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39


Continuing our experiments with the (2,2-difluorocyclopropyl) methyl benzoate, the near-optimal HPLC separation using a traditional CSP as well as the optimised SFC conditions with the CCO-C4 phase is compared in Figure 6.


Only a partial separation was achieved using HPLC, and it was difficult to optimise further since the separation degraded with minute changes in temperature and solvent composition. Therefore, the separation was hard to scale for purification especially at the low column temperature. Using SFC with the fluorinated phase, not only did we achieve complete enantioseparation using pure CO2


Figure 5. Chromatograms depicting the effects of temperature and pressure on the separation of (2,2-difluorocyclopropyl) methyl benzoate on the CCO-F4 phase as the conditions move from the yellow to blue regions indicated in the Figure 4 inset.


conditions, but the separation was 4 times faster than the best conditions developed using HPLC. Since only a small percentage of methanol was added post- column to facilitate fraction collection, a significant savings in solvent and time were realised upon scale-up to SFC purification using the CCO-F4 phase.


Driven by the success of the first two prototype CCO-F4 and CCO-F2 phases, we decided to replace the methyl moiety of the CCO-F4 phase with trifluoromethyl groups thus enhancing the fluorine content of the CSP. Okamoto et al. reported that chiral recognition is dependent on the position, type and the number of substituents on the aromatic ligand of the polysaccharide CSP.


Figure 6. Analytical chromatograms of the separation of (2,2-difluorocyclopropyl) methyl benzoate using a) HPLC with a Chiralcel OJ-H column; and b) SFC with a CCO-F4 column.


no separation was achieved even after reducing the solvent percentage to 1% on either the CCO-F4 or CC4 phases despite using the full range of the typical polar protic solvent (alcohols) or aprotic solvents normally used in SFC. Realising that the compound may be too lipophilic for use with the addition of any organic solvent, the use of pure CO2


as the modifier was also explored.


to mimic a weakly polar solvent and thus enable successful chiral recognition by


Elimination of the co-solvent and lowering the temperature of the mobile phase to 10ºC resulted in a discrete separation of the enantiomers. As shown in Figure 5, further optimisation through variation of the temperature and pressure parameters enabled us to change the density of the CO2


SFC. Based on these results, we determined that a condition of 10ºC and 200 bar outlet pressure provided the best separation of the enantiomers.


The basis for this phenomenon can be explained using the isopycnic plot developed by Tarafdar and Guichon and shown in the inset of Figure 4 [29,30]. This plot demonstrates the density variations with changes in pressure and temperature. The typical region of operation for SFC is highlighted in yellow, which represents a modestly dense fluid (0.8 – 0.95 g/mL). By moving towards the blue region, the density of CO2


approaches that of a liquid (> 1.0 g/ mL), which in this case enables sufficient solubility of the compound to induce interactions with the CSP.


Figure 7. Separations of miconazole and verapamil using the CCO-F4-CF3 fluorinated phase. Column dimension is 4.6mm I.D. x 250mm length containing 5-µm particles maintained at 25°C. The mobile phase consists of CO2


and percentage co-solvent listed in


each chromatogram delivered at a flow rate of 3.0mL/ min with 160 bar outlet pressure.


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