Potential Of Capillary Zone Electrophoresis Research Articles

A study on the alkaline hydrolysis of isatin-β-thiosemicarbazone by capillary electrophoresis with enhanced sample loadability

An analytical potential of capillary zone electrophoresis (CZE) with enhanced sample loadability (a 200nL injection volume) in determination of alkaline hydrolysis products of isatin-β-thiosemicarbazone (IBT), a compound with important biological activity, has been studied. The CZE separation conditions for a complete resolution of transformation products, i.e. 2-aminophenylglyoxalate, 2-(2-aminophenyl)-2-semicarbazonoethane, anthranilate and E-Z geometric isomers of 2-(2-aminophenyl)-2-thiosemicarbazonoethane, have been optimized. CZE separations with UV detection at 240nm were performed using glycine running buffer at high pH (9.2) and containing an uncharged β-cyclodextrin as a complexing agent. High sensitivity (with detection limits ranging from 0.1 to 1.2μM), good repeatability (RSD of migration times less than 0.4% and 0.4–3.4% RSD of peak areas) and linearity over two orders of magnitude were achieved for the compounds studied. The employed CZE method, characterized by simple sample handling (only dilution step needed) and total analysis time of less than 15min, has been applied successfully to time monitoring of the transformation of IBT in alkaline media. Under optimized CZE conditions, the effect of pH of reaction media, implemented by different concentration of NaOH (0.1–100mM), on the course of the alkaline hydrolysis of IBT was studied in this respect, as well.

Comparison of potential of capillary zone electrophoresis and Malvern's improved laser Doppler velocimetry for characterisation of silica nanomaterials in aqueous media

Two silica certified reference nanomaterials containing 50- and 100-nm particles were used for comparing electrophoretic mobility measurements by capillary zone electrophoresis and Malvern’s improved laser Doppler velocimetry under the same experimental conditions. Phosphate buffer solutions with concentrations of 2.5 and 5.0 mM, at pH 8.5 and 6.2 were used for preparation of dispersions. These dispersions were stable sufficiently to perform measurements. The values of absolute electrophoretic mobility determined by a Malvern’s improved laser Doppler velocimetry were found to be lower than those determined by a capillary zone electrophoresis. In addition, the results of size measurements by dynamic light scattering and scanning electronic microscopy were compared. The reasons for differences in measured values are discussed.

Potential of capillary zone electrophoresis for estimation of humate acid-base properties

Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA s) and chernozem (HA ch) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA s fractions obtained by salting-out with ammonium sulfate at a saturation of 0–40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of “humic hump” was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5–7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA s fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA s with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the p K a of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 × 10 −5 cm 2 V −1 s −1 can be used for approximate estimation of HA isoelectric point. The data were discussed and agreement with the random coil structural model has been shown.

Caracterização de ácidos húmicos de diferentes origens por eletroforese capilar de zona

O objetivo deste trabalho foi caracterizar ácidos húmicos (HA) de diferentes origens por eletroforese capilar de zona (CZE) e avaliar a influência da concentração do tampão borato nesta caracterização. Os resultados mostraram que HA de diferentes origens podem ser rapidamente caracterizados por CZE. Por esta técnica, a caracterização de HA é devido a diferenças na razão carga/tamanho e das propriedades dos componentes individuais, conseqüência de suas diferentes origens.

Direct determination of phosphate esters in concentrated nitrate media by capillary zone electrophoresis

The potential of capillary zone electrophoresis (CZE) for the determination of dibutyl phosphate (DBP) and monobutyl phosphate (MBP), two degradation products of the tributyl phosphate extractant used in the nuclear fuel reprocessing industry, was evaluated. Analysis conditions were optimised, taking particularly into account that many determinations had to be performed in concentrated aqueous nitrate or nitric acid solutions. Separations were therefore carried out using the counter-electroosmotic mode with cathodic detection in a pH 8.3 electrolyte containing a suitably selected chromophore, salicylate, to ensure the indirect UV detection of the analytes. Various aspects of the method, including its sensitivity, working range, repeatability, and rapidity, were examined. Quantification of both phosphate esters was achieved in less than 3 min at concentrations ranging from 2×10 −6 to 10 −3 mol l −1 in samples containing no macro-component. The lower end of this range increased to 5×10 −6 mol l −1 for MBP and 1.5×10 −5 mol l −1 for DBP in samples containing 5×10 −2 mol l −1 of sodium nitrate, thus enabling their determination in solutions containing nitrate or nitric acid at concentrations up to, respectively, 10 000 and 3000 times higher than the target analyte concentration. This simple, fast and reliable method is routinely applicable to aqueous samples with no other preliminary treatment than a proper dilution; analysis was also performed in organic matrices after a prior extraction. The method was validated by an excellent correlation with the standard DBP analysis technique, gas chromatography (GC). In order to develop appropriate chemical treatments to destroy these compounds, the method was applied to the monitoring of DBP and MBP degradation by hydrogen peroxide in 1 mol l −1 nitric acid solutions.

