Capillary Zone Electrophoretic Separation Research Articles

Optimized sample buffer for dispersed, high-resolution capillary zone electrophoretic separation of Escherichia coli B

Capillary zone electrophoresis (CZE) is a powerful tool for high resolution chemical separations. Applying CZE to microbial samples may facilitate a deeper understanding of bacterial physiology and behavior. However, the study of complex microbial samples has been limited by the uncontrolled hetero-aggregation of bacterial cells under an applied electric field. We tested a wide range of sample buffers and buffer additives for the optimization of bacterial CZE separations using a 20 mM Tris–HCl background electrolyte. By modifying the sample buffer, but not the background electrolyte, we retain constant separation conditions, which aids in the comparison of the sample buffer additives. We report optimized methods for automated CZE separation and simultaneous fractionation of Escherichia coli B, which is one of the two most widely used wild-type strains. A modified sample buffer containing neutral salts and the addition of glycerol produced a 20-fold increase in loading capacity and a reduction in peak width/broadening of 86% in comparison to previously reported work. In addition, the glycerol-modified sample buffer appears to reduce the persistent aggregation and adhesion to the capillary walls during electrophoretic separations of complex environmental microbiota.

Microchip-Based Electrophoretic Separations with a Pressure-Driven Backflow.

It is well known that the resolving power of capillary zone electrophoretic separations may be improved with an increase in the applied electric field strength and separation time. While large electric fields may be realized in short analysis channels commonly employed in microfluidic systems, this experimental design is not suitable for achieving long separation times. In this chapter, we describe the use of a steady and/or a periodic pressure-driven backflow to increase the separation time in short microchannels thereby enabling the analysis of closely related species on microchip devices. The reported backflow was realized in our assays using an on-chip pressure-generation capability that relied on the partial blockage of electroosmotic flow around a junction of two glass channel segments having different depths. Although the noted strategy led to additional band broadening in the system, the resolving power of our device was observed to substantially improve upon introduction of the reported steady/periodic pressure-driven backflow for analysis channels shallower than 5μm.

Ultraviolet Detection of Monosaccharides: Multiple Wavelength Strategy to Evaluate Results after Capillary Zone Electrophoretic Separation

Ultraviolet Detection of Monosaccharides: Multiple Wavelength Strategy to Evaluate Results after Capillary Zone Electrophoretic Separation

Separation and Determination of Cold Medicine Ingredients by Capillary Zone Electrophoresis Using Sulfated β‐Cyclodextrin as an Electrolyte Modifier and Chiral Selector

AbstractCapillary zone electrophoretic separations of cold medicine ingredients, including acetaminophen (AC), dextromethorphan (DMF) and racemates of phenylpropanolamine (PPA) and chlorpheniramine maleate (CPM) using randomly sulfated‐β‐CD (S‐β‐CD) as an electrolyte modifier and a chiral selector were investigated. The results indicate that S‐β‐CD is an excellent chiral selector and a suitable electrolyte modifier as well for the separation of those cold medicine ingredients. Influences of S‐β‐CD concentration and buffer concentration on the separation were examined. Baseline separation of these cold medicine ingredients with 1.0 % (w/v) S‐β‐CD could be simultaneously and successfully achieved within 11.8 minutes. In addition, S‐β‐CD could also act as a chiral selector for enantioseparation of PPA and CPM. A high enantioselectivity was obtained for these two analytes. Linear relationships between the peak area and its concentration for the calibration curves of AC and DMF were obtained (correlation coefficients: 0.9987 for AC, 0.9965 for DMF, respectively). The relative standard deviations of the migration time and peak area of AC and DMF were 0.19, 2.44 % and 0.34, 2.99 %, respectively. Detection limits were 0.93 and 2.57 μg/mL for AC and DMF, respectively. Recoveries of AC and DMF ranging between 102.42 and 97.28 % were observed. The proposed method was successfully applied to the determination of AC and DMF in cold medicines.

