Capillary Electrochromatography Conditions Research Articles

A chiral metal-organic framework {(HQA)(ZnCl2)(2.5H2O)}n for the enantioseparation of chiral amino acids and drugs

Chiral metal-organic frameworks (CMOFs) with enantiomeric subunits have been employed in chiral chemistry. In this study, a CMOF formed from 6-methoxyl-(8S,9R)-cinchonan-9-ol-3-carboxylic acid (HQA) and ZnCl2, {(HQA)(ZnCl2)(2.5H2O)}n, was constructed as a chiral stationary phase (CSP) via an in situ fabrication approach and used for chiral amino acid and drug analyses for the first time. The {(HQA)(ZnCl2)(2.5H2O)}n nanocrystal and the corresponding chiral stationary phase were systematically characterised using a series of analytical techniques including scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, circular dichroism, X-ray photoelectron spectroscopy, thermogravimetric analysis, and Brunauer-Emmett-Teller surface area measurements. In open-tubular capillary electrochromatography (CEC), the novel chiral column exhibited strong and broad enantioselectivity toward a variety of chiral analytes, including 19 racemic dansyl amino acids and several model chiral drugs (both acidic and basic). The chiral CEC conditions were optimised, and the enantioseparation mechanisms are discussed. This study not only introduces a new high-efficiency member of the MOF-type CSP family but also demonstrates the potential of improving the enantioselectivities of traditional chiral recognition reagents by fully using the inherent characteristics of porous organic frameworks.

Covalent organic framework incorporated chiral polymer monoliths for capillary electrochromatography

A covalent organic framework, Schiff base network-1 (SNW-1), was synthesized and incorporated into cellulase based poly(glycidyl methacrylate-co-ethylene dimethacrylate) (cellulase@poly(GMA-EDMA-SNW-1)) monolith to afford a novel chiral stationary phase for capillary electrochromatography (CEC). SNW-1 is attractive as a stationary phase for CEC because it not only features high surface areas but also provides conjugate structures and abundant amine groups to give π-π electrostatic stacking and hydrogen bonding property. Incorporation of SNW-1 into monolithic column could improve the column efficiency and increase the interactions between the tested racemates and the stationary phase thus significantly improved their CEC separation. The obtained monoliths were characterized by scanning electron microscopy, elemental analysis and nitrogen adsorption. Moreover, effects of SNW-1 concentration, immobilization pH of cellulase and CEC conditions were also investigated. Under the optimized conditions, the cellulase@poly(GMA-EDMA-SNW-1) monolith exhibited excellent enantioseparation performance for eight pairs of different classes of chiral drugs including β-blockers, antihistamines and anticoagulants. Satisfactory repeatability was achieved with relative standard deviations for intra-day, inter-day and column-to-column runs less than 4.5%, and batch-to-batch runs less than 6.8%. The experiment results reveal that the combination of the versatile features of monoliths and unique properties of SNW-1 could be a promising strategy for chiral separation.

Development of Amine Capillary Column Applied to the Analysis of Basic Compounds by Electrochromatography

An amine capillary column was developed in a fused-silica capillary as stationary phase and applied to separation of basic compounds by capillary electrochromatography (CEC). The functionalized capillary was prepared by inmobilization of 3-Aminopropyl)triethoxysilane (APTES). The CEC conditions including APTES and buffer concentration, pH and applied voltage were investigated to obtain the optimal CEC system for the separation of three anesthetics as basic test molecules. The capillary column provided an efficiency of up to 20,000 plates/m. Lidocaine, ketamine and xilacine were baseline separated under the running conditions with 10 mM Na2HPO4 pH 9.0 as BGE. The applied voltage was 5 kV temperature was set at 25oC and UV detection was performed. The resolutions were 4.97 and 1.53 for ketamine, lidocaine and xilacine, respectively. The column used in CEC mode showed better separation of the anesthetics compared with those used in the capillary zone mode. The comparison with reversed stationary phases used in HPLC in terms of resolution (Rs), sensitivity (LOD), efficiency (N), precision (RSD), asymmetry (T) was also performed. This study provided an alternative way for the CEC separation of basic compounds and demonstrated the improvement in the chromatographic parameters.

