Electrokinetic Capillary Chromatography Research Articles

Capillary electrokinetic chromatography for chiral separation of potential SARS-CoV-2 3CL protease inhibitors.

In this work, a capillary electrophoresis method was developed as a quality control tool to determine the enantiomeric purity of a series of five chiral compounds evaluated as potential severe acute respiratory syndrome coronavirus 2 3CL protease inhibitors. The first cyclodextrin tested, that is, highly sulfated-β-cyclodextrin, at 6% (m/v) in a 25mM phosphate buffer, using a capillary dynamically coated with polyethylene oxide, at an applied voltage of 15kV and a temperature of 25°C, was found to successfully separate the five derivatives. The limits of detection and quantification were calculated together with the greenness score of the method in order to evaluate the method in terms of analytical and environmental performance. In addition, it is noteworthy that simultaneously high-performance liquid chromatography separation of the enantiomers of the same compounds with two different columns, the amylose tris(3,5-dimethylphenylcarbamate)-coated and the cellulose tris(3,5-dichlorophenylcarbamate)-immobilized on silica stationary phases, was studied. Neither the former stationary phase nor the latter was able to separate all derivatives in a mobile phase consisting of n-heptane/propan-2-ol 80/20 (v/v).

Effect of liposome composition on β-blocker interactions studied by capillary electrokinetic chromatography.

Liposome capillary electrokinetic chromatography was used to investigate the interactions between three β-blockers of different hydrophobicity and various liposome solutions. The studied β-blockers comprised alprenolol, propranolol, and carvedilol. The composition of the liposome solutions, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phos-phoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine, and cholesterol in various molar ratios, was designed by a response surface methodology-central composite design approach. Subsequently, after conducting the liposome capillary electrokinetic chromatography experiments and determining the retention factors from the electrophoretic mobilities of the compounds, and further calculating the distribution coefficients, an analysis of variance was performed. After extracting the statistical models, optimal operational conditions were obtained based on the developed models. To further investigate the interactions between the β-blockers and the liposomes, nanoplasmonic sensing experiments were carried out on two different liposome systems. The overall results demonstrate the strong influence of cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine on the distribution coefficients.

Capillary electrokinetic chromatography for studying interactions between β-blockers and Intralipid emulsion

Toxicity of β-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of β-blockers of different hydrophobicity’s (log KD values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed β-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log KD values of the β-blockers. The binding and adsorption constants also show that less hydrophobic β-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of β-blockers is worth further investigation.

Chiral separation by capillary electrokinetic chromatography with hydrophobic deep eutectic solvents as pseudo-stationary phases

This study demonstrated for the first time that hydrophobic deep eutectic solvents (HDESs) can be used in capillary electrophoresis (CE) for chiral separations. We found that the an HDES methyltrioctylammonium chloride:octanoic acid (N8881Cl:OctA) can exist in the form of nano-sized microdroplets in CE background electrolyte solutions, and show hydrophobic effects as a new type of pseudo-stationary phase (PSP) during CE separation. When used in combination with various cyclodextrin (CD)-type chiral selectors, the presence of N8881Cl:OctA significantly improved the enantioresolutions of several model drugs. Moreover, the migration time of the enantiomers can also be reduced when an anionic CD (e.g., carboxymethyl-β-cyclodextrin (CM-β-CD)) was used. Critical factors influencing the chiral separations were systematically investigated including the HDES concentration, hydrogen-bond acceptor (HBA)/hydrogen-bond donor (HBD) ratio, CD concentration, buffer pH, and applied voltage, etc. An insight into chiral recognition mechanism with HDES is provided for reference. A comparison of the chiral CE performance of HDESs with traditional surfactants was also performed to demonstrate their superiority as a new type of PSP.

