On-line coupling of partial filling-capillary zone electrophoresis with mass spectrometry for the separation of clenbuterol enantiomers

The on-line coupling of capillary zone electrophoresis with mass spectrometry (CZE-MS) for the separation of enantiomers is hampered by the presence of nonvolatile chiral selectors such as cyclodextrins in the separation buffer. This problem can be overcome by use of the partial filling technique where only a part of the capillary is filled with the separation buffer containing chiral selectors. Since the electroosmotic flow is almost completely suppressed at acidic pH, that dimethyl-beta-cyclodextrin is neutral, no free cyclodextrin would reach the MS detector when using a partially filled capillary. By this method, clenbuterol enantiomers were successfully resolved and separated from salbutamol (internal standard) in aqueous solution and in plasma samples. A solid-phase extraction (SPE) was used for the preparation of plasma samples before analysis.     

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography (CEC) is achieved with open-tubular capillaries (o-CEC), with packed capillaries (p-CEC) or with monolithic capillaries. In o-CEC, capillaries are coated with a thin film containing cyclodextrin derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as chiral selectors. In p-CEC, typical chiral HPLC stationary phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins, glycoproteins, macrocyclic antibiotics, quinine-derived and 'Pirkle' selectors, polyacrylamides and molecularly imprinted polymers are used as chiral selectors. Chiral monolithic stationary phases prepared by in situ polymerization into the capillary were also developed for electrochromatographic enantiomer separation.     

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.     

Capillary electrophoresis using copolymers of different composition as physical coatings: a comparative study

In this work, a comparative study on the use of different polymers as physically adsorbed coatings for CE is presented. It is demonstrated that the use of ad hoc synthesized polymers as coatings allows tailoring the EOF in CE increasing the flexibility of this analytical technique. Namely, different polymers were synthesized at our laboratory using different percentages of ethylpyrrolidine methacrylate (EpyM) and N,N-dimethylacrylamide (DMA). Thus, by modifying the percentage of EpyM and DMA monomers it is possible to manipulate the positive charge of the copolymer, varying the global electrical charge on the capillary wall and with that the EOF. These coated capillaries are obtained by simply flushing a given EpyM-DMA aqueous solution into bare silica capillaries. It is shown that by using these coated capillaries at adequate pHs, faster or more resolved CE separations can be achieved depending on the requirements of each analysis. Moreover, it is demonstrated that these coated capillaries reduce the electrostatic adsorption of basic proteins onto the capillary wall. Furthermore, EpyM-DMA coatings allow the reproducible chiral separation of enantiomers through the partial filling technique (PFT). The EpyM-DMA coated capillaries are demonstrated to provide reproducible EOF values independently of the pH and polymer composition with%RSD values lower than 2% for the same day. It is also demonstrated that the coating procedure is reproducible between capillaries. The compatibility of this coating protocol with CE in microchips is discussed.     

Evaluation of balhimycin as a chiral selector for enantioresolution by capillary electrophoresis

The glycopeptide antibiotic balhimycin and its haloanalogue bromobalhimycin were evaluated as chiral selectors for enantioresolution by capillary electrophoresis. In order (i) to eliminate the adsorption of the glycopeptide antibiotics on the capillary wall, (ii) to shorten the separation time and (iii) to improve the detection sensitivity, a combined approach of the dynamic surface coating technique, the co-electroosmotic flow electrophoresis technique and the partial filling technique was employed for the enantioresolution of 16 acidic racemates. The effect of experimental parameters (plug length of the partial filling solution containing the chiral selector, selector concentration and buffer pH) on enantiorecognition was investigated. Furthermore, the enantiorecognition ability imparted by balhimycin, bromobalhimycin and vancomycin were compared. For most tested compounds, the highest enantiorecognition was obtained with balhimycin as chiral selector. Only in the case of the enantioresolution of tiaprofenic acid, vancomycin showed a superior enantiorecognition.     

Penicillin G acylase as chiral selector in CE using a pullulan-coated capillary

In the present study, penicillin G acylase (PGA), an enzyme belonging to the family of hydrolases, has been investigated as chiral selector in CE using the partial filling technique. Owing to the strong disposition of PGA to be adsorbed by the inner capillary wall, permanently coated capillaries were used to diminish both the protein-wall interactions and the EOF. In particular, the silica surface of the capillary was chemically coated by an antiadhesive and an hydrophilic layer of pullulan, a high-molecular-mass homopolysaccharide. The coating procedure consisted in the silanization with glycidoxypropyltrimethoxysilane and the subsequent coupling of the hydroxyl groups of pullulan onto the silanized capillary. Using this approach, a significant EOF suppression was obtained within a wide pH range (pH 3.0-9.0); this result was very important in order to find the suitable conditions for the application of partial filling technique. The optimization of partial filling was carried out by considering the effects of different experimental conditions (buffer pH, PGA concentration, and loading duration), on the migration time and enantioresolution of rac-ketoprofen. Under the selected conditions as: 100 mM sodium phosphate buffer (pH 5.5) containing 240 microM of PGA (partial filling of 120 s at a pressure of 50 mbar), a series of acidic compounds resulted to be enantioresolved in about 10 min. The long-term stability of the proposed coating was evaluated; more than 100 injections were performed without significant loss of reproducibility.     

