Direct UV detection of underivatized amino acids using capillary electrophoresis with online sweeping enrichment

This is an original report proposed a CE method for direct analysis of the underivatized amino acids using UV detection with relatively higher sensitivity, which was based on coordination interactions between amino acids and Cu (II) ions. In addition, an online sweeping preconcentration technique was easily combined to improve the detection sensitivity. Satisfying separations of the amino acids were obtained under optimized conditions: 50 mmol/L CuSO₄–0.05% HAc–H₂O (pH 4.5), and the separation voltage of 15 kV. The LODs for the analytes ranged from 0.1 to 0.5 μmol/L. The linearity of detection for all analytes was two orders of magnitude with the correlation coefficients greater than 0.99. The repeatability was displayed with an RSD less than 3% for migration time and peak height (n = 5). Moreover, some amino acids in real samples of human saliva and green tea were analyzed by this direct UV detection CE method with acceptable sensitivity.     

Hydrophobicity‐aided potentiometric detection of catecholamines,beta‐agonists,and beta‐blockers in a mixed‐solvent capillary electrophoresis system

A series of cationic drug‐like substances with distinct basicity, hydrogen‐bonding ability, and hydrophobicity, including three catecholamines, two beta‐agonists, and thirteen beta‐blockers, was successfully detected in a capillary electrophoresis system using an end‐capillary coupled potentiometric sensor consisting of a PVC‐based liquid membrane deposited directly on a 100 μm diameter copper rod. The electrophoretic separation was performed on a 72 cm×75 μm id uncoated fused‐silica capillary with an acidic background electrolyte containing phosphoric acid in a water–acetonitrile mixture, pH* 2.8. Samples were injected electrokinetically at 5.0 kV for 10 s and a running voltage of 19.5 kV was applied. Excluding the bufuralol/practolol pair, baseline separation of all substances was achieved in the developed CE system within 9 minutes. A linear relationship (R² 0.8752) between the sensitivity of the applied potentiometric detector and the parameter log P characterising the hydrophobicity of the analytes was demonstrated. The best observable limits of detection (LODs) were obtained for the highly hydrophobic substances, i. e. bufuralol (8.10×10^–8 M injected concentration, S/N = 3), propranolol, alprenolol, and clenbuterol (ca. 1.10×10^–7 M). In the case of hydrophilic catecholamines and carbuterol their LODs with potentiometric detection were lowered by a factor of almost one thousand, reaching a value of 6.6×10^–5 M.     

Capillary electrophoresis using core‐based hyperbranched polyethyleneimine (CHPEI) static‐coated capillaries

With unique 3‐D architecture, the application of core‐based hyperbranched polyethyleneimine (CHPEI), as a capillary coating in capillary electrophoresis, is demonstrated by manipulation of the electroosmotic mobility (EOF). CHPEI coatings (CHPEI5, M_w ≈? 5000 and CHPEI25, M_w ≈? 25 000) were physically adsorbed onto the inner surface of bare fused‐silica capillary (BFS) via electrostatic interaction of the oppositely charged molecules by rinsing the capillaries with different CHPEI aqueous solutions. The EOF values of the coated capillaries were measured over the pH range of 4.0–9.0. At higher pH (pH >6) the coated capillary surface possesses excess negative charges, which causes the reversal of the EOF. The magnitudes of the EOF obtained from the coated capillaries were three‐fold lower than that of BFS capillary. Desirable reproducibility of the EOF with % RSD (n = 5) ? 2 was obtained. Effect of ionic strength, stability of the coating (% RSD = 0.3) and the dependence of the EOF on pH (% RSD = 0.5) were also investigated. The CHPEI‐coated capillaries were successfully utilized to separate phenolic compounds, B vitamins, as well as basic drugs and related compounds with reasonable analysis time (<20 min) and acceptable migration‐time repeatability (<0.7% RSD for intra‐capillary and <2% RSD for inter‐capillary).     

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C⁴D‐LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2‐[4‐(2‐hydroxyethyl)piperazin‐1‐yl]ethanesulfonic acid, 15 mM 2‐amino‐2‐hydroxymethyl‐propane‐1,3‐diol, and 2 mM 18‐crown‐6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double‐opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C⁴D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.     