Validated Stability Indicating Assay of Gemifloxacin and Lomefloxacin in Tablet Formulations by Capillary Electrophoresis

A capillary zone electrophoretic (CZE) method has been developed for the determination of gemifloxacin and lomefloxacin in pharmaceutical tablet formulations. The CZE separation was performed using a 75 µm×35 cm fused silica capillary under the following conditions: 25°C; applied voltage, 12 kV; 25 mM H3PO4‐NaOH running buffer (pH 8.5). The detection wavelength was 254 nm. Flumequine was used as internal standard. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision, specificity, and robustness. The calibration was linear from 5 to 50 µg mL−1 for gemifloxacin and 10 to 60 µg mL−1 for lomefloxacin, and the limit of detection and quantification were 2.93, 4.91 µg mL−1, and 3.87, 8.93 for gemifloxacin and lomefloxacin, respectively. Recoveries ranging from 94.4–108.6% were obtained for both drugs. The method was successfully applied to the determination of gemifloxacin and lomefloxacin in pharmaceutical tablets. Excipients present in the tablets and degraded products from different stress conditions did not interfere in the assay.

Environmental formaldehyde analysis by active diffusive sampling with a bundle of polypropylene porous capillaries followed by capillary zone electrophoretic separation and contactless conductivity detection

Compared to other volatile carbonylic compounds present in outdoor air, formaldehyde (CH 2O) is the most toxic, deserving more attention in terms of indoor and outdoor air quality legislation and control. The analytical determination of CH 2O in air still presents challenges due to the low-level concentration (in the sub-ppb range) and its variation with sampling site and time. Of the many available analytical methods for carbonylic compounds, the most widespread one is the time consuming collection in cartridges impregnated with 2,4-dinitrophenylhydrazine followed by the analysis of the formed hydrazones by HPLC. The present work proposes the use of polypropylene hollow porous capillary fibers to achieve efficient CH 2O collection. The Oxyphan ® fiber (designed for blood oxygenation) was chosen for this purpose because it presents good mechanical resistance, high density of very fine pores and high ratio of collection area to volume of the acceptor fluid in the tube, all favorable for the development of air sampling apparatus. The collector device consists of a Teflon pipe inside of which a bundle of polypropylene microporous capillary membranes was introduced. While the acceptor passes at a low flow rate through the capillaries, the sampled air circulates around the fibers, impelled by a low flow membrane pump (of the type used for aquariums ventilation). The coupling of this sampling technique with the selective and quantitative determination of CH 2O, in the form of hydroxymethanesulfonate (HMS) after derivatization with HSO 3 −, by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE–C 4D) enabled the development of a complete analytical protocol for the CH 2O evaluation in air.

Determination of carnosic acid and rosmarinic acid in sage by capillary electrophoresis

A simple and rapid capillary electrophoresis method was developed for the identification and quantitative determination of two antioxidative compounds – carnosic acid and rosmarinic acid – in the extracts of commercial Sage ( Salvia officinalis) tea-bags. Capillary zone electrophoretic separation of carnosic and rosmarinic acids was performed using 40 mmol/l borate, at pH 9.6 as the running buffer. Weighed sage samples were extracted from tea-bags by sonification and the extracts were directly injected without any purification and pre-separation process. Coumarin was used as internal standard for quantitation and the limits of detection for carnosic acid and rosmarinic acid were obtained as 2.79 and 3.18 μg/ml, respectively using UV detection at 210 nm.

Sulfur speciation by capillary zone electrophoresis: Determination of dithionite and its decomposition products sulfite, sulfate and thiosulfate in commercial bleaching agents