The role of deep eutectic solvents in chiral capillary electrokinetic chromatography: A comparative study based on α-cyclodextrin chiral selector

• The first investigation of the effect of HBAs of DESs on chiral EKC. • The comparison of the performance of chiral and achiral DESs in chiral EKC. • The comparison of the performance of DESs and ILs as additives in chiral EKC. • The objective evaluation of the feasibility of DESs as additives in chiral EKC. Deep eutectic solvents (DESs) are emerging as potential candidates as additives/modifiers in chiral electrokinetic chromatography (EKC). However, the true role of DESs in the chiral separation process is far from being fully elucidated. Since most previous studies have been restricted to using choline chloride as the hydrogen-bond acceptor (HBA) in DESs, here we first evaluated the effect of five types of DESs (containing different HBAs) on the enantioseparation performance of an α-CD-based EKC system. The results demonstrate that these DESs are capable of moderately improving the enantioresolutions of several model drugs. A series of comparative experiments indicate that the improvement is mainly due to the contribution of the HBAs, rather than the selected hydrogen-bond donors (HBDs). Considering that most of these DESs are achiral, we further prepared five chiral DESs, aiming to investigate whether there is a synergistic effect between α-cyclodextrin and chiral DESs. However, these chiral DESs did not show obvious superiority compared with achiral DESs. This conclusion is different from the use of achiral or chiral ionic liquids (ILs) as additives in chiral EKC, in which chiral ILs usually exhibit significantly better enantioseparation performance than achiral ILs. Another important conclusion of this work is that there seems to be no significant difference between using DESs and their individual components, which is also different from the performance of most previously reported ILs. This conclusion indicates that the DESs may be virtually completely dissociated into their initial components (HBAs + HBDs) in the separation medium due to the destruction of their hydrogen-bond network. Therefore, a question which deserves particular attention of researchers in this field is raised here: whether it is absolutely necessary to use DESs in chiral EKC since adding HBAs and HBDs separately seems to have the same function?

Separation of planar chiral ferrocenes by capillary electrokinetic chromatography and liquid chromatography

Capillary electrokinetic chromatography (CEKC) and liquid chromatography (LC) methods were explored for the enantiomeric separation of six unsymmetrically disubstituted ferrocene derivatives. In normal-phase mode liquid chromatography, the use of n-heptane, ethanol or isopropanol with 1% n-butylamine as mobile phase on six polysaccharide-based columns, allowed to fully separate the enantiomers of three compounds out of the six (i.e 7-chloro-N-(2-((dimethylamino)methyl)ferrocenyl)quinolin-4-amine (ferroquine) (compound 1), 1-[(1S)-(1-Aminoethyl)]-2-(diphenylphosphino)ferrocene (compound 5) and 1-[(1R)-1-(Dicyclohexylphosphino)ethyl]-2-(diphenylphosphino)ferrocene (compound 6). Among the columns used, the Lux i-Cellulose-5 was the most effective. In capillary electrokinetic chromatography, a phosphate buffer of 25 mM concentration and pH equal to 2.5 was chosen as background electrolyte, leading to cationic ferrocene derivatives. The addition of neutral cyclodextrins was undertaken first and native β- or γ-cyclodextrins were found to resolve the enantiomers of two derivatives. Then, 15 mM of anionic cyclodextrins were added to the background electrolyte. The use of SBE-β-CD, S-β-CD or S-γ-CD have allowed the separation of the enantiomers for most of the ferrocene derivatives studied with high resolution values in short migration time. For instance, for 1-(R)-2-(Diphenylphosphino)ethyldi-tert-butylphosphine ferrocene (compound 2), the migration times were less than 2 minutes and the resolution value was equal to 3.52 in short-end mode with 15 mM S-β-CD, at 25 kV and 25°C. Finally, a dual cyclodextrins system was tested using 15 mM of S-β-CD plus 15 mM HP-γ-CD in the phosphate buffer. This system allowed the improved separation of two ferrocene derivatives with an unusual resolution value equal to 41.5 in long-end mode. Overall, CEKC showed better enantioseparating power of the six chiral ferrocenes studied than liquid chromatography.

Hydrophobic deep eutectic solvents as pseudo-stationary phases in capillary electrokinetic chromatography: An explorative study

