毛细管电泳中影响径向电场控制电渗的主要因素

利用自制的二维电场毛细管电泳系统研究了不同因素对径向电场控制电渗能力的影响,发现缓冲液的ｐＨ值、浓度、种类以及管壁表面状态、管径等对电渗的电场调控有关键性的影响。有趣的是,添加剂不影响电场的调控能力,而杯芳烃涂层毛细管却能提高电渗对径向电场的响应能力。利用这种涂层效应有可能实现较高ｐＨ值下电渗的电场调控。     

Enhancement of electroosmotic flow using zwitterionic additives

Zwitterionic additives provide a means of altering the EOF without increasing conductivity. The magnitude of the EOF in a bare silica capillary increased by as much as 69% upon addition of 500 mM of zwitterion to the running buffer. The EOF enhancement increases linearly with the zwitterion concentration. With zwitterionic additives of the form +NH3-(CH2)n-COO-, the magnitude of the EOF increase is directly related to the number of methylene groups, (n), which ranges from n = 1 to 7. The endgroups on the zwitterions also affect the EOF enhancement. The effect of Z1-methyl (+N(CH3)3CH2CH2CH2SO3-) on EOF was not a function of either the buffer cation or pH. The EOF enhancement is a function of the dielectric increment of the additive and the nature of the amine functionality.     

Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4 x 10(-4) cm2 V(-1) s(-1) The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte pKa values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Analysis of galactoglucomannans from spruce wood by capillary electrophoresis

The aim of this study was to setup a method for detection and quantification of monosaccharide components in technical galactoglucomannas (T-GGM) from spruce wood using capillary zone electrophoresis (CZE). CZE technique was optimised regarding borate buffer concentrations, EOF modifier application, and system pH. Aqueous solution of T-GGM was chemically hydrolysed by sulphuric acid, in an autoclave. In this way obtained monosaccharides were derivatized with 4-amino benzoic acid ethyl ester via reductive amination using sodium cyanoborohydride. The results of the optimisation procedure showed that the borate buffers at lowest concentrations (100 and 200 mM) with acetonitrile addition as EOF modifier gave the optimal measurement results, as it showed sufficient separation at relatively short migration times. The amounts of single monosaccharide components in the T-GGM samples obtained by the optimised CZE procedure were practically the same in comparison to the results of the well established HPLC-anion exchange chromatography. On the basis of this research, it was concluded that the capillary zone electrophoresis is an efficient analytical procedure for the characterisation of galactoglucomannans derived from softwoods.     

The impact of buffers and surfactants from micellar electrokinetic chromatography on matrix-assisted laser desorption ionization (MALDI) mass spectrometry of peptides. Effect of buffer type and concentration on mass determination by MALDI-time-of-flight mass spectrometry

This paper describes the effect of various buffers, surfactants, and organic additives commonly encountered in capillary zone electrophoresis and micellar electrokinetic chromatography on the molecular weight determination of peptides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Signal-to-noise ratio generally decreased with increasing buffer concentration without affecting mass accuracy, but the type of buffer was also important. Good spectra were obtained with an ammonium acetate buffer up to a concentration of 500 mM without impacting ionization of either peptides or other mobile phase constituents. Ionization of organic additives, such as anionic surfactants, non-ionic surfactants, and cyclodextrins was buffer dependent and presented a problem when the mass of the additive was in the range of the peptide mass. Brij-35, Tween-80, and cyclodextrins all produced prominent spectra of their own in the presence of sodium or potassium containing buffers, but not with ammonium acetate. Cationization of these neutral species with sodium or potassium ions allowed them to acquire a positive charge and produce spectra. In contrast, the ammonium ion appears to be a poor cationizating agent. Ionization of neutral surfactants was suppressed in ammonium acetate without impacting the spectra of peptides. Ammonium acetate buffers containing 30 mM sodium dodecyl phosphate also gave spectra with good signal intensity and no interference from the surfactant. Suppression of peptide ionization in MALDI was a problem when methanol, tetrabutyl amine, or poly(vinyl alcohol) were used with either ammonium acetate, sodium phosphate, and N-(2-hydroxyethyl)piperazine-N-(2-ethansulfonic acid).     