In this paper, a capillary zone electrophoretic (CZE) method was developed for the separation of the sulfur species dithionite (S 2O 4 2−), sulfite (SO 3 2−), sulfate (SO 4 2−) and thiosulfate (S 2O 3 2−). A carrier electrolyte (pH 7.0) containing 1.5 mmol L −1 pyromellitic (PM) acid, 10 mmol L −1 Tris(hydroxymethyl)-aminomethane (Tris), 0.5 mmol L −1 diethylenetriamine (DETA) and 0.1% (v/v) formaldehyde (as stabilizer for S 2O 4 2− and SO 3 2−) allowed the determination of the sulfur anions after 9 min CZE separation with indirect UV detection at 214 nm. The addition of 0.1% (v/v) formaldehyde to the sample solution stabilizes dithionite and sulfite as HOCH 2SO 2 − and HOCH 2SO 3 − anions. The procedure was applied for the determination of dithionite and its decomposition products sulfite, sulfate and thiosulfate in commercial formulations of bleaching agents. Dithionite was found to be the major component of the commercial formulations in concentrations between 30.80 and 33.30% (w/w). As anticipated, sulfite, sulfate and thiosulfate were found to be present as decomposition or by-products in the commercial formulations at concentrations of 14.30–14.80, 5.20–5.70 and 0.30–0.40% (w/w), respectively. The results were found to be in good agreement with those of polarographic and spectrophotometric determinations.

Capillary Zone Electrophoretic Separation of Four Novel Compounds Active Against Human Immunodeficiency Virus Type 1

The separation of four novel small molecules active in the treatment of acquired immunodeficiency syndrome (AIDS), is presented. The molecules were synthesized by computer-aided design with the property of inhibiting the binding of the human immunodeficiency virus type 1 trans-activator of transcription (HIV-1 Tat) protein to the trans-activation response region (TAR) RNA required for HIV-1 transcription, and thus blocking of the HIV replication cycle. The active compounds were separated to baseline by capillary zone electrophoresis (CZE) under optimized conditions with 0.1 mol L−1 ammonium acetate solution (pH 3.8) as running buffer and UV detection. The method will be used study the interactions between the anti-HIV-1 active molecules and HIV TAR RNA and for rapid screening of these potential drugs for early drug discovery.

Analysis of calcitonin and its analogues by capillary zone electrophoresis and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry.

Capillary zone electrophoretic (CZE) separations and mass spectrometric analysis of salmon calcitonin and related analogues were performed to generate electrophoresis and mass fingerprints for quality control of the recombinant polypeptide pharmaceutical salmon calcitonin. The calcitonins and their corresponding tryptic digests were successfully separated by CZE at low pH in fused silica capillaries dynamically modified with poly-cationic polymers. The poly-cationic modified inner surface of the fused silica capillaries generated a strong anionic electroosmotic flow (EOF). Analytes of negative, neutral, and positive charge were all swept through the capillary toward the positive electrode. Compared to Polybrene-coated capillaries, capillaries coated with PEI showed a markedly slower but much more stable electroosmotic flow. The migration order of the analytes was predicted by comparing approximate values of the charge to (molecular mass)2/3 ratios. The predicted migration order was confirmed by off-line analysis of CZE fractions with matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Capillary zone electrophoresis of phenol and methylphenols at high pH in methanol

AbstractCapillary zone electrophoresis of phenol and its three methyl‐substituted derivatives (cresols) was investigated in water and methanol as solvents for the electrolyte. In water the acid strengths of o‐cresol and p‐cresol are too similar to allow their capillary zone electrophoretic separation. In pure methanol the differences in acid strength are amplified and all three cresols and phenol were clearly separated with use of pH 13–14 buffers prepared from maleic acid. The dependence of electrophoretic mobilities on pH in MeOH was investigated, and a slight difference was observed between theoretically calculated mobilities and measured mobilities. Maleate background electrolytes provided highly repeatable separations of the analytes. High pH medium in methanol appears to be a good choice for capillary zone electrophoretic separation of weak acids with similar acidity in water. The effective separation of 2,4‐xylenol and 2,6‐xylenol was also demonstrated.