Open tubular capillary electrochromatography of underivatized amino acids using Rh(III) tetrakis(phenoxyphenyl)porphyrinate as wall modifier

The separation of 17 “common” underivatized amino acids was attempted by open tubular capillary electrochromatography (OT-CEC) in fused-silica capillaries coated with Rh(III) tetrakis(phenoxyphenyl)porphyrinate (Rh(III)TPP(m-OPh)₄OAc) using sodium phosphate and Tris–phosphate buffers as background electrolytes (BGEs). The OT-CEC separation of amino acids was compared with that obtained by capillary zone electrophoresis in bare fused-silica capillaries using the same BGEs. The amino acids were not derivatized and the UV-absorption detection was set at 200 nm. Depending on the experimental conditions at least 15 amino acids were separated. The best separations were obtained in a Rh(III)TPP(m-OPh)₄OAc-coated capillary in 50 mM Tris–100 mM phosphate buffer at pH 2.25. Separation of the critical triplet Val–Ile–Leu was always at least indicated being better at higher BGE concentrations. Regarding the sensitivity of the method, lower concentration limits of detection (LODs) in the coated capillary were obtained for Thr, Gly, Tyr, and Val; the other amino acids exhibited lower LODs in the uncoated capillary. The separation of acidic amino acids was not achieved.     

Electrokinetic separation of peptides and proteins using a polyvinylamine-coated capillary with UV and ESI-MS detection

In order to accomplish the analysis of peptides and proteins by capillary electrophoresis, Lupamin, a high-molecular-weight linear polyvinylamine (PVAm) polymer, was introduced to modify the inner wall of fused-silica capillaries by physical absorption. Thanks to the high density of positively charged amino groups in Lupamin under acidic conditions, not only is a strong reversed electroosmotic flow generated in the coated capillary but the adsorption of analytes on the inner wall of the capillary is also efficiently eliminated. It has been demonstrated that the Lupamin-coated capillary can be used to advantage for the rapid analysis of amino acids, peptides, and proteins with good resolution and peak shape by capillary electrophoresis. In order to evaluate the basic feature of a Lupamin-coated capillary, electroosmotic flows generated by a Lupamin coating layer under different conditions including pH, coating time, concentration, and the composition of electrolytes on Lupamin-coated and uncoated capillaries were investigated. Furthermore, electrospray ionization-mass spectrometry (ESI-MS) detection was carried out for the analysis of amino acids and peptides.     

Chiral capillary electrophoresis with cationic pyrrolidinium‐β‐cyclodextrin derivatives as chiral selectors

New single‐isomer, cationic β‐cyclodextrins, including mono‐6‐deoxy‐6‐pyrrolidine‐β‐cyclodextrin chloride (pyCDCl), mono‐6‐deoxy‐6‐(N‐methyl‐pyrrolidine)‐β‐cyclodextrin chloride (N‐CH₃‐pyCDCl), mono‐6‐deoxy‐6‐(N‐(2‐hydroxyethyl)‐pyrrolidine)‐β‐cyclodextrin chloride (N‐EtOH‐pyCDCl), mono‐6‐deoxy‐6‐(2‐hydroxymethyl‐pyrrolidine)‐β‐cyclodextrin chloride (2‐MeOH‐pyCDCl) were synthesized and used as chiral selectors in capillary electrophoresis for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids. The unsubstituted pyCDCl exhibited the greatest resolving ability. Most analytes were resolved over a wide range of pH from 6.0 to 9.0 with this chiral selector. In general, increasing pH led to a decrease in resolution. The effective mobilities of all the analytes were found to decrease with increasing CD concentration. The optimal concentration for most carboxylic acids and dansyl amino acid was in the range 5–7.5 mM and >15 mM for hydroxycarboxylic acids. ¹H NMR experiments provided direct evidence of inclusion in the CD cavity.     

