Enantiomeric separations of primary amino compounds by capillary electrochromatography with monolithic chiral stationary phases of chiral crown ether-bonded negatively charged polyacrylamide gels

A novel enantiomeric separation method by capillary electrochromatography with chiral crown ether-bonded negatively charged polyacrylamide gels is presented. Two kinds of chiral crown ether derivatives, (+)-tetraallyl 18-crown-6 carboxylate and (+)-18-crown-6 tetracarboxylic acid 2-allyl ester were synthesized and allowed to covalently bind to a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, respectively. The gel was placed in fused-silica tubing, the walls of which had been activated with a bifunctional reagent to make the resulting gel bind covalently to the inner surface. Enantiomeric separations of 12 primary amino compounds were achieved using these columns and mobile phases of 200 mM triethanolamine-300 mM boric acid buffers with high efficiencies of up to 135000 plates m(-1). Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 13 months with intermittent use for 3 months followed by storage at room temperature for 10 months. The result of the optical purity test of alanine-2-naphthylamide is also described.     

Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.     

Amylose-3,5-dimethylphenylcarbamate immobilized on monolithic silica stationary phases for chiral separations in capillary electrochromatography

Chiral monolithic silica capillary columns were prepared by immobilization of amylose-3,5-dimethylphenylcarbamate (ADMPC) bearing a small fraction of 3-(triethoxysilyl)propyl residues through intermolecular polycondensation of the triethoxysilyl groups. The obtained columns were used for chiral separations in capillary electrochromatography (CEC). The effects of the silica monolith nature and the used chiral selector concentration on the resulting enantiomeric separations were investigated. Fifteen chiral compounds, including acidic, neutral, and basic substances were evaluated and twelve showed partial or baseline separation at some of the different conditions tested. These results demonstrated the promising applicability of ADMPC-immobilized monolithic silica columns in CEC enantioseparations, but also revealed the need for further improvements on the level of baseline separations and efficiencies.     

Enantiomeric separations by capillary electrochromatography using a macrocyclic antibiotic chiral stationary phase.

Racemic mixtures of tryptophan and dinitrobenzoyl leucine have been successfully resolved by capillary electrochromatography (CEC) using the macrocyclic antibiotic teicoplanin, covalently bonded to a 5 microns silica support. Modification of a previously published packing procedure was required to pack reliable capillaries, capable of performing enantiomeric separations. Good levels of enantioselectivity were obtained in all cases, with optimised separations being performed in less than 6 min. Retention times, resolution and reproducibility are discussed.     

分子印迹技术在毛细管电色谱中的应用

分子印迹技术是制备具有分子识别功能聚合物，即分子印迹聚合物（MIPs)的一种新技术；毛细管电色谱（CEC）是一个具有发展前途的色谱新技术。将分子印迹技术和毛细管电色谱两种新技术相结合，优势互补，具有极大的发展潜力。本文对分子印迹技术在毛细管电色谱中的应用，以及各类MIPs-CEC毛细管柱的制备方法进行了较为全面的综述，引用文献52篇。     

Investigation of capillary electrochromatography with brush-type chiral stationary phases

Fused-silica capillaries (100 microm ID) were packed with the (3R, 4S)-Whelk-O chiral stationary phase (CSP) bonded on 3.0 microm silica particles. The enantiomers of 41 neutral analytes containing stereogenic centers, axes or planes were examined by packed capillary electrochromatography. More than 30 of these were cleanly resolved, owing to the selectivities and efficiencies afforded by this CSP. High reproducibility with no indication of diminished performance was observed using the same capillary for hundreds of runs (including intermediate change of the buffer system) over a period of several weeks. Acetate, 2-(N-morpholino)ethanesulfonic acid, or phosphate buffers, each modified with either acetonitrile or methanol, were used as mobile phases. The influence of buffer concentration, modifier amount, temperature, applied voltage, and pH on performance of the brush-type CSP was investigated.     

