« CHROMATOGRAPHY
application include many cases of multiple achiral impurities in some samples as well as the limitation in achiral selectivity under mobile phase conditions necessary to achieve the chiral separation.
In this
light, it is interesting to consider the possibilities available if additional fl exibility and technology advancements could be incorporated. More diverse achiral selectivity may be accessible with new achiral phases available for SFC. Further work in automated optimization of separations through varying achiral and chiral column lengths with appropriate method development and modeling should also benefi t this application.
Figure 5. Preparative SFC stacked injection separation traces of Diperodon plus caff eine with chiral column only (upper left), coupled achiral + chiral column (lower left), and fraction 1
repurifi cation chromatogram (right), necessary after insuffi cient separation from caff eine in the fi rst, chiral-only stage.
data shown lower in the fi gure was obtained. Here the caff eine peak is fully resolved from enantiomer peaks within the three-minute cycle time. With this method, caff eine as well as each enantiomer peak were resolved in one preparative step.
Four injections completed
the equivalent small quantity sample purifi cation in both cases. The coupled mode process was completed in 53 fewer minutes than the standard two-stage process in this example.
Notwithstanding this case, for any sample showing chiral and achiral selectivity with a coupled column method, relevant data and sample properties must be assessed to determine the likely feasibility of coupled-mode preparative separation. If the cycle time relative to the chiral-only separation is signifi cantly longer, considering this diff erence and sample size will determine whether the coupled process or two- stage process is more time-consuming. Stacked injection processing (purifi cation) is usually the most time-consuming component unless a sample is small. The coupled-mode process also includes time to setup and run the achiral column screening component following chiral isocratic method development.
Additional time-consumers
in the two-stage process may include additional analytical method development time if, for example, reversed-phase is required to remove the interfering impurity.
additional time also needed to evaporate the fractions obtained. Conclusions
The methodology described provides a procedure for advantageously isolating chemically pure enantiomers in a readily accessible way for most chiral separations laboratories using SFC. This experiment described a selection process to fi nd a useful, complementary set of achiral columns to facilitate one-step chiral and achiral purifi cation for chemically impure racemates. Other columns than those described here may be useful depending on the compounds being separated so any lab attempting this may need to make a relevant selection. We have found for our laboratory that in roughly 20% of impure chiral samples we can fi nd a coupled separation method which eliminates the need for a two-stage purifi cation. Barriers to more general
Following both stages there is Author Biography
Manuel Ventura, Ph.D., is a Principal Scientist and leader of the Separations group supporting Therapeutic Discovery Chemistry at the South San Francisco site of Amgen, Inc. He began his career at Pfi zer, Inc. where he was key developer of a novel SFC/MS interface applied to high-throughput library analysis. Dr. Ventura has continued developing new platforms and processes utilizing SFC throughout his career and has contributed numerous presentations and publications. Manuel received his Ph.D. in analytical chemistry from the University of Texas at Austin in 1997.
References
1. White., C.J. J. Chromatogr. A, 1074, 163 (2005). 2. Welch, C.J. (2007) Chiral Chromatography in Support of Pharmaceutical Process Research, in Preparative Enantioselective Chromatography, Blackwell Publishing Ltd, Oxford, UK (Ed. G. B. Cox.)
3. Miller, L., Potter, M. J. Chromatogr. B, 875, 230 (2008). 4. 5. 6. 7.
9. 10. 12.
De Klerck, K., Mangelings, D., Vander Heyden, Y. J. Pharm. Biomed. Anal. 69, 77-92 (2012). Phinney, K.W., Sander, L.C., Wise, S.A. Anal. Chem., 70, 2331-2335 (1998). Alexander, A.J., Staab, A. Anal. Chem., 78 (11), 3835-3838 (2006). Zeng, L., Xu, R., Zhang, Y., Kassel, D.B. J. Chromatogr. A, 3080-3088 (2011).
8. Wang, Y., Xue, X., Xiao Y, Zhang, F., Xu, Q. Liang, X. Rapid Commun. Mass Spectrom., 15(22), 2067-75 (2001).
Zhang, X., Towle, M.H., Felice, C.E., Flament, J.H. and Goetzinger, W.K. J. Comb. Chem., 8, 705-714 (2006).
McClain, R., Przybyciel, M. LCGC Asia Pacifi c, 15(4), 26-33 (2012).
11. Welch, C.J., Biba, M., Gouker, J.R., Kath, G., Augustine, P., Hosek, P. Chirality, 19, 184- 189 (2007).
Berger, T.A., Smith, J., Fogelman K., Kruluts, K. Am. Lab., 34, 14-20 (2002).
www.americanpharmaceuticalreview.com | | 95 Acknowledgements
The author acknowledges the eff orts contributed by Dhanashri Bagal and Brent Murphy enabling these experiments to be performed and reported.
The author also acknowledges Wolfgang Goetzinger for
test compound provision, as well as Larry Miller and Kyung Gahm for editorial support.
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