Poly(N,N‐dimethylacrylamide‐co‐4‐(ethyl)‐morpholine methacrylamide) copolymer as coating for CE

In this work, a new physically adsorbed coating for CE is presented. This coating is based on a poly(N,N‐dimethylacrylamide‐co‐4‐(ethyl)‐morpholine methacrylamide) (DMA/MAEM) copolymer synthesized in our laboratory. It is demonstrated that the direction and magnitude of the EOF in CE can be modulated by varying the composition of the DMA/MAEM copolymer and the type and pH of the BGE. Moreover, the DMA/MAEM coating provides %RSD_n = 5 values for migration times lower than 0.9% for the same capillary and day, whereas the %RSD_n = 25 obtained for the interday assay was lower than 2.9%. The stability of the coating procedure is also tested between capillaries obtaining %RSD_n = 15 values lower than 2.9%, demonstrating that this physically adsorbed copolymer gives rise to a stable and reproducible coating in CE. Finally, the usefulness of this new cationic copolymer as CE coating is demonstrated through different applications. Namely, it is demonstrated that the CE separation of basic proteins, nucleotides and organic acids is achieved in a fast and easy way by using the DMA/MAEM coated capillary. The use of fused bare silica capillaries did not allow the separation of these compounds under the same analytical conditions. These results demonstrate that this type of coating in CE provides the option of using BGEs that are useless when utilized together with bare silica capillaries making wider the application and possibilities of this analytical technique.     

Covalent bonding of homochiral metal‐organic framework in capillaries for stereoisomer separation by capillary electrochromatography

In this work, a [Cu(mal)(bpy)]?H₂O (mal, l ‐(?)‐malic acid; bpy, 4,4′‐bipyridyl) homochiral metal‐organic frameworks (MOFs) was synthesized and used for modifying the inner walls of capillary columns by utilizing amido bonds to form covalent links between the MOFs particles and capillary inner wall. The synthesized [Cu(mal)(bpy)]?H₂O and MOFs‐modified capillary column were characterized by X‐ray diffraction, thermogravimetric analysis, particle size distribution analysis, nitrogen absorption characterization, FTIR spectroscopy, SEM, and energy‐dispersive X‐ray spectroscopy (EDX). The MOFs‐modified capillary column was used for the stereoisomer separation of some drugs. The LODs and LOQs of six analytes were 0.1 and 0.25 μg/mL, respectively. The linear range was 0.25–250 μg/mL for ephedrine, 0.25–250 μg/mL for pseudoephedrine, 0.25–180 μg/mL for d ‐penicillamine, 0.25–120 μg/mL for l ‐penicillamine, 0.25–180 μg/mL for d ‐phenylalanine, and 0.25–160 μg/mL for l ‐phenylalanine, all with R² > 0.999. Finally, the MOFs‐modified capillary column was applied for the analysis of active ingredients in a real sample of the traditional Chinese medicine ephedra.     

Non-aqueous electrolytes for isotachophoresis of weak bases and its application to the comprehensive preconcentration of the 20 proteinogenic amino acids in column-coupling ITP/CE–MS

Isotachophoresis (ITP) has long been used alone but also as a preconcentration technique for capillary electrophoresis (CE). Unfortunately, up to now, its application is restricted to relatively strong acids and bases as either the degree of (de)protonation is too low or the water dissociation is too high, evoking zone electrophoresis. With the comprehensive ITP analysis of all 20 proteinogenic amino acids as model analytes, we, here, show that non–aqueous ITP using dimethylsulfoxide as a solvent solves this ITP shortcoming. Dimethylsulfoxide changes the pH regime of analytes and electrolytes but, more importantly, strongly reduces the proton mobility by prohibiting hydrogen bonds and thus, the so-called Zundel–Eigen–Zundel electrical conduction mechanism of flipping hydrogen bonds. The effects are demonstrated in an electrolyte system with taurine or H⁺ as terminator, and imidazole as leader together with strong acids such as oxalic and even trifluoroacetic acid as counterions, both impossible to use in aqueous solution. Mass spectrometric as well as capacitively coupled contactless conductivity detection (C⁴D) are used to follow the ITP processes. To demonstrate the preconcentration capabilities of ITP in a two-dimensional set-up, we, here, also demonstrate that our non-aqueous ITP method can be combined with capillary electrophoresis–mass spectrometry in a column-coupling system using a hybrid approach of capillaries coupled to a microfluidic interface. For this, C⁴D was optimized for on-chip detection with the electrodes aligned on top of a thin glass lid of the microfluidic chip.     

