Reversed-phase Capillary Electrochromatography Research Articles

Nanoparticle-Based Continuous Full Filling Capillary Electrochromatography/Electrospray Ionization-Mass Spectrometry for Separation of Neutral Compounds

Highly efficient reversed-phase capillary electrochromatography (CEC) separations (plate numbers up to 700 000/m), with electrospray ionization mass spectrometry detection were achieved utilizing novel dextran-coated polymer nanoparticles as a pseudostationary phase. A continuous full filling (CFF) technique in which nanoparticles are continuously introduced into the capillary was employed for separation of neutral analytes (dialkyl phthalates), utilizing an orthogonal electrospray interface to prevent nanoparticles from entering the mass spectrometer. CFF-CEC benefits from that an entirely fresh column is employed for every analysis, avoiding carryover effects associated with stationary-phase contamination. The highly efficient separations obtained were accomplished by optimizing the organic modifier concentration in the electrolyte and by using a high nanoparticle concentration (5 mg/mL), to improve interparticle mass transfer and gain sufficient retention. Nanoparticles, with an average diameter of 600 nm, were prepared by polymerization of methacrylic acid and trimethylolpropane trimethacrylate, which in turn were coated with dextran. These nanoparticles formed stable suspensions in electrolytes having broad ranges of polarities, enabling straightforward optimization of the reversed-phase conditions.

Two-Dimensional Microcolumn Separation Platform for Proteomics Consisting of On-Line Coupled Capillary Isoelectric Focusing and Capillary Electrochromatography. 1. Evaluation of the Capillary-Based Two-Dimensional Platform with Proteins, Peptides, and Human Serum

In this report, an on-line coupling of capillary isoelectric focusing (CIEF) to capillary electrochromatography (CEC) is developed via a nanoinjector valve for performing two-dimensional (2D) proteomics separation. CIEF constitutes the first separation dimension, while CEC operates as the second separation dimension. Besides the orthogonal migration mechanisms of the two capillary-based separation modes, which lead to a 2D system whose overall peak capacity is the product of the peak capacity of the individual modes, the solvent of the CIEF mode is a weak eluent for the reversed-phase CEC (RP-CEC) mode, thus, allowing the transferring of focused fractions from CIEF to CEC without inducing band broadening, and instead zone sharpening would result. In fact, the transferred focused protein fraction from the CIEF column to the CEC column will stay tightly adsorbed to the inlet top of the CEC column until it will be eluted and separated into its protein components with a hydro-organic mobile phase. The theoretical peak capacity of the CIEF-CEC 2D platform is estimated at n(CIEF) (= 560) x n(CEC) (= 97) = 54 320. This peak capacity is more than needed for proteomics profiling. Also, only a fraction of this peak capacity is needed when looking at heart cuts for performing subproteomics. The 2D platform described here offers the convenience to generate the needed peak capacity to solve a given proteomic separation problem. This is facilitated by the RP-CEC dimension, which ensures rapid isocratic separation of proteins and peptides and rapid solvent change and column equilibration and avoids lengthy gradient elution. The RP-CEC column is based on neutral C17 monolith, which offers high separation efficiency and relatively high column permeability. To the best of our knowledge, the proposed 2D platform combining CIEF and CEC is reported for the first time for proteins and proteomics.

Octyl-functionalized hybrid silica monolithic column for reversed-phase capillary electrochromatography

Hybrid silica monolithic stationary phase functionalized with octyl groups was synthesized by a two-step acid/base-catalyzed hydrolysis/co-condensation of tetraethoxysilane (TEOS) and n-octyltriethoxysilane (C 8-TEOS). The influences of determining factors in the sol–gel process such as the monomer ratio and water content on the monolith formation were systematically investigated. An increase in the TEOS/C 8-TEOS ratio in the polymerization mixture shifted the pore size distribution towards smaller pore diameter with larger pore volume. The optimal TEOS/C 8-TEOS volume ratio was found to be 90/50, under which condition the median pore diameter of the monolith was around 1.0 μm with pore volume of 3.25 cm 3/g. The chromatographic characteristics of the monolithic column prepared with the optimized fabrication condition were studied. Some aromatic compounds including alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and phenols were successfully separated on the octyl-functionalized silica monolithic column with high column efficiency up to 180,000 plates/m.

