Chiral Separations

September 2, 2008
Patricia Van Arnum
Pharmaceutical Technology
Volume 32, Issue 9

The selection of the chiral stationary phase is an important consideration in separating enantiomers when using high-performance liquid chromatography, supercritical fluid chromatography, and simulated moving bed chromatography.

Methods for chiral separations are an important part of achieving the desired enantioselectivity of a given active pharmaceutical ingredient. Selection of the stationary phase in a chromatographic approach is crucial tool for optimizing the separation process.

The function of chiral stationary phases

Polysaccharide-based chiral stationary phases (CSPs) are widely used for both analytical and preparative enantioselective chromatography because of their wide range of applicability and high loading capacity, explains Geoffrey B. Cox, vice-president of technology at Chiral Technologies (West Chester, PA). Derivatized polysaccharides, just as the native cellulose or amylose polymer on which they are based, form a helical tertiary structure, which engenders a chiral (asymmetric) environment that interacts differently with each of the two enantiomers as they adsorb on the stationary phase, he explains. Computer modeling of the structure of the chiral polymer shows that the helices are characterized by chiral grooves around the polysaccharide backbone. The polar groups in the solutes interact with the central, polar polysaccharide core mainly through hydrogen-bonding interactions that align the molecules in the grooves. The remainder of the solute molecule interacts with the groups with which the polysaccharide is derivatized, either sterically or through p-p interactions. Because both the solute and the environment are chiral, the adsorbed enantiomers experience different energies of absorption and the chromatographic separation is achieved.

High-performance liquid chromatography (HPLC), simulated moving bed chromatography (SMB), and supercritical fluid chromatography (SFC) take advantage of the differential absorption, meaning that one enantiomer moves more quickly through the column than the other, explains Cox. In HPLC, the stationary phase is held stationary in a column while the mobile phase passes through. The enantiomers emerge separately from the column each at a unique retention volume. SFC is broadly similar except that the mobile phase is based on carbon dioxide, which reduces the solvent consumption in the process while speeding the separations due to the low mobilephase viscosity.

In SMB, the system is arranged to mimic a countercurrent separation, where the two phases are moved in opposite directions. This makes for a more efficient use of both the stationary and mobile phases and in addition is a continuous process as opposed to the batch operation of HPLC. HPLC and SFC are generally used in the early stages of development when up to a few kilograms of purified enantiomer is required while SMB is typically used in larger scale-up for Phase II through manufacturing of the drug product due to its intrinsically lower cost.

Types of chiral stationary phases

Cox points out that there are several CSP types as explained below.

Pirkle type. These are CSPs based on the work of Prof. Pirkle and that use small molecules as the chiral selector. These CSPs are designed to give a variety of different interactions, usually hydrogen bonding and p-p acceptor or donor plus perhaps some steric interaction to give the desired three-point contact of the enantiomer with the chiral phase. Where the fit of the solute to the stationary phase is good, these CSPs have high selectivity and good loading capacity (e.g., the Whelk-O phase, which was developed for the separation of naproxen enantiomers). Often, these phases do not have a broad applicability and are able to resolve relatively few racemic compounds, notes Cox.

Cyclodextrins. These CSPs work on the basis of inclusion of the solute molecule into the basket-like structure of the cyclodextrin, says Cox. As with the polysaccharides, the ring of carbohydrate molecules forms a chiral environment that may be modified by the derivatization of the free hydroxyl groups. The cyclodextrins are used as mobile phase additives in electrophoretic separations but can also be used as a stationary phase for HPLC. The main disadvantage of the cyclodextrin CSPs is one of low loading capacity.

Macrocyclic antibiotics. Several CSPs based on macrocyclic antibiotics (e.g., vancomycin and teicoplanin) have been developed. These CSPs have a reasonably wide application range, although they are less generally useful than the polysaccharide-based media and in some cases (such as for amino acids) have been found to be complementary to the polysaccharide CSPs, explains Cox. Questions have been raised about their use for preparative chromatography of pharmaceutical products, given their antibiotic origins.

Template CSPs. CSPs have been developed for specific separations by the production of a "template" phase. This consists of the polymerization of the phase around molecules of the solute—or of a close analog—that is required to be retained, explains Cox. Removal of the template molecule results in a stationary phase with cavities of exactly the right dimensions for the desired enantiomer and into which the undesired enantiomer does not comfortably fit. Such CSPs can demonstrate extremely high selectivity (sometimes too great to be useful for analytical use) but typically have capacities too low to be of interest for preparative work.

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.