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, (

)-thalidomide gives the desired pharmaceutical effects, while (

)-thalidomide is highly teratogenic. It was later assumed that pure (

)-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 CO

, 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 CO

. 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 

 (HPGC) (9); this was done with little attention as GC was already a well-established analytical technique. SFC relies on the use of CO

 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 CO

, 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 

 performed the first chiral application using SFC (12). The addition of organic modifier and small percentages of additives to the CO

-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).

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).

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 CO

 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.

 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, CO

 presents advantageously low critical parameters, that is, T

 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.

 is a nonpolar solvent, with an elution strength similar to pentane and hexane. The solvation power of CO

 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 CO

 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).

 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).

, 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 CO

 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 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).

Detectors based on UV–vis absorbance belong to the most common detection techniques used with SFC, since CO

 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 CO

. 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 CO

 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).

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 CO

 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 CO

-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 

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.

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 

R. Jayakumar, R. Vadivel, and N. Ananthi, 

M. Reist, P.-A. Carrupt, E. Francotte, and B. Testa, 

M. Gumustas, S.A. Ozkan, and B. Chankvetadze, 

K. De Klerck, D. Mangelings, and Y. Vander Heyden, 

V. Pilařová, K. Plachká, M.A. Khalikova, F. Svec, and L. Nováková, 

E. Klesper, A.H. Corwin, and D.A. Turner, 

Supercritical-fluid chromatography in packed and open-tubular columns

 (1991). doi:https://doi.org/10.6100/IR357895

M. Novotny, S.R. Springston, P.A. Peaden, J.C. Fjeldsted, and M.L. Lee, 

P.A. Mourier, E. Eliot, M.H. Caude, R.H. Rosset, and A.G. Tambute, 

Introduction in Chiral Sep. by Liq. Chromatograhpy Relat. Technol., 

H.Y.Aboul-Enein and I. Ali, Eds. (Marcel Dekker, Inc., New York, USA, 2003), pp. 1–20.

Recognition Mechanisms of Chiral Selectors: An Overviewin Chiral Sep.

, 1–33 (2019). doi:10.1007/978-1-4939-9438-0_1

D. Wolrab, M. Kohout, M. Boras, and W. Lindner, 

J.M. Płotka, M. Biziuk, C. Morrison, and J. Namieśnik, 

N. de Koster, C.P. Clark, and I. Kohler, 

Y. Vander Heyden, D. Mangelings, N. Matthijs, and C. Perrin, in 

Chiral Separations in Handb. Pharm. Anal. by HPLC

, S. Ahuja and M.W. Dong, Eds. (Elsevier Inc., 2005), pp. 447–498.

C.M. Galea, Y. Vander Heyden, and D. Mangelings, in 

N. Kolderová, B. Jurásek, M. Kuchař, W. Lindner, and M. Kohout,

C. Brunelli, Y. Zhao, M.-H. Brown, and P. Sandra, 

J.A. Blackwell, R.W. Stringham, and J.D. Weckwerth, 

Y. Zhao, G. Woo, S. Thomas, D. Semin, and P. Sandra,

B. van de Velde, D. Guillarme, and I. Kohler, 

A. Dispas, H. Jambo, S. André, E. Tyteca, and P. Hubert, 

R.M. Smith, O. Chienthavorn, N. Danks, and I.D. Wilson, 

D. Guillarme, V. Desfontaine, S. Heinisch, and J.-L. Veuthey, 

 are with the Centre for Analytical Sciences Amsterdam, in Amsterdam, in The Netherlands. 

 are also with the van ‘t Hoff Institute for Molecular Science, at the University of Amsterdam, in Amsterdam, in The Netherlands. 

 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.

Articles

Peer Reviewed

With the substantial developments carried out over the past years in instrumentation, columns, and detector hyphenation, the interest in chiral supercritical fluid chromatography (SFC) has been steadily growing in various fields. In the first part of this review article, the theoretical advantages, technological developments, and common practices in chiral SFC are discussed. 

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

Choosing a liquid chromatography (LC) column for a particular application can be a surprisingly challenging task. On the one hand, column manufacturers provide us many options to choose from, including particle types, pore sizes, particle sizes, and different lengths and diameters. On the other hand, we usually do not have time to experimentally evaluate many combinations of these parameters, and sometimes we end up picking something similar to the columns that are already available. The “kinetic plot” is a powerful graphical tool that can help leverage the best available theory to help us understand how different combinations of parameters (that is, particle size, length, among others) will perform in regard to the time needed to get to a particular column efficiency (and thus resolution), and therefore make well-informed decisions when choosing columns.