Capillary Electrochromatography on Methacrylate Based Monolithic Columns: Evaluation of Column Performance and Separation of Polyphenols

Fused-silica capillary columns (100 µm I.D.) englobing a porous monolithic stationary phase were prepared by in situ copolymerization of 2-ethylhexyl methacrylate, ethylene glycol dimethacrylate and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) in the presence of a porogenic mixture containing 1-propanol, 1,4 butanediol and water. The influence of the monomers ratio and the porogen solvent composition as well as the content of AMPS in the polymerization mixture on column total porosity and efficiency was investigated to attain minimum HETP values for the reversed-phase capillary electrochromatography separation of bioflavonoids. For the most promising column, the van Deemter plots, in both μ-HPLC and CEC, were also evaluated. In CEC the reduced plate height was found almost constant (1.6–2.0) within the range of linear mobile phase velocity between 0.2–2.0 mm s−1. The chemical and mechanical stabilities of the monolithic column over a wide range of buffer pH (2-10) and time were satisfactory. Furthermore, the effects of mobile phase parameters, such as buffer concentration and organic modifier content, on the electroosmotic flow were studied systematically. CEC separations of standard mixtures of polyphenols, including flavonols, flavanones and flavanones-7-O-glycosides, were accomplished in less than 8 min. The CEC separation of the major flavanone glycoside constituents in the extract from a freshly squeezed grapefruit juice was also reported.

The Development of Reversed-Phase Capillary Electrochromatography for the Separation of Steroids

This paper describes the preparation and optimization of packed capillary columns for reversed-phase separation of steroids with CEC. The fabrication of on-column frits is considered to be the most important step for obtaining a reproducible packed column for CEC separation. Porous silicate frits were generated in a fused-silica capillary by heating the silica gel/sodium hydroxide solutions electrically. The optimized conditions involve silica gel (10.8%), sodium hydroxide (5.8%), and heating time (5 sec) with heating voltage (5V) for obtaining a 100-μ end-frit that can withstand pressure over 6000 psi. A HPLC pump was utilized to pack the 5-μm ODS particle slurry into the capillary column. The ODS packed capillaries were then utilized for the separation of four anabolic cholesterols with a capillary electrophoresis system without pressurization of the column. The reproducibility of the packed columns was evaluated by measuring the relative standard deviations of four steroids. The relative standard deviations of migration time for column-to-column, day-to-day, and run-to-run are less than 7%, 2%, and 1% for four steroids, respectively.

Hybrid organic-inorganic phenyl monolithic column for capillary electrochromatography

A novel hybrid organic-inorganic silica-based monolithic column possessing phenyl ligands for reversed-phase (RP) capillary electrochromatography (CEC) is described. The monolithic stationary phase was prepared by in situ co-condensation of tetraethoxysilane (TEOS) with phenyltriethoxysilane (PTES) via a two-step catalytic sol-gel procedure to introduce phenyl groups distributed throughout the silica matrix for chromatographic interaction. The hydrolysis and condensation reactions of precursors were chemically controlled through pH variation by adding hydrochloric acid and dodecylamine, respectively. The structural property of the monolithic column can be easily tailored through adjusting the composition of starting sol solution. The effect of PTES/TEOS ratios on the morphology of the created stationary phases was investigated. A variety of neutral and basic analytes were used to evaluate the column performance. The CEC columns exhibited typical RP chromatographic retention mechanism for neutral compounds and had improved peak shape for basic solutes.