Theoretical and nomenclatural considerations of capillary electrochromatography with monolithic stationary phases

During the past decade, CEC has been one of the few novel achievements in the field of separation science attracting a wide interest. The technology progress permitted the realization of the long-sought idea to employ an electroosmotically driven flow through the columns improving the separations in terms of both resolution and efficiency. The early practical obstacles related to the use of conventional bead-packed columns have been solved by the introduction of continuous beds, also known as monoliths. Hitherto, various synthesis approaches have been successfully developed producing monolithic beds in situ in capillary columns, sharing similar physical structure built up of tiny particles (in the sub-microm range) that are covalently linked together and to the capillary wall. Parallel with the practical column technology studies, the theory of electrochromatography has been continuously developed, focusing on such basic issues as EOF characterization, separation efficiency, and peak dispersion effects. This review provides a short introduction to the theory of CEC with special attention to monolithic separation beds. The paper also summarizes the latest achievements in CEC and discusses the nomenclature, EOF characteristics, and some specific advantages of monolithic column technology.     

On-line concentration of neutral and charged species in capillary electrochromatography with a methacrylate-based monolithic stationary phase

A method involving self-concentration, on-column enrichment and field-amplified sample stacking for on-line concentration in capillary electrochromatography with a polymer monolithic column is presented. Since monolithic columns eliminate the frit fabrication and the problems associated with frits, the experimental conditions could be more flexibly adjusted to obtain higher concentration factor in comparison with conventional particulate packed columns. With self-concentration effect, the detection sensitivity of benzene and hexylbenzene is improved by a factor of 4 and 8, respectively. With on-column enrichment and ultralong injection, improvement as high as 22,000 times in detection sensitivity of benzoin is achieved. Furthermore, a combination of the three above-mentioned methods yields up to a 24,000-fold improvement in detection sensitivity for caffeine, a charged compound. Parameters affecting the efficiency of on-line concentration are investigated systematically. In addition, equations describing on-line concentration process are deduced.     

Enantiomeric separation of acidic compounds of pharmaceutical interest by capillary electrochromatography employing glycopeptide antibiotic stationary phases

Enantiomeric separation of some selected acidic compounds of pharmaceutical interest belonging to the group of non-steroidal anti-inflammatory drugs were separated by capillary electrochromatography employing silica based glycopeptide antibiotic stationary phases, namely vancomycin or a teicoplanin derivatives (Hepta-Tyr). The vancomycin stationary phase allowed to achieve the chiral resolution of some racemic studied compounds only using mobile phases containing ammonium formate at a relatively low pH 2.5-3.5 and acetonitrile. Employing the teicoplanin derivative stationary phase, good enantiomeric resolution was achieved eluting with mobile phases containing sodium phosphate pH 6-acetonitrile. Enantiomers were moved to the detector because a relatively high reversed electroosmotic flow (due to the positive charge of the stationary phase) and to the electrophoretic mobility of analytes.     

Investigation of mixed-mode monolithic stationary phases for the analysis of charged amino acids and peptides by capillary electrochromatography

The potential of N,N-dimethylacrylamide-piperazine diacrylamide-based monolithic stationary phases bearing sulfonic acid groups for electroosmotic flow generation is investigated for the separation of positively charged amino acids and peptides. The capillary columns were used under electrochromatographic but also under purely chromatographic (nano-HPLC) conditions and the separations interpreted as the result of possible chromatographic and electrophoretic contributions. The stationary phases were found to be mechanically stable up to pressures of 190 bar and chemically stable towards a wide variety of organic and hydro-organic mobile phases. In order to investigate the retention mechanism, the salt concentration and the organic solvent content of the (hydro-)organic mobile phase were varied in a systematic manner, taking three aromatic amino acids (phenylalanine, tryptophan, histidine) as model analytes. The respective contributions of electrostatic and hydrophobic and/or hydrophilic interactions were further investigated by varying the charge density and the hydrophobicity of the standard stationary phase. The former was done by varying the amount of charged monomer (vinylsulfonic acid) added during synthesis, the latter by (partially) replacing the interactive monomer (N,N-dimethylacrylamide) by other more hydrophobic monomers. A mixed mode retention mechanism based primarily on electrostatic interactions modified in addition by "hydrophilic" ones seems most suited to interpret the behavior of the amino acids, which stands in contradistinction to the previously investigated case of the behavior of neutral analytes on similar stationary phases. Finally the separation of small peptides was investigated. While the separation of Gly-Phe and Gly-Val was not possible, the separation of Phe-Gly-Phe-Gly and Gly-Phe but also of the closely related Gly-His and Gly-Gly-His could be achieved.     

