Supercritical Fluid Chromatography for Chiral Analysis, Part 1: Theoretical Background

The quantification of the enantiomers of racemic substances is of great importance in the development and regulation of pharmaceutical compounds. Active ingredients are often chiral; typically, only one of the stereoisomers has the desired pharmacokinetic and/or pharmacodynamic properties. Therefore, the stereoisomer distribution of chiral drug products must be characterized and evaluated during the drug discovery and development pipeline. Moreover, various chiral drugs present a stereoselective metabolism, highlighting the need for appropriate analytical strategies for the stereoselective analysis of metabolites, for example, in clinical and environmental studies. Due to its ease of use, robustness, and transferability, chiral liquid chromatography (LC) is the most common approach used in pharmaceutical analysis. Compared with LC, supercritical fluid chromatography (SFC) allows higher linear flow velocities while maintaining high chromatographic efficiency, often enabling the reduction of analysis time. In addition, SFC provides enhanced or complementary chiral selectivity and avoids or reduces toxic solvents, such as those used in normal-phase LC. In the first part of this review article the theoretical advantages, technological developments, and common practices in chiral SFC are discussed. This will be followed by a contribution discussing recent applications in pharmaceutical, clinical, forensic, and environmental analysis.

Chirality is of great importance in the development and regulation of new pharmaceutical compounds (1). When a racemic pharmaceutical compound, or drug in general, is introduced into the human body, its pharmacological activity may be different than expected due to a varying interaction between stereoisomers with their intended biological target—such as a receptor or an enzyme (2). Cells, organisms, and living systems are chiral; interactions with enantioselective ligands therefore result in different outcomes. Stereoisomers are composed of the same type of atoms and functional groups. However, in most cases, one of the stereoisomers has a better fit with the drug target and shows the desired activity; it is therefore considered the active pharmaceutical ingredient (API). The other stereoisomer behaves differently—that is, it shows different, none, or higher activity, and may form a potential threat to the human body. Although drugs can be produced and administered as single enantiomers with high enantiomeric excess (> 99%), the enzymes present in the human body or the configurational instability of the chiral centres can be the cause of a rapid configurational inversion of the pure enantiomers, as well as of their chiral metabolite(s), resulting in a different stereoisomer ratio (3). The converted stereoisomers are usually inactive towards the target but can also exhibit their own biological activity, which may be potentially toxic or lead to unwanted side effects. An example is the in vivo chiral inversion of thalidomide (4), which caused a worldwide tragedy of congenital disabilities in the 1960s through the intoxication of the embryo or foetus (2). Indeed, (R)-thalidomide gives the desired pharmaceutical effects, while (S)-thalidomide is highly teratogenic. It was later assumed that pure (R)-thalidomide could prevent the teratogenic effects. However, its chiral centre was found to be unstable in the human body, leading to a rapid inversion into a racemic mixture catalyzed by enzymes, such as human serum albumin (4).

Owing to the innovative technologies developed in the 1980s for pure enantiomer production and the advances in chiral selective bioanalysis, the industry and regulation agencies became aware that many of the developed drugs are actually racemic mixtures of stereoisomers (5). This caused a switch in the drug development pipeline because the synthesis of pure enantiomer drugs was considered both beneficial and crucial to preventing dangerous and/or unexpected side effects. By administering one single drug enantiomer, a lower dose is typically required, toxicity is minimized, and complex drug-drug interactions are reduced (6).

The stereoisomers present in a pharmaceutical formulation must be individually characterized to be approved by regulatory agencies. Enantiomers have identical chemical and physical properties, highlighting the need for state-of-the‑art analytical technologies to separate, detect, identify, and possibly quantify these synthesized drugs to determine their purity. Different analytical techniques can be used for enantioselective separation. Gas chromatography (GC), capillary electrophoresis (CE), and liquid chromatography (LC) are the preferred techniques, with LC being the method of choice for non-volatile analytes due to its ease of use, simplicity, and advanced instrumentation (7). LC does have some limitations, such as a relatively long equilibrium, long analysis time, and relatively slow diffusion process. These limitations have increased the interest in developing separation techniques that could further improve the performance of chiral separation methods, such as supercritical fluid chromatography (SFC) (7). Due to the low viscosity and high diffusivity of supercritical CO₂, higher mobile phase velocities can be used—without compromising efficiency— with lower back pressure. Since the substantial developments of modern instrumentation at the beginning of the 2010s, significant improvements have been made, leading to the first commercialized advanced SFC instruments displaying higher robustness, reproducibility, and sensitivity (8). In addition, the introduction of novel column dimensions with sub-2-μm (ultrahigh-performance supercritical fluid chromatography [UHPSFC]) and core–shell particles has led to significantly improved efficiencies and analysis speed. In combination with the unique features of supercritical fluids, highly efficient separations are achievable using relatively high flow rates and low back pressures. Other advantages are shorter analysis time, faster equilibration time, reduced costs, and a lower environmental impact due to the use of CO₂. This makes SFC a promising alternative for enantiomeric selective separation of chiral products.