In last month’s installment of “LC Troubleshooting,” we discussed the basic idea of a “kinetic plot” and how we can ultimately use this graphical tool to make informed decisions when choosing columns, and understand why a column might not be delivering expected performance improvements. The fixed-length kinetic curve is a transformation of the same information in a conventional van Deemter curve with the added benefit that the kinetic plot can also easily take into account pressure limits of a liquid chromatography (LC) system (1). This month, we continue this discussion by first considering different ways of constructing fixed-length kinetic curves, either from experimental data or from data obtained from other sources, and then going on to show how kinetic performance limit (KPL) curves can be obtained by extrapolation starting from a single set of 

 data (that is, a van Deemter curve). Finally, we discuss the effect of particle size on the KPL curves, and show how different particle sizes are optimal for different ranges of analysis time and plate numbers.

Construction of Fixed-Length Kinetic Curves

Figure 1 shows the relationship between the conventional van Deemter curve representation and the dependence of the plate height on the mobile phase velocity. This relationship was discussed in detail in Part I of this series, but it is repeated here because it plays a central role in the continuing discussion. Here, 

 is the column efficiency (plate number), 

 is the mobile phase velocity. It is critically important to understand that all the data in the figure corresponds to a column with a fixed length, and that moving along each of the curves corresponds to a change in the flow rate, and thus, a change in the pressure drop across the column.

There are multiple methods to obtain the fixed-length kinetic curve in Figure 1b:

 can be obtained directly from experiments with a single column where the flow rate is varied.

 values from a van Deemter curve, they can be used to calculate N and t

 can be calculated using parameters reported in the literature that have been extracted from experimental data measured elsewhere.

The first two methods are conceptually straightforward and don’t require further explanation here. Making the experimental measurements in a way that yields high quality data does require attention to some details. Readers interested in this topic are referred to the tutorial by Andrés and others (2).

The third method can be approached by starting from a reduced van Deemter curve, which is the dimensionless form of the relationship between plate height (

 to give the dimensionless “reduced plate height”:

Similarly, the velocity is converted to a “reduced velocity” (

 is the diffusion coefficient of the analyte in the mobile phase:

The reduced velocity is also called the “Peclet number” in chemical engineering. The advantage of relating the plate height to the mobile phase velocity in this reduced format is that it enables the prediction of the effect of particle size and diffusion coefficient on plate height, and thus plate number. The reduced form of the van Deemter equation is then written as:

The typical values for the A, B, and C parameters are 1, 3, and 0.1 for fully porous particles and 0.8, 3, and 0.06 for superficially porous particles (SPPs). Typical values for the diffusion coefficient of small molecules under LC conditions are approximately 5×10

 is between 1 and 50. If we first calculate 

 using equation 1, the fixed length kinetic curves can obtained in the same way as method 2 after a column length is chosen.

Extrapolation from a Fixed-Length Kinetic Curve to the KPL

As we discussed in Part I of this series, one of the practical limitations of the conventional van Deemter curve is that it does not explicitly account for pressure limitations of a given column or LC system. In principle, one could address this limitation by making plate number measurements with columns of different lengths at different flow rates, and then plot a series of van Deemter curves or fixed-length kinetic curves, as shown in Figure 3 of the previous installment. However, this process is tedious and expensive (in both time and money). Instead, similar results can be obtained by extrapolation of the 

 data obtained for a single column length. The principle that underlies this extrapolation is that the plate height, which is a length independent measure of the column performance, will remain constant if one operates at the same flow rate (or velocity) but uses a column with a different length. For example, if an experiment at a flow rate of 1 mL/min on a column yields 10,000 plates in a t0 time of 1 min, then operating a column twice as long at the same flow rate will yield 20,000 plates, but at the cost of an increase in analysis time by a factor of two (that is, 

 = 2 min). The extent to which one can increase the column length at a fixed flow rate depends on the pressure drop—that is, one can only increase the length until the maximum pressure limit of the system, column, or the user’s preferred maximum operating pressure is reached. Of course, there will be some errors made in making these extrapolations (for example, secondary effects on the observed plate number from extra-column dispersion, viscous heating, pressure effects on mobile phase density, viscosity and retention cannot be accounted for this way); however, the extrapolation results are generally accurate enough to support method development and troubleshooting (3). Readers interested in more detail on this topic are referred to the supplementary material of (4) and the experimental work described in (5).