Development and application of polymeric monolithic stationary phases for capillary electrochromatography

Monolithic columns for capillary electrochromatography are receiving quite remarkable attention. This review summarizes results excerpted from numerous papers concerning this rapidly growing area with a focus on monoliths prepared from synthetic polymers. Both the simplicity of the in situ preparation and the large number of readily available chemistries make the monolithic separation media a vital alternative to capillary columns packed with particulate materials. Therefore, they are now a well-established stationary phase format in the field of capillary electrochromatography. A wide variety of synthetic approaches as well as materials used for the preparation of the monolithic stationary phases are presented in detail. The analytical potential of these columns is demonstrated with separations involving various families of compounds and different chromatographic modes.     

Macroporous monolithic chiral stationary phases for capillary electrochromatography: New chiral monomer derived from cinchona alkaloid with enhanced enantioselectivity

A new chiral monomer derived from cinchona alkaloid, namely O-9-(tert-butylcarbamoyl)-11-[2-(methacryloyloxy)ethylthio]-10,11-dihydroquinine 1, was employed for the preparation of enantioselective monolithic capillary columns by an in situ copolymerization with 2-hydroxyethyl methacrylate 2 (HEMA), ethylene dimethacrylate 3 (EDMA) in the presence of cyclohexanol and 1-dodecanol as porogens (UV or thermal initiation of azobisisobutyronitrile (AIBN) as radical initiator). The porous properties and the electrochromatographic behavior of the new chiral monoliths were comparatively evaluated with previously described analogs obtained from O-9-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine 4 as chiral monomer. Despite close structural and physicochemical similarities of the both chiral monomers, the pore distribution profiles of the resulting monoliths were shifted typically towards larger pore diameters with the new monomer 1. Once more, it was confirmed that a low cross-linking (10 wt% related to total monomers) and a pore diameter of about 1 microm in the dry state provides the best electrochromatographic efficiency as a result of lower resistance to mass transfer (smaller C-term contribution to peak broadening) and more homogeneous flow profile (smaller A-term). Most importantly, as expected the new poly(1-co-HEMA-co-EDMA) monoliths showed enhanced enantioselectivities and in addition faster separations as compared to poly(4-co-HEMA-co-EDMA) analogs, which represents a significant improvement. Further, the elution order was reversed owing to the pseudoenantiomeric behavior of quinine- and quinidine-derived monomers. Fluorescence-labeled 9-fluorenylmethoxycarbonyl (FMOC), dansyl (DNS), 7-dimethylaminosulfonyl-1,3,2-benzoxadiazol-4-yl (DBD), carbazole-9-carbonyl (CC) amino acids could be separated with resolution values between 2 and 4 (with efficiencies typically between 100,000 and 200,000 plates/m) and fluorescence detection (variable wavelength fluorescence detector in-line with UV) yielding routinely a gain in detection sensitivities up to two orders of magnitude without specific optimization of the conditions with regards to fluorescence efficiency.     

Capillary electrochromatography with monolithic stationary phases. 4. Preparation of neutral stearyl-acrylate monoliths and their evaluation in capillary electrochromatography of neutral and charged small species as well as peptides and proteins

A neutral, nonpolar monolithic capillary column having a relatively strong electroosmotic flow (EOF) yet free of electrostatic interactions with charged solutes was developed for the reversed-phase capillary electrochromatography (RP-CEC) of neutral and charged species including peptides and proteins. The neutral nonpolar monolith is based on the in situ polymerization of pentaerythritol diacrylate monostearate (PEDAS) in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. PEDAS plays the role of both the cross-linker and the ligand provider, generating a macroporous nonpolar monolith having C17 chains as the chromatographic ligands. Despite the fact that the neutral PEDAS monolith is devoid of fixed charges, the monolithic capillary columns exhibited a relatively strong EOF due to the ability of PEDAS to adsorb sufficient amounts of electrolyte ions from the mobile phase. The adsorbed ions imparted the neutral PEDAS monolith the zeta potential necessary to support the EOF required for mass transport across the monolithic column. The absence of fixed charges on the surface of the neutral PEDAS monolith and in turn the adsorption sites for electrostatic attraction of charged solutes allowed the rapid and efficient separations of proteins and peptides at pH 7.0, with an average plate number of 255,000 and 121,000 plates/m, respectively. To the best of our knowledge, this constitutes the first report on the separation of proteins at neutral pH by RP-CEC using a neutral monolithic column.     