Potential of capillary electrophoresis for the monitoring of the stability of placental alkaline phosphatase

Alkaline phosphatase (AP) is a potential therapeutic agent in the treatment of sepsis. In this paper the potential of capillary zone electrophoresis (CZE) for the monitoring of the degradation of placental alkaline phosphatase (PLAP) was investigated. To induce degradation PLAP samples were exposed to high temperatures, low and high pH and freeze-drying. The samples were then analyzed by CZE and enzymatic activity assay. Upon exposure to temperatures above 65 °C, PLAP lost its activity exponentially over time, while CZE revealed both a linear decrease of the area of the main peak and a rise of degradation products. At acidic pH the enzyme appeared to lose its activity. CZE revealed a decrease of the area of the main peak, but no degradation products could be detected. At pH 12 the enzymatic activity and the area of the main peak both decreased linearly over time and, in addition, formation of degradation products could be detected by CZE. Activity and CZE profile of PLAP remained unchanged upon freeze-drying in the presence of inulin. Prolonged storage of freeze-dried samples at room temperature caused a slight decrease of enzymatic activity, while the potential formation of oligomers was revealed by CZE analysis. The examples in this study show that, in combination with activity assays, CZE can provide useful complementary information, especially on the status of the protein and the presence of degradation products.

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 m M sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C 18 cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l −1 and detection limits were between 1.1 and 2.4 mg l −1.

Separation and simultaneous determination of quinolone antibiotics by capillary zone electrophoresis

The potential of capillary zone electrophoresis has been investigated for the separation and quantitative determination of some quinolone antibiotics. The influence of different conditions, such as the nature and concentration of the electrophoretic electrolyte, on migration time, peak symmetry, efficiency and resolution was studied. A buffer consisting of 100mm HEPES adjusted to pH 8.5 containing 10% (v/v) acetonitrile was found to furnish a very efficient and stable electrophoretic system for the separation of exoxacin, ciprofloxacin, ofloxacin, oxolinic acid, nalidixic acid and pipemedic acid. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.25–40 μg mL−1; detection limits were approximately 0.25 ng mL−1. It was demonstrated that the method can be used for the simultaneous determination of these six antibiotics in serum and urine samples.

Capillary electrophoresis in the analysis of humic substances: Facts and artifacts

Humic substances, extracted as mixtures from soil and surface waters according to their solubility in acids and bases, are relatively high-molecular-mass polyelectrolytes containing aromatic, aliphatic and heterocyclic subunits. The degree of ionization of their phenolic and carboxylic groups is governed by the capillary electrophoresis (CE) buffer pH. In CE, fulvic acids exhibit a consistent and characteristic set of sharp peaks (phenolic acids), extending from a humic “hump” whose average electrophoretic mobility (AEM) depends on humic structure and buffer composition; humic acids give only the “hump”. Special attention must be given to the interpretation of CE electropherograms when fingerprinting humic substances with borate buffers because observed peaks do not necessarily indicate distinct humic fractions, but may be artifacts caused by the interaction of borate ions with 1,2- and 1,3-diols present in the humic mixtures. Depending on the molarity of borate ions in the separation buffer, humic acids exhibit electropherograms with sharp peaks extending from the “humic hump” and corresponding to borate complexes. The potential of capillary zone electrophoresis for the comparison of electropherogram patterns is illustrated for the Suwannee River reference humic substances extracted according to the recommendations of the IHSS compared with a fraction of the same source concentrated with the more recent reverse osmosis (RO) technique. The Suwannee River RO fraction behaved like the extracted Suwannee River humic acid fraction under these experimental conditions.

Capillary zone electrophoretic determination of C 2C 18 linear saturated free fatty acids with indirect absorbance detection

The potential of capillary zone electrophoresis for the separation of linear saturated fatty acids in their free form was evaluated. With increasing chain length, difficulties arise from decreasing analyte solubility in aqueous media, increasing occurrence of analyte aggregation and decreasing separation selectivity between successive homologues. A solution was to use electrolytes containing cyclodextrins (CDs) and methanol. The separation of C 2C 14 fatty acid homologues differing in only one carbon atom was achieved in less than 10 min using a purely aqueous electrolyte with trimethyl-β-CD as an additive. The separation of C 7C 18 homologues was completed in under 20 min with electrolytes containing up to 60% methanol, in addition to the aforementioned CD. In the presence of the CD, analyte solubility is enhanced through the inclusion of the alkyl chain of the acid in the CD cavity, while separation selectivity is improved because the stability constants of the inclusion complexes increase with increasing chain length of the acid. The lack of a suitable chromophore moiety was circumvented through the optimization of indirect absorbance detection conditions. p-Anisate was selected as the chromogenic species. The minimum detectable concentrations are of the order of (1–2)·10 −6 mol l −1 (0.2–0.5 ppm) and detection linearity was established over at least three orders of magnitude of concentration. The quantitative analysis of a coco oil extract sample is presented, showing results almost identical with those obtained by gas chromatography. Owing to the close values for response factors resulting from the indirect detection principles, a rapid percentage composition can be obtained by corrected peak area normalization.

Determination of inositol phosphates in fermentation broth using capillary zone electrophoresis with indirect UV detection

The potential of capillary zone electrophoresis for the fast monitoring of a fermentation process in which inositol phosphates are enzymatically hydrolyzed has been investigated. The developed analysis consists of capillary zone electrophoresis combined with indirect UV detection, using 1-naphthol-3,6-disulfonic acid as the chromophore. The total analysis of all six inositol phosphates covering a concentration range of 0–500 μ M takes only 13 min.

Separation of metallothionein isoforms by capillary zone electrophoresis

The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 μmI.D. × ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 m M Tris-HCl buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 m M Tris- HCl, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 μg/ml −1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1–10 μg MT ml −1. The successful use of two solvent extraction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.