Chiral separations in food analysis

Chromatography—concepts and contrasts : By J.M. Miller, Wiley, New York, 1988, 297 pp., price ca. US$ 53.15, ISBN 0-471-84821-2.

Chiral drugs exert pharmacological effects through strict matching with chiral biological macromolecules and chiral recognition. Each enantiomer has different pharmacological activities, metabolic processes and rates, as well as toxicity pharmacokinetic characteristics owing to the difference in its interactions with the chiral environment. Therefore, method development for the resolution of chiral drugs is of great significance for the synthesis of chiral drugs and for quality control during the production process. Molecularly imprinted polymers (MIPs) are prepared by using a target molecule as the template. MIPs demonstrate highly specific recognition properties toward the target molecule since they have specific spatial molecular structures and functional groups. Hence, MIPs are particularly suitable for the separation and purification of chiral drugs. Capillary electrochromatography (CEC) offers the advantages of high separation efficiency and high selectivity owing to the dual separation mechanisms including capillary electrophoresis and liquid chromatography. By using MIPs as the stationary phases for CEC, the advantages of the two technologies can be combined to achieve efficient separation of chiral drugs. MIPs were first applied to CEC for chiral resolution in 1994, and since then, there have been notable advances in this field. The four main chiral separation modes in CEC involve the use of MIPs as the stationary phases of open tubular, packed, and monolithic columns, and as the pseudostationary phase in the separation medium. This review summarizes the research progress of these four methods and reveals the potential of MIPs in chiral resolution by CEC. The advantages and disadvantages of these methods are commented. MIPs as the stationary phases of packed columns can allow for chiral separation. However, the preparation of packed columns in narrow capillaries is difficult. In addition, frits must be prepared at the ends of the capillaries to seal the MIPs. The frits lead to the formation of bubbles during the CEC analysis, thus resulting in poor repeatability and stability. These problems can be overcome by using MIP-based open tubular columns. Furthermore, conditioning of open tubular columns is easy and less time-consuming. However, open tubular columns have limited capacity. MIP-based monolithic columns have greater capacity than do open tubular columns, and frits are not required in this case. However, in situ preparation of MIPs monolith in narrow capillaries is still challenging. The application of MIPs to chiral CEC can also be realized by using them as pseudostationary phases (additives) in the separation medium, and this allows for ease of operation. Moreover, the amount of MIPs introduced into the capillary can be accurately controlled. Thus, the batch-to-batch reproducibility can be improved, but this has the disadvantage of increased MIP consumption. In order to further expand the potential of MIPs in chiral CEC, the following aspects must be considered. First, improvement of the preparation method. In most reported MIP-based-chiral CEC techniques, the peaks of the imprinted molecules show severe tailing, and this problem must be resolved. Improving the mass transfer rate of the prepared MIPs may be a suitable solution in this regard. Second, development of new functional monomers. A functional monomer is an indispensable component in the preparation of MIPs. New functional monomers can be prepared according to the "three-point interaction" rule. Third, selection of template molecules. A single enantiomer of chiral drugs is used as the template molecule to prepare chiral MIPs. The method is not suitable for the preparation of MIPs of chiral drugs for which a single enantiomer is difficult to obtain. Therefore, appropriate choice of the template molecules for these drugs is imperative. Fourth, discussion of chiral separation mechanism. The mechanism of interaction between the template molecules and MIPs needs to be explored further, in order to obtain theoretical guidance for the design and preparation of chiral MIPs.

New psychoactive substances represent serious social and health problem as tens of new compounds are detected in Europe annually. They often show structural proximity or even isomerism, which complicates their analysis. Two methods based on ultra high performance supercritical fluid chromatography and ultra high performance liquid chromatography with mass spectrometric detection were validated and compared. A simple dilute-filter-and-shoot protocol utilizing propan-2-ol or methanol for supercritical fluid or liquid chromatography, respectively, was proposed to detect and quantify 15 cathinones and phenethylamines in human urine. Both methods offered fast separation (<3min) and short total analysis time. Precision was well <15% with a few exceptions in liquid chromatography. Limits of detection in urine ranged from 0.01 to 2.3ng/mL, except for cathinone (5ng/mL) in supercritical fluid chromatography. Nevertheless, this technique distinguished all analytes including four pairs of isomers, while liquid chromatography was unable to resolve fluoromethcathinone regioisomers. Concerning matrix effects and recoveries, supercritical fluid chromatography produced more uniform results for different compounds and at different concentration levels. This work demonstrates the performance and reliability of supercritical fluid chromatography and corroborates its applicability as an alternative tool for analysis of new psychoactive substances in biological matrixes.