Figure 2 illustrates the concept of extrapolation from a single fixed-length curve to establish the KPL curve for a given particle size. Each dashed black line connects a point on the fixed-length curve (blue) to a point on the KPL curve (red), where the two points correspond to the same mobile phase velocity (and flow rate). Starting from the blue curve, the column length is increased until the pressure drop across the column reaches a prescribed limit (400 bar in this case). Repeating this process for each point on the blue curve results in the complete red curve, where each point corresponds to a different column length, but the same pressure maximum (Δ

) of 400 bar. This process is where the idea of “Kinetic Performance Limit” comes from—it is the limit in the sense that more efficiency cannot be obtained by making the column longer because doing so would exceed the pressure limit (4).

To calculate the KPL curve starting from the fixed-length curve, an equation is needed that relates the column pressure drop to the mobile phase velocity, its viscosity, 

, and the column length. This is given by:

This equation is straightforward as it easy to understand that the column pressure drop increases with column length, velocity (or flow rate) and the mobile phase viscosity. The parameter, 

, is called the column permeability (the subscript 0 indicates it is based on the 

 velocity) and is a measure for how “easily” the mobile phase can flow through the column. Its value is proportional to the square of the particle size (

); smaller particles yield lower column permeability and thus higher pressure drops. The factor Φ

 is the column flow resistance parameter; typical values are around 800 for fully porous particles, and 650 for superficially porous ones. When the column pressure drop is plotted as a function of 

, typically a straight line is found with a slope equal to L·

Two different approaches can then be used to calculate the KPL. The first one is based on the plate height data, and it involves direct conversion of the 

 values (either measured or calculated; see methods 2 and 3 in the first section) to 

 values that lie on the KPL curve (that is, 

 and NKPL) (6). Equations 5 and 6 are used for these calculations:

The second approach starts from the experimentally designed 

 that were obtained using a column with length 

, and the experimentally determined column pressure drop Δ

. In this case, a column length extrapolation factor, λ, is calculated for each flow rate using equation 7:

This λ factor is the physical representation of the extrapolation shown in Figure 2 because it is a measure of how much longer the column can be made relative to the experimental column length 

 used to make the fixed-length curve. Therefore, it also represents how much the plate number and analysis time will increase on the KPL, yielding the following simple expressions to determine the KPL curve:

Once the red KPL curve has been constructed by extrapolation from the dark blue curve shown in Figure 3, it is useful to think about the significance of the points on the KPL line. Starting from the right-most point on the dark blue fixed-length curve (which corresponds to the van Deemter optimum velocity), if we fix the flow rate and extrapolate this, it takes us along the light blue line, ending in the light blue circle on the KPL curve. Here, we see that the column length is now 40 cm and a remarkable plate number of 56,700 can be obtained, which is 8-fold higher than the plate number of 7180 achievable at the same flow rate but with the much shorter column length of 5 cm. The pressure drop when operating the 5 cm column this way is just 51 bar, significantly under-utilizing the available pressure of the instrument. In other words, moving from the dark blue to the light blue circle represents capitalizing on the available instrument capability.

On the other hand, if the plate number of 7200 at the optimum in the fixed-length curve (dark blue circle) is already sufficient for the resolution needed, then this result can be obtained much faster by following the green arrow down to the green circle on the KPL curve. Here, we see that the same plate number is obtained, but with an analysis time savings of about 57% (

 of 0.12 compared to 0.28 min). In this case, both the flow rate and column length are increased on the way to reaching the KPL curve. The flow rate is increased to reduce the analysis time, but the column length must also be increased to compensate for the increase in plate height on the high-velocity side of the van Deemter optimum so that the plate number stays constant.

Finally, if the analysis time is tolerable at the optimum in the fixed-length curve (dark blue circle; 

 = 0.28 min.), then a much higher plate number can be obtained in the same time by following the black arrow to the right until the KPL curve is reached at the black circle. In this case, both the flow rate and column length are increased again. The flow rate must be increased so that the same analysis time can be maintained even when the column length is increased. Ultimately, by fully using the available pressure capability of the instrument, the plate number can be doubled from 7200 to 14,400 without any increase in analysis time.

