Contemporary Analysis of Chiral Molecules

October 1, 2016
Eric Francotte
Special Issues
Volume 29, Issue 10
Page Number: 31–37

The first high performance liquid chromatography (HPLC) column for enantioselective chromatography was introduced commercially in 1981. This chromatographic mode has now become the method of choice for the analysis of chiral pharmaceutical compounds, making previous approaches, such as optical rotation, almost completely obsolete. However, supercritical fluid chromatography (SFC) has been gaining increasing recognition as a complementary technique to HPLC for pharmaceutical enantioselective analysis. Gas chromatography (GC) and capillary electrophoresis (CE) remain very useful for particular applications.

Eric Francotte, FrancotteConsulting, Separation Sciences, Basel, Switzerland.

The first high performance liquid chromatography (HPLC) column for enantioselective chromatography was introduced commercially in 1981. This chromatographic mode has now become the method of choice for the analysis of chiral pharmaceutical compounds, making previous approaches, such as optical rotation, almost completely obsolete. However, supercritical fluid chromatography (SFC) has been gaining increasing recognition as a complementary technique to HPLC for pharmaceutical enantioselective analysis. Gas chromatography (GC) and capillary electrophoresis (CE) remain very useful for particular applications.

Analysis of enantiomers is a critical topic in the life science, food, fragrance, and environment industries, and is also key in asymmetric synthesis and catalysis. Enantioselective chromatography has evolved as a powerful tool for the analysis of chiral compounds and is now established as the method of choice in almost all academic and industrial laboratories dealing with chiral molecules. Over the past 35 years intense efforts have been made to obtain reliable tools for this purpose and various methods and techniques have been developed and optimized for the accurate determination of the chiral composition of enantiomeric mixtures (Table 1).

The techniques in Table 1 do not all have the same importance but all have been applied. While the formation of diastereoisomers prior to analysis was the common practice in the past, today the respective enantiomers of almost any type of small chiral molecules can be directly “discriminated”. Among all techniques, liquid chromatography (LC) and supercritical fluid chromatography (SFC) are now the most used methods for enantioselective analysis in the pharmaceutical environment. Gas chromatography (GC) is still used, but more for volatile substances and amino acids. There are numerous applications of capillary electrophoresis (CE), including drugs, but most of these applications have been performed in academic environments. It is used less in the pharmaceutical industry, but might still be useful and even the best method for particular applications. Spectroscopic methods, such as nuclear magnetic resonance (NMR) spectroscopy, vibrational circular dichroism (VCD) spectroscopy, and mass spectrometry (MS), are also occasionally used, but not to the extent of the chromatographic methods.

As many chromatographic techniques are now available for the analysis of chiral molecules, the choice is usually driven by the application, that is, for volatile compounds, GC might be preferred, while for preparative purposes, LC or SFC will be favoured.

The types of application covered by the enantioselective analytical techniques are quite broad and the major ones are listed in Table 1. This article focuses on the contemporary enantioselective analytical techniques with an emphasis on pharmaceutical chiral compounds and, therefore, only the most contemporary approaches are discussed.


Analysis of Chiral Molecules by HPLC

Over the past 30 years, HPLC on chiral stationary phases has evolved as a powerful method to separate enantiomers. HPLC is applicable to enantiomers of almost all types of small chiral molecules, it is compatible with almost all kinds of functional groups (basic, acidic, neutral), and it can usually be applied directly without previous chiral derivatization to diastereoisomers. In the classical pharmaceutical environment, it concerns about 50% of the molecules, the others being achiral or more complex molecules, such as small and large peptides, monoclonal antibodies, and antibody–drug conjugates.