Separation of structurally related synthetic peptides by capillary zone electrophoresis

The separation of two different sets of synthetic peptides has been investigated by high-performance capillary zone electrophoresis utilising naked, fused silica capillaries. The effects of electrolyte pH, buffer concentration, capillary length and electric field strength on the separation efficiency and selectivity were systematically varied, with the highest resolution achieved with buffer electrolytes of low pH and relatively high ionic strength. Under optimised separation conditions utilising the “short end injection” separation approach with negative electric field polarity, a series of eight structurally-related synthetic peptides were baseline resolved within 4 min without addition of any modifier of the background electrolyte with separation efficiencies in the vicinity of 600 000 theoretical plates/m. Further significant enhancement of separation efficiencies could be achieved by taking advantage of the “long end injection” approach with positive electric field polarity. The outcome of these experimental variations parallels the “sweeping” effect that has been observed in the capillary electrochromatographic and micellar electrokinetic separations of polar molecules and permits rapid resolution of peptides with focusing effects. In addition, small changes in the electrolyte buffer pH and concentration were found to have a significant impact on the selectivity of synthetic peptides of similar intrinsic charge. These observations indicate that multi-modal separation mechanisms operated under these conditions with the unmodified fused silica capillaries. This study, moreover, documents additional examples of peptide-specific multi-zoning behaviour in the high-performance capillary zone electrophoretic separation of synthetic peptides.

Assay of riboflavin in sample wines by capillary zone electrophoresis and laser-induced fluorescence detection.

To routinely assay the concentration of riboflavin (RF) in wines, a rapid and sensitive method was developed and evaluated. The method is based on a simple sample preparation, capillary zone electrophoretic separation and laser-induced fluorescence detection (CZE-LIF). Sample preparation required only dilution and filtration. Under optimized conditions, the limit of detection of riboflavin was 0.5 micro g/L, using a hydrodynamic sample introduction of 10 s at 54 mbar. The method was fully validated: the recovery of RF in wines was >95%. The concentrations of RF within the three sample types of Italian wines investigated here ranged from 69 to 151 micro g/L with a mean value (+/-SD) of 112 +/- 25 micro g/L, from 74 to 193 micro g/L with a mean value of 115 +/- 45 micro g/L, and from 156 to 292 micro g/L with a mean value of 226 +/- 40 micro g/L, for white, rosé, and red wines, respectively. Such an accurate and highly sensitive CZE-LIF method represents a powerful improvement over previous methods in terms of sensitivity, simplicity, and efficiency. It is well suited to satisfy the demands for accurate and sensitive detection with minimal sample preparation and cleanup.

Determination of codeine and its metabolite in human urine by CE with amperometric detection

The amperometric detection of codeine and its metabolite, morphine following capillary zone electrophoretic separation is described. A carbon-disk electrode used as working electrode for two analytes exhibited a good response at 0.90 V (vs. Ag/AgCl). The optimal conditions of separation and detection were pH 8.4 phosphate buffer solution (0.06 mol l −1), 12 kV for the separation voltage, sample injection at 12 kV for 10 s. The linear ranges were from 3.1×10 −7 to 2.5×10 −5 mol l −1 for codeine and 1.9×10 −7 to 1.5×10 −5 mol l −1 for morphine with a correlation coefficient of 0.9996 and 0.9999, respectively. The detection limits for codeine and morphine were 1.6×10 −7 and 6.8×10 −8 mol l −1. The method developed was successfully applied to the determination of codeine and its metabolite, morphine in the human urine without any other sample pretreatment except filtration, and the results were satisfactory.

Use of β-cyclodextrin in the capillary zone electrophoretic separation of the components of clandestine heroin preparations

The present paper describes the methodological optimization and validation of a capillary zone electrophoresis method for the rapid determination of heroin, secondary products and additives present in clandestine heroin samples, by using 20 m M β-cyclodextrins in phosphate buffer, pH 3.23. Applied potential was 15 kV and separation temperature was 24°C; detection was by UV absorption at 200 nm wavelength. Heroin samples were first dissolved in CHCl 3–MeOH (96:4, v/v) and injected by pressure (0.5 p.s.i., 3 s; 1 p.s.i.=6894.76 Pa) after evaporation of the organic mixture and reconstitution in aqueous buffer. Under the described conditions, phenylethylamine (internal standard), morphine, monoacetylmorphine, heroin, acetylcodeine, papaverine, codeine and narcotine were baseline resolved in less than 10 min. The limit of detection was better than 1 μg/ml for each analyte. The study of the intra-day and day-to-day precision showed, in terms of migration times, RSDs ≤0.71% and, in terms of peak areas, RSDs ≤3.2%. Also, the evaluation of linearity and analytical accuracy of the method provided good results for all the analytes investigated, thus allowing its application to real cases of seized controlled drug preparations.