Ionic liquids monolithic columns for protein separation in capillary electrochromatography

A series of ionic liquids (ILs) monolithic capillary columns based on 1-vinyl-3-octylimidazolium (ViOcIm+) were prepared by two approaches (“one-pot” approach and “anion-exchange” approach). The effects of different anions (bromide, Br−; tetrafluoroborate, BF4−; hexafluorophosphate, PF6−; and bis-trifluoromethanesulfonylimide, NTf2−) on chromatography performance of all the resulting columns were investigated systematically under capillary electrochromatography (CEC) mode. The results indicated that all these columns could generate a stable reversed electroosmotic flow (EOF) over a wide pH range from 2.0 to 12.0. For the columns prepared by “one-pot” approach, the EOF decreased in the order of ViOcIm+Br−>ViOcIm+BF4−>ViOcIm+PF6−>ViOcIm+NTf2− under the same CEC conditions; the ViOcIm+Br− based column exhibited highest column efficiencies for the test small molecules; the ViOcIm+NTf2− based column possessed the strongest retention for aromatic hydrocarbons; and baseline separation of four standard proteins was achieved on ViOcIm+NTf2− based column corresponding to the highest column efficiency of 479000Nm−1 for cytochrome c (Cyt c). These results indicated that the property of ILs based columns could be tuned successfully by changing anions, which gave these columns potential to separate both small molecules and macro biomolecules.

Synthesis and study of the properties of PLOT columns based on new dendritic polymers for the separation of proteins by capillary electrochromatography

Capillary electrochromatography (CEC) is actively used for biomedical studies. Therefore, the search for new materials for stationary and pseudostationary phases for CEC that allow the control of the selectivity of separation, modification of quarts capillary walls, an increase in efficiency, and the reduction of the limits of analyte detection is a promising line of research. PLOT columns in this work have been synthesized using new dendritic polymers that are water soluble oligosaccharide derivatives of hyperbranched polyethyleneimine (PEI-Mal). The obtained coating has been characterized by the value of electroosmotic flow (EOF), and the influence of the composition and pH of a buffer electrolyte on the separation of proteins under CEC conditions with PLOT columns has been studied.

Methacrylate monolithic columns functionalized with epinephrine for capillary electrochromatography applications

Epinephrine-bonded polymeric monoliths for capillary electrochromatography (CEC) were developed by nucleophilic substitution reaction of epoxide groups of poly(glycidyl-methacrylate-co-ethylenedimethacrylate) (poly(GMA-co-EDMA)) monoliths using epinephrine as nucleophilic reagent. The ring opening reaction under dynamic conditions was optimized. Successful chemical modification of the monolith surface was ascertained by in situ Raman spectroscopy characterization. In addition, the amount of epinephrine groups that was bound to the monolith surface was evaluated by oxidation of the catechol groups with Ce(IV), followed by spectrophotometric measurement of unreacted Ce(IV). About 9% of all theoretical epoxide groups of the parent monolith were bonded to epinephrine. The chromatographic behavior of the epinephrine-bonded monolith in CEC conditions was assessed with test mixtures of alkyl benzenes, aniline derivatives and substituted phenols. In comparison to the poly(GMA-co-EDMA) monoliths, the epinephrine-bonded monoliths exhibited a much higher retention and slight differences in selectivity. The epinephrine-bonded monolith was further modified by oxidation with a Ce(IV) solution and compared with the epinephrine-bonded monoliths. The resulting monolithic stationary phases were evaluated in terms of reproducibility, giving RSD values below 9% in the parameters investigated.

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography

A silica-based monolithic stationary phase prepared by the sol–gel process in a 100 μm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, ( S)- N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 μm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N, N, N′, N′-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N, N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.