Micellar electrokinetic capillary chromatography as a powerful tool for pharmacological investigations without sample pretreatment: a precise technique providing cost advantages and limits of detection to the low nanomolar range.

A number of pharmaceuticals (e.g., acetaminophen, salicylic acid, sulfamethoxazole, theophylline, tolbutamide and trimethoprim) have been determined in human plasma by micellar electrokinetic chromatography (MEKC), without sample pretreatment, using underivatized fused-silica capillaries. The total analysis time was only 10 min. A sodium dodecyl sulfate (SDS)-containing borate buffer (60 mM with 200 mM SDS) at pH 10 was used. Between runs, proteins adsorbed to the capillary wall are removed by rinsing with SDS buffer and either acetonitrile (e.g., 50% v/v) or isopropanol (e.g., 10% v/v). Other rinsing procedures are discussed (salts, enzyme-containing solutions, organic solvents, sodium hydroxide, hydrofluoric acid). The separation system is tested in a concentration range between 10 ng/mL and 100 microg/mL; a detection limit of about 20 ng/mL can readily be obtained. The sensitivity was substantially improved using isopropanol as buffer additive. A day-to-day precision for relative peak areas of 1-2% relative standard deviation (RSD, n > 40) was reached in the upper concentration range. Under repeatability conditions, these values could also be obtained for low microg/mL concentrations. Thus, not only drug monitoring but also pharmacokinetic investigations from blood plasma become possible without further sample pretreatment.     

Enantiomer separation of miconazole by capillary electrophoresis with dual cyclodextrin systems.

A cyclodextrin-modified micellar capillary electrophoretic method (MECC) was developed using mixtures of beta, cyclodextrins (beta-CD) and mono-3-O-phenylcarbamoyl-beta-CD as chiral additives for the chiral separation of miconazole with the dual CDs systems. The enantiomers were resolved using a running buffer of 50 mmol/L borate pH 9.5 containing 15 mmol/L beta-CD and 15 mmol/L mono-3-O-phenylcarbamoyl-beta-CD containing 50 mmol/L sodium dodecyl sulfate and 1 mol/L urea. A study of the respective influence of the beta-CD and the mono-3-O-phenylcarbamoyl-beta-CD concentration was performed to determine the optimal conditions with respect to the resolution. Good repeatability of the method was obtained.     

Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.     

Dynamic capillary coatings for electroosmotic flow control in capillary electrophoresis

Control of the electroosmotic flow (EOF) is critical for achieving optimal separations by capillary electrophoresis. For instance, manipulation of the EOF can yield either high resolution separations or rapid analyses. Dynamic capillary coatings are a simple and cost-effective approach to altering the EOF. The normal EOF can be slowed using buffer additives such as Mg²⁺ and hexamethonium which ion exchange onto the surface silanols to lower the effective wall charge. Alternatively, cationic polyelectrolytes or cationic surfactants can be used to establish a cationic coating on the capillary wall, which results in a reversed EOF. Practical considerations such as pH stability and reproducibility obtainable with an EOF modifier will be discussed.     