Highly sensitive tITP‐CZE determination of l‐histidine and creatinine in human blood plasma using field‐amplified sample injection with mobility‐boost effect

2D computer simulation revealed that amino acids and weak electrolytes were cationized because of the migration of counter‐ion from a BGE zone to a sample zone, which encouraged electrokinetic injection (EKI) of these analytes (by the mobility‐boost (MB) effect). To investigate the effects of kinds and concentrations of counter‐ions on the MB effect and the analyte amount injected into the capillary, experiments, and 1D computer simulations were performed. When acetate was used as the counter‐ion, the LODs (S/N = 3) of l ‐histidine and creatinine, respectively, reached 0.10 and 0.25 nM because of the concentration effect by transient ITP (tITP). The concentrations of l ‐histidine and creatinine in human blood plasma obtained using the proposed method were agreed with those obtained using the conventional methods. The proposed method can be applied to the analysis of amino acids and weak bases that have similar pI and pK_a to l ‐histidine and creatinine.     

In‐situ photopolymerized polyhedral oligomeric silsesquioxane‐derived monolithic capillary columns with quinidine functionality for enantioseparation by nano‐liquid chromatography

The successful fabrication of monolithic capillary columns for enantiomer separations was achieved within vinylized fused silica capillaries via fast “one‐pot” photo‐initiated free radical polymerization reaction. A mixture consisting of polyhedral oligomeric silsesquioxane, O‐[2‐(methacryloyloxy)ethylcarbamoyl]‐10,11‐dihydroquinidine was copolymerized in the presence of n‐butanol, ethylene glycol and photo‐initiator 2,2‐dimethoxy‐2‐phenylacetophenone. The morphology of the resultant polymeric hybrid inorganic‐organic material and its permeability as well as porosity can be controlled by adjusting the composition of the monomers and binary porogenic solvent. The chromatographic characteristics of the columns have been investigated. Separation factors of N‐acetyl‐phenylalanine (Ac‐Phe) and dichlorprop dropped with decrease of chiral functional monomer. Permeability was better when the macroporogen ethyleneglycol was present at higher concentrations during the polymerization. In general, the chiral compounds were well separated (dichlorprop: α = 1.53, R_s up to 4.14; Ac‐Phe: α = 1.36, R_s up to 2.69) by nano‐HPLC with an optimized enantioselective monolithic capillary column which can be prepared within a few minutes.     

Rapid determination of p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values of 20 amino acids by CZE with UV and capacitively coupled contactless conductivity detections

A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK _a) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C⁴D) was used as an additional detection mode. The C⁴D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK _a determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK _a values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK _a values were in accordance with the literature. Figure a UV (1) and C⁴D (2) detectors placed on-line on the CE capillary. b Curve of effective mobility as a function of pH for histidine     

Ionic liquids as stationary phases in gas chromatography: Determination of chlorobenzenes in soils

The present research focuses on the evaluation of different ionic liquid (IL) stationary phases in gas chromatography. The different IL columns were evaluated in terms of peak resolution (R_s) and peak symmetry for the separation of the chlorobenzenes. The determination of chlorobenzenes in soil samples by means of the optimal IL stationary phase (SLB‐IL82) is proposed as an application. Soil pretreatment was based on a simplified quick, easy, cheap, effective, rugged, and safe extraction procedure and a large injection volume via a programed temperature vaporizer working in solvent vent mode. The retention time of the chlorobenzenes increased as the polarity of the IL column decreased. SLB‐IL82 is the stationary phase that provides the best values as regards R_s and asymmetry factor. Soil sample blanks were spiked with the analytes before subjecting the sample to the extraction process. The existence of a matrix effect was checked and the analytical characteristics of the method were determined in a fortified garden soil sample. The method provided good linearity, good repeatability and reproducibility values, and the LODs were in the 0.1–4.7 μg/kg range. Two fortified soil samples were applied to validate the proposed methodology.     

Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ¶β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol.     

