Multiple chiral centers
Separations of molecules with multiple chiral centers are more difficult since chiral stationary phases are normally good
at separating enantiomers but not at separations of diastereoisomers, explains Cox. Separations of molecules with multiple
centers can, however, be achieved by careful selection of operating conditions. One such example is the separation of the
various isomers of nadolol. This separation was achieved using 0.1% of ethanesulfonic acid in the hexane-ethanol mobile phase
with "Chiralpak AD-H" (Chiral Technologies) as a stationary phase.
Preparative scale separations of molecules with multiple chiral centers are rare, mostly because the maximum recovery of any
one enantiomer represents only 25% of the sample and also because it is difficult to adjust the selectivity to attain a high
loading of the product, says Cox. These separations are typically done at small load. Usually a more profitable approach,
certainly at the larger scale, is to separate the diastereoisomeric pairs by some other technique, perhaps crystallization
or achiral chromatography, and subsequently to isolate the enantiomers from each of the diastereoisomers.
Separations at a larger scale are carried out by SMB, which is essentially a binary separator. This technique is suited to
the separation of enantiomers. When using SMB, the enantioselective separation should be performed at the best place in an
optimized synthesis. This process involves the optimization of the chemical processes, studies on the CSP lifetime, optimization
of the mobile-phase components and composition, which take into account the viscosity, flash points, and threshold limit
values of the solvent components, and optimization of the chromatographic technology. The implementation of the final process
into the cGMP environment involves qualification, validation. and production data for the separation.
Supercritical fluid chromatography
SFC used with chiral stationary phases also is a way to resolve enantiomers. With SFC, most of the liquid solvent is replaced
by pressurized carbon dioxide, and only a small percentage of an organic solvent is required to solubilize the compound and
serve as a cosolvent with the carbon dioxide.
In 2007, Regis Technologies introduced two polysaccharide-coated columns for use in SFC and HPLC analyses for chiral separations.
The columns are: "RegisCell," which has a (tris-(3,5- dimethylphenyl) carbamoyl cellulose) selector, and "RegisPack," which
has a (tris-(3,5-dimethylphenyl) carbamoyl amylose) selector. The amylose and cellulose coatings have a different orientation
in space (mirror images) and, therefore, provide subtle differences in the separation of chiral compounds, explains Ted Szczerba,
technical director with Regis. "These columns provide enantiomeric separations of a wide range of racemate classes rather
than for the separation of a specific group of chiral compounds," Szczerba says. "Therefore, both columns can serve as a starting
point for developing a chiral separation method."
SFC is considered a normal-phase technique, and recent research evaluated whether it was feasible to use nonpolar columns
under SFC conditions to separate compounds that closely eluted or co-eluted by gradient and isocratic reverse phase (RP)-HPLC
(1). Specifically, Regis scientists evaluated the effects on resolution (Rs) when operating reverse phase columns under SFC
conditions. They used nonpolar columns (C18, C8, and phenyl). They also separated closely eluting compounds (4-ethoxyacetanilide,
sulfamethoxazole, benzocaine, m-cresol, acetophenone, m-nitroaniline, caffeine, acetaminophen, and trans-stillbene oxide) under a variety of conditions (evaluated temperature, pressure, percent co-solvent and flow rate). The research
showed the benefit of SFC using reverse phase columns as an alternative technique to RP-HPLC (1).
For recent advances in asymmetric synthesis, see "Achieving Enantioselectivity".
1. E. Pullen et al., "Evaluation of Nonpolar Reversed-Phase Columns Under Supercritical Conditions," LCGC North America: The Peak, July 2008, 8–18.