Besides finding the optimal kinetic performance conditions at which one can operate a particular stationary phase support (for example, a given particle size), as shown in Figure 3, the kinetic plot methodology also enables comparison of different stationary phase supports. Figure 4 shows a comparison of KPL curves for three different particle sizes (same analyte and mobile phase composition in each case) calculated using method 3 discussed above for a pressure limit of 400 bar. We see that the smallest particles (1.7 μm) show the best kinetic performance for short analysis times (lower part of the plot) and low efficiencies (left part of the plot), whereas the larger particles (5 μm) are the better choice for high efficiencies at long analysis times, and the 3.5 μm particles mostly fill up the gap in between. The reason for this behavior is that columns with small particles can be operated at high velocities (flow rates) without much loss in performance (that is, the shallow C-term slope in the van Deemter curve means that plate height does not increase much as the velocity is increased). However, it comes at the cost of higher pressure drops (since 

As a result, only relatively short columns can be operated within the pressure limit, resulting in low efficiencies but only requiring short analysis times. This process results in the 1.7 μm particle curve occupying the bottom left region of the kinetic plot. On the other hand, when using large particles, long columns (that is, longer than 15 cm, so coupled columns are required) can be operated at reasonable velocities, which enables very high plate numbers (>100,000 plates) at the cost of much longer analysis times. It is interesting to note that each of the KPL curves approach a vertical asymptote. The practical significance of this occurrence is that the asymptote represents a plate number limit that cannot be exceeded for that particle size, even if the column length is increased.

In this installment of “LC Troubleshooting,” we have continued the discussion of kinetic plots, including how to construct them from either experimental data or data from other sources, the conceptual meaning of the KPL, and the effect of particle size on KPL curves. In the next installment in this series, we will draw all of these concepts together and discuss how kinetic plots can be used for practical column selection and troubleshooting situations where column performance is not consistent with our expectations and will include our introduction of a web-based calculator application that leverages the theory summarized here.

A. Andrés, K. Broeckhoven, and G. Desmet, 

, 20–34 (2015). https://doi.org/10.1016/j.aca.2015.08.030.

, 323–339 (2021). https://doi.org/10.1002/jssc.202000779.

K. Broeckhoven, D. Cabooter, F. Lynen, P. Sandra, and G. Desmet,

, 2787–2795 (2010). https://doi.org/10.1016/j.chroma.2010.02.023.

D. Cabooter, F. Lestremau, A. de Villiers, K. Broeckhoven, F. Lynen, P. Sandra, and G. Desmet,

, 3895–3903 (2009). https://doi.org/10.1016/j.chroma.2009.02.079.

, 4058–4070 (2005). https://doi.org/10.1021/ac050160z.

 received his Ph.D. in 2010 from the Vrije Universiteit Brussel (VUB), in Brussels, Belgium. Following postdoctoral research at VUB and work as a visiting researcher in the separation processes laboratory at ETH Zurich, he became a research professor at VUB in 2012. He was subsequently promoted to Assistant Professor and then to his current position as Associate Professor in 2017.



is the editor of “LC Troubleshooting”. Stoll is a professor and the co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota, USA. He is also a member of LCGC’s editorial advisory board. Direct correspondence to: amatheson@mjhlifesciences.com

LC Troubleshooting

A kinetic plot is a powerful tool, but how do you construct one—from either experimental data or data from other sources? We explain.

But Why Doesn’t It Get Better?  Kinetic Plots for Liquid Chromatography, Part 2:  Making and Interpreting the Plots

Over the winter break at the university where I work, I was ordering consumables for the upcoming semester. As I was browsing the web pages at a major vendor for glass inlet liners, I was again impressed by the many glass inlet liner options and geometries available for split and splitless inlets, still by far the most used today. In this instalment, we will review the processes that happen in the inlet when a sample is injected, and use this to make some sense of the many available inlet liner configurations. We will generate some best practices and guidelines in selecting an inlet liner, but we will see that there is no one single liner that fits all situations. Selecting the best inlet liner should be a step in all method development involving split or splitless injections.

