Chiral separation screening has become a widely accepted approach for the rapid identification of an appropriate chiral stationary
phase for use in more focused enantioseparation optimization. A set of extended screens encompassing various chromatographic
modes using high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) is presented. These
multimodal screens are tailored to meet the specific and changing needs of customers as distributed along the drug development
process within a typical pharmaceutical development setting. In addition, a systematic column testing and regeneration approach
is presented and emphasized for maintaining the reliability of column and screen data. Comprehensive screening systems created
to serve such an expansive customer base, actively supported with manufacturer-recommended column suitability and regeneration
procedures, are not well represented in the literature.
Of the myriad analytical tests required to support a pharmaceutical candidate during the drug development process, the determination
of a rugged and reliable enantioseparation method for a chiral compound is frequently the most challenging requirement. An
arsenal of techniques is available for this task, including capillary electrophoresis (CE), supercritical and subcritical
fluid chromatography (SFC), capillary electrochromatography (CEC), gas chromatography (GC), and high performance liquid chromatography
(HPLC). Depending on specific factors such as compound structure, chemical and physical properties, and sample matrix, each
respective technology possesses relative advantages and disadvantages pertaining to speed, efficiency, and sample compatibility.
Regardless of the technique used for enantiomer analysis, significant cost and expertise are inherent in the process. Herein
is one distinct advantage among many for choosing HPLC. Since HPLC instrumentation is a mainstay of chromatography, one usually
only needs to invest in the chiral columns to pursue an HPLC chiral separation. Admittedly, the columns can be costly, but
not as expensive as the financial outlay that might be required to set up dedicated instrumentation using ancillary techniques.
Another advantage of HPLC is its general familiarity among scientists. Most analytical chemists understand basic HPLC and
can quickly use their familiar instrumentation toward the chiral separation. The proportion of scientists in a typical laboratory
setting who are skilled in the competing techniques (mentioned above) is almost always notably less compared to those familiar
with traditional HPLC.
An efficient approach to meeting the chiral separation demands imposed across the drug development arena should involve the
various techniques aforementioned when they offer an advantage. However, the further a chiral molecule progresses along the
drug development pipeline, the more the likelihood increases that traditional chiral HPLC will surface as the analysis technique
of choice at compound registration. As the reliability of instrumentation supporting other separation techniques continues
to improve, an increased presence of non-HPLC enantiomer analysis methodology will certainly permeate the downstream drug
Presently, the superiority of chiral analysis via HPLC for most analytes continues to focus most of the development within
this well-honed technique. As a result, a great number of HPLC chiral columns have been marketed in support of enantioseparation
development. Even though certain chiral stationary phases (CSPs) are known to be more effective for selected compound classes,
separation modes, or sample matrices, the search for the most suitable chiral selector for a given chiral analyte remains
primarily a trial and error process. All is not blind, however. Ever-improving data repositories can provide direction in
the search. Such chiral application databases can provide separation methods for compounds possessing structural similarity
to those being studied by the chromatographer (1). Although these tools are certainly helpful for providing direction in the
screening effort, the search for the ultimate enantioseparation remains time-consuming, inefficient, and expensive. An inadequate
and noncomprehensive screening paradigm can result in a scientist unknowingly settling for an unnecessarily inferior separation
system. The number of CSPs, when combined with compatible mobile phases, becomes exponential and moves beyond the scope of
the typical time and resource-limited laboratory.
As a pragmatic response, the industry has generally approached HPLC chiral separation development with the use of various
tailored efforts, usually built upon a brute screening approach using the more promising CSPs. Such screens are sometimes
augmented by a flow scheme or decision tree offering specific guidance depending on the nature of the analyte (acid, base,
neutral) or sample matrix (aqueous, nonaqueous). Ultimately, the experimental process is driven by the needs of the customer.
A brief survey of published literature over the past decade reveals that the majority of HPLC enantioseparation screens are
suited toward rapid development of methodology that can also be used at the preparative scale (2–12,14–16).
Within the realm of chiral preparative chromatography, analyte solubility and mobile phase volatility rise in importance to
nearly the level of the quality of the enantioseparation itself. The use of polysaccharide-based chiral selectors with normal
phase or polar-organic eluents frequently offers all of the above. Not surprisingly, many of the published chiral HPLC screens
use columns of this class (5–10), a few of which are now off patent and available as "clones" with potential cost savings
(11). While many in the industry utilize these CSPs in both normal-phase and polar-organic modes, the analogous polysaccharide
coated columns designed for use in reversed-phase mode are occasionally substituted for polar-organic analysis (3,12,13).
Some screens add a π-electron acceptor/π-electron donor CSP to their polysaccharide column screening repertoire (7,9,14).
Literature reviews conducted by Akin and colleagues (17), Beesley (11), and Manglings and Vander Heyden (4) confirm the dominance
of these CSPs, especially in the early stages of drug development. In the last few years, screens incorporating immobilized
polysaccharide derivatives have been reported. Because of their immobilized nature, these columns allow for the use of a much
broader range of solvents, notably enhancing capabilities, especially in polar-organic mode (8,10,15). The authors of this
publication can report that more recently marketed immobilized CSPs also offer promise (18–20).
Chiral separation screening success using the predominately polysaccharide-style column set described above is unmatched,
and remains a logical domain from which to operate, especially if preparative applications are in mind. Some chiral separation
screens increasingly use SFC technology to take advantage of the technique's speed and "green" label earned because of its
reduced solvent consumption (9–11). For laboratories primarily concerned with the quality of the enantiomeric separation at
analytical scale, without preparative aspirations in mind, the choice of chiral selector and mode is greatly increased. The
chromatographer's toolkit is greatly expanded once the reversed-phase mode is considered in tandem with compatible polysaccharide
CSPs and other more specialized selectors indicative of this separation mode.
Samples dissolved in biological matrices, as well as more polar compounds with enhanced solubility in aqueous-based diluents,
are excellent candidates for reversed-phase chiral methodology. Laboratories routinely tasked with chiral purity analysis
are frequently already oriented toward standard achiral purity determination, much of which is conducted in the reversed-phase
mode. In one aspect, the distomer (undesired enantiomer) could simply be considered as yet another impurity, albeit a special
one. The advantage of using chiral analysis in reversed-phase mode, compatible with parallel achiral analysis in the same
mode, is obvious. In such cases, instrument conversion to normal-phase solvents is avoided, as well as the tendency to "dedicate"
an instrument to said chromatographic mode.