Chiral Supercritical Fluid Chromatography

History of Supercritical Fluid Chromatography: SFC was first introduced almost 60 years ago by Klesper et al. under the terminology high pressure gas chromatography (HPGC) (9); this was done with little attention as GC was already a well-established analytical technique. SFC relies on the use of CO₂ in the mobile phase, with relatively low critical pressure and temperature (7.38 MPa and 31.1 °C, respectively) needed to reach supercritical conditions (10). In the 1980s, Lee and co-workers introduced open-tubular capillary SFC (cSFC) (11). Due to the nonpolar nature of CO₂, cSFC was mainly limited to the petrochemical industry and was quickly replaced with packed-column SFC (pSFC). pSFC was brought to light in the 1970s by modifying a high performance liquid chromatography (HPLC) system with a back pressure regulator. In 1985, Mourier et al. performed the first chiral application using SFC (12). The addition of organic modifier and small percentages of additives to the CO₂-based mobile phase enabled the analysis of more polar compounds, increasing its application range.

In general, all stationary phases available for LC can also be used for SFC, which is also true for chiral stationary phases. Though there are a great number of LC columns, SFC-specific stationary phases were introduced in the 2000s (2-ethylpyridine [2-EP] phase), aimed at improving the peak shape for basic compounds. 2012 marked the most important instrumentation breakthrough for pSFC with (i) the commercialization of hybrid ultrahigh-pressure liquid chromatography (UHPLC)–SFC systems with vastly improved back pressure regulators, flow rate stability, and automation capabilities; and (ii) the introduction of UHPSFC, which enabled the use of modern columns with sub-2‑µm particles. The chronological evolution of SFC is illustrated in Figure 1 (9).

Chiral Selectors: Unlike diastereomers, enantiomers cannot be separated using standard (achiral) column stationary phases due to their identical physicochemical properties. Chiral separation using chromatographic techniques is achieved using either an indirect or a direct approach (13,14).

The indirect approach is based on the derivatization of the enantiomers using a chiral derivatizing agent (CDA) with high enantiomeric purity (enantiomeric excess) to form two diastereomers. Diastereomers have distinct physicochemical properties and can be resolved by achiral LC methods. However, the purity of the derivatization agent and possible problems with the yield and/or racemization during the derivatization reaction can influence the estimation of the enantiomeric purity of the compounds studied.

For this reason, the most common strategy used in SFC and LC is the direct approach, using stationary phases in which the selector has one or more chiral elements (chiral stationary phases [CSPs]) (15). The chiral selectivity provided by the CSPs is based on the difference in the chiral recognition between the chiral selectors and the enantiomers. Indeed, due to their different spatial configurations, the enantiomers experience different bonding energies and fit differently into the chiral reaction sites (14). Therefore, one enantiomer fits better and interacts with the binding site of the chiral selector, while the other only binds partially and experiences weaker interactions. If this difference is pronounced enough, the two enantiomers can be separated from each other (16).

Chiral Stationary Phases:

In chiral SFC, the CSP is the most critical parameter to achieve chiral resolution. In the past decades, hundreds of different CSPs have been developed, covering a large variety of chiral structures. However, only a limited number of those stationary phases are commercially available. This large variety of enantiomeric stationary phases is needed because there is no universal chiral selector developed yet that is capable of separating a diverse mixture of enantiomeric isomers (17). Most CSP columns are specially developed for LC purposes but have been successfully employed for SFC as well. It is difficult to select the adequate column for a specific chiral selective separation without preliminary experiments. Therefore, multiple columns and mobile phase combinations are usually experimentally screened to obtain the most suitable enantiomeric selectivity. The CSPs are assigned to different categories based on the type of chiral selector. Chiral columns commercially available include (immobilized) polysaccharides, cyclodextrins, macrocyclic glycopeptides, “Pirkle”-type, ion-exchange-type, and crown ethers (18,19) (Figure 2).