Capillary gas chromatography (GC) has always suffered from some fundamental challenges regarding sample introduction into the chromatograph. First, the liquid sample must be transferred from a syringe or other sampling device to an open tubular column that most often has an inside diameter smaller than the syringe. Second, the injected sample must be vaporized and homogeneously mixed with the carrier gas stream. The sample/carrier gas mixture must be quantitatively and reproducibly transferred into the open tube of the capillary column. Finally, the inlet must be large enough to accommodate the full vapour volume generated when the liquid sample is evaporated. As described in the classical book by Grob, 

Split and Splitless Injection in Capillary Gas Chromatography

, the many processes involved with solving these challenges can be very complex, to the tune of this book being nearly 500 pages long (1).

A final challenge involved in transferring a liquid sample into a capillary column is that the entire flow path must be inert. In his book, Grob describes that much research has been done over the years to ensure that the flow path is inert and does not react with or adsorb compounds from the samples. The flow path must also be easy to clean, as it will eventually collect nonvolatile contaminants resulting from sample degradation. All of these challenges led to the development of glass inlet liners to provide an inert flow path that can accommodate the other challenges with vaporization of the sample, mixing with the carrier gas, and transferring that mixture to the column. Figure 1 shows a split/splitless inlet from above, with the top flanges, including the septum and septum nut, removed. Note that the inlet block and gas flow lines are all metal, which can be highly reactive with organic compounds when heated, and would be very difficult to clean. The inlet liner is glass, and usually fits loosely into the inlet body to allow for carrier gas to flow both through the inside of the liner and around the outside. An O-ring provides a gas-tight seal when the flanges and nuts are replaced. Details of the flow paths for split (and splitless) inlets and on general troubleshooting and maintenance of inlet systems are discussed in most texts including gas chromatography and on ChromAcademy, 

’s online learning platform (2). As a general maintenance and troubleshooting rule, the glass inlet liner and O-ring should be replaced together on a regular schedule, depending on the cleanliness of the samples being analyzed.

This highlights the need for a removable and replaceable liner for the flow path that also solves the challenges mentioned above. Even more challenging, a liner that works for one sample may not work for another. For the best performance, options must be tested and evaluated. In the remainder of this discussion, we will examine principles of split and splitless injections that inform the choice of liner, beginning with some overriding concerns that govern all liners.

The Two Overriding Concerns: Inertness and Volume

Regardless of the injection technique being used, the inlet liner must be inert to the samples, and must have large enough volume to accommodate the vapour generated when the sample is injected into the heated inlet and vaporized. Depending on the solvent, inlet temperature, and inlet pressure, typical sample solvents used in gas chromatography may generate anywhere from 200 µL to over 1 mL of vapour when evaporated in the inlet. If the volume inside the inlet liner is not large enough, vapour can backflow into the carrier gas lines. Since that vapour will also contain vaporized analytes, those analytes can condense in the gas lines and eventually become eluted as ghost peaks or baseline drift. Solvent vapour volume calculators for common solvents and inlet conditions are available online (3,4). These calculators can be used as guides for choosing the needed volume for the inlet liner. Generally, the chosen liner should have a higher internal volume than provided by the calculator. Table 1 shows vapour volumes generated using the two calculators for some common conditions. Excessive vapour volume occurs in relatively few situations, usually with polar solvents, high injection volumes, or low head pressure. Note that increased head pressure solved the problem with excessive evaporation of water.

The inlet liner must also be inert to any reactive compounds, both analytes and interferences. Nearly all inlet liners for split and splitless inlets are made from glass, which by itself is not deactivated and can have free silanol groups and other surface contaminants that can react with or adsorb analytes. One of the impressive options seen at the vendors’ websites is the choice of deactivation methods, which also should be matched to the expected reactivity of the analytes. Not surprisingly, deactivated inlet liners come at a higher individual price. Being an academic researcher, when developing a method, I used to start with the most inexpensive option and move to the more expensive deactivated liners as needed. Today, I simply purchase deactivated liners up front, so I do not have to find out the hard way, usually through poor peak shapes or poor quantitative reproducibility, that I need to change to a more inert inlet liner. I also purchase deactivated liners from the vendors. I no longer attempt to deactivate them myself.

Next, we discuss glass liner choices for some specific situations, the most common split and splitless injections, and some special cases, including solid-phase microextraction (SPME) and programmed temperature vaporization (PTV) systems.