Hundreds of chiral stationary phases (CSPs) have been commercialized for HPLC, but the practical experience of the last 25 years has led to a ‘“natural” selection of the phases, and it is generally recognized that a relatively limited number of columns cover at least 90% of all applications (Figure 1). These “all-rounder” phases consist of polysaccharide derivatives, particularly from cellulose and amylose. It has already been shown more than 40 years ago that cellulose triacetate (CTA) has unique chiral recognition properties in enantioselective chromatography (1), and the very first enantioselective pharmaceutical application on this polysaccharide phase was reported more than 30 years ago (2). However, it is the process elaborated by Yoshio Okamoto and his group in Japan that has made possible the preparation of robust and high-performing polysaccharide-based CSPs, which have been commercialized and are suitable for validated analytical processes (3). Other column manufacturers have produced generic versions of these polysaccharide-based phases. Most of the reported applications were performed in the normal-phase mode, but the CSPs can also be used in the reversed-phase mode.

While normal-phase mode is generally applied to determine enantiomeric excesses of synthetic samples or for preparative purposes, reversed-phase conditions are usually preferred for samples obtained from biological matrices (stereoselective metabolism, toxicity, and chiral stability). Figure 2 shows a typical example of chiral separation, illustrating the increasing complexity of the investigated drugs (with multiple chiral centres) and the possibility offered by the available tools to achieve the separation of the four stereoisomers under normal-phase conditions or under polar organic conditions, which are more appropriate to investigate aqueous samples on polysaccharide-based CSPs.

As a result of the complexity of the mechanism of interaction with polysaccharide-based phases, it is almost impossible to predict the chiral separation on these phases. Small variations of the structure of the analyte, of the polysaccharide CSP, or of the mobile-phase composition can dramatically affect the separation, and even cause the inversion of the elution order of the enantiomers. Therefore, in most cases, method development is required to identify appropriate conditions to separate the enantiomers. Many factors can influence the separation, and it is common practice to screen a broad combination of CSPs and mobile phases. Numerous strategies have been proposed by different groups for screening chiral stationary phases (4–6). However, because of the rapidly evolving market for chiral columns, the setup has to be regularly adjusted. The screening is usually performed on single columns, which are successively tested, but this mode is not very efficient, particularly if a large number of chiral substances need to be processed. This is still the most common approach, but it is now widespread to use switching valves sequentially connected to the different columns in an automated fashion, which has the advantage of being a relatively low-cost setup.

The screening process can be accelerated by using an isochronal setup (7), but the use of HPLC systems capable of working in parallel clearly shows the highest effectiveness (8). Our group developed this strategy several years ago, and it is currently used as the standard procedure for chiral method development (8). Under the standard setup, using columns of 15 cm × 0.46 cm and a flow rate of 0.8 mL/min (20 min per run), up to 20 different conditions (CSP or mobile phases) can be tested each hour. The system is totally automated. The parallel setup might also be useful if the analysis of many samples of the same molecule is required, for example, for the screening of catalysts in asymmetric synthesis, for samples from biological investigations, or for samples from formulation optimization studies. In these cases, the same CSP is used on all channels of the parallel unit.

The necessity of running efficient parallel screening approaches for chiral method development has become even more essential since the introduction of the immobilized polysaccharide phases. Indeed, with this new generation of chiral columns, it is possible to apply strong solvating modifiers like ethyl acetate, tetrahydrofuran, dichloromethane, chloroform, or dimethoxymethane that are not tolerated by the classical non‑immobilized polysaccharide phases, considerably increasing the potential to improve the separation by modulating the mobile phase. This is a great advantage because it could help to optimize the retention times and selectivity, which are crucial parameters for enantioselective analysis.


Various approaches have been developed for the preparation of immobilized polysaccharide phases (9), including a particularly simple process that our group developed (7,8,10) and which has led to the introduction of several CSPs of this type on the market (11,12). Screening approaches incorporating these phases have been described (8,13).

