A novel diazoresin/poly(N‐vinyl aminobutyric acid) covalent capillary coating for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of poly(N‐vinyl aminobutyric acid) (PVAA) obtained from hydrolyzed polyvinylpyrrolidone was demonstrated using photosensitive diazoresin (DR) as a coupling agent. A layer‐by‐layer self‐assembled film of DR and PVAA based on ionic bonding was first fabricated on the inner wall of capillary, then ionic bonding was converted into covalent bonding after treatment with UV light through a unique photochemical reaction of DR. The covalently bonded coatings suppressed protein adsorption on the inner surface of the capillary, and thus a baseline separation of lysozyme, cytochrome c, BSA, amyloglucosidase, and myoglobin was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVAA coatings, the covalently linked DR/PVAA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of the highly toxic and moisture‐sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make covalently coated capillaries for CE.     

Novel covalently coated diazoresin/polyvinyl alcohol capillary column for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of PVA was demonstrated using photosensitive diazoresin (DR) as coupling agents. Layer‐by‐layer self‐assembly film of DR and PVA based on hydrogen bonding was first fabricated on the inner wall of capillary, then the hydrogen bonding was converted into covalent bonding after treatment with UV light through the unique photochemistry reaction of DR. The covalently bonded coatings suppressed basic protein adsorption on the inner surface of capillary, and thus a baseline separation of lysozyme, cytochrome c and BSA was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVA coatings, the covalently linked DR/PVA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of highly toxic and moisture‐sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make the covalently coated capillaries for CE.     

In‐capillary approach to eliminate SDS interferences in antibody analysis by capillary electrophoresis coupled to mass spectrometry

Capillary electrophoresis is an important technique for the characterization of monoclonal antibodies (mAbs), especially in the pharmaceutical context. However, identification is difficult as upscaling and hyphenation of used methods directly to mass spectrometry is often not possible due to separation medium components that are incompatible with MS detection. Here a CE‐MS method for the analysis of mAbs is presented analyzing SDS‐complexed samples. To obtain narrow and intensive peaks of SDS‐treated antibodies, an in‐capillary strategy was developed based on the co‐injection of positively charged surfactants and methanol as organic solvent. For samples containing 0.2% (v/v) of SDS, recovered MS peak intensities up to 97 and 95% were achieved using cetyltrimethylammonium bromide or benzalkonium chloride, respectively. Successful removal of SDS was shown in neutral coated capillaries but also in a capillary with a positively charged coating applying reversed polarity. The usefulness of this in‐capillary strategy was demonstrated also for other proteins and for antibodies dissolved in up to 10% v/v SDS solution, and in other SDS‐containing matrices, including the sieving matrix used in a standard CE‐SDS method and gel‐buffers applied in SDS‐PAGE methods. The developed CE‐MS approaches enable fast and reproducible characterization of SDS‐complexed antibodies.     

Improvement of reproducibility and sensitivity of CE analysis by using the capillary coated dynamically with carboxymethyl chitosan

Analysis reproducibility and detection sensitivity of capillary electrophoresis (CE) are often questioned by applied scientists, which has hindered its application as a routine method. To address these issues, a simple, precise, and reproducible dynamic coating method was developed by applying carboxymethyl chitosan (CMC) dynamic coating on fused silica capillary. The proposed coating was accomplished by simply rinsing the capillary with CMC solution for 1 min in between runs, with no regeneration procedure or buffer additives needed. Electroosmotic flow could be well controlled by adjusting the pH of background electrolyte, and the adsorption of analytes onto the capillary inner wall was effectively eliminated. The main parameters of the coating condition were optimized, and extensive applications of these CMC-dynamically coated capillaries in CE separations were then firmly confirmed. By using proteins, aristolochic acids, and inorganic anions as model analytes, the coating showed a good stability, high reproducibility, as well as improved sensitivity. Baseline separations could be obtained with high efficiency. The reduced adsorption was impressively effective for basic proteins, with an average plate number of 90,000/m for each protein, apart from the good resolution on the chromatogram. A high sensitive detection of α-lactalbumin was achieved with a limit of detection (S/N = 3) of 3.5 nM, and the number of theoretical plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with nonaqueous CE and CE-mass spectrometry proved to be practical. All results showed that the CMC-dynamically coated capillary has special properties and obvious superiority over the uncoated ones for CE analysis.     