Optimization of the Separation of Triazines, Metabolites, and Phenylurea Herbicides in Mixture by Reversed Phase Capillary Electrochromatography

The separation of 10 different phenylurea (PHU) and triazine (TRZ) herbicides in mixture was optimized by reversed phase capillary electrochromatography (CEC) by studying the effect of several physico‐chemical parameters such as the mobile phase buffer type, pH and concentration, the acetonitrile concentration, and the separation capillary length. Two different buffer systems were investigated, namely ammonium 2‐morpholinethanesulfonic acid (MES) and acetate buffers in the pH range 5–7. In MES mobile phases, the triazines herbicides elution order and separation was strongly influenced by the pH. The separation of atrazine and metobromuron compounds was difficult to obtain and was only achieved using a column of 62 cm total length. The separation of all the compounds in mixture was obtained in 5 mM ammonium acetate mobile phase pH 6.0 containing 75% of acetonitrile. Under these conditions, the method was linear in the range 2.5–50.0 µg/mL and exhibited a detection limit of 1.25 µg/mL. By slightly lowering the mobile phase acetonitrile content, the co‐separation of the 10 herbicides with the atrazine N‐dealkylated metabolites was also successfully achieved.

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.

Application of Reversed Phase Short End-Capillary Electrochromatography to Herbicides Residues Analysis

Reversed phase capillary electrochromatography was applied to the determination of phenylurea herbicides residues in different samples of vegetables and vegetable processed food. The use of 40% acetonitrile mobile phase content provided a large elution window and strongly reduced the presence of matrix interferences in real samples. The compounds were baseline resolved within 14 min with 97612–113312 N/m range by using the short end injection method. The method was repeatable and linear and applied to the analysis of spiked soya milk and canned soya shoots samples extracted after a reduced sample pretreatment procedure. The analysis of vegetables showed some difficulties arising from the complexity and the strong variability of the matrix.

Analysis of flavanone-7- O-glycosides in citrus juices by short-end capillary electrochromatography

The separation of the major flavanone-7- O-glycoside constituents of Citrus was carried out by isocratic reversed phase capillary electrochromatography using a 75 μm i.d. silica fused column packed with 5 μm ODS silica gel. In comparison to HPLC mode, capillary electrochromatography resolution of flavanone glycosides was obtained with a high selectivity factor. Optimum separation conditions were found using a mixture of ammonium formate (pH 2.5)–acetonitrile (8:2, v/v) as the mobile phase by the short-end injection mode. Under these conditions all the investigated flavanones were baseline-resolved within short analysis time (i.e. between 5 and 10 min). A study, evaluating the intra- and inter-day repeatability as well as limit of detection and method linearity, was developed in accordance with the analytical procedures for method validation. The developed method was applied for the quantitative analysis of flavanone glycosides in commercial fruit juices (sweet orange, lemon and grapefruit).

Indirect resolution of beta-blocker agents by reversed-phase capillary electrochromatography.

The indirect resolution of five beta-adrenoreceptor blocking agents (propranolol, oxprenolol, pindolol, metoprolol, and atenolol) using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), and capillary electrochromatography (CEC) is reported. Three octadecylsilanized (ODS) silica gel-based stationary phases, differing in particle diameter and carbon surface density for suitability of determination of the enantiomeric composition of these substances in drugs and biological fluids, were studied. Under optimum separation conditions, all the investigated compounds were baseline-resolved within acceptable analysis times (i.e., between 10-16 min). The resolution values ranged between 1.80 and 1.14, and the separation factors were in no case less than 1.07. The possible use of the developed CEC method for the determination of enantiomeric composition of beta-adrenoreceptor blocking agents in drug substances was investigated. A study, in which the enantiomers of metoprolol were examined regarding specificity, reproducibility, limit of detection, and ruggedness, was developed in accordance with some analytical procedures for method validation.

Method for Dead Time Determination in Reversed-Phase Capillary Electrochromatography with Methacrylate-Based Stationary Phases

A method for dead time determination in reversed-phase capillary electrochromatography (RP-CEC) with methacrylate-based monolithic stationary phases has been presented based on homologue series method. The dead times measured with homologue series method and thiourea, a commonly used electroosmotic flow (EOF) marker are compared under various conditions, such as the type and the concentration of organic modifier, ionic strength, and applied voltage. Results show that the former one is larger than the latter one. Under high ionic strength and low applied voltage, EOF velocity measured by thiourea is close to that determined by homologue series method.