Chiral recognition ability of an (S)-naproxen- imprinted monolith by capillary electrochromatography.

The racemic naproxen was selectively recognized by capillary electrochromatography (CEC) on an (S)-naproxen-imprinted monolith, which was prepared by an in situ thermal-initiated polymerization. The recognition selectivity of a selected monolith strictly relied on the CEC conditions involved. The factors that influence the imprinting selectivity as well as the electroosmotic flow (EOF), including the applied voltage, organic solvent, salt concentration and pH value of the buffer, column temperature, and surfactant modifiers were systematically studied. Once the column was prepared, the experiment results showed that the successful chiral recognition was dependent on CEC variables. For example: the recognition could be observed in acetonitrile and ethanol electrolytes, while methanol and dimethyl sulfoxide (DMSO) electrolytes had no chiral recognition ability. The buffer with pH values of 2.6 or 3.0 at a higher salt concentration had chiral recognition ability. Column temperatures of 25-35 degrees C were optimal. Three surfactants, sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and polyoxyethylene sorbitan monolaurate (Tween 20), can improve the recognition. Baseline resolution was obtained under optimized conditions and the column efficiency of the later eluent (S)-naproxen was 90 000 plates/m.

Stationary and mobile phases in capillary electrochromatography (CEC)

AbstractThis review describes the state of the art of capillary electrochromatography (CEC). Properties of and interactions between stationary and mobile phases applied in CEC are described and discussed; developments in stationary phases, partly also monolithic stationary phases, are reviewed. Special attention is paid to the comparison of the behaviour of stationary and/or mobile phases under CEC versus HPLC conditions with respect to variables such as particle and pore size of the stationary phase, mobile phase composition, and temperature. These issues are discussed throughout the paper. A number of applications in CEC is presented as well.

Capillary electrochromatography with monolithic stationary phases: III. Evaluation of the electrochromatographic retention of neutral and charged solutes on cationic stearyl-acrylate monoliths and the separation of water-soluble proteins and membrane proteins

This article, which is closely related to part II, is concerned with the evaluation of the retentive properties of cationic stearyl-acrylate monoliths (i.e. cationic C 17 monoliths) over a wide range of elution conditions with various uncharged and charged solutes including proteins. The retention parameters for charged solutes including the retention factor k ∗ observed under capillary electrochromatography conditions and the velocity factor k ∗ ep, which reflects the electrophoretic process, were measured for weak, moderate and strong basic compounds. These retention parameters allowed the assessment of the respective contributions from electrophoretic and partitioning separation mechanisms. The cationic C 17 monoliths exhibited sufficient hydrophobic interactions with relatively weak basic solutes. Moderate and strong bases showed migration behaviors dominated by their relatively strong electrophoretic mobility with marginal chromatographic partitioning. At low pH, the cationic C 17 monoliths allowed the separation of proteins with minimum electrostatic interactions between proteins and the cationic sites on the surface of the stationary phase. The utility of the cationic C 17 monoliths was demonstrated in the rapid and efficient separation of two crude extracts of membrane proteins, namely galactosyl transferase and cytochrome c reductase. Short capillary columns (8.5 cm effective length) of the cationic C 17 monoliths allowed rapid and efficient separations of neutral and charged pesticides and metabolites, phenylthiohydrantoin amino acids and proteins at the time scale of seconds at relatively high flow velocity.

Recent progress in enantiomeric separation by capillary electrochromatography.

Recent progress in enantiomeric separations by capillary electrochromatography (CEC) is reviewed. The development of simple and robust CEC column technologies plays an important role for popularization of CEC. During the last several years, various approaches for the preparation of enantioselective columns have been reported. Currently, the monolithic column technology (continuous beds) represents the most advanced approach for the preparation of CEC columns. The development of new chiral stationary phase used for CEC is another important issue in this field. Fundamental investigations on electrochromatographic behaviors of various CSPs are necessary in order to understand the separation mechanism and thus improve the separation performance. Some chiral stationary phases performed better under nonaqueous CEC conditions than reversed-phase conditions. Coupling CEC with mass spectrometry (MS) provides a powerful tool for enantiomeric separation. Finally, some applications of enantiomeric separation by CEC are summarized.