Polysaccharide-based stationary phases are the most common phases used in chiral SFC due to their numerous advantages, including wide application range, repeatability, high loadability, and a large diversity of phases commercially available. With polysaccharide stationary phases, the compounds form hydrogen bonds with the carbamate linkages between the side chains and the helical polysaccharide backbone. The helical polysaccharide backbone gives rise to steric restrictions that may inhibit access of one of the enantiomers to hydrogen‑bonding sites, enabling the separation between enantiomers (20). However, underivatized cellulose and amylose show insufficient enantioselectivity due to their dense helical structure. Therefore, electron donor or electron-withdrawing groups have been added onto the polysaccharide backbone. Tris-(3,5-dimethylphenylcarbamate)-derivatives of cellulose and amylose are the most popular phases, with a large diversity of columns commercially available. Most polysaccharide-based selectors are coated onto a silica support. However, this method suffers from poor polar solvent compatibility used in normal- and reversed-phase conditions and is more prone to selector bleeding. For this reason, immobilized chiral selectors have been developed, showing in most cases similar selectivity to their coated counterpart and extending the solvent range applicable during method development (20).

Cyclodextrins are composed of six to 12 D-(+)-glucopyranose units connected through α-1, 4-linkages with a hydrophobic interior cavity and a hydrophilic exterior surface displaying hydroxyl groups. The most commonly used cyclodextrins are α-, β-, and γ-cyclodextrins, which contain six, seven, and eight glucopyranose units, respectively. Cyclodextrins are frequently linked to silica support using a spacer. Chiral recognition is based on the inclusion of the (bulky) hydrophobic group of the analyte into the hydrophobic interior of the cyclodextrin, as well as dipole-dipole interactions and hydrogen bonding with the hydroxyl groups located at the surface of the cyclodextrin (20). Recently, modified cyclodextrins (for example, phenylcarbamate) have been developed and commercialized, demonstrating improved enantioselectivity and a wider range of applicability.

Similar to cyclodextrins, crown ethers allow for enantioselective separation based on the analyte entering the chiral cavity of the crown ether to form complexes (21). Crown ethers are macrocyclic polyethers that can form selective complexes with several cationic species via ion-dipole interactions and/or hydrogen bonds (22). Specifically, crown ether-based CSPs are well-suited for the separation of racemic compounds containing a primary amino group (23,24).

Chiral separations using selectors based on immobilized macrocyclic glycopeptides, such as vancomycin, teicoplanin, teicoplanin aglycone, and ristocetin, have also been reported (25). These glycopeptides contain numerous chiral centres and enable the separation of enantiomers showing different functionalities and polarities (20). The selectors are bonded onto the silica support and remain stable under typical SFC conditions but long re-equilibration times may be necessary. The primary interactions occurring between the target analytes and macrocyclic glycopeptides include hydrogen bonding, dipole-dipole interactions, π-π interactions, hydrophobic interactions, and steric repulsion (26).

Pirkle-type chiral stationary phases contain a low-molecular-weight chiral molecule that is covalently bound to the silica support. Racemates are resolved via similar interactions to those occurring with macrocyclic glycopeptides. These phases usually favour nonpolar solvents, which makes them compatible with SFC. Pirkle-type selectors show some interesting advantages, including the separation of a wide range of compounds, column durability, and compatibility with many solvents. Moreover, selectors with opposite configurations (for example, both enantiomeric configuration) are available, allowing methods to be developed in which the elution order can be inverted depending on the configuration of the selector. This characteristic is particularly important when investigating chiral impurities, where having the enantiomeric impurity eluting before the main enantiomer may be beneficial (20).

Ion-exchange CSPs have been developed specifically for the enantiomeric separation of ionic species, such as acids and amines and, recently along with the introduction of zwitterionic chiral selectors, underivatized amino acids (19,27–29). Besides the ion-pairing mechanism, additional electrostatic interactions must also be present (H-bonding, π-π, steric, and Van der Waals) to enable enantioseparation (19). For anionic chiral molecules, quinine and quinidine are used as weak anion exchange selectors. For cationic species, a syringic acid derivative is used as a strong cation exchange phase (19). Enantioselectivity and peak shapes can be enhanced with the addition of acidic or basic additives stimulating the elution of ionic molecules and, in the case of basic additives, shielding of free silanol groups to reduce peak tailing. Retention factors within these ion-exchange columns can be adjusted by changing concentration and the type of counterion, while the enantioselectivity of the chiral phase is not or is only minimally affected (28).