An ideal split injection requires that the sample be injected as quickly as possible, vaporized as quickly as possible, and mixed homogeneously with the carrier gas, with the mixture transferred rapidly to the column head. Rapid injection minimizes band broadening and is the primary cause of the sharp peaks characteristic of split injection combined with temperature programming. To assist in vaporization, split liners have a large volume and often have obstructions, baffles, or packing (glass wool or adsorbent) to facilitate vaporization. Figure 2 shows a typical cup-design glass inlet liner and two glass wool packed liners for split injections. I usually start with the cup design, developed by Jennings and often referred to as the “Jennings cup” (5). The cup and associated tubing provide a flow path that assists with vaporization by the large surface area and mixing by the twisted flow path. In Figure 2, the cup is highlighted by the yellow oval. Glass wool and baffles provide a less expensive alternative (especially glass wool for samples with a “dirty” matrix), but both can be challenging with reactive analytes; if active analytes are involved, I recommend the additional expense of deactivated liners. If you look closely at Figure 2(c), you will see some contamination in the glass wool, providing evidence of the main challenge of using glass wool.

Splitless injection is a much slower process than split. A rapid injection with the syringe is followed by a slow evaporation and transfer of the vaporized sample to the column head. To reduce the band broadening caused by the slow sample transfer to the column, temperature programming is almost always used with splitless injections. The inlet liner for splitless injections must be a trade-off between adequate internal volume to accommodate solvent vapour and reasonable transfer time for the vapour phase solvent/solute/carrier gas mixture to the column. The classical liner is a straight glass tube with about 2 mm inside diameter, as seen in Figure 3(a). A splitless liner usually has a smaller internal volume than for split. This is partially compensated by slower vaporization, but the liner volume should still be confirmed using a vapour volume calculator. The tube ends may be tapered or goosenecked to assist with column installation or syringe insertion. The liner may also be packed with glass wool or an adsorbent material to facilitate solvent and sample vaporization.

Most injections using solid-phase microextraction (SPME) are a special application of a split/splitless inlet in which no solvent is used. Since no solvent is used there is no need for a large volume to accommodate solvent vapour. Since there is no need for a large volume, a small, 0.75 mm inside diameter straight tube inlet liner is most commonly used. One advantage of SPME injections is that there is no solvent, so there is no need for the inlet liner to have a large internal volume; in fact, the smallest possible volume is best to reduce band broadening during the injection process. Figure 3(b) shows a typical glass liner for SPME. Note the vary narrow inside diameter, which is possible since there is no solvent vapour, and the taper at one end, which provides a guide for the needle to fit more easily into the small diameter. In the early days of SPME, my group wrote on the details of optimizing SPME injections using the split/splitless inlet (6). The narrow diameter liner becomes more critical as the analytes become more volatile. For semi-volatile analytes, a traditional straight splitless liner will often work.

You may have a programmed temperature vaporization (PTV) or multimode inlet that is capable of several injection techniques in a single platform. The basics of these inlets were discussed in a previous “GC Connections” instalment (7). If temperature programmed inlets are used, the inlet and the inlet liner must have a low thermal mass for rapid heating and cooling, so inlet liners are typically large internal volume with thin glass walls. If large volume injections are performed, the inlet liner usually contains an adsorbent to accommodate the large volume of injected liquid solvent.

Basic characteristics of inlet liners for specific injections methods are summarized in Table 2, along with my recommendations for a starting point in method development.

When I am choosing inlet liners, I start simple. For split injections, I start with a classical cup design, which, while a little more expensive than some of the other options, provides a large space for vapour expansion and a high heated surface area to aid in sample vaporization. Also, the cup prevents an accidental “lucky shot” in which the liquid stream from the syringe shoots straight into the column. For splitless, I prefer to start with a 2 mm inside diameter straight tube, with no glass wool or other obstructions. I then consider options for a modified flow path, such as glass wool, should my evaluation of the data dictate, usually by seeing unsatisfactory quantitative reproducibility for repeated injections of standards. The challenge with choosing a glass inlet liner is that there is no solution that fits all analyses and situations. Once a starting point is chosen, if the results are not satisfactory, alternatives must be selected and tried by experimentation. Finally, when choosing a glass liner for a specific application, the classic first steps in method development particularly apply: check the applications literature, and contact the vendor for advice as well as ask someone who knows.