Enantiomeric separations of chiral polychlorinated biphenyls on three polysaccharide-type chiral stationary phases by supercritical fluid chromatography

Enantiomeric separations of 18 chiral polychlorinated biphenyls (PCBs) were investigated on three polysaccharide-type chiral stationary phases (CSPs; Sino-Chiral OJ, Chiralpak IB, and Chiralcel OD) by supercritical fluid chromatography (SFC). With these commonly used polysaccharide CSPs, 17 PCBs except PCB 135 (R(S) = 0.81) were well resolved (R(S) > 1.5) under appropriate mobile phases and temperatures. Using Sino-Chiral OJ, 14 PCBs could be baseline-separated, while only one and nine PCBs could be completely separated using Chiralpak IB and Chiralcel OD, respectively. The influence of column temperature was studied for the optimization of resolution, as well as for the type and percentage of organic modifier in the mobile phase. The resolution decreased as the temperature increased in the range of 26-40 °C in which the enantiomeric separations were an enthalpy-driven process. The addition of modifiers in the mobile phase decreased the resolution of the PCB enantiomers, but it clearly shortened their retention time. These separation results indicate that SFC is a promising chromatographic technique for chiral separation and enantiopure standard preparation.     

Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.     

Nanochitosan crosslinked with polyacrylamide as the chiral stationary phase for open-tubular capillary electrochromatography

Nanoparticles exhibiting favorable surface-to-volume ratios create efficient stationary phases for electrochromatography. New nanomaterials derived from chitosan (CS) were immobilized onto modified capillaries for use as the chiral stationary phase (CSP) in open-tubular electrochromatography. This immobilization was achieved through the copolymerization of glycidyl methacrylate-modified nano-CS with methacrylamide (MAA) and bis-acrylamide crosslinkers (forming the MAA-CS capillary) rather than the attachment of nano-CS to the copolymer of glycidyl methacrylate, MAA, and bis-acrylamide (forming the MAA+CS capillary). The completed MAA-CS capillary and its precursors were examined by SEM and ATR-IR measurements. Before separating chiral samples, the MAA-CS capillary was characterized by electroosmotic flow measurements at varying pH values, concentrations, and volume percentages of organic modifiers in the running buffers. Tryptophan enantiomers were well separated by the MAA-CS capillary, whereas no enantioselectivity was observed in the MAA+CS capillary. With the addition of 80% MeOH into the phosphate buffer, the chiral separation of (±)-catechin was accomplished in a normal-phase mode. However, the new CSP has its limitations, as only two groups of α-tocopherol stereoisomers were separated.     

Recent developments in the field of monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions to the rapidly growing area of monolithic columns based on both silica and synthetic polymers for capillary electrochromatography and chip electrochromatography, with a focus on those published during the year 2004. A wide variety of both modified approaches to the "old" monoliths and new monoliths have been reported despite the very short period of time covered. This demonstrates that monolithic stationary phases have become a well-established format in the field of electrochromatography. The simplicity of their preparation as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials.     

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

Separation strategy for acidic chiral pharmaceuticals with capillary electrochromatography on polysaccharide stationary phases

The effect of five factors on the capillary electrochromatographic enantioseparation of acidic compounds was studied using an experimental design. The studied factors were pH, acetonitrile content in the mobile phase, temperature, buffer concentration, and applied voltage. These experiments allowed defining a generic separation strategy applicable on acidic compounds with chemical and structural diversity. The starting screening conditions consist of a 45 mM ammonium formate electrolyte at pH 2.9 mixed with 65% acetonitrile, an applied voltage of 15 kV, and a temperature of 25 degrees C. The screening phase occasionally can be followed by an optimization procedure. Evaluation of the proposed strategy pointed out that it allows achieving baseline resolution within a relatively short time when a beginning of separation is obtained at the starting conditions. This strategy revealed enantioselectivity for 11 compounds out of 15, of which 10 could be baseline-separated after the proposed optimization steps.