Recent Progress in Chiral Stationary Phase Development and Current Chiral Applications

Chiral separations remain a decided area of interest, particularly in the pharmaceutical and agrochemical fields. Although high performance liquid chromatography (HPLC) remains a strong choice for separations because of its robustness, transferability, and instrument availability, the use of chiral supercritical fluid chromatography (SFC) continues to expand in analytical and preparative techniques. Several chiral stationary phases continue to enjoy wide use because of their broad application in both HPLC and SFC.

Chiral separations continue to be of great interest because of the prevalence of racemates in markets such as the pharmaceutical and agrochemical (pesticide) industries. In fact, a review of the importance of pharmaceutical chiral separations in single-enantiomer patent cases was recently published (1), and another review estimates that about 30% of pesticides are chiral with about half of these having multiple chiral features (2). The individual pesticide enantiomers may exhibit different effects on the environment. Although the separation of enantiomers can be challenging because of their identical physical and chemical properties in an achiral environment, chiral stationary phases (CSPs) have greatly facilitated enantioseparations in high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Research on specialized separation techniques using novel CSPs, particularly derivatized polysaccharides and cyclodextrins, continues for the resolution of specific individual enantiomers, and chiral separation on commercially available CSPs remains a mature and widely used technique with some new entries to the market. Polysaccharide and macrocyclic glycopeptides CSPs continue to be the most widely used commercial chiral phases, with cyclodextrins, cyclofructans, π-complex, and protein-based CSPs also finding use. For non-HPLC separations such as gas chromatography (GC) and capillary electrophoresis (CE), cyclodextrins continue to dominate. Enantioseparations of larger, more-complex molecules with multiple chiral centers have increased as biotech continues to grow, meaning that more compounds must be resolved simultaneously, and chiral separations of more-polar molecules are needed, especially for the agrochemical and pharmaceutical fields.

There are plenty of resources and information available for performing chiral separations, both from the commercial suppliers of CSPs and in the literature. There are numerous reviews of the widely used CSPs, including recent reviews of cellulose and polysaccharide-based CSPs (3), protein and glycoprotein CSPs (4), macrocyclic antibiotic CSPs (5), cyclodextrin CSPs (6,7), and chiral ion- and ligand-exchange CSPs (8). Reflecting the burgeoning interest in SFC chiral separations of pharmaceuticals, several reviews specific to SFC have recently been published (9–12).

The State of Current CSPs

Chiral separation continues to be the primary technique of choice, with many companies seeing an increase of about 20% for both analytical and preparative enantioseparations in their laboratories. The market continues to enjoy growth and maturation as older technologies are replaced by newer and improved technologies. New CSPs continue to be introduced to the market, including the zwitterionic phases, Chiralpak Zwix(+) and Zwix(-) from Chiral Technologies, a new immobilized crown-ether phase, Chiralpak CR-I also from Chiral Technologies, and an immobilized ovomucoid phase, C18-Ovo-5-120 from Separation Methods Technologies. Companies such as YMC America, Inc., Chiral Technologies, Diacel, and Separation Methods Technologies are continuing to expand their offerings of immobilized polysaccharide-derived CSPs because these columns offer greater stability, can be used with a wider variety of mobile phases, and are useful in both liquid chromatography (LC) and SFC applications.

One apparent trend in the market is toward the increased use of SFC for chiral separations. The advantages of SFC are the reduced environmental impact and operating costs with increased throughput. Although SFC has traditionally found more use in preparative-scale chiral separations, where the waste reduction and decreased solvent use is attractive to industry, these immobilized CSPs are also seeing increased use in analytical applications as well. SFC is currently receiving a lot attention from the pharmaceutical industry for screening and method development of chiral separations (13–15).

Polysaccharide CSPs

As in previous years, chiral separations achieved by polysaccharide CSPs in HPLC account for approximately one-third of all HPLC chiral separations in the literature, and most of these separations are carried out on commercially available columns. The most commonly used phase on cellulose or amylose continues to be 3,5-dimethylphenyl carbamate, which includes immobilized columns such as Chiralpak IA (Chiral Technologies), Chiralpak IB, Lux Cellulose-1 (Phenomenex), and coated phases such as Chiralcel OD-H (Chiral Technologies), Kromasil CelluCoat (Akzo Nobel), and AmyCoat (Akzo Nobel). Because the conformation of the polymer is influenced by how the stationary phase is attached to the packing material, coated and immobilized columns can exhibit different selectivity. Chloro-substituted polysaccharide CSPs have also found much use, including tris(3,5-dichlorophenyl carbamate) (Chiralpak IC), tris(3-chloro-4-methylphenyl carbamate) (Lux Cellulose-2), tris(5-chloro-2-methylphenyl carbamate) (Chiralpak AY-H), and tris(3-chloro-4-methylphenyl carbamate) (Chiralcel OZ-H). The selectivity of the Chiralpak IC column toward different compounds is demonstrated in Figure 1.

Figure 1: The separation of three compounds using a 250 mm × 4.6 mm, 5-μm dp Chiralpak IC column (Chiral Technologies). Mobile phase: 70:30:0.1 (v/v/v) n-hexane–ethyl acetate–diethylamine. Adapted from reference 24.

A reversed-phase study using tris(chloromethylphenyl carbamate) derivatives of cellulose and amylose concluded that using these CSPs in screening protocols yields higher success rates in achieving baseline separations with shorter screening times (16). An updated generic separation strategy in normal-phase HPLC was reported using the following commercial CSPs: Lux Cellulose-1, Lux Cellulose-2, Lux Amylose-2, Lux Cellulose-4 and Chiralpak AD-H, Chiralcel OD-H, and Chiralcel OJ-H (17). Reversed-phase screening strategies for HPLC with Chiralpak IA, Chiralpak IB, and Chiralpak IC with applications compatible with liquid chromatography–mass spectrometry (LC–MS) were also reported (18). Simplified screening protocols for chiral separations in HPLC and SFC on Chiralpak IA, IB, IC, and ID in reasonable time frames and high success rates were recently published (14).