The main advantage of ion-exchange selectors in SFC is the in situ formation of acids and volatile salt additives, which stimulate, as additional additive and counterion, the replacement process within the ion-exchange mechanism. These compounds are generated by pressurizing CO₂ together with a polar protic modifier (methanol) and a base additive (amine). As these reactions are in equilibrium, the formation of these additives can be controlled by changing the percentage of polar organic modifier, the concentration and types of basic additives, and the applied temperature and pressure for supercritical fluid conditions.

Composition of the Mobile Phase: CO₂ is by far the most used mobile phase in SFC, although other fluids have also been explored, including lower alkanes, xenon, and ammonia (10). Indeed, CO₂ presents advantageously low critical parameters, that is, T_c = 31 °C and P_c = 74 bar. Moreover, CO₂ shows low toxicity, low environmental impact, high detector compatibility, high inertness, and is very cost-effective. The mild critical parameters also allow for the analysis of thermally degradable compounds.

Due to the symmetry of its bonding, CO₂ is a nonpolar solvent, with an elution strength similar to pentane and hexane. The solvation power of CO₂ can be controlled through variation of pressure and temperature, both affecting density, but this is often not sufficient for the elution of more polar compounds. Therefore, the analysis of polar compounds requires the addition of organic modifiers and other additives in the mobile phase. The proportion of organic modifier can vary between 1–50% (v/v) in both isocratic and gradient modes; in chiral separations, the proportion is in the range of 10–20% to enhance enantiomeric resolution (26). In this case, the limited elution strength favours the chiral recognition process because the enantiomers remain for a more extended period within the chiral environment. The addition of modifiers increases the critical pressure and temperature values substantially (30). Therefore, supercritical states may not be reached and the SFC separations are commonly performed in the subcritical state (27). Modifiers added to the mobile phase favour the separation because they ensure the deactivation of active sites on the surface of the column supporting material (30). Furthermore, they enhance the selectivity, as H-bond and dipole-dipole interactions are formed between the solvent and the analyte.

Protic organic modifiers, such as methanol, ethanol and isopropanol, are preferred in SFC separations compared with aprotic solvents. These hydrogen bond donor solvents prevent the hydrogen bond acceptor silanol groups acting on the hydrogen bond donor analytes. These adverse interactions may otherwise affect the separation and cause peak distortion. Alcohols are seen as the most beneficial modifier for SFC analysis, with methanol being the most popular (31), as it shows the highest eluotropic strength and gives better efficiency than ethanol or isopropanol (32). Protic solvents provide optimal coverage of the (free) silanol groups and show good separation efficiency. Aprotic solvents (for example, acetonitrile) are rarely used, as their hydrogen bonding capability is insufficient. Although these types of solvents improve resolution between polar protic enantiomers, they cause long retention times and peak distortions (26). Moreover, they are associated with potential adsorption onto the stationary phase and, therefore, change the sterically positioning of the chiral selector. The enhancement in resolution observed with acetonitrile is due to fewer analyte interactions compared with alcohols; for instance, acetonitrile limits interactions initiated by the hydroxy groups of native and derivatized polysaccharide‑based CSPs (26). This allows easier access to the chiral cavity of the chromatographic selector and enhances chiral resolution for this type of analyte.

Non-conventional solvents such as dichloromethane, ethyl acetate,
and tetrahydrofuran have been studied as well. In this context, the chiral stationary phase must be robust and compatible with these solvents. Pirkle-type or immobilized CSPs are both suited due to their high resistance to strong elution solvents. In SFC, these “exotic” solvents are mostly used in dedicated applications where traditional modifiers do not provide adequate separations or are not compatible with the analytes of interest, such as alcohol-sensitive molecules.