Research into the application of deep eutectic solvents (DESs) in capillary electrophoresis (CE) is still in its infancy. Even so, a controversy has already arisen over the “true role” of DESs in CE. This is because the DESs employed previously were all hydrophilic, and it is therefore reasonable to speculate that they may be virtually completely dissociated into its initial components (hydrogen-bond acceptor (HBA) and hydrogen-bond donor (HBD)) in aqueous CE buffers. Since there seems to be no essential difference in CE performance between adding a DES and adding its two constituents separately (HBA and HBD) in a running buffer, the significance of using DESs in these CE systems has not been clearly established. In this context, this study established the first DES-based CE system in which a DES really worked as a single entity. To achieve this goal a hydrophobic DES (HDES), (−)-menthol:octanoic acid, was employed to act as a pseudo-stationary phase (PSP) in electrokinetic chromatography (EKC) mode. We found that the HDES is capable of forming stable aggregates (micro-droplets) which can act as a PSP in the running buffer. The novel HDES-type PSP exhibited impressive selectivities toward several groups of analogues, homologues, and isomers via hydrophobic mechanism. It can be used either solely for direct EKC separation, or in combination with traditional PSPs (e.g., cyclodextrins). As a proof-of-concept study, we have demonstrated that the synergistic effect generated in an “HDES + Hydroxypropyl-γ-cyclodextrin” dual-PSP system allowed an excellent separation of a simulated complex mixture consisting of 13 aromatic acids.

Synthesis and characterization of carnitine-based ionic liquids and their evaluation as additives in cyclodextrin-electrokinetic chromatography for the chiral separation of thiol amino acids

In this study, new chiral ionic liquids based on the non-protein amino acid L-carnitine as cationic chiral counterpart and several anions (bis(trifluoromethane)sulfonimide (NTf2−), L-lactate− or Cl−) as counterions were synthesized. Moreover, three different salts based on L-carnitine were also synthesized and the other three were commercially acquired and used for comparison. The synthesized ionic liquids and salts were characterized by nuclear magnetic resonance, fourier transform infrared spectroscopy, high-performance liquid chromatography-mass spectrometry, and elemental analysis. Subsequently, they were used as additives to establish a γ-CD-based dual system for the enantiomeric separation of cysteine and homocysteine (previously derivatized with fluorenylmethoxycarbonyl chloride) by capillary electrokinetic chromatography. The effect of the nature of the anionic counterions and the presence of different substituents on the L-carnitine molecule on the chiral separation of both amino acids was investigated. The enantioseparations obtained with each dual system studied were compared in terms of the enantiomer effective mobilities (μeff) and the effective electrophoretic selectivity (αeff). Practically, all the dual systems evaluated exhibited substantially improved enantioseparations for the two amino acids compared with the single γ-CD system.

Quality by Design Assisted Optimization of a Chiral Capillary Electrokinetic Chromatographic Method for the Separation of Amlodipine Enantiomers Using Maltodextrin as Chiral Selector.

Analytical-method development based on design of experiment has been applied for optimizing the enantioseparation of amlodipine by chiral capillary electrokinetic chromatography using maltodextrin as the chiral selector. The effect of different factors on the enantioresolution quality was screened. Three separation factors, namely maltodextrin concentration, pH of the background electrolyte and applied voltage were selected as independent variables. The number of experiments was reduced while maximizing the information content using design of experiment. Based on a full-quadratic design that included three variables on three levels, the total design space could be reduced to fifteen factor combinations using a D-optimal algorithm. The aim of the experiment was to find the optimal factor combinations with respect to resolution. The maltodextrin concentration (7.5–10% w/v) demonstrated the strongest effect on the resolution followed by pH (2–4) of the background electrolyte and the applied voltage (15–20 kV). An increase in the maltodextrin concentration was found to result in a greater stereoselectivity, represented by the higher resolution values (Rs ≥ 1.5). The separation conditions in the proposed method were feasible to be adjusted within the applied range with an acceptable resolution.

Enantioselective analysis of the methamphetamine precursors ephedrine and pseudoephedrine by capillary electrokinetic chromatography using cyclodextrins as chiral selectors

Ephedrine and pseudoephedrine can be used as precursors for the illicit synthesis of stimulant drugs, such as methamphetamine and methcathinone. For this reason, an enantioselective capillary electrokinetic chromatography method was developed for the separation of ephedrine and pseudoephedrine enantiomers. After testing two different charged or chargeable cyclodextrins (β-cyclodextrin sulphate and carboxymethyl-β-cyclodextrin) and a few mixtures as possible chiral selectors, a mixture of 3 mM carboxymethyl-β-cyclodextrin and 3 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin in pH 3.4 acetate buffer was used as the enantioselective background electrolyte and the separation was carried out in an uncoated silica capillary (75 µm internal diameter, 48.6 cm total length, 40.0 cm effective length). Analytical method performance was then evaluated in terms of linearity, precision, selectivity and accuracy with good results. Proof of concept application to mixtures simulating illicit methamphetamine (containing small amounts of ephedrine and pseudoephedrine as possible residual synthesis impurities) provided satisfactory results for the identification and quantitation of the four analyte diastereomers.