The glass inlet liner is one of the most important, yet least understood and most often ignored, components of a gas chromatographic experiment. It is the critical link between sample preparation and separation by the column. The best sample preparation and the best column can be ruined by a poor choice of glass liner. Unfortunately, there is no “killer app” for choosing an appropriate glass inlet liner. Users are best served by making an intelligent first choice, usually simple, and appropriate to the injection technique (split or splitless) being used, thinking about the expected chemistry of their samples and standards and making changes, such as from cup to baffles to inserting glass wool, as analytical and chromatographic performance dictate. Not all glass inlet liners are alike, and not all will solve every problem. For best performance, they must be studied by experiment.

Split and Splitless Injection for Quantitative Gas Chromatography: Concepts, Processes, Practical Guidelines, Sources of Error

 (John Wiley and Sons, Hoboken, New Jersey, USA, 4th Ed., 2001).

“GC Sample Introduction” ChromAcademy, https://www.chromacademy.com/channels/gc-training-courses/instrumentation/gc-sample-introduction/ (accessed February 2022).

Agilent Solvent Vapour Calculator, https://www.agilent.com/en/support/gas-chromatography/gccalculators (accessed February 2022).

Restek Solvent Expansion Calculator, https://www.restek.com/en/tools-and-calculators/tools/solvent-expansion-calculator/ (accessed February 2022).

 is the Founding Endowed Professor in the Department of Chemistry and Biochemistry at Seton Hall University, and an Adjunct Professor of Medical Science. Direct correspondence to: amatheson@mjhlifesciences.com

GC Connections

We review the processes that happen in the inlet when a sample is injected and use this to make some sense of the many available inlet liner configurations. 


Split/Splitless Inlets in GC: What’s Up with All Those Different Glass Inlet Liners?

In January, Steve Lehotay from the U.S. Department of Agriculture provided an update on the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method, originally developed for multiresidue pesticide analysis. Like any method, QuEChERS had some deficiencies, which were addressed with the new QuEChERSER (adding “efficient” and “robust” to the acronym) mega-method. Meanwhile, the European Union (EU) Reference Laboratories developed a new method for the multiresidue analysis of highly polar pesticides, called the 

. This month, we take a look at QuPPe, comparing and contrasting it with QuEChERS, and noting how we can learn from previously developed methods as we strive for improvements.

Steve Lehotay (1) engaged us with a summary of the current state of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) multiresidue extraction method in our last “Sample Preparation Perspectives” column. Lehotay and Anastassiades developed QuEChERS approximately 20 years ago as a multiclass, multiresidue method, primarily for determining pesticides in produce. Because the method developed employed a wide range of flexibility (that is, it was simple and developed for the analytical instrumentation widely used at the time), modifications to the approach led to it becoming a model for the extraction and chromatographic sample preparation of more diverse analysis needs, such as for veterinary drugs, environmental contaminants, and mycotoxins. The still growing popularity of QuEChERS is evidenced by close to 4500 peer-reviewed publications since its inception.

The simplicity and flexibility inherent in the QuEChERS method also results in its limitations. For example, the minimal or “just enough” clean-up steps avoiding indirect matrix effects make the approach both applicable as a template for an array of analyses and provide some limits on the range of residue polarities amenable to the technique. Additionally, analytical instrumentation, such as liquid chromatography–tandem mass spectrometry (LC–MS/MS), has improved dramatically since the turn of the century. With this in mind, Lehotay updated the technique to include “efficient and robust”, and updated the method name to 

, which was the primary topic of the January column (1). The QuEChERSER upgrade accommodates a broader scope of polar and nonpolar analytes, creating what Lehotay describes as a mega-method. A comparison of the workflows for QuEChERS and its offspring QuEChERSER is shown in Figure 1. From these schematics, the major differences between the methods can be gleaned.

Despite the improvements afforded by QuEChERSER, there is still a need for the simple and effective extraction of residues of polar pesticides incurred in produce and other foodstuff. Working with the European Union (EU) Reference Laboratories for Residues of Pesticides, Michelangelo Anastassiades, co‑inventor of the original QuEChERS method, created a new method that was aimed specifically at the highly polar pesticides not amenable to extraction via QuEChERS (or QuEChERSER) (2), which he called the 

. The workflows for the QuPPe family of methods is shown in Figure 2. Although starting with goals similar to those used with the original QuEChERS extraction, what is immediately evident in these workflows is that Anastassiades learned and reapplied concepts from the previous development of QuEChERS and added his knowledge of chemistry in the development of QuPPe.

