Page 313 - Chiral Separation Techniques

12.3 Advantages of SFC for Chiral Separations 305

methods for all four compounds related to synthesis of an antiviral drug candidate
[35]. Successful LC methods with the same CSP could only be developed for two of
the candidate compounds. Samples for pharmaceutical analysis rarely consist only
of the target analyte. Ideally, achiral and chiral analysis can be performed concur-
rently, and the increased resolution power of SFC reduces the likelihood that unex-
pected sample components will interfere with the analysis.

12.3.2 Rapid Method Development

Method development remains the most challenging aspect of chiral chromatographic
analysis, and the need for rapid method development is particularly acute in the
pharmaceutical industry. To complicate matters, even structurally similar com-
pounds may not be resolved under the same chromatographic conditions, or even on
the same CSP. Rapid column equilibration in SFC speeds the column screening pro-
cess, and automated systems accommodating multiple CSPs and modifiers now per-
mit unattended method optimization in SFC [36]. Because more compounds are
likely to be resolved with a single set of parameters in SFC than in LC, the analyst
stands a greater chance of success on the first try in SFC [37]. The increased reso-
lution obtained in SFC may also reduce the number of columns that must be evalu-
ated to achieve the desired separation.

12.3.3 Column Coupling

In some instances, a single CSP may not provide the desired separation. Compounds
having multiple chiral centers often present analytical challenges because co-elution of
enantiomers and diastereomers can occur. Drug formulations typically contain excipi-
ents, preservatives, or other active ingredients that can cause chromatographic interfer-
ences. Serial coupling of multiple chiral columns or coupling of achiral and chiral
columns resolves some of these issues, and this is readily performed in SFC as a con-
sequence of the reduced pressure-drop across the column. Mobile phase compatibility
issues, often a problem when coupling columns in LC, are eliminated in SFC. Coupling
of multiple CSPs of the same type was used as a brute force approach by Mathre et al.
to resolve the four possible stereoisomers of a drug candidate [38]. Kot et al. reported
serial coupling of three different CSPs in SFC. The column triplet provided a multipur-
pose chromatographic system for the enantioresolution of a wide variety of racemic
compounds [39]. This approach should be used with caution, however, because oppos-
ing selectivities of the coupled CSPs may effectively cancel out a separation that would
normally be achieved on a single CSP [40]. The separation of components of a cough
syrup on a Chiralpak AD CSP alone, and on a coupled achiral/chiral column system is
illustrated in Fig. 12-3. Peak overlap of the two active ingredients occurred when only
the chiral column was used. No changes in chromatographic parameters were necessary
to accommodate the coupled column system [41]. Column coupling in SFC can provide
tremendous advantages over LC for challenging separations.