Evaluation of carbon nanostructures as chiral selectors for direct enantiomeric separation of ephedrines by EKC

Single-walled nanotubes and multi-walled nanotubes (MWNTs) have been evaluated as chiral selectors for the enantiomeric separation of ephedrines by using EKC with surfactant-coated carbon nanotubes. The analysed compounds were (+/-)-ephedrine, (+/-)-norephedrine and (+/-)-N-methylephedrine. The potential of those carbon nanostructures as chiral selectors has been evaluated by changing different experimental variables such as pH, addition of organic modifiers, potential and injection time. The capability of MWNTs to resolve enantiomeric mixtures was demonstrated by using partial filling of the capillary with concentrated surfactant-coated MWNTs. Differences in the enantioselectivity were discussed.     

Dependence of chiral separations on the amount of cyclodextrins as selectors, employing the partial filling technique in capillary zone electrophoresis

Summary The enantioselectivity, resolution factor and migration times for some local anaesthetic drugs were evaluated at different selector concentrations and plug lengths with different cyclodextrins as chiral selectors using the partial filling technique. The resolution remained constant when equal amounts of the chiral selector at a concentration below the optimal concentration were introduced in the capillary. However, the selectivity factor increased with increasing concentration of the chiral selector.     

Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis

A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.     

New adsorbed coatings for capillary electrophoresis

New acrylic polymers bearing oxirane groups were synthesized to be used in the production of coated capillaries. A fully automated coating procedure was devised based on the use of diluted water solutions of these polymers. The whole procedure required less than 30 min. The new polymers rapidly adsorbed from water onto the capillary wall, thus suppressing electroosmotic flow (EOF) to a negligible value. The adsorbed coatings were stable for hundreds of hours at high pH, temperature, and in the presence of 8 M urea. Efficient separations of acidic and basic proteins were achieved in the new phases.     

Capillary methods for drug analysis

This review gives an overview of the applicability of capillary electrophoresis, capillary liquid chromatography, capillary electrochromatography, and their derived techniques to analyze drug impurity mixtures, formulations, biological samples, and chiral compounds. For each application type, a few examples are given to illustrate the potential of the capillary technique. Details are provided about the capillaries used, chiral selectors, and stationary and mobile phases.     

Non-covalent capillary coatings for protein separations in capillary electrophoresis

Adsorption of proteins, particularly basic proteins onto fused silica capillaries severely degrades capillary electrophoretic performance. This review provides a synopsis of the fundamentals underlying protein adsorption and its impact on CE performance. The efficacy of small molecule background electrolyte additives, surfactants, physically adsorbed polymers (dynamic and static), and successive multiple ionic-polymer layer coatings are evaluated using a number of performance metrics. Peak efficiency and migration time reproducibility are used as measures of reversible protein adsorption, while protein recovery, electroosmotic flow reproducibility and step changes in the baseline are used as indicators of irreversible protein adsorption.     

Evaluation of poly([2‐(acryloyloxy)ethyl]trimethylammonium chloride) cationic polymer capillary coating for capillary electrophoresis and electrokinetic chromatography separations

Capillary electrophoresis and electrokinetic chromatography are typically carried out in unmodified fused‐silica capillaries under conditions that result in a strong negative zeta potential at the capillary wall and a robust cathodic electroosmotic flow. Modification of the capillary wall to reverse the zeta potential and mask silanol sites can improve separation performance by reducing or eliminating analyte adsorption, and is essential when conducting electrokinetic chromatography separations with cationic latex nanoparticle pseudo‐stationary phases. Semipermanent modification of the capillary walls by coating with cationic polymers has proven to be facile and effective. In this study, poly([2‐(acryloyloxy)ethyl]trimethylammonium chloride) polymers were synthesized by reversible addition‐fragmentation chain transfer polymerization and used as physically adsorbed semipermanent coatings for capillary electrophoresis and electrokinetic chromatography separations. An initial synthesis of poly([2‐(acryloyloxy)ethyl]trimethylammonium chloride) polymer coating produced strong and stable anodic electroosmotic flow of –5.7 to –5.4 × 10⁻⁴ cm²/V⋅s over the pH range of 4–7. Significant differences in the magnitude of the electroosmotic flow and effectiveness were observed between synthetic batches, however. For electrokinetic chromatography separations, the best performing batches of poly([2‐(acryloyloxy)ethyl]trimethylammonium chloride) polymer performed as well as the commercially available cationic polymer polyethyleneimine, whereas polydiallylammonium chloride and hexadimethrine bromide did not perform well.     