Graphene oxide coated capillary for chiral separation by CE

This article describes a new application of graphene oxide (GO) in CE based on the coating of fused silica capillary for chiral separation. The coated capillary was characterized by SEM, energy dispersive X‐ray spectroscopy, and Raman spectra. The results indicated that the capillary was coated with GO. Chiral separations were carried out in the GO‐coated capillary for the ephedrine–pseudoephedrine (E‐PE) isomers and β‐methylphenethylamine (β‐Me‐PEA) isomers at the optimal buffer conditions without any chiral selector by CE. The precision and reproducibility of GO‐coated capillary were investigated, and the RSDs of migration time (n = 6) for the E‐PE isomers were 1.35–1.41%, and 0.97–3.50% for β‐Me‐PEA isomers, respectively. The LOD for E‐PE isomers and β‐Me‐PEA isomers was 3 μM and 18 μM, respectively.     

Surface initiated polymerization of a cationic monomer on inner surfaces of silica capillaries: Analyte separation by capillary electrophoresis versus polyelectrolyte behavior

[2‐(Methacryloyl)oxyethyl]trimethylammonium chloride was successfully polymerized by surface‐initiated atom transfer radical polymerization method on the inner surface of fused‐silica capillaries resulting in a covalently bound poly([2‐(methacryloyl)oxyethyl]trimethylammonium chloride) coating. The coated capillaries provided in capillary electrophoresis an excellent run‐to‐run repeatability, capillary‐to‐capillary and day‐to‐day reproducibility. The capillaries worked reliably over 1 month with EOF repeatability below 0.5%. The positively charged coated capillaries were successfully applied to the capillary electrophoretic separation of three standard proteins and five β‐blockers with the separation efficiencies ranging from 132 000 to 303 000 plates/m, and from 82 000 to 189 000 plates/m, respectively. In addition, challenging high‐ and low‐density lipoprotein particles could be separated. The hydrodynamic sizes of free polymer chains in buffers used in the capillary electrophoretic experiments were measured for the characterization of the coatings.     

CE‐MS analysis of heroin and its basic impurities using a charged polymer‐protected gold nanoparticle‐coated capillary

The first application of charged polymer‐protected gold nanoparticles (Au NPs) as semi‐permanent capillary coating in CE‐MS was presented. Poly(diallyldimethylammonium chloride) (PDDA) was the only reducing and stabilizing agent for Au NPs preparation. Stable and repeatable coating with good tolerance to 0.1 M HCl, methanol, and ACN was obtained via a simple rinsing procedure. Au NPs enhanced the coating stability toward flushing by methanol, improved the run‐to‐run and capillary‐to‐capillary repeatabilities, and improved the separation efficiency of heroin and its basic impurities for tracing geographical origins of illicit samples. Baseline resolution of eight heroin‐related alkaloids was achieved on the PDDA‐protected Au NPs‐coated capillary under the optimum conditions: 120 mM ammonium acetate (pH 5.2) with addition of 13% methanol, separation temperature 20°C, applied voltage ?20 kV, and capillary effective length 60.0 cm. CE‐MS analysis with run‐to‐run RSDs (n=5) of migration time in the range of 0.43–0.62% and RSDs (n=5) of peak area in the range of 1.49–4.68% was obtained. The established CE‐MS method would offer sensitive detection and confident identification of heroin and related compounds and provide an alternative to LC‐MS and GC‐MS for illicit drug control.     

Capillary electrophoresis of intact basic proteins using noncovalently triple‐layer coated capillaries

The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH‐independent and highly reproducible EOF. The PB–DS–PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: α‐chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125 000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple‐layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G₁ showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody–antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB–DS–PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.     

Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.     