Explanation of the Formation of Complexes between Representatives of Oxazolidinones and HDAS-β-CD Using Molecular Modeling as a Complementary Technique to cEKC and NMR.

Molecular modeling (MM) results for tedizolid and radezolid with heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD) are presented and compared with the results previously obtained for linezolid and sutezolid. The mechanism of interaction of chiral oxazolidinone ligands belonging to a new class of antibacterial agents, such as linezolid, tedizolid, radezolid, and sutezolid, with HDAS-β-CD based on capillary electrokinetic chromatography (cEKC), nuclear magnetic resonance (NMR) spectroscopy, and MM methods was described. Principles of chiral separation of oxazolidinone analogues using charged single isomer derivatives of cyclodextrin by the cEKC method were presented, including the selection of the optimal chiral selector and separation conditions, complex stoichiometry, and binding constants, which provided a comprehensive basis for MM studies. In turn, NMR provided, where possible, direct information on the geometry of the inclusion complexes and also provided the necessary structural information to validate the MM calculations. Consequently, MM contributed to the understanding of the structure of diastereomeric complexes, the thermodynamics of complexation, and the visualization of their structures. The most probable mean geometries of the studied supramolecular complexes and their dynamics (geometry changes over time) were determined by molecular dynamics methods. Oxazolidinone ligands have been shown to complex mainly the inner part of cyclodextrin, while the external binding is less privileged, which is consistent with the conclusions of the NMR studies. Enthalpy values of binding of complexes were calculated using long-term molecular dynamics in explicit water as well as using molecular mechanics, the Poisson–Boltzmann or generalized Born, and surface area continuum solvation (MM/PBSA and MM/GBSA) methods. Computational methods predicted the effect of changes in pH and composition of the solution on the strength and complexation process, and it adapted the conditions selected as optimal during the cEKC study. By changing the dielectric constant in the MM/PBSA and MM/GBSA calculations, the effect of changing the solution to methanol/acetonitrile was investigated. A fairly successful attempt was made to predict the chiral separation of the oxazolidinones using the modified cyclodextrin by computational methods.

Chiral Capillary Electrokinetic Chromatography: Principle and Applications, Detection and Identification, Design of Experiment, and Exploration of Chiral Recognition Using Molecular Modeling.

This work reviews the literature of chiral capillary electrokinetic chromatography from January 2016 to March 2021. This is done to explore the state-of-the-art approach and recent developments carried out in this field. The separation principle of the technique is described and supported with simple graphical illustrations, showing migration under normal and reversed polarity modes of the separation voltage. The most relevant applications of the technique for enantioseparation of drugs and other enantiomeric molecules in different fields using chiral selectors in single, dual, or multiple systems are highlighted. Measures to improve the detection sensitivity of chiral capillary electrokinetic chromatography with UV detector are discussed, and the alternative aspects are explored, besides special emphases to hyphenation compatibility to mass spectrometry. Partial filling and counter migration techniques are described. Indirect identification of the separated enantiomers and the determination of enantiomeric migration order are mentioned. The application of Quality by Design principles to facilitate method development, optimization, and validation is presented. The elucidation and explanation of chiral recognition in molecular bases are discussed with special focus on the role of molecular modeling.

Electrophoretic separation of multiple chiral center analyte with a three cyclodextrins mixture.

A capillary electrokinetic chromatography method (CEKC) was developed for complete stereoisomeric separation of a neutral, hydrophobic, multiple chiral center dihydropyridone analogue, a drug candidate proposed in type 2 diabetes treatment. A background electrolyte comprising three cyclodextrins was found to successfully separate the eight isomers. First an anionic cyclodextrin, the SBE-β-CD, was selected to allow the chiral separation of our neutral compound and partial resolutions of the eight isomers were obtained. Then, the effects of different parameters such as the nature and concentration of the other cyclodextrins added and pH of the buffer were examined. Finally, a triple CD-system consisted of 15 mMSBE-β-CD plus 15 mMγ-CD and 40 mMHP-γ-CD in a 50mM borate background electrolyte at pH 10, was found to successfully separate the eight isomers. Last, the selectivity and limits of detection and quantification were evaluated for this optimized method.

