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38 August / September 2016 Instrumentation


Analyses were performed using an Agilent 1260 SFC/MS system consisting of a binary pump, SFC control module, UV/DAD detector, and column compartment with an internal 2-position, 6-port valve, and 6120 MSD with an APCI source (Agilent, Inc, Santa Clara, CA, USA). A CTC HTS PAL autosampler (Leap Technologies, NC, USA) was equipped with a 10 µL syringe and a 5 µL fixed loop with a control method to vent CO2


from the loop prior to sample


introduction. The effluent of the SFC is split to the MSD using a 3-way tee (Valco, Houston, TX, USA) and a 50 cm long, 50 µm I.D. PEEKsil capillary tubing (Upchurch Scientific, Oak Harbor, WA, USA). All data were acquired using Agilent 64-bit ChemStation (Version C.01.05).


Analysis Conditions


Figure 3. Example of separations using the a) CCO-F4 and b) CCO-F2 fluorinated phases. Each column 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.


ChromegaChiral CC4 column (4-chloro- 3-methylphenyl carbamate), was also purchased from ES Industries, Inc. in the same dimensions and particle size to be used for comparative purposes.


All compounds used for this study (Figure 2) were also purchased from Sigma-Aldrich except for (2,2-difluorocyclopropyl) methyl benzoate (PubChem CID #101586031), and a single Pfizer proprietary compound, both of which were


synthesised in-house.


HPLC grade methanol, acetonitrile, and isopropanol (J.T. Baker, Phillipsburg, NJ, USA); ammonium formate and ammonia (Fisher Scientific, Pittsburgh, PA, USA); and bulk grade carbon dioxide (AirGas West (Escondido, CA, USA) were used in this study. The CO2


was


purified and pressurised to 1500 psig using a custom booster and purifier system from Va- Tran Systems, Inc. (Chula Vista, CA, USA).


The mobile phase consisted of CO2 and


the percentage co-solvent listed in each chromatogram, delivered at a flow rate of 3.0mL/min with 160 bar outlet pressure and the column temperature maintained at 25°C.


Results


While fluorinated CSPs have been researched and reported to demonstrate higher chiral recognition abilities than traditional polysaccharide CSPs, their evaluation for selectivity was limited to a small number of chiral probes and then used only in HPLC [22]. This research attempts to determine whether fluorinated phases are suitable for chiral SFC applications with the goal of addressing separation inefficiencies for halogenated compounds. Using several different standards, we were able to show that the CCO-F4 and CCO-F2 phases could achieve chiral recognition, and the structures of these prototype phases are shown in Table 1. In Figure 3 both columns appear to demonstrate versatility against the range of compounds used confirming the finding of Yashima and Chankvetadze, who have extensively studied similar column substrates [19,20,23]. The separations of miconazole and the racemic Pfizer compound (both of which are halogenated), provided some initial encouragement because the phases, while different from those previously reported, have demonstrated favourable preliminary results.


Figure 4. Chromatograms of (2,2-difluorocyclopropyl) methyl benzoate using standard SFC conditions with 1% acetonitrile as modifier on the CCO-F4 (top) and CC4 (bottom) phases. Inset: Isopycnic plot for carbon dioxide indicating the density profile at various temperatures and pressures. Yellow region represents normal operating conditions in SFC. Blue region represents rarely used region in SFC. Adapted from Tarafder [29].


Evaluating the columns against our target compound, (2,2-difluorocyclopropyl) methyl benzoate, we were initially discouraged that no separation was achieved using standard conditions and a range of modifier solvents. In Figure 4 with acetonitrile as the co-solvent,


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