For racemic substances that cannot be resolved on polysaccharide-based CSPs, a few other columns are generally tested in a secondary screening. All these phases have been designed by bonding small chiral molecules to silica gel. These phases include an amino tetrahydrophenanthrene-based phase (14), the cyclodextrin-based CSPs (15), the cinchona alkaloid ionic phases (16), the cyclofructan (17), the macrocyclic peptides (18), and glycoprotein phases (19), which together share about 10% of the applications of the enantioselective analyses performed in the pharmaceutical field. The quinine‑based phase (20), the α-1-acid glycoprotein (AGP) (or orosomucoid) (21), and the cyclofructan (22) phases might exhibit excellent chiral recognition power for polar or ionic compounds, which often fail on polysaccharide-based CSPs. Figure 3 shows the analytical separation of the enantiomers of free amino acids on the new quinine-based ionic CSP (20).

Thousands of pharmaceutical applications have been reported attesting to the effectiveness of the enantioselective HPLC technique. Modern enantioselective HPLC is very efficient (≤5-μm particle size) and an enantiomeric ratio lower than 99.9/0.1 can usually be easily determined. The method is also routinely applied for validated processes (23,24) and for enantioselective drug metabolism assessment (25). Figure 4 shows a recent application of the simultaneous quantification of the enantiomers of the drug lacosamide and its chiral impurity on an immobilized polysaccharide-based phase (24). Another typical application of a high enantiomeric excess (ee) determination for the drug Pemetrexed also exemplifies that even polar analytes can be well resolved on polysaccharide‑based phases (26).

It must also be mentioned that a considerable number of enantioselective methods are developed in the context of preparative separations or asymmetric synthesis of drugs and drug intermediates.
For this purpose the polysaccharide‑based phases dominate again, with more than 95% of the applications in the pharmaceutical industry.

Recently, it has been shown that ultrafast chiral HPLC can be achieved within a few seconds, but no practical applications have been reported so far (27–28).

Analysis of Chiral Molecules by SFC

Intensive research activities on supercritical fluid chromatography (SFC) in open tube capillaries and in packed columns have been performed since the early 1980s (29), but it took many years until the technique was really adopted. The poor acceptance was mainly a result of the limited robustness and low reliability of the available instrumentation. From the beginning of the new millennium, the situation radically changed with the significant improvement of the new SFC instruments, and it was, in fact, preparative enantioselective chromatography that rescued packed SFC (pSFC). Enantioselective separation has been the major SFC application for about 10 years and has now been adopted by most pharmaceutical companies for this purpose.

pSFC is very similar to normal-phase HPLC-it “just” uses liquid CO2 as the major component of the mobile phase. However, in its supercritical state, CO2 exhibits unique properties that gives it some advantages in terms of chromatographic performance because of the low viscosity of the fluid. Other advantages include speed, efficiency, safety, costs, and environmental impact (green technology) (30).

Even though SFC is usually achieved under sub-critical conditions, most of these advantages remain. These reasons have contributed to the rapid establishment of packed SFC as a powerful technique for enantioselective analysis. Moreover, the switch from LC to SFC was helped because the same “chiral” columns can be used for either LC or SFC.

The suitability of SFC for enantioselective analysis of drugs has been reviewed in a series of review articles and book chapters, containing an extended list of drug applications (31–36). However, enantioselective separations that can be achieved on a particular CSP by HPLC do not necessarily work under SFC conditions (and vice versa). This is not surprising because the mobile phases are different and such effects are observed in enantioselective HPLC on polysaccharide-based stationary phases when slight modifications of the modifier composition are performed. Based on our own experience, about 10% of the chiral pharmaceuticals are better or only resolved by HPLC, while the same proportion is better resolved under SFC conditions.

As for LC, any resolution of a new racemic compound in SFC requires method development. Several groups have reported on their own process flow (13,37–40), but because the chiral column market is constantly evolving, the setup has to be regularly adapted, even though the preferred columns remain those based on polysaccharides. Similar to HPLC, a parallel setup with eight columns operating simultaneously can be used for screening CSPs in SFC (40,41). The instrument permits the rapid identification of optimal conditions (chiral column, mobile phase) to perform the enantiomeric separations. Using a standard configuration with columns of 15 cm × 0.46 cm, a flow rate of 3 mL/min (7 min for each run), and a gradient of 5–40% modifier within 5 min, up to 60 different conditions can be tested within 1 h. In the primary screening, eight polysaccharide-based CSPs have been selected (40,41). Analogously to enantioselective HPLC, almost any combination of CO2 with organic modifiers is feasible with the immobilized polysaccharide-based CSPs because they tolerate most organic solvents (40,42).