A capillary zone electrophoretic method for the study of formation of a covalent conjugate between microcystin LR and protein phosphatase 2A.

The study of conjugate formation between microcystin (MCYST)-LR and protein phosphatase (PP) 2A, which was isolated from bovine kidney and mouse brain, was achieved by using a highly efficient capillary zone electrophoretic (CZE) separation method. The MCYST-LR-PP2A conjugate (from bovine kidney) was resolved from its precursor after just 15 min of incubation. Moreover, the migration time, and, hence, the total analysis time, was less than 5 min. While the present findings of the time lag between conjugate formation and full inhibition are not novel, the CZE method does provide an alternative tool to HPLC with a higher separation efficiency to yield data for kinetic and mechanistic studies of the enzyme-toxin interaction. The CZE data reported here were found not to be adequately described by a first-order kinetic model. Moreover, the CZE method, which involves the use of a low ionic strength aqueous buffer, does not suffer from the drawback of the use of denaturing organic solvents such as those used in HPLC.

Novel three-dimensional capillary electrophoresis system for complex and trace analysis

A novel triple column capillary electrophoresis system is described. Design specifications facilitate method development and analyses by providing on-line, selective, pre-concentration and clean-up of both high (ml) and low (μl) volumes of specific analytes in two dimensions and separation via an additional third dimension. The system described additionally provides four distinct detection capabilities via both contactless conductivity and UV. The addition of a third dimension to the previously reported “coupled-column” systems, and further modifications made, has allowed for optimal identification, separation, and quantitation of micro-components in complex mixtures. The ability to perform both capillary zone electrophoretic and isotachophoretic separations on-line and in any combination enhances the scope for rapid analytical method development and analysis of complex or trace sample components.

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 4·10 −4 cm 2 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 p K a 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.

Capillary zone electrophoretic separation of neutral species of chloro- s-triazines in the presence of cationic surfactant monomers

Chloro- s-triazines are difficult to separate by capillary zone electrophoresis (CZE), due to their low p K a values. However, these analytes can be effectively separated by CZE in the presence of cationic surfactant monomers, such as tetradecylammonium bromide (TTAB) and dodecyltrimethylammonium bromide (DTAB). The separation mechanism based on a 1:1 binding of analytes to cationic surfactant monomers is proposed. The binding constants of chloro- s-triazines to cationic surfactant monomers are estimated. The results show that the strength of the interactions of these analytes with TTAB monomers is considerably strong, whereas that of the corresponding analyte with DTAB monomers is about 12- to 14-fold weaker. A linear correlation of binding constants with log P ow (the logarithm of the partition coefficient of analytes between 1-octanol and aqueous phases) indicates that the migration order of these chloro- s-triazines depends primarily on their hydrophobicity. Moreover, the skewed peaks of chloro- s-triazines observed may reveal the occurrence of adsolubilization of these analytes in the adsorbed cationic surfactant layer on the capillary surface.

Capillary zone electrophoretic separation and determination of imidazolic antifungal drugs

Capillary zone electrophoresis (CZE) was adapted to the simultaneous determination of a mixture of three imidazolic antifungal drugs. Separation was achieved by using a fused-silica capillary column with an acetic acid–Tris buffer at pH 5.18 and UV detection at 196 nm. Several electrophoretic parameters were investigated: pH and buffer concentration, applied voltage, temperature and injection conditions. The optimized CZE method was applied to the individual determination of ketoconazole, clotrimazole and econazole in pharmaceutical forms, after a previous single extraction step in methanol, with recoveries of 98.00, 99.96 and 99.58% respectively. The antifungal drugs can be determined at a concentration level lower than 1.0·10 −7 M.