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species.

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.

Capillary electrochromatography with monolithic-silica columns. II. Preparation of amphiphilic silica monoliths having surface-bound cationic octadecyl moieties and their chromatographic characterization and application to the separation of proteins and other neutral and charged species.

Three different synthetic routes have been introduced and evaluated for the preparation of amphiphilic silica-based monoliths possessing surface-bound octadecyl ligands and positively charged groups. The amphiphilic silica monoliths (designated as cationic C18-monoliths) have been designed for use in reversed-phase capillary electrochromatography (RP-CEC) with hydro-organic mobile phases. These amphiphilic stationary phases yielded anodic electroosmotic flow (EOF) over a wide range of mobile phase pH. The magnitude of EOF remained constant up to pH 4.0 and then decreased at pH > 4.0 due to the ionization of silanol groups and the subsequent decrease in the net positive surface charge density of the amphiphilic monoliths. The cationic C18-monoliths exhibited reversed-phase chromatography (RPC) behavior toward non-polar solutes (e.g., alkyl benzenes), which parallels that observed with octadecyl-silica (ODS) monoliths. On the other hand, the amphiphilic stationary phases exhibited both non-polar and polar interactions toward slightly polar solutes such as anilines and PTH-amino acids. CEC retention factor k* and velocity factor k*e, which reflects the contribution of the electrophoretic mobility, were evaluated for charged solutes such as anilines and proteins.

Separation of negatively charged nonsteroidal anti-inflammatory drugs by reversed-phase capillary electrochromatography

Reversed-phase capillary electrochromatography in a 5-μm C 18 fully packed capillary was employed to optimize the separation of negatively charged nonsteroidal anti-inflammatory drugs. The effect of the physico-chemical parameters and different analysis modes on the separation of 2-arylpropionic acids was studied and evaluated. The mobile phase composition, buffer type, concentration and pH differently influenced the peak efficiency and resolution, selectively modulating the analytes interaction with the stationary phase. The use of zwitterionic MES or acetate mobile phases strongly modulated the analytes migration order and peak efficiency. The optimum experimental conditions were found in MES buffer, pH 5.0, containing the 75% acetonitrile–methanol (1:1). All the analytes were baseline separated in a mixture in less than 13 min with peak efficiencies in the range of 78 500–84 200 N/m. Under these conditions the analytes were negatively charged and their effective electrophoretic mobilities played a role in the separation. The analysis of different pharmaceutical preparations containing anti-inflammatory drugs, e.g. drops and tablets, is also presented after a very simple sample pretreatment.

Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip.

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example.

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.

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column.

The mixed mode of reversed phase (RP) and strong cation-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280,000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for t(0) and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.

Bonded-phase photopolymerized sol-gel monoliths for reversed phase capillary electrochromatography

A porous chemically modified photopolymerized sol-gel (PSG) monolith enhanced the capillary electrochromatographic separations of two test mixtures, one containing eight alkyl phenyl ketones and the other containing thiourea and three polyaromatic hydrocarbons. Derivatization of the PSG surface with silane coupling reagents resulted in bonded phases of pentafluorophenylpropyldimethyl, pentafluorophenyl, 3,3,3-trifluoropropyl, n-octadimethyl, perfluorohexyl, and aminopropyl. The fabrication of the bonded-phase PSG column is easy to do with the silanization reaction proceeding at room temperature for not longer than 60 minutes. The hydrophobicity of the PSG was altered without degrading its chromatographic performance. The bonded-phase PSG monoliths have higher stability at pH values below 4 as compared to the parent (underivatized) PSG. Separations of different mixtures containing nucleosides, positively charged peptides, and taxol derivatives illustrate the potential of bonded-phase PSG columns for the analyses of biologically and pharmaceutically important compounds. We report column efficiencies of up to 180 000 plates/meter and retention factors as large as 28.6 for decanophenone.

Bonded-phase photopolymerized sol-gel monoliths for reversed phase capillary electrochromatography

Bonded-phase photopolymerized sol-gel monoliths for reversed phase capillary electrochromatography