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE‐MS

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein–capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N’,N’‐tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N‐(2‐hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly‐(TEDETAMA‐co‐HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE‐UV and CE‐MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer‐coated capillary was shown through the analysis of lysozyme in a cheese sample.     

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil^® and Cervarix^®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil^®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.     

Stable neutral double hydrophilic block copolymer capillary coating for capillary electrophoretic separations

Quaternized diblock copolymer, poly(N‐methyl‐2‐vinylpyridinium iodide‐block‐ethylene oxide), was successfully used as a neutral, dynamic coating to suppress the electroosmotic flow. The block copolymer consisted of two polymers that were linked covalently together. The cationic block (poly(N‐methyl‐2‐vinylpyridinium iodide)) was bound efficiently to the negatively charged capillary wall via electrostatic interactions, and the hydrophilic block (ethylene oxide) stabilized the system and created a neutral capillary surface with ultralow electroosmotic flow (+2.0 ± 4.5 × 10^?10 m²/Vs). The main advantages of the coating were simple and fast preparation, easy regeneration and automation, and stable electroosmotic flow. To emphasize the potential of this type of coating its stability was measured at a wide pH range demonstrating a high stability in the pH range of 4.0–10.5 and lifetime up to 8 days. The successful studies carried out with beta‐blockers, basic proteins, and lipoproteins proved the suitability of the coating for the separation of different sized analytes. Furthermore, the neutral coating developed is useful in a wide range of protein analysis and biological interaction studies under physiological condition.     

Polymeric ionic liquid-coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.     

Nano‐amylose‐2,3‐bis(3,5‐dimethylphenylcarbamate)‐silica hybrid sol immobilized on open tubular capillary column for capillary electrochromatography enantioseparation

The chiral organic‐inorganic hybrid materials can exhibit a high loading, and the chiral selector nanoparticles can create efficient stationary phases for open‐tubular capillary electrochromatography (OT‐CEC). Hence, a novel protocol for the preparation of an OT column coated with nano‐amylose‐2,3‐bis(3,5‐dimethylphenylcarbamate) (nano‐ABDMPC)‐silica hybrid sol through in situ layer‐by‐layer self‐assembly method was developed for CEC enantioseparation. By controlling the assembly cycle number of nano‐ABDMPC‐silica hybrid sol, a homogeneous, dense and stable coating was successfully prepared, which was confirmed by SEM and elemental analysis. As the main parameter influencing the chiral separating effect, the nano‐ABDMPC bearing 3‐(triethoxysilyl)propyl residues concentration was investigated. The experimental results showed that 10.0 mg/mL nano‐ABDMPC bearing 3‐(triethoxysilyl)propyl residues coated OT capillary column possessed chiral recognition ability toward the six enantiomers (phenylalanine, tyrosine, tryptophan, phenethyl alcohol, 1‐phenyl‐2‐propanol, and Tröger's base) at some of the different conditions tested. Additionally, the coated OT column revealed adequate repeatability concerning run‐to‐run, day‐to‐day and column‐to‐column. These results demonstrated the promising applicability of nano‐ABDMPC‐silica hybrid sol coated OT column in CEC enantioseparations.     

Influence of polyelectrolyte capillary coating conditions on protein analysis in CE

CE of biomolecules is limited by analyte adsorption on the capillary wall. To prevent this, monolayer or successive multiple ionic‐polymer layers (SMILs) of highly charged polyelectrolytes can be physically adsorbed on the inner capillary surface. Although these coatings have become commonly used in CE, no systematic investigation of their performance under different coating conditions has been carried out so far. In a previous study (Nehmé, R., Perrin, C., Cottet, H., Blanchin, M. D., Fabre, H., Electrophoresis 2008, 29, 3013–3023), we investigated the influence of different experimental parameters on coating stability, repeatability and peptide peak efficiency. Optimal coating conditions for monolayer and multilayer (SMILs) poly(diallyldimethylammonium) chloride/ poly(sodium 4‐styrenesulfonate) coated capillaries were determined. In this study, the influence of polyelectrolyte concentration and ionic strength of the coating solutions, and the number of coating layers on coating stability and performance in limiting protein adsorption was carried out. EOF magnitude and repeatability were used to monitor coating stability. Coating ability to limit protein adsorption was investigated by monitoring variations of migration times, time‐corrected peak areas and separation efficiency of test proteins. The separation performance of polyelectrolyte coatings were compared with those obtained with bare silica capillaries.     

Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

A lamp‐based fluorescence detection (Flu) system for CE was extended with a wavelength‐resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe‐Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut‐off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal‐intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE‐WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged‐polymer coated capillary. Using the 3‐D data set, signal averaging over time and emission‐wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.     

Co‐electroosmotic capillary electrophoresis of basic proteins with 1‐alkyl‐3‐methylimidazolium tetrafluoroborate ionic liquids as non‐covalent coating agents of the fused‐silica capillary and additives of the electrolyte solution

The paper reports the results of a study carried out to evaluate the use of three 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as non‐covalent coating agents for bare fused‐silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co‐EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co‐electroosmotic CE is obtained with the 1‐butyl‐3‐methylimidazolium tetrafluoroborate coated capillary and 100 mM acetate buffer (pH 4.0) containing 4.4 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as the BGE.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

The coating of smart pH‐responsive polyelectrolyte brushes in capillary and its application in CE

A novel pH‐responsive coating technique was developed and applied to CE successfully in this paper. The coating was formed by bonding mixed opposite charge poly(acrylic acid) and poly(2‐vinylpyridine) randomly onto the inner wall of a silica capillary. The coating processes were first characterized by ellipsometry and atomic force microscopy at macroscale and microscale, respectively. Measurements of EOF were implemented to confirm the coating. Direction and velocity of EOF became controllable from negative to positive, showing a perfect sigmoidal curve as the coating net charges alternated by the pH of BGE. The control of the EOF makes it possible to analyze different kinds of small molecules, peptides, and proteins successfully in the same capillary. Results showed that the stability and reproducibility for separations of fluoroquinolone standards were satisfactory for more than a hundred separations. A series of basic and acidic protein standards were separated with admirable efficiency and minimal adsorption using both polarities. The separation of tryptic BSA digest showed that the prepared capillary has immense potential in analyzing a single sample with both acidic and basic separations, which achieved the expectation in proteomics study by CE‐MS.     

Preparation and characterisation of dual-layer latex-coated columns for open-tubular capillary electrochromatographic preconcentration of cations combined in-line with their separation by capillary electrophoresis

A dual-layer ion-exchange latex-coated column was prepared and characterised for on-capillary preconcentration of cations using an open-tubular ion-exchange CEC format. After preconcentration, the analyte cations were eluted with a transient isotachophoretic gradient and separated by CE. The latex double layer was established by first coating the negatively charged wall of the capillary with a layer of cationic quaternary ammonium anion-exchange Dionex AS5A latex particles (60 nm diameter), and then coating a layer of anionic sulphonated cation-exchange Dionex CS3 latex particles (300 nm diameter) onto the underlying AS5A layer. The adhesion of layers is based on electrostatic attractions. Several dual-layer capillaries were characterised for their EOF and ion-exchange capacity and this showed that coatings could be prepared reproducibly by a simple flushing procedure. The dual-layer columns exhibited a moderate, pH-independent EOF (ca. 26 x 10(-9 )m2V(-1)s(-1)) and an ion-exchange capacity of 57 microequiv./g (or 2.69 nequiv./column). Using an 8 cm length of coated capillary combined with a 72 cm length of untreated capillary, a method for on-line preconcentration and separation of monovalent organic bases, alkali metal ions and alkaline earth metal ions by CE was developed. Recoveries for the preconcentration step were 48% for 4-methylbenzylammonium, 43% for benzylammonium, 30-32% for alkali metal ions and 71-75% for alkaline earth cations. In all cases, recoveries were reproducible with RSDs being less than 6.2%. The influences of the ion-exchange selectivity coefficient of the analyte and the sample-loading rate on analyte recovery were also examined. The proposed method was utilised for the determination of alkaline earth cations and low microM detection limits were obtained.