Organic modifiers alone may not be sufficient to achieve optimal (enantioselective) separation, as their interaction with the free silanol groups affects the elution and peak shape of polar and ionic compounds (30). The presence of additives in the mobile phase is therefore recommended to further improve the separation. Several mechanisms responsible for the improved performance have been discussed, such as covering of the free silanol groups, changes in mobile phase polarity, enhanced solvation power, lower ionization suppression, and formation of ion-pairing complexes. Acidic additives such as formic acid, acetic acid, or trifluoroacetic acid are conventionally used for the separation of acidic compounds, while bases such as diethylamine, triethylamine, or isopropylamine are used for basic compounds. Such additives are often used in combination to optimize the separation of various acidic, neutral, and basic compounds simultaneously. Typically, additive concentrations of 0.1–2% (v/v) result in significantly improved SFC separation, selectivity, and peak shape. Due to the acidic nature of CO₂ in combination with methanol (carbonic acid formation), some additives may not always be necessary for the separation of acidic compounds due to in situ additive formations (28). Organic salt additives such as tetramethylammonium acetate, ammonium acetate, and ammonium formate are used as well to improve separation and detection. Notably, ammonia salts and other volatile species bring interesting advantages, as they are compatible with mass spectrometry (MS) and can be easily removed from the mobile phase after separation (8).

Blackwell et al. found that any additive (acid, neutral, or basic) improves separation efficiency in SFC, mainly due to additional covering of silanol groups (33). Effects on retention factors and selectivity of compounds are significant as well, including chiral selectivity of enantiomeric species. Additives show stronger adsorption onto the CSP surface than methanol, affecting the recognition processes of chiral molecules (33). Thus, the spatial arrangement of the analytes is of great importance, that is, whether it enables stronger or weaker enantioselective interactions. However, it is still unclear why these additives provided improved separations in some studies but were deleterious in other applications (31).

Due to its poor miscibility in CO₂, water cannot be used as a primary polar modifier to separate polar compounds (34). However, it represents an interesting additive in combination with an organic modifier and using a concentration ranging from 1–10%. The polarity of water is higher than alcohols; moreover, water can form two hydrogen bonds. In combination with the analyte partitioning capabilities, the solvating power and the elution strength for polar compounds are enhanced. These effects are most significant in combination with highly polar modifiers. Moreover, the presence of water in the mobile phase improves the compatibility of the SFC eluent with MS. In chiral separation, and similar to other additives, water affects the recognition process of the chiral molecules, modifying the separation efficiency (33,35). The mechanism responsible for the improved separation efficiency using water in SFC is not yet fully understood (27).

Detectors for Chiral Supercritical Fluid Chromatography

The detectors traditionally used with LC and GC are, with minor adjustments, also compatible with SFC systems. Whether a detector is suitable for a specific application is mainly dependent on the composition of the mobile phase (13). While flame ionization detection (FID) is only applicable for neat CO₂ mobile phases, detectors such as ultraviolet–visible light (UV–vis), aerosol-based detectors, and MS are common, as they are compatible with the use of organic modifier and volatile additives.

Non-Chiral Detectors: Non-chiral detectors used in chiral SFC analysis belong to two main groups, pre-decompression and post-decompression detectors, which can both be used in the same system. In pre-decompression techniques, the detector is usually located after the SFC column and before the back pressure regulator (BPR). Alternatively, the post-decompression detection takes place after the BPR in gas phase, or, in the presence of an organic modifier, in a mixture of gas and liquid phase (36).

Pre-Decompression Detectors: Detectors based on UV–vis absorbance belong to the most common detection techniques used with SFC, since CO₂ does not absorb light above 190 nm. However, the addition of organic modifiers to the mobile phase may alter the UV–vis cut-off as observed with methanol and 2-propanol, which both have a UV–vis cut-off of 205 nm. The UV–vis cell design is similar in SFC to that in conventional HPLC, except that the cell must be able to withstand the high pressures generated by the compressed CO₂. UV–vis sensitivity in SFC is generally lower than in LC. Indeed, the solvating power of the mobile phase has a direct impact on the UV–vis absorbance of the analyte, as well as the refractive index. As the solvating power of CO₂ is dependent on the pressure and temperature, the system needs to keep these parameters as steady as possible to ensure low UV–vis background noise (37). Hence, newly designed BPRs with lower pressure fluctuations have been developed to improve the SFC-UV–vis noise (38).

Fluorescence detection has superior selectivity and sensitivity compared with UV–vis detection, but is limited to compounds exhibiting fluorescence (39). In SFC, fluorescence detection is performed using a high-pressure cell, similar to UV detection. Similar to what is observed with UV–vis detectors, pressure and temperature fluctuations also have a significant impact on the background noise. Moreover, mobile phase modifiers and additives can affect excitation or emission wavelength, as well as cause quenching of the fluorescence signal (39).

