Study of physically absorbed stationary phases for open tubullar capillary electrochromatography.

A novel method based on the adsorption of positively charged compounds on the wall of a fused-silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run-to-run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590000/m and the average one above 250000/m. Stored at 2-8 degrees C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.     

Capillary electrochromatography using fibers as stationary phases.

Fiber-packed capillary columns have been evaluated in chromatographic performance in capillary electrochromatography (CEC). The change of electroosmotic flow (EOF) velocity and selectivity using different kinds of fiber materials was examined. Although the EOF velocity among the different fiber packed columns was almost the same, retention of parabens was larger on the Kevlar-packed column than on the Zylon-packed one, and was larger on the as-span-type fiber-packed column than on the high-modulus-type packed one. Using 200 microm ID x 5 cm Kevlar packed column combined with a 100 microm ID x 20 cm precolumn capillary and a 530 microm ID x 45 cm postcolumn capillary, the separation of three parabens within 30 s was achieved. Other compounds were also separated in a few minutes by the fiber-packed CEC method.     

Capillary electrochromatography with bare silicas of different pore sizes as stationary phases

Bare silica can be used with reversed phase eluents for the chromatographic separation of basic analytes. It provides high surface charge density within a certain pH range, thus generating a high electroosmotic flow (EOF) when applied in electrochromatography. The influence of pore size on EOF velocity and mass transport is demonstrated. High EOF and fast mass transfer were encountered with 100 nm and 200 nm material and related to a pore perfusion mechanism. On a silica with 200 nm average pore size at pH 7, an EOF velocity of 2 mm/s was obtained at 600 V/cm. Silicas with pore diameters between 6 nm and 200 nm, corresponding to surface areas between 500 m/g and 10 m/g (data calculated from inverse size exclusion chromatography experiments), were used for CEC and HPLC separation of strongly basic solutes. On separation of tricyclic antidepressants by CEC, “normal” and “abnormal” efficiencies were achieved and were found to vary with the charge density within the separation column.     

Capillary electrochromatography in anion-exchange and normal-phase mode using monolithic stationary phases

Hydrophilic macroporous weak and strong anion-exchange stationary phases have been prepared in a monolithic format within untreated fused-silica capillaries by the simple thermally or UV-initiated polymerization of 2-dimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of a binary porogenic mixture of dodecanol and cyclohexanol. The tertiary amino functionalities were then alkylated in situ to afford strong anion-exchangers. These new monolithic stationary phases with optimized porous properties were used for the CEC separation of various organic anions. Thus, a mixture of 2-substituted propionic acid drugs (profens) was separated in 13 min and high column efficiencies of up to 231,000 plates/m were achieved. The separation of substituted benzoic acids indicates that the selectivity results primarily from the anion-exchange interactions, while electrophoretic migration contributes only slightly. In addition, these hydrophilic anion-exchangers are also able to separate weakly acidic, neutral and basic compounds such as phenols, xanthines and aromatic amines in normal-phase electrochromatographic mode.     

Capillary liquid chromatography and capillary electrochromatography using silica hydride stationary phases

A hydride-based octadecyl stationary phase on both 4.0 and 1.8 microm silica particles is tested in both the capillary LC and the pressurized capillary electrochromatography (pCEC) modes. These two materials are compared to standard C18 stationary phase made by organosilanization and to the hydride material packed into a convention 4.6mm I.D. column. The performance of the capillary columns is evaluated in terms of analysis times for various mixtures as well as efficiency. Of particular interest are the differences between the LC mode where only laminar flow is present and pCEC operation where a flat electrodriven flow profile is superimposed on the parabolic pressurized flow. Differences in performance between columns packed with 4.0 and 1.8 microm particle silica are also evaluated.     

Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.     

Sol-gel stationary phases for capillary electrochromatography

A review is presented on the current state of the art and future trends in the development of sol-gel stationary phases for capillary electrochromatography (CEC). The design and synthesis of stationary phases with prescribed chromatographic and surface charge properties represent challenging tasks in contemporary CEC research. Further developments in CEC as a high-efficiency liquid-phase separation technique will greatly depend on new breakthroughs in the area of stationary phase development. The requirements imposed on CEC stationary phase performance are significantly more demanding compared with those for HPLC. The design of CEC stationary phase must take into consideration the structural characteristics that will provide not only the selective solute/stationary phase interactions leading to chromatographic separations but also the surface charge properties that determine the magnitude and direction of the electroosmotic flow responsible for the mobile phase movement through the CEC column. Therefore, the stationary phase technology in CEC presents a more complex problem than in conventional chromatographic techniques. Different approaches to stationary phase development have been reported in contemporary CEC literature. The sol-gel approach represents a promising direction in this important research. It is applicable to the preparation of CEC stationary phases in different formats: surface coatings, micro/submicro particles, and monolithic beds. Besides, in the sol-gel approach, appropriate sol-gel precursors and other building blocks can be selected to create a stationary phase with desired structural and surface properties. One remarkable advantage of the sol-gel approach is the mild thermal conditions under which the stationary phase synthesis can be carried out (typically at room temperature). It also provides an effective pathway to integrating the advantageous properties of organic and inorganic material systems, and thereby enhancing and fine-tuning chromatographic selectivity of the created hybrid organic-inorganic stationary phases. This review focuses on recent developments in the design, synthesis, characterization, properties, and applications of sol-gel stationary phases in CEC.     