Simultaneous determination of eleven preservatives in cosmetics by micellar electrokinetic chromatography

A new method for the simultaneous quantitation of 11 preservatives-imidazolidinyl urea, benzyl alcohol, dehydroacetic acid, sorbic acid, phenoxyethanol, benzoic acid, salicylic acid, and four parabens (methyl, ethyl, propyl, and butyl)-in cosmetics by micellar electrokinetic capillary chromatography was established and validated. The separation was performed using an uncoated fused-silica capillary (50 pm id x 60.2 cm, effective length 50 cm) with a running buffer consisting of 15 mmol/L sodium tetraborate, 60 mmol/L boric acid, and 100 mmol/L sodium dodecyl sulfate. A 1:10 dilution of the running buffer was used as the sample buffer to extract the cosmetic samples. The key factors, such as the concentration and pH of the running and sample buffers, which influence quantitative analysis of the above 11 preservatives in cosmetic samples, were investigated in detail. The linear ranges of the calibration curves for imidazolidinyl urea and the other 10 preservatives were 50-1000 and 10-200 mg/L, respectively. The correlation coefficients of the standard curves were all higher than 0.999. The recoveries at the concentrations studied ranged from 93.0 to 102.7%. RSDs were all less than 5%. The new method with simple sample pretreatment met the needs for routine analysis of the 11 preservatives in cosmetics.     

Reproducible protein analysis by CE using linear polyacrylamide-coated capillaries and hydrochloric acid rinsing

Hydrochloric acid was investigated as a rinsing reagent to remove adsorbed proteins from linear polyacrylamide-coated capillaries for electrophoresis. Three model proteins were used, namely cytochrome c as a basic protein, beta-lactoglobulin as an acidic protein, and beta-casein as a more easily denaturing protein. In order to regenerate capillary surfaces, they have been rinsed for 5 min with 2 M hydrochloric acid, 5 min with water, and then 30 min with buffer after every tenth run. It was found important to perform this regeneration procedure on time. The obtained results show good repeatability of the apparent EOF mobility with percentage RSDs below 3% (n = 60) in various cases. These good results were mainly confirmed in long-term series with more than 200 runs each. Only very high concentrations (175 microM) of beta-lactoglobulin and beta-casein at pH 3.5 gave RSD% values above 5%. For these conditions, the further test of 85% m/m phosphoric acid as rinsing reagent showed a good repeatability of the apparent EOF mobilities as well.     

Cationic gemini surfactant as dynamic coating in CE for the control of EOF and wall adsorption

The cationic gemini surfactant ethylene bis(1-dodecyldimethylammonium) dibromide was used as a dynamic coating to control EOF and prevent wall adsorption of basic proteins in CE for the first time. This gemini surfactant shows a more powerful capability in EOF reversal than traditional single-chained surfactant. The gemini surfactant reverses the EOF at a concentration level even less than 0.01 mM, and the EOF magnitude is affected by surfactant concentration, pH, ionic strength, and ions added in buffer. Highly efficient and rapid protein separation (N > 300,000) was obtained with buffer containing 2 mM gemini surfactant under pH ranging from 3 to 6. The effects of surfactant and buffer concentration on protein separation were investigated in detail. Under the optimal conditions, good repeatability (RSD of migration time <0.6% for run-to-run and <2.5% for day-to-day assays) and recovery (>90%) of tested proteins were obtained. This new dynamic coating is also suitable for biosample analysis.     

Chiral separation of six diastereomeric flavanone-7-O-glycosides by capillary electrophoresis and analysis of lemon juice

Inexpensive and disposable polyester microchips were fabricated through photolithographic and wet-chemical etching procedure, followed by replication using an imprinting method at room temperature. Laboratory-scale laser-induced fluorescence equipment was employed as a detection system. The generation of electroosmotic flow (EOF) on the polyester channels was discussed in this paper. Surfactants in the running buffer had a significant effect on the EOF depending on their types. The epsilon potential of the electric double layer formed by adsorbing sodium lauryl sulfate molecules on the wall of polyester channels seemed to be constant within the buffer pH investigated. EOF could also be suppressed to zero by adding polyoxyethylene 23 lauryl ether into the running buffer. The separation of two laser dyes was obtained using polyester chips through both micellar electrokinetic chromatography and capillary zone electrophoresis. The polyester channels modified with 10-undecen-1-ol exhibited a dramatically high-separation efficiency compared with the conventional fused-silica capillary tubes.     