Prediction of Migration Times of Organic Ions in Capillary Zone Electrophoresis

The electrophoretic mobility ratio (R value) of any two ions is constant and independent of the capillary type and electrophoretic conditions if their electrical charges and hydration radii are constant. The use of strong acid salts and quaternary ammonium salts is therefore proposed for the determination of R values. Such analytes are called markers. The following determinations can be carried out: (i) the determination of the migration time corresponding to the electroosmotic flow (EOF) in any capillary under any electrophoretic condition by measuring the migration times of two markers in the condition studied (useful when the EOF is weak); (ii) the determination of the migration time of an analyte in any capillary by knowing the migration time of the markers in the capillary studied. If the pH is changed and the ionization of the analyte is pH dependent, the resulting migration time for the analyte can be calculated. The constancy of the mobility ratios of seven markers was checked experimentally at eight different pH values (between pH 3 and 10), at three temperatures, and for two buffer concentrations. The predicted and experimental migration times were also compared in two different types of capillaries.     

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R _s = 1.80 for naproxen to R _s = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R _s value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R _s = 0.89).     

Coelectroosmotic capillary electrophoresis of phenolic acids and derivatized amino acids using N,N-dimethylacrylamide-ethylpyrrolidine methacrylate physically coated capillaries

Two different families of compounds, i.e., phenolic and amino acids have been separated by capillary electrophoresis using a physically adsorbed polymer as capillary coating. The polymer used was N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EpyM) and it provided an stable coating by only flushing the capillary with a DMA-EpyM aqueous solution for 2 min between runs. The usefulness of this procedure has been demonstrated through the fast analysis of different families of solutes. Two different detection systems, diode-array detector and laser-induced fluorescence, have been used to determine phenolic acids and derivatized amino acids with fluorescein isothiocyanate, respectively. The main factors affecting reversal of electroosmotic flow (EOF) such as pH, type and concentration of buffer, and concentration and influence of organic solvents, as well as all the instrumental conditions were studied and optimized for both families of compounds.     

Amino Acid Ionic Liquids as Chiral Ligands in Ligand‐Exchange Chiral Separations

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1‐alkyl‐3‐methylimidazolium L ‐proline (L ‐Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers—dl ‐phenylalanine (dl ‐Phe), dl ‐histidine (dl ‐His), dl ‐tryptophane (dl ‐Trp), and dl ‐tyrosine (dl ‐Tyr)—were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R_s)=3.26–10.81 for HPLC; R_s=1.34–4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L ‐Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L ‐Pro are consequently attached to the support surface, thus inducing an ion‐exchange type of retention for the dl ‐enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand‐exchange‐based chiral separation. It also reveals the tremendous application potential of this new type of task‐specific ILs.     

Adsorptive BSA Coating Method for CE to Separate Basic Proteins

A novel and simple coating method was developed by coating bovine serum albumin (BSA) onto the inner surface of a fused-silica capillary, to avoid the adsorption of analytes during CE. The advantage presented here was that the coating process is more simple, fast, stable, and reproducible. The coated capillary avoided the adsorption of analytes onto the inner surface of a fused-silica capillary and might be a promising candidate for separation of complex biological samples with further development. Meanwhile, the efficiencies of the coated capillary were evaluated by EOF, chromatographic peak shape, and theoretical plate number (N m^?1) of RNase A. The optimal coating conditions were obtained from the results. The pH value of coating buffer PB was 4.2, the standing time was 12 h at 4 °C, and the coating concentration of BSA was 1.5 mg mL^?1. The stability of the coating on the inner wall of the capillary and the reproducibility of the coated capillaries were good. The theoretical plate number values of RNase A were over 1.3 × 10⁵ (N m^?1) in the coated capillary. After successive electrophoresis for 48 h using the coated capillary, the RSD values of EOF and the theoretical plate number were 4.14 % and 9.14 %, respectively. In addition, the RSD values of EOF and the theoretical plate number (N m^?1) in the coated capillaries were 13.19 % and 8.96 %, respectively. Finally, the coated capillary was successfully applied to separate the mixture of four basic proteins (RNase A, lysozyme, trypsin and myoglobin).