Polar stationary phases for capillary electrochromatography

This review article summarizes the variety of polar stationary phases that have been employed for capillary electrochromatographic separations. Compared with reversed-phase stationary phases, the polar alternatives provide a completely different retention selectivity towards polar and charged analytes. Different types of polar stationary phases are reviewed, including the possible retention mechanisms. Electrochromatographic separations of polar solutes, peptides, and basic pharmaceuticals on polar stationary phases are presented.     

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.     

Polymeric monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions of a number of groups working in the rapidly growing area of monolithic columns for capillary electrochromatography (CEC), with a focus on those prepared from synthetic polymers. Monoliths have quickly become a well-established stationary phase format in the field of CEC. The simplicity of their in situ preparation method 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. A wide variety of approaches as well as materials used for the preparation of the monolithic stationary phases are detailed. Their excellent chromatographic performance is demonstrated by numerous separations of different analytes.     

Capillary electrochromatography with monolithic stationary phases. II. Preparation of cationic stearyl-acrylate monoliths and their electrochromatographic characterization

A novel cationic monolithic stationary phase based on the co-polymerization of pentaerythritol diacrylate monostearate (PEDAS) with a selected quaternary amine acrylic monomer was designed for performing capillary electrochromatography at high flow velocity. While PEDAS functioned as both the ligand provider and the cross-linker, the quaternary amine acrylic monomer was introduced to control the magnitude of the electroosmotic flow (EOF). The fabrication of the cationic stearyl-acrylate monolith (designated as cationic C17 monolith) with controlled porosity was achieved by free radical polymerization using the initiator 2,2'-azobisisobutyronitrile in the presence of a ternary porogenic solvent composed of cyclohexanol, ethylene glycol and water. Four different quaternary amine acrylic monomers were investigated in order to find the optimum monomer for achieving maximum electroosmotic flow (EOF) velocity. Both photo- and thermally-initiated polymerization proved effective in producing the cationic C17 monolith, and the best monolith was achieved when [2-(acryloyloxy)ethyl]trimethyl ammonium methyl sulfate (AETA) was used as the quaternary amine acrylic monomer. Although the zeta potential of the resulting cationic C17 monolith is positive with respect to water, the magnitude and direction of the EOF was markedly affected by the nature of the electrolyte in the mobile phase. Consequently, anodal, zero or cathodal EOF was observed depending on the nature of the electrolyte, and this was attributed to the adsorption of the ionic components of the electrolyte on to the solid stationary phase, which is characterized by its amphiphilic nature consisting of C17 chains, ester functions, hydroxyl groups and quaternary amine moieties. Optimized PEDAS-AETA monoliths yielded columns with high separation efficiency and allowed rapid separations on the time scale of seconds to be achieved with short capillaries.     

Polymer-brush stationary phases for open-tubular capillary electrochromatography

Synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes from the inside of silica capillaries by surface-initiated atom transfer radical polymerization (ATRP) yields unique stationary phases for open-tubular capillary electrochromatography (OT-CEC). Although PHEMA brushes have only a small effect on the separation of a set of phenols and anilines, derivatization of PHEMA with ethylenediamine (en) allows baseline resolution of several anilines that co-elute from bare silica capillaries. Derivatization of PHEMA with octanoyl chloride (C8-PHEMA films) affords even better resolution in the separation of a series of phenols and anilines. Increasing the thickness of C8-PHEMA coatings by a factor of 2 enhances resolution for several solute pairs, presumably because of an increase in the effective stationary phase to mobile phase volume ratio. Thus, this work demonstrates that thick polymer brushes provide a tunable stationary phase with a much larger phase ratio than is available from monolayer wall coatings. Through appropriate choice of derivatizing reagents, these polymer brushes should allow separation of a wide range of neutral molecules as well as compounds with similar electrophoretic mobilities.     

Monolithic enantiomer-selective stationary phases for capillary electrochromatography

Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.     

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.     

Monolithic stationary phases for liquid chromatography and capillary electrochromatography

A monolithic stationary phase is the continuous unitary porous structure prepared by in situ polymerization or consolidation inside the column tubing and, if necessary, the surface is functionalized to convert it into a sorbent with the desired chromatographic binding properties [J. Chromatogr. A 855 (1999) 273]. Monolithic stationary phases have attracted considerable attention in liquid chromatography and capillary electrochromatography in recent years due to their simple preparation procedure, unique properties and excellent performance, especially for separation of biopolymers. This review summarizes the preparation, characterization and applications of the monolithic stationary phases. In addition, the disadvantages and limitations of the monolithic stationary phases are also briefly discussed.     

Capillary electrochromatography with a silica column with a dynamically modified cationic surfactant.

A novel mode of capillary electrochromatography (CEC), called dynamically modified silica-capillary electrochromatography, is described in this paper. The column packed with bare silica was dynamically modified with long chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added into the mobile phase. CTAB ions were adsorbed onto the surface of bare silica, and the resulted hydrophobic layer on the silica gel was used as the stationary phase. Using the dynamically modified silica column, neutral solutes were separated by CEC. The highest number of theoretical plates obtained was about 71,500/m and the relative standard deviations for t0 and capacity factor of toluene were 4.7% and 4.9% for 20 consecutive runs, respectively. The separation mechanism of neutral solutes and the influence of mobile phase composition on the separation was investigated. The separation of nitrogen-containing solutes was carried out with this mode and the peak tailing of basic solute was effectively eliminated because the adsorption of basic solute on silica was blocked by the preferred adsorption of CTAB.     

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.     

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。     

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 microm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 microm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C6 ligands had been introduced.     

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.