The influence of surface treatments on the electroosmotic flow in micellar electrokinetic capillary chromatography

Summary An investigation was carried out to study the electroosmotic flow (EOF) in micellar electrokinetic capillary chromatography (MECC). Fused silica capillaries were treated with various reagents to investigate the parameters influencing the magnitude and reproducibility of the EOF. The results for untreated and treated columns were collected and compared under the same experimental conditions. Furthermore, experiments with and without sodium dodecylsulfate (SDS) addition to the buffer were carried out to elucidate the influence of this surfactant on the EOF. The mechanisms behind the effects of these modifications on the EOF and the stability of the coatings on the column wall are discussed.     

Control of electroosmotic flow in nonaqueous capillary electrophoresis by polymer capillary coatings

In aqueous capillary electrophoresis the electroosmotic flow (EOF) can be strongly suppressed or eliminated by coating the capillary surface silanols either by buffer additive adsorption or chemical modification. Hydrophilic coatings, e.g., polyvinyl alcohol (PVA) proved to be most efficient for EOF control in applications like DNA analysis. In nonaqueous capillary electrophoresis (NACE), however, the EOF cannot be totally suppressed with these capillaries and coating efficiency turned out to be solvent-depending. In this paper, fused-silica capillaries with monomeric and polymeric coatings differing in hydrophobicity and chemical properties (vinyl, vinyl acetate, vinyl alcohol and acrylates with different alkyl chain length) were investigated. Besides studying the EOF characteristics with different organic solvents and water, gas chromatography (GC) measurements were carried out to probe the silanol reduction via ether retention and the surface hydrophobicity by retention of nonane. Good correlations between GC results and EOF magnitude could be found. It could be demonstrated that the polymeric coating has to be solvatized by the buffer solvent to reduce the EOF. The PVA coating was optimal for aqueous systems but not effective for some nonaqueous buffers. On the other hand, polyvinyl acetate and polyethyl acrylate as polymeric coatings proved to be optimal to reduce the EOF in NACE.     

卤代乙酸及其结构相近化合物的高效毛细管电泳分离

氟、氯、溴等卤代乙酸是结构非常相近的离子型化合物,对它们的分离测定比较困难。用高效毛细管电泳法在碱性或酸性缓冲液条件下可将它们分离。在酸性缓冲液条件下,可提高有机酸分离的选择性。较低的操作电压有利于提高阴离子的分离度,而改变温度对分离度的影响不大。     

A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with TB buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments (11 and 8 base pairs) became sharper and were detected. Relative standard deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.     

Characterization of discontinuous buffer junctions using pH indicators in capillary electrophoresis for protein preconcentration

An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30 min. To study the formation of pH junctions in CE, a pH indicator, bromothymol blue, is used in this work to reveal the pH changes at the discontinuous buffer boundary. Bromothymol blue (BTB) exhibits a drastic change in its visible absorption spectrum (300-600 nm) going from the acidic to basic pH conditions, and is therefore ideal for visualizing the changes in pH at the junctions created by various buffer combinations. Preconcentration of myoglobin was performed in discontinuous buffers containing BTB. Major differences in the BTB absorption profiles were identified from buffer systems that differ significantly in preconcentration performance, which in turn, allowed for the identification of ideal buffers for sample preconcentration. Up to 2000-fold preconcentrations of myoglobin were achieved in the buffer systems studied in this work. In addition, the role of the electroosmotic flow (EOF) on the preconcentration performance was investigated. A low EOF was found to be desirable, as the pH junction could stay longer in the capillary for accumulation of proteins. The pH junction also displayed characteristics to resist bandbroadening. Potential laminar flow resulted from the mismatched residual EOFs under the two pH conditions within the discontinuous buffers appeared to have minimal effect on the preconcentration. In fact, external applied pressure can be used to control the migration of the pH junction without compromising the protein preconcentration.