Column Coupling to Solve a Challenging API Separation Using the Whelk-O 1 Chiral Stationary Phase

The Pirkle Type Whelk-O® 1 CSP was used several years ago to solve a challenging chiral separation for early phase drug development. Of all the commercially available columns tested, only the Whelk-O ® 1 CSP exhibited stereoeselective resolution for the particular active pharmaceutical (API) under investigation. Using a 250 × 4.6 mm (R,R)-Whelk-O® 1 column in a 5 μm particle size format, a suitable "fit for purpose" method was developed to accomplish this enantiomeric resolution. The drawback for this method was that the enantiomeric separation required using a 0.5 mL/min flow rate and a column temperature of 10 °C. The method run time was 150 min. While this method was suitable for Phase I investigations, the drug has progressed further in development and a more efficient and more readily transferable chiral method was desired. Using the Regis chiral screening service and coupling two Whelk-O® 1 columns, this desire was achieved.

Figure 1: Representative chromatogram from original method. 70% Hexanes/30% Ethanol/Methanol (4/3 v/v) with 0.1% TFA at 10 °C and 0.5 mL/min.

Transferring a method which requires maintaining the analytical column at 10 °C can be a difficult process. This requirement challenges the limitation of many of the commercial column chillers on the market today. Many of them will maintain a column "15 °C below ambient". Of course, "ambient" can vary widely from lab to lab, let alone country to country. A prime emphasis for method redevelopment would be a room temperature separation. A second goal was to shorten the run times.

Figure 2: Representative chromatogram from Regis column screen service. 70% Hexanes/30% 30mM Ammonium Acetate in Ethanol at 25 °C and 2.0 mL/min.

A racemic mixture was submitted to Regis for rescreening this compound using the current commercial polysaccharide and Pirkle chiral stationary phases. Unlike the original screen for this API, the Regis screen looked at both LC and SFC conditions. For this molecule, the Whelk-O®1 CSP was still the only CSP to exhibit suitable stereoselectivity. Also for this molecule, SFC offered no significant advantages in efficiency. The screen optimization was an improvement, but the resolution was not fully adequate.

Figure 3: Representative chromatogram of two Regis (R,R) Whelk-O®1, 5 μm Kromasil, 25 cm μ 4.6 mm columns connected in series using ~ 3 in PEEK tubing (0.005 μ 1/16 in). Mobile Phase: 70% Hexane/30% 30 mM Ammonium Acetate in 52.5/45/2.5 Methanol/Ethanol/2-Propanol. Flow Rate: 1.0 mL/min.

After exhausting all other possibilities in CSP and mobile phase selection, the only answer for this separation was to increase the theoretical number of plates via column coupling.

Conclusion

Taking advantage of the screening results from the Regis Chiral screening service as well as the robustness of the Whelk-O®1 column packing, the original method was effectively reoptimized to a method requiring ambient column temperature, 1.0 mL/min flow rate and 60 min run time. The new method is more chromatographically efficient and as importantly, can be readily transferred to external laboratories.

Regis Technologies, Inc.

8210 Austin Ave, Morton Grove, IL 60053

tel. (847)583-7667; fax (847)967-1214

Website: www.registech.com