Comparison of column packing techniques for capillary electrochromatography

Four different column packing techniques were used to pack capillary columns for comparison in capillary electrochromatography (CEC); the techniques were slurry pressure packing, using supercritical CO2, electrokinetic packing, and packing with centripetal forces. Capillary columns of 50 μm ID were packed with each technique and evaluated under electrochromatographic conditions. An initial evaluation suggested that among the packing techniques, supercritical CO2 packing and packing with centripetal forces produced the most efficient packed capillaries. However, examining the various protocols in detail revealed that experience of the practitioner and familiarity with packing equipment are critical factors in column fabrication. Upon further investigation of the packing techniques and optimization of the column fabrication protocol, columns packed by the four packing techniques showed similar performance when operated under CEC conditions. Efficiencies of 270,000–300,000 plates/m were observed for 50 μm ID capillary columns packed with 3 μm packing material.

β-CYCLODEXTRIN-MODIFIED MONOLITHIC STATIONARY PHASES FOR CAPILLARY ELECTROCHROMATOGRAPHY AND NANO-HPLC CHIRAL ANALYSIS OF EPHEDRINE AND IBUPROFEN

ABSTRACT Chiral monolithic capillary columns for reversed-phase capillary electrochromatography (CEC) and for nano-HPLC were prepared by linking β-cyclodextrin modifier into the acrylic monolithic phase. Columns with the physically and chemically bonded β-cyclodextrin derivatives were tested under CEC and nano-HPLC conditions; enatioselective separation of (−)-ephedrine/(+)-pseudoephedrine and (+/−)-ibuprofen was successfully performed. The separation efficiency of CEC and HPLC was examined and compared; resolution of (+/−) ibuprophen was 2.45 and 2.97 for CEC and HPLC respectively, number theoretical plates of thiourea were 41,600 N/m in CEC.

Capillary electrochromatography analysis of hormonal cyclic and linear peptides.

The retention behavior of linear and cyclic peptides has been studied by capillary electrochromatography (CEC) with a variety of different n-alkyl silica reversed-phase sorbents and also with mixed-mode phases containing both strong cation-exchange (sulfonic acid) and n-alkyl groups bonded onto the silica surface, using eluents ranging from pH 2.0 to pH 5.0. Depending upon the amino acid sequence, electrochromatographic retention of the peptides was strongly affected by the composition of the eluent, its pH value, and the choice of sorbent packed into the capillaries. The dominant separation processes operating with these charged analytes could be modulated inter alia by the content of organic modifier, acetonitrile, in the eluent, with peptide resolution predominantly arising from electrophoretic migration processes at high acetonitrile content. As the concentration of acetonitrile was decreased, chromatographic retention processes became more pronounced. With the n-alkyl silica CEC columns used in this study, silanophilic interactions between the sorbents and the charged peptides could be suppressed by increasing the molarity of the buffer and by adjusting the pH of the eluent to lower values. On the other hand, electrostatic interactions between basic peptides and the surface of strong cation-exchanger, mixed-mode materials can be suppressed at low pH values by using higher ionic strength conditions in the eluent. Different selectivity behavior was achieved with desmopressin and the other peptides with Spherisorb C18/SCX and Hypersil mixed-mode materials when an identical eluent composition of 60% (v/v) acetonitrile with 7.6 mM triethylammonium phosphate, pH 3.0, was used. These findings confirm that the surface charge density of the sorbent fulfills an important role in the modulation of peptide selectivity in CEC. These studies also confirm that the dependency of the logarithm of the CEC retention coefficients, i.e., log Kcec, of a peptide separated with n-octadecyl silica sorbents under CEC conditions, on the volume fraction, psi, of the organic solvent modifier, acetonitrile, within the range of 0.20 < or = psi < or = 0.60, can be approximated by a linear relationship. Moreover, these studies show that the selectivity differences of peptides separated by CEC with nonpolar sorbents in packed capillary systems can be discussed in terms of semiempirical dependencies that link peptide retention behavior with their molecular descriptor properties, e.g., their hydrophobicity, surface charge anisotropy, surface area, molecular mass and intrinsic charge, and thus to their corresponding linear free energy relationships.