Cyclodextrin-Based CSPs

Although no new cyclodextrin based CSPS have been brought to market lately, the use of cyclodextrins still accounts for over one-fourth of all publications in chiral HPLC in recent years. The bonded cyclodextrin–based CSPs are popular because of their robustness, wide selectivity, and ability to enantioseparate in the reversed and polar organic phases. The mechanical stability of the cyclodextrin CSPs lends itself to preparative-scale use as well.

Macrocyclic Glycoprotein CSPs

The macrocyclic glycoprotein CSPs continue to fill a broad and useful niche in chiral HPLC because of their unique versatility and broad selectivity, and are unsurpassed in the enantioseparation of chiral amino acids. Sigma-Aldrich offers a Chirobiotic method development kit containing V2, T, R, and TAG columns for screening in polar ionic, polar organic, reversed-phase, and normal-phase modes. A review of macrocyclic glycopeptide-based CSPs in HPLC methods for amino acid enantiomers and related analogues was published in 2010 (19) and recently updated (5).

Cyclofructan CSPs

The recently released cyclofructan-based Larihc CSPs from AZYP, a new supplier of novel chiral and achiral phases for HPLC, hydrophilic-interaction chromatography (HILIC) and SFC, also available from Supelco/Sigma-Aldrich, continue to have great application in chiral HPLC separations. (Larihc is "chiral" spelled backwards.) These columns include Larihc CF6-P, considered the "king column" for separation of racemic primary amines, Larihc CF6-RN, which separates nonprimary amines, and Larihc CF7-DMP, the only commercialized cyclofructan 7 column, which shows complementary enantioselectivity to the CF6-RN CSP.

Figure 2: Examples of separations of pharmaceutical compounds on CF6 and CF7 CSPs. Adapted from reference 25.

Larihc CF6-P is the only column that separates primary amines in a nonaqueous solvent using the polar organic mode. The Larihc CF6-P was reported to be useful in the separation of chiral illicit drugs and controlled substances, with the other Larihc CSPs also yielding enantioseparations (20). A comparison of separations of 46 chiral reagents to determine enantiomeric purity using Larihc, Cyclobond (Supelco/Sigma-Aldrich), and Chirobiotic CSPs was recently reported, with this being the first use of Larihc CSP for this purpose (21). Recent reports indicate that the Larihc CF6-P CSP is broadly applicable to the separation of nonamine containing racemates as well (20–22).

All Larihc CSPs are reported to work well in SFC because of their use in the normal-phase or polar organic phase modes. A comparison of dimethylphenyl carbamate cyclofructan 7 CSP use in SFC and HPLC was recently made (23) and the retention and enantiodiscrimination properties and the effect of different SFC modifiers was reported.

Figure 3: Chromatograms showing chiral separations in normal-phase, polar organic phase, and reversed-phase modes on CF6-RN CSP. Adapted from reference 26.

Chiral Separations Today

Although the chiral market is maturing and stable, perhaps the biggest need in the field remains an increased overall understanding of chiral methodologies and more practical training. Because information is largely supplied by vendors to newcomers to chiral HPLC, novices can sometimes stumble around for a while wading through vendor literature and advice. In addition to an understandable sales objective, vendors occasionally misunderstand the customers' end-to-end chiral operation, so promised increases are sometimes not realized at the laboratory level. Furthermore, increased performance is also sometimes not realized because of low-tech reasons that may be related more to laboratory layout and operation, rather than the lack of latest technology and equipment. Newcomers should make use of the short courses on chiral HPLC that give a wealth of unbiased information to the participants and are offered at major conferences such as Eastern Analytical Symposium (EAS), The Pittsburgh Conference (Pittcon), and Chirality.

Increased selectivity remains a challenge in chiral separations. With increased selectivity, loading can be increased in preparative chiral separations, which will provide the greatest cost savings. With significant cost savings in the separation process, companies can shift from using chiral selective synthesis for chiral purification to purification by chromatography. Although HPLC is used for preparative-scale separations, SFC continues to gain ground as the most cost-effective way to purify enough material for further studies.

The market is maturing, but technology has not moved forward in any revolutionary way. Chiral HPLC still lacks (and may always lack) a CSP that can operate in all modes for enantioseparation of molecules from low to high polarity.

Acknowledgments

We gratefully thank Daniel W. Armstrong, Elena Eksteen, Hafeez Fatunmbi, J.T. Lee, and Gary Yanik for their suggestions in the preparation of this manuscript.

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Timothy J. Ward is a professor of chemistry and associate dean of sciences at Millsaps College (Jackson, Mississippi). Ward received his BS degree from the University of Florida and his PhD from Texas Tech University. Dr. Ward served as chair of the International Symposium on Chirality in July 2007, in San Diego, California. His research interests include chiral separations, the development of analytical LC and CE methods, and their application to pharmaceutical and archaeological analysis.

Timothy J. Ward

Karen D. Ward is an instructor at Millsaps College. She received her BS degree from Texas A&M University and her MS from Texas Tech University. Ms. Ward previously worked in the pharmaceutical industry at the Analytical Environmental Research Division at Syntex Pharmaceuticals in Palo Alto, California. Direct correspondence to: wardtj@millsaps.edu

Karen D. Ward