Post-Decompression Detectors:

An evaporative light scattering detector (ELSD) is a universal detector commonly used in LC, where the mobile phase is first completely evaporated prior to detection of dried analyte particles using scattered light. The fast evaporation of CO₂ aids in the nebulization process but the decompression also results in a cooling effect, which limits the nebulization and evaporation processes. Sufficient heating is therefore required to counteract this cooling effect when using SFC-ELSD. Compared with FID, ELSD is less prone to background noise, as it tolerates organic modifiers, but some non-volatile SFC additives can lead to baseline noise.

Due to its high sensitivity and selectivity, as well as the possibility for obtaining structural information, MS represents the most powerful detection technique in combination with chiral SFC. Similar to open-cell detectors, SFC–MS shows the advantage of fast nebulization, as the CO₂-based mobile phase quickly evaporates at the capillary outlet. This makes SFC suitable for different atmospheric-pressure ionization sources. However, when combined with SFC, advanced interfaces for a controlled depressurization between the column outlet and the MS detector are required, maintaining the separation efficiency while providing optimal MS detection. Similar to ELSD and charge-aerosol detection, a make-up solvent or “sheath” liquid should be added prior to detection to minimize analyte precipitation. Figure 3 shows the five current main interfaces developed for SFC–MS, two of which are commercially available (36). These interfaces are called i) “direct coupling”, ii) “pre-UV and BPR splitter without sheath pump”, iii) “pressure control fluid”, iv) “pre-BPR splitter with sheath pump”, and v) “BPR and sheath pump with no splitter”, as summarized by Guillarme et al. (40).

Additionally, in chiral separation, chiral detectors play an important role because they allow selective detection of chiral molecules and give further information about the compound structure. There are two main chiral detection techniques available: optical rotation (OR) and circular dichroism (CD) detectors (41). An OR detector measures the difference in the refractive index of enantiomers, while the CD detector measures the difference in optical absorption. As OR is based on the assessment of the refractive index, it is the more general detector but also suffers from baseline instability due to alteration of the refractive index with changing conditions (temperature, density, mobile phase composition). In contrast, the baseline of a CD is as stable as an UV–vis detector (42). In terms of molecular information, an OR detector provides the optical polarity of the enantiomer (+ or -), while the CD detector simultaneously provides circular dichroism and UV absorbance data, and enables the identification of the CD polarity of each enantiomer.

Discussion and Conclusion

Substantial developments in the field of SFC and chiral SFC have been made over the past decade, allowing for highly efficient and sensitive analysis of a large diversity of (chiral) analytes. These developments include improved BPR, continuous flow, and detection techniques implementing pressure-resistant flow cells or sophisticated MS interfaces. Additionally, the introduction of hybrid SFC/UHPLC and UHPSFC systems with sub-2-μm porous and core–shell particles have provided significant improvements in separation efficiency, while enabling the use of relatively high flow rates.

These optimal features place SFC as a competitive technique for enantioselective separations in addition to conventional LC, which nowadays remains the most widely used approach for chiral analysis. Both techniques provide comparable enantiomeric resolutions, as demonstrated in various studies. The chiral separation mechanisms are difficult to compare, as specific interactions for chiral recognition are formed in a slightly different manner in SFC compared with LC, mainly due to the presence of solvent-stationary phase interactions. Depending on the mobile phase composition, chiral stationary phases show different polarities and spatial configurations between LC and SFC. This may lead to different retention factors and/or elution orders, making chiral SFC complementary to chiral LC. Nevertheless, SFC can resolve enantiomers 3 to 5 times faster, which opens possibilities for high‑throughput analysis. Moreover, particularly compared with normal‑phase LC-based chiral separations, chiral SFC significantly reduces the consumption of toxic solvents, which leads to environmental, financial, and safety benefits. Table 1 summarizes the relevant advantages and drawbacks of chiral SFC, supported by recent literature. An overview of recent literature describing applications using chiral SFC in pharmaceutical, clinical, forensic, and environmental analysis will follow in a future issue of LCGC Europe.

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Gerry Roskam, Bas van de Velde, Andrea Gargano, and Isabelle Kohler are with the Centre for Analytical Sciences Amsterdam, in Amsterdam, in The Netherlands. Roskam and Gargano are also with the van ‘t Hoff Institute for Molecular Science, at the University of Amsterdam, in Amsterdam, in The Netherlands. Roskam, van de Velde, and Kohler are also with the Division of BioAnalytical Chemistry in the Amsterdam Institute of Molecular and Life Sciences, at Vrije Universiteit Amsterdam, in Amsterdam, in The Netherlands.