Application of hydrophobic anion-exchange phases in capillary electrochromatography

Capillary electrochromatography (CEC) requires stationary phases that enable appropriate electroosmotic propel under various conditions. Analyte retention can be controlled through hydrophobic or electrostatic interaction with the packing material. The development and characterization of new strong anion-exchange materials with additional hydrophobic moieties (SAX/C 18 mixed-mode phases) is described. The synthesis was based on polymer encapsulation of porous silica. The phases were systematically characterized by means of elemental analyses, HPLC frontal analyses and CEC experiments. The studies focused on the influence of various parameters (e.g., pH, kind of buffer, capillary wall) on the electroosmotic flow (EOF). Phases with high anion-exchange capacity generated a fast and constant EOF over a wide pH range. Long-time stability of EOF and hydrophobic retention under CEC conditions were demonstrated within the course of 100 consecutive injections. The applicability of the SAX/C 18 phases in appropriate buffer systems is demonstrated for neutral, acidic and basic compounds.

Assessment of Electroosmotic Perfusion in Capillary Chromatographic Columns Using Electrical Conductivity

The electrical conductivity of capillary electrochromatography (CEC) columns packed with macroporous particles has been investigated. Columns were prepared with commercially available octadecylsilane-coated 7 μm diameter particles (Nucleosil) having nominal pore diameters of 100, 300, 500, 1000, and 4000 Å, and operated under typical CEC conditions. The conductivity of the 100 Å column was in agreement with that predicted from theory for nonporous spheres, indicating that intraparticle current was negligible. Columns packed with the wide-pore media (1000 Å and 4000 Å), in contrast, yielded conductivity values over 2-fold greater than the 100 Å. The electroosmotic contribution to current flow in these columns was deemed insignificant on the basis of theoretical modeling and the experimental data. It was therefore concluded that the increased column conductivity of the wide-pore packed columns was the result of intraparticle current transport. These results further suggest that wide-pore packings are more permeable to fluid flow and thus can provide maximum gains in efficiency due to electroosmotic perfusion when electrical double layer thickness is small relative to the median pore diameter of the packing.

Column technology for capillary electrochromatography

Column technologies for capillary electrochromatography (CEC) are reviewed. To achieve high efficiency, the inner diameters of open-tubular and packed columns should be less than 25 and 200 μm, respectively. To obtain acceptable separation speed under typical CEC conditions (e.g. 30 kV, 1 mm s −1 electroosmotic flow velocity, and 2–4×10 −8 m 2 V −1 s −1 electroosmotic mobility) the column lengths for open-tubular and packed columns should be less than 120 and 60 cm, respectively. Capillary CEC columns are generally classified into three types: packed, open-tubular, and continuous-bed or monolithic. The various column preparation procedures and the advantages and disadvantages of each column type are discussed in detail.

Fritless capillary electrochromatography.

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.

Capillary electrochromatography in columns packed by gravity.Preliminary study

Chromatographic material was delivered into fused silica capillary columns mainly by the use of sedimentation by gravity. After sedimentation of the packing material, the columns were pressurized at 8000 psi and evaluated for capillary electrochromatography (CEC). The capillary columns were tested using a commercially available capillary electrophoresis unit. Under typical CEC conditions, the capillary columns packed by gravity showed reduced plate heights below 2 with a reproducibility of less than 9% RSD.