Contribution of supercritical fluid chromatography to serially coupling columns for chiral and achiral separations

Capillary electrochromatography (CEC) has been realised as a combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) allowing the separation of analytes based on their differing adsorption and migration behaviour. There is a considerable interest in developing new methodologies for CEC, which uses chromatographic beads or monoliths as stationary phases or alternatively employs open tubular (OT) capillaries relying on surface modifications. Besides the bulk flat flow profile of the mobile phase in CEC columns allowing an enhanced resolution, due to the influence of the applied electrical field there are other possibilities to provide a higher resolution with better detection. As an additional newly means for peak sharpening and sample enrichment isotachophoresis and a “sweeping mechanism” could be envisaged, which both take advantage of the inhomogeneous electric field pattern in the capillary by choosing appropriate leading and terminating background electrolytes (BGEs). Transient isotachophoresis would be another technique to provide an increased plate height in the separations, using an optimized sample matrix, e.g. containing a relatively high acetonitrile content, with the same leading and terminating BGEs. In this thesis, a new method to generate a “sharpening zone”, which is similar to the concept of transient isotachophoresis, was established using an aqueous sample matrix containing an organic solvent. Target analytes within this zone could be concentrated whilst they migrated through the capillary. A novel technique was developed that used the sharpening zone for sweeping and concentrating several unresolved analyte peaks allowing an enhanced peak resolution. As the aim was to concentrate only some part of the analyte zone containing compounds of interest, the position and width of the sharpening zone needed to be well controlled. Various techniques to tune the width and position of the sharpening zone were thus developed, involving modifying the organic modifier content in the BGE, applying pressure additionally to the electroosmotic flow (EOF) and modifying the inner surface of the capillaries. This work also critically evaluated some related existing knowledge and theoretical approaches in CEC including the concept of tuneable system eigenpeaks (noncomigrating or ghost peaks caused by waves of an electric field in the capillary). Although peak broadening caused by eigenpeaks (which often suppresses resolution in CEC) has been well described for aqueous or pure non-aqueous BGEs, a systematic study of eigenmobility in aqueous/acetonitrile BGEs had not been reported. The experiments related to the eigenmobility led to a new approach to tune the eigenpeak position in chromatograms whereby a selective sample peak sharpening mechanism was elaborated using the sharpening zone. The novel approach was demonstrated for the separation of various peptides in the CE mode and for the resolution of incompletely resolved chiral amino acids in CEC. These investigations furthermore led to the development of an appropriate stationary phase, which was produced using techniques for an in-column synthesis of a molecular imprinted polymer (MIP). Well prepared MIPs are capable of specifically enhancing the retention of target analytes, herein focusing on enantio-selective isolation of chiral molecules. Since such a MIP was rather rigid, which often caused capillary clogging when used as a conventional monolithic stationary phase, this MIP was incorporated into porous layer open tubular (PLOT) capillaries. Numerous experimental difficulties were overcome with respect to the vinylisation of long fused-silica capillaries and for the polymerisation of the MIP within using thermal- and photo-polymerisations. A method for the characterization of such MIP-PLOT capillaries was elaborated involving the Langmuir adsorption theory. The new columns were evaluated using chromatography, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and CEC analysis. All experiments were designed according to the principles of Green Chemistry. These concepts involved the use of small scale analysis, simulation studies reducing the number of performed experiments, systematic studies with low amounts of solvents and materials, and optimisation of synthesis methods to reduce time, energy and reagent consumption. The theories, methods and results presented in this thesis will, in the future, allow further targeted developments of novel analytical tools for a green, sensitive, selective and improved resolution analysis of chemical and biological compounds including chiral compounds, peptides and proteins.

The challenge to develop enantioselective analytical methods to quantify chiral compounds in food and environmental matrices is an actual and imperative issue. It is well known that enantiomers may differ in their biological activities, but the stereochemistry and the discrimination of enantiomers are frequently ignored in analytical workflows. However, the knowledge about their proportion is crucial to guarantee food and environmental safety. In this sense, chiral analysis is a valuable tool to evaluate food quality and genuineness, to determine the geographical origin of a certain sample aiming to find out fraud or adulteration, to investigate toxicity and bioaccumulation of environmental contaminants (e.g., pharmaceuticals and pesticides). This chapter highlights the importance of chiral analysis in food matrices and its relationship with the environmental contamination. The last advances in chiral analysis by liquid chromatography (LC), gas chromatography (GC), and supercritical fluid chromatography (SFC) coupled to mass spectrometry (MS) are presented, with a special remark in the type of chiral column used for chromatography and the proposed workflow in the method development. Some of the most recent applications in chiral analysis of food and beverages, environmental monitoring of surface water, wastewater, soils, biodegradation studies, and contamination of foodstuff are presented and critically discussed.Graphical AbstractKeywordsChiral chromatographyChiral stationary phaseEnantiomersPesticidesPharmaceuticals

Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography (2019–2022)

Capillary electrochromatography applied to the separation of enantiomers utilizing packed capillary columns with silica-vancomycin. A tutorial

The development of the hyphenation of high-performance liquid chromatography (HPLC) and other chromatographic methods (supercritical fluid chromato-graphy (SFC), gel-permeation chromatography (GPC), gas chromatography (GC) and capillary electrophoresis (CE)) with nuclear magnetic resonance (NMR) is reviewed. The focus is not only on application (e.g. pharmaceutical chemistry, food chemistry, polymer analysis, fuel chemistry), but also on technical improvements (e.g. flow rate, flow cell, probe, solvent suppression). The increasing importance and overwhelming analytical possibilities of on-line coupling of HPLC with NMR is demonstrated. The literature up to March 1995 is considered.

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.

Chiral analysis in food science

Ionic liquids (ILs) are salts formed by a bulky nitrogen-or phosphorous-containing dissymmetrical organic cations such as alkyl-imidazolium, pyridinium or pyrrolidinium, ammonium or phosphonium and a variety of organic anions: triflate, dicyanamide, acetate, trifluoroacetate, trifluoromethylsulfate or inorganic: bromide, chloride, nitrate, perchlorate, chloroaluminate, tetrafluoroborate, or hexafluorophosphate. The number of possible combinations of anion-cation amounts 10 18 , which makes this class of substances one of the biggest in chemical chest of substances. Properties of ILs depend on their structure. In order to obtain ionic liquid of desirable properties, with the specific melting point, viscosity, density, miscibility with water and organic solvents, one should select appropriate ions. For this reason ionic liquids are often called "designer" solvents. There are certain common characteristics which definitely distinguish this class of chemical compounds. They have the ability of dissolving inorganic and organic compounds, including enzymes, simultaneously retaining their activity. They are stable chemically and thermally. The difference between the boiling point and of melting is of 300 C. They possess low vapor pressure, which is why ionic liquids don't produce volatile environmental pollution. The most specific feature of these compounds is the low melting temperature -below 100 C. If melting point is below room temperature (25 C), the IL is called room-temperature ionic liquid (RTIL). ILs found applications as new non-molecular types of solvents with unique properties in different areas of chemistry like organic synthesis, electrochemistry, extraction, spectroscopy and mass spectrometry. The growing interest in ILs has been observed also in chromatographic methods: gas chromatography (GC), capillary electrophoresis (CE) and liquid chromatography (LC). From a point of view of separation techniques, an important property of ILs is a possibility of generating strong donor-acceptor interactions and ability of undergoing coulombian interactions (chromatography), good electrical conduction (capillary electrophoresis), the great viscosity, the surface tension, the thermal stability (gas chromatography), excellent solvent properties (extraction). Worth noting are critical reviews emphasizing specific advantageous of ILs application in chromatographic and electromigration techniques (Buszewski & Studziska,

Capillary electrochromatography is a miniaturized technique offering some advantages over conventional ones in separation science. It combines the best features of capillary zone electrophoresis (high efficiency of separation) and liquid chromatography (high selectivity). Because of the low flow rates, minute volumes of mobile phases can be applied, and therefore, it can be considered a green analytical technique. Capillary electrochromatography has been successfully used to separate many classes of compounds, including peptides, proteins, drugs, antibiotics, pollutants, enantiomers, etc. In this overview chapter, the main features of this technique are summarized. Its potential for the separation of enantiomers of chiral compounds and the main chiral stationary phases employed are presented. Finally, some selected applications in this field, reported in the literature in the period 2020-2024, are presented.

Drug chirality has attracted attention as it will influence the pharmacological activities of racemic drugs. In order to get optically pure drugs, both analytical and preparative grade chiral separation technology has been developed. Since the first introduction of anionic moiety into β-cyclodextrin by Stalcup et al, the highly enantioseparation abilities of anionic β-cyclodextrin towards basic racemates have attracted chromatographers attention. Endeavors were then made on the synthesis and application of anionic beta-cyclodextrin for enantioseparation. However, positive charged β-cyclodextrin derivatives were rarely investigated. Lesser work was reported on enantioseparations with cationic β-cyclodextrin derivatives as chiral additives in reversed phase liquid chromatography or capillary electrophoresis whereas no work reported about the usage of cationic β-cyclodextrin derivatives as chiral stationary phases. In our work, cationic β-cyclodextrin derivatives are either physically coated or chemically bonded onto the surface of 5 pm spherical silica and applied as chiral stationary phases in HPLC and SFC. The cationic β-cyclodextrin derivatives have shown strong chiral separation abilities towards high medical value racemates and some propriety racemates.

Recent developments in supercritical fluid chromatography – mass spectrometry: Is it a viable option for analysis of complex samples?