Hundreds of pharmaceutical applications have been published and reported over the last 10 years and surely many more have been performed, in particular within the pharmaceutical companies that apply SFC for hundreds of new chiral molecules every year.

Although enantioselective SFC is now well adopted and even the method of choice in drug discovery in most pharmaceutical companies, there is only a very limited number of validated methods (43,44) because the SFC technique has not yet been fully embraced by the pharmaceutical development community. However, considering the availability of robust and high performing analytical instruments, and the recent publication of numerous papers demonstrating the possibilities of SFC for pharmaceutical investigations in a good laboratory practice/manufacturing (GLP/GMP) environment, it can be anticipated that this situation will rapidly change in the next few years (45,46).


Analysis of Chiral Molecules by GC

GC was a major technique for enantioselective analysis for two decades (1980–2000). It was researched intensively and reached a high level of advancement (47). Numerous stationary phases have been developed and a few have been commercialized. The most used phases are based on amino acids and cyclodextrins (Figure 1). GC is particularly appropriate for volatile molecules, which is not often the case for chiral drug compounds. However, it is regularly applied to determine the purity of volatile pharmaceutical intermediates and it remains a standard approach for enantioselective analysis of amino acids. Nevertheless, the introduction of the new cinchona alkaloid ionic phases, which show a wide application range, might change this in the future (20). These latter ionic phases have the advantage that they do not require a derivatization of the amino acids before analysis.

GC is very efficient and generally shows a high separation power. Very low selectivity values (alpha values lower than 1.1) are sufficient to achieve accurate ee determinations. Easy coupling of GC to MS was also a major advantage compared to normal-phase chiral separation for many sensitive and biological applications. The usefulness of GC for enantioselective analysis has been recently reviewed by C. Morrisson (48).

Recent applications include the chiral analysis of nonprotein amino acids (49) or pesticides in humans (50). The latest examples of validated methods are the enantioselective analysis for (L)-pidolic (51) and the chiral GC–MS method for the determination of free D-amino acids ratio in human urine for gestational diabetes studies (52).

Similar to LC and SFC, chiral GC requires method development. Although it is usually done by screening GC columns one by one, Schafer and colleagues proposed a parallel chromatography screening system (53). Interestingly, most of the current applications are still performed on the GC phases developed decades ago, but there is still some research ongoing to develop new and more efficient chiral GC columns. Recent developments include the utilization of cellulose derivatives in coated open tubular capillaries, which has been reported by Zhang and colleagues (54). The chiral nematic mesoporous silica column exhibits very good chiral recognition abilities for a broad variety of racemic compounds.

Enantioselective GC remains a popular technique for the analysis of chiral compounds and is the second technique in importance after LC.

Analysis of Chiral Molecules by Capillary Electrophoresis (CE) and Micellar Electrokinetic Chromatography (MEKC)

From the mid-1980s, enantioselective capillary electrophoresis and micellar electrokinetic chromatography have been the focus of intensive research. CE and MEKC have the ability to separate according to charges and are true orthogonal methods of analysis because they work through different mechanisms compared to GC, LC, or SFC.

Much academic work has been performed in CE and MEKC, and there is a vast amount of original papers on this topic. Various reviews compiling extensive lists of pharmaceutical applications are available (55–57). Interestingly, as for the other separation techniques, the chiral selectors derived from saccharides have developed as the most widely applicable. The cyclic oligosaccharides cyclodextrins clearly cover the majority of the applications. The method has undoubtedly some advantages in terms of cost and practical operation.