Chiral capillary electrophoresis and nuclear magnetic resonance investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors

In the present work, synthetic cyclohexa- and cycloheptapeptides previously singled out by a combinatorial chemistry approach have been evaluated as chiral selectors in capillary electrophoresis. By applying the countercurrent migration technique and employing a new adsorbed coating, a series of dinitrophenyl amino acids as well as some chiral compounds of pharmaceutical interest have been evaluated for enantiorecognition. The results thus obtained led to a deeper investigation of the chiral discrimination process, by carrying out nuclear magnetic resonance (NMR) studies on selected cyclopeptide-analyte complexes. These studies shed light on the chemical groups involved in the analyte-selector interaction and provided useful information for a wider application of these cyclopeptides in the separation of other drug enantiomers.     

Chiral separations by open tubular capillary electrokinetic chromatography.

The inner walls of 50 microns fused-silica capillaries are etched by ammonium hydrogendifluoride and then modified by the silanization/hydrosilation method with a chiral selector. Three different types of selectors were evaluated: lactone, beta-cyclodextrin and naphthylethylamine. Each of the bonded chiral stationary phases provided at least partial separation for one type of racemic solute. These results confirm that bonded organic moieties on the etched inner wall of a capillary can provided sufficient solute-bonded phase interactions to influence the retention of molecules driven through a capillary by electroosmosis or a combination of electroosmosis and electrophoretic mobility.     

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.

     

Cyclodextrin-NH-MIL-53 open tubular stationary phase for capillary electrochromatography enantioseparation

A novel chiral group functionalized metal-organic framework, Cyclodextrin-NH-MIL-53, was synthesized and modified on the inner wall of a capillary column via a post-synthetic process. The prepared chiral metal-organic framework was utilized as a chiral capillary stationary phase and used in an open-tubular capillary electrochromatography method to enantioseparate several racemic amino acids. Excellent enantioseparation of five pairs of enantiomers was obtained in this chiral separation system (Resolutions of D/L-Alanine = 16.844, D/L-Cysteine = 3.617, D/L-Histidine = 9.513, D/L-Phenylalanine = 8.133, and D/L-Tryptophan = 2.778). The prepared Cyclodextrin-NH-MIL-53 and the Cyclodextrin-NH-MIL-53-based capillary columns were characterized by scanning electron microscopy, X-ray diffraction, Fourie-transform infrared spectroscopy, and circular dichroism. The chiral capillary electrochromatography conditions, such as separation conditions, amount of Cyclodextrin-NH-MIL-53, and electroosmotic flow, were optimized. This research is estimated to present a novel insight and method for the design and use of metal-organic framework-based capillaries for enantioseparation.     

Automated coating procedures to produce poly(ethylene glycol) brushes in fused‐silica capillaries

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in‐capillary surface‐initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4−8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4−8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.     

微波辅助合成法快速制备替考拉宁开管毛细管电色谱柱

采用微波辅助合成技术,快速制备了以替考拉宁为固定相的开管毛细管电色谱柱。在pH 4.0~7.0的范围内比较了空管与替考拉宁修饰柱的电渗情况,表明替考拉宁开管毛细管电色谱柱有效地降低了电渗。用该色谱柱分离了多种手性对映体,均达到基线分离,体现了替考拉宁开管毛细管电色谱柱良好的分离性能。以DL-色氨酸考察了柱子的稳定性和重现性,结果显示采用微波辅助合成技术制得的替考拉宁开管毛细管电色谱柱具有良好的稳定性和重现性。     

毛细管电泳新型手性分离体系研究进展

在现代分离科学中,手性化合物的分离分析一直是研究的重点和难点。相比于高效液相色谱(HPLC)、气相色谱(GC)等传统色谱分析方法,毛细管电泳(CE)技术凭借其高效率、低消耗、分离模式多样化等诸多优势,已经发展成为手性分离研究领域最有应用前景的分析方法之一。近年来,研究人员在CE手性分析方法的构建过程中,基于毛细管电动色谱(EKC)、配体交换毛细管电泳(LECE)、毛细管电色谱(CEC)等各种基础电泳模式,不断地对传统手性分离体系进行优化和改造,构建出了许多高性能的新型手性CE分离体系。如利用各类功能化离子液体以"手性离子液体协同拆分""手性离子液体配体交换""离子液体手性选择剂"等模式设计出多种基于离子液体的CE手性分离体系;利用纳米材料独特的尺寸效应、多样性、可设计性等特点,直接或与传统手性选择剂有机结合构建CE手性分离体系。此外,金属有机骨架材料修饰、低共熔溶剂修饰、非连续分段式部分填充等各式新颖的CE手性分离体系也都被研究人员成功开发,并表现出较大的发展潜力。该综述将对近年来(尤其是2015～2019年)此类新型CE手性分离体系的发展状况进行梳理,并结合相应的手性识别机理研究和手...