Assessing the Interactions of Auristatin Derivatives with Mixed Phospholipid-Sodium Dodecyl Sulfate Aggregate Dispersions.

The aim of this study was to assess what properties of the pseudostationary phases in electrokinetic capillary chromatography affect the interactions between monomethyl auristatin E (MMAE) and hydrophilically modified structural analogues thereof with various lipophilic phases. MMAE is a widely used cytotoxic agent in antibody–drug conjugates (ADC), which are used as selective biopharmaceutical drugs in the treatment of cancers. MMAE and its derivatives are highly lipophilic, yet they fail to interact with biomimicking phosphatidylcholine–phosphatidylserine liposomes. To reveal what properties affect the interaction of the auristatin derivatives with cell plasma membrane-mimicking vesicles, capillary electrokinetic chromatography was used with four different types of micellar and vesicular pseudostationary phases: pure vesicles, mixed vesicles, mixed micelles, and pure micelles. Vesicular phases were composed of pure phospholipids [dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC)] and phospholipid–surfactant mixtures [sodium dodecyl sulfate, (SDS) with DMPC and DLPC] while the micellar phases comprised pure surfactant (SDS) and surfactant–phospholipid mixtures (SDS–DMPC and SDS–DLPC). In addition, differential scanning calorimetry and dynamic light scattering were used to monitor the aggregate composition. Our data shows that the interaction between hydrophobic auristatin derivatives and hydrophobic pseudostationary phases critically depends on the type, size, and hydrogen bonding capability of the pseudostationary phases.

Evaluation of cyclodextrin‐micellar electrokinetic capillary chromatography with pyrrolidinium‐based ionic liquid surfactant as a pseudostationary phase for chiral separation

AbstractIonic liquids have drawn increasing attention in separation science recently. As a new promising brand of surfactants, ionic‐liquid‐based surfactants have their unique properties and superior aggregation behavior, which can be introduced as micelle‐forming surfactants in micellar electrokinetic capillary chromatography mode. There have been few reports of the applications of ionic‐liquid‐based surfactants for micellar electrokinetic capillary chromatography enantioseparation. In this study, pyrrolidinium‐based ionic liquids surfactants were applied for the first time for chiral separation with hydroxypropyl‐β‐cyclodextrin as a chiral selector by micellar electrokinetic capillary chromatography. As observed, the established micellar electrokinetic capillary chromatography systems with ionic‐liquid‐based surfactants could provide better separation of tested chiral drugs than conventional non‐ionic liquid cationic surfactant. In addition, parameters such as the alkyl chain length, concentration of ionic‐liquid‐based surfactants, type and proportion of organic modifier, concentration of chiral selector and composition and pH of buffer solution were systematically investigated to optimize the cyclodextrin‐micellar electrokinetic capillary chromatography system.

Evaluation of the enantioselectivity of capillary electrokinetic chromatography using ethanediamine-bonded poly (glycidyl methacrylate) microspheres as the pseudostationary phases.

In this work, a new capillary electrokinetic chromatography (EKC) approach using ethanediamine-bonded poly (glycidyl methacrylate) (Ami-PGMA) microspheres as pseudostationary phases (PSPs) for enantioseparation with a polysaccharide, chondroitin sulfate E (CSE), as the chiral selector. The CSE@Ami-PGMA EKC system was applied to enantioseparate basic drugs, and distinct improved separations of tested enantiomers were obtained while comparing with the single CSE system (the resolution increased from 0.41 to 1.26 for nefopam, from 1.24 to 2.15 for laudanosine, and from 0.92 to 2.36 for amlodipine). The Ami-PGMA microspheres were fully characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopy, and the results showed Ami-PGMA microspheres were uniform and spherical in size (1μm). Several principal parameters were systematically investigated, and the optimal chiral separations were obtained with Tris/H3 PO4 (20mM, pH2.4, and 3.4 for NEF) containing 2.5% (w/v) CSE and 20-μg Ami-PGMA microspheres in 20°C. Subsequently, the concentrations of Ami-PGMA microspheres and CSE were proved to be the dominant factors for the separation in the CSE@Ami-PGMA EKC system by Statistical Product and Service Solutions (SPSS).