Although numerous applications for a wide variety of chiral drugs have been reported, most of the published works have been performed in academic environments and CE has not reached the level of acceptance of enantioselective LC analysis in the pharmaceutical industry. The technique is still suffering from its lack of robustness and relatively low sensitivity, even though some progress has been made to improve this (58). The question of the limited integration of CE in the pharmaceutical industry was recently discussed (59) and this trend is reflected by the small number of CE publications from this sector over the last three years. Nevertheless, a few practical applications have been achieved in the industrial pharmaceutical environment, including validated processes with UV or MS detection (60–63).

Analysis of Chiral Molecules by Spectroscopic Methods

Enantioselective analyses by spectroscopic methods have drawn the attention of many research groups for a long time. In particular, NMR using a chiral solvating agent (CSA) (64) has been, besides optical rotation (αD), a standard approach for the determination of optical purity before 1980, at a time where no chiral stationary phases were available. NMR using CSA is a simple method and does not need previous derivatization of the analyte with a chiral molecule to form diastereoisomers. It was much used until the end of the century, but because of its limited accuracy (± 3%) NMR has been progressively replaced by chromatographic techniques. The value of the technique has recently been reviewed (65) and applications to pharmaceutical drugs are still regularly published (66–68). A number of new chiral solvating agents showing excellent chiral discrimination properties were also developed (69), giving accuracy values approaching those of chromatography on chiral stationary phases but still lower (about 1% of the minor enantiomer). Nevertheless, it is definitely less broadly applicable than LC or GC.

MS has also been applied to determine ee (70), and the promise of the method has recently been reviewed (71). The feasibility of using an MS instrument solely for chiral recognition has clearly been demonstrated and a first practical application for the quantitative determination of the chiral purity of an antibiotic drug by flow-injection MS/MS has been reported by Wu and colleagues two years ago (72). However, this approach is not broadly applicable and it is likely that it will not reach the level of popularity of the chromatographic techniques.

VCD has also been reported to be a suitable technique for ee determination (73,74) and might be useful in particular cases (75,76), but it cannot compete with more generally applicable techniques for daily use.


Enantioselective Sensors

The development and application of enantioselective sensors is a topic of increasing interest and might possibly become a useful technique for ee determination. Progress in this field has recently been reviewed (77). Very recent applications include the discrimination of the enantiomers of amino acids or ascorbic acid by applying electrochemical processes (78–80) or the enantiomers of naproxen by recording change in the optical properties (colorimetry) (81). Although this approach requires the development of a specific sensor for each particular application, it can be of great interest in diagnostic or environmental analysis and has the advantage that it can be miniaturized.


A broad variety of chromatographic and spectroscopic approaches are available for the enantioselective analysis and determination of the chiral purity of chiral drugs. Among all approaches, chromatography is clearly the preferred technique. Methods are easy to elaborate with a relatively limited number of chiral stationary phases, mainly made from polysaccharide derivatives in LC or SFC, and from cyclodextrins or amino acids in GC, as the chiral selectors. Interestingly, the number of chiral selectors that have emerged as the most widely applicable is relatively limited, whatever the technique, and even more fascinating is that the great majority are composed of or contain a large amount of saccharides. Compared to the chromatographic methods, spectroscopic approaches are usually less accurate and are not so generally applicable, even though their usefulness has been demonstrated in some cases.


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Eric Francotte received his Ph.D. in organic chemistry from the University of Louvain in Belgium, and spent two years as a postdoctoral fellow at the University of Geneva in Switzerland. He joined former Ciba-Geigy (Novartis) in 1980 where he established a centre of expertise for the development and application of chiral polymers for the chromatographic resolution of chiral compounds. After 10 years he moved to Novartis pharma as an executive director and was responsible for a global technology platform dealing with separation sciences. His major achievements include the invention of an innovative process to immobilize polysaccharide derivatives as chiral stationary phases. Several phases arising from this technology have been marketed as the result of an external collaboration and are considered as the new gold standards in enantioselective chromatography. He was also a pioneer in the implementation of new chromatographic technologies, such as SMB and SFC. He is the author or co-author of numerous publications, patents, and editor of several books. He has chaired various international symposia in the field of preparative chromatography, chirality, and SFC. He is now an independent consultant for separation science.