A series of four QuPPe methods are described, one each for most plant-based commodities and honey; cereals, pulses, nuts, and oily seeds; liver, kidney, muscle, and milk; and animal fat. In these methods, the sample is weighed and water may also be added. The acidified method is used for the extraction, and heating (fats) or cooling (lipids and proteins) is used to remove biological macromolecules. The clean-up process is performed prior to LC–MS/MS or ion chromatography (IC)–MS/MS. Ethylenediaminetetraacetic acid (EDTA) is added, as necessary, to the complex metal ions. Isotopically labelled internal standards are added at the beginning of the procedures to compensate for volume deviations, analyte loss, or matrix effects on recovery rates. Where internal standards are not possible, quantification by standard addition is used. Because of the highly polar nature of the target analytes, plastic vials are used to minimize loss of analyte because of surface adsorption onto glass vials, though some carryover is still possible depending on the analyte. Because of the simplicity of the method, strong matrix effects are frequently observed. Prior to QuPPe, samples are ground to less than 500 μm and homogenized to a free-flowing mixture.

During the development of QuPPe, the analytical team spiked a host of highly polar pesticides (Table 1) at levels of 0.005–20 mg/kg per sample and conducted matrix-matched and matrix isotope-labelled internal standards (ILIS) calibrations with interlaboratory calibrations. The mean recoveries reported were between 70–120%, with a 0–20% relative standard deviation. Spiked samples on to a number of foods of plant- and animal-origin were reported, including citrus fruits, pome fruits, stone fruits, soft and small fruits, dried fruit, root and tuber vegetables, leek plants, fruiting vegetables, cabbages, leafy vegetables and herbs, stem vegetables, legumes or pulses, cereals, oily seeds, nuts, animal tissues, whole fat and skimmed milk, honey, eggs, and animal fats.

Given the relative newness of the method, reports of QuPPe are just starting to emerge, though the method is accepted by the EU. Perusal of the Scopus database shows 21 literature reports of QuPPe from 2016 through the end of 2021, with two to six papers per year.

One of the first literature reports (3) of glufosinate in samples of plant origin used multiwalled carbon nanotubes for cleanup, and reported linearity from 10–500 μg/kg for 12 sample matrices, with detection limits of 0.3–3.3 μg/kg. In one manuscript (4), a fusion of the QuPPe and QuEChERS methods was reported for the isolation of 45 nonpolar and polar residues, with a broad range of log 

ow (over 10 orders). A parallel, orthogonal hydrophilic interaction liquid chromatography (HILIC) and reversed‑phase LC approach was used. Elsewhere, HILIC was observed to provide good retention and selectivity of highly polar herbicides extracted from plant-based foods (5). Importantly, fewer matrix effects were found when cleanup was performed with chitosan or graphene. Another report (6) added the primary‑secondary amine (PSA) during the clean-up step and used direct analysis in real time (DART)–MS, comparing orbital trap MS and quadrupole time-of-flight (QTOF) mass analyzers, for the analysis step.

Although still an emerging technique, the lessons learned from QuEChERS were re-applied in developing a simple method for the extraction of highly polar pesticides from samples of plant and animal origin. This method, and potentially modified versions, bears watching as an important tool for multiresidue monitoring.

M. Anastassiades, A.-K. Wachtler, D.I. Kolberg, E. Eichhorn, H. Marks, A. Benkenstein, S. Zechmann, et al., “Quick Method for the Analysis of Highly Polar Pesticides in Food Involving Extraction with Acidified Methanol and LC- or IC-MS/MS Measurement”, version 12 (published on EURL-SRM website on 23 July 2021). URL: https://www.eurl-pesticides.eu/docs/public/tmplt_article.asp?CntID=887&LabID=200&Lang=EN

Y. Han, L. Song, P. Zhao, Y. Li, N. Zhou, Y. Qin, X. Li, and C. Pan, 

J. Robles-Molina, B. Gilbert-López, J. F. García-Reyes, and A. Molina-Díaz.

This month we’ll take a look at Quick Polar Pesticides (QuPPe), comparing and contrasting it with QuEChERS and noting how we can learn from previously developed methods as we strive for improvements.


March 2022 | LCGC Europe

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

But Why Doesn’t It Get Better? Kinetic Plots for Liquid Chromatography, Part 2: Making and Interpreting the Plots

Split/Splitless Inlets in GC: What’s Up with All Those Different Glass Inlet Liners?

Quick Polar Pesticides (QuPPe): Learning From and Expanding Upon Others