CE with multi-walled carbon nanotubes (MWCNTs). Part II. SDS coated functionalized MWCNTs as pseudo-stationary phases in nanoparticle EKC – Retention behaviors of small and large solutes

In this study, functionalized multi-walled carbon nanotubes (MWCNTs), namely hydroxylated MWCNTs (MWCNT-OH), carboxylated MWCNTs (MWCNT-COOH) and sulfonated MWCNTs (MWCNT-SO3H) coated with sodium dodecyl sulfate (SDS) were demonstrated as effective pseudo-stationary phases (PSPs) in the separation of various species by the nanoparticle capillary electrokinetic chromatography (NPEKC) mode of capillary electrophoresis (CE). Due to the significant increase in their surface charge density in the presence of SDS, the three SDS coated MWCNTs yielded high performance separation for herbicides, barbiturates, dansyl-DL-amino acids (Dns-AAs), dipeptides and proteins by NPEKC. In addition, high resolution tryptic peptide maps of three standard proteins including myoglobin, cytochrome C and lysozyme were readily obtained. The three PSPs systems yielded high plate numbers that spanned a wide range of values depending on the type of species. The values of the observed selectivity factors (i.e., α values) were significantly different among the three PSPs for solutes that underwent strong interactions with the SDS coated functionalized MWCNTs while for negatively charged solutes (e.g., Dns-AAs) of the same charge sign as the PSPs the α values were about the same on the three different PSPs indicating weak association with the PSPs and signaling separation based chiefly on the differences in electro-migration arising from differences in charge-to-mass ratios.

Enantioselective recognition of radezolid by cyclodextrin modified capillary electrokinetic chromatography and electronic circular dichroism

A method for the enantioseparation of radezolid (RAD), an analogue of a truly new class of antibacterial agents, oxazolidinones, was developed based on capillary electrokinetic chromatography using a cyclodextrin as a chiral pseudophase (CD-cEKC). The mechanism of RAD separation, together with its precursor, were investigated to directly define the relationship between the oxazolidinone structure and the complexation process. During the development of the method, anionic single isomer cyclodextrins were tested. They were ranked in order from hydrophilic to hydrophobic as follows: heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin (HS-β-CD), heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD) and heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD). Experiments were performed at pH values of 2.5, 6.6, 8.2 and 9.6. The cyclodextrins that had an acetyl or methyl group at the C2 and C3 positions, referred to as HDAS-β-CD and HDMS-β-CD, respectively, exhibited partial and baseline separation of enantiomers in a low pH buffer. However, higher temperatures were required for HDAS-β-CD and acetonitrile addition was required for HDMS-β-CD. During the experiments, different organic solvents, varying in their amphiprotic or aprotic nature, were tested. The best results for the separation of enantiomers using the CD-cEKC method were obtained with 40mM HDMS-β-CD dissolved in a 50mM phosphate buffer (pH 2.5) with the addition of acetonitrile (65:35, v/v) at 27°C, reversed polarity and a voltage equal to 28kV. The apparent binding constants for each enantiomer to HDAS-β-CD or HDMS-β-CD were calculated. Finally, the stereochemistry of (S) and (R)-RAD and the behaviour of selected complex formations were established using electronic circular dichroism.

Distribution of local anesthetics between aqueous and liposome phases

Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (KD) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25°C, the distribution constants were determined at 37 and 42°C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant.

Advances in the Use of Cyclodextrins as Chiral Selectors in Capillary Electrokinetic Chromatography: Fundamentals and Applications

Capillary electrokinetic chromatography is generally recognized as a versatile and robust capillary electromigration technique for the separation of enantiomers. In this mode, one or more chiral selectors are added to the background electrolyte acting as pseudostationary phases. Within the various chiral selectors that have been applied to enantioseparations in capillary electrokinetic chromatography, cyclodextrins are by far the most often used selectors because of their versatility, structural variety and commercial availability. This is reflected in the large number of applications of cyclodextrins to analytical enantioseparations that have been reported between January 2012 and July 2016, the period of time covered by this review. Many of these applications cover aspects of life sciences such as drug analysis, bioanalysis or food analysis. Despite the large number of commercially available cyclodextrins, new derivatives have been developed in order to achieve altered enantioselectivities or to further broaden the application range. Cyclodextrins have also been used to demonstrate the validity of theoretical models of electromigration as well as complex formation equilibria in enantioseparations. Finally, recent studies for an understanding of the molecular basis of the chiral recognition between cyclodextrins and the analytes are discussed.