate of 3.0mL/min with 200 bar outlet pressure. Dipole

Utilising the optimal conditions determined in Figure 9, separation of the (2,2-difluoro- cyclopropyl) methyl benzoate enantiomers was achieved on the three stable columns. It is interesting to note that the retention and selectivity (under these conditions) appear to support the assertion that it is both the direction and the magnitude of the electron withdrawing/donating effect that enhances the separation [17,24,32-35]. For instance, the dipole contributions between F and Cl for methane are 1.51D and 1.56D respectively, which could account for the drastic differences in retention and the modest enhancement in selectivity. Since the dipole moment for each specific phase cannot be measured or calculated at this time, these values can only be indicative of the relative differences in magnitude of the dipole effects.

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