毛细管电泳中获得稳定电渗流的毛细管预处理方法

报道了一种毛细管电泳分析中获得重复性分析结果的毛细管柱预处理方法。通过采用有机溶剂的碱性溶液对毛细管柱进行预冲洗,可得到内壁均一的能产生稳定电渗流的毪 细管术,实现了强极性有机化合物如硝基酚的毛细管区带电泳分析。     

Speciation of inorganic and methylated arsenic compounds by capillary zone electrophoresis with indirect UV detection. Application to the analysis of alkali extracts of As2S2 (realgar) and As2S3 (orpiment)

A capillary zone electrophoresis (CZE) method with indirect UV detection was developed to simultaneously separate inorganic and organic arsenic compounds including arsenite (iAsIII), arsenate (iAsV), monomethylarsonate and dimethylarsenic acid (DMAV). 2,6-Pyridinedicarboxylic acid (PDC) and n-hexadecyltrimethylammonium hydroxide (CTAOH) were selected to compose a background electrolyte (BGE), where PDC was used as chromophore and CTAOH functioned as electroosmotic flow (EOF) modifier to reduce/eliminate EOF. The choice of detection wavelength, the optimization of BGE pH, and effects of applied electric field strength and temperature on separation were further investigated. The limits of detection for the targeted analytes were between 0.19 and 0.23 ppm as molecule. Good linearity of more than three orders of magnitude was obtained. Repeatability of migration times and peaks areas were 0.8-1.7 and 3.4-6.9% R.S.D.; whereas reproducibility were 1.2-2.2 and 3.6-7.1% R.S.D., respectively. The established CZE method was then applied to analyze the alkali extracts of realgar (As2S2) and orpiment (As2S3). The main components in both alkali extracts were identified to be iAsIII and iAsV.     

Optimization for quantitative determination of four flavonoids in Epimedium by capillary zone electrophoresis coupled with diode array detection using central composite design

Herba Epimedii (family Berberidaceae), Ying-Yang-Huo in Chinese, is a famous Chinese herbal medicine. Flavonoids are thought to be the major active components in it. A capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of four flavonoids including icariin, epimedin A, epimedin B and epimedin C in Epimedium. The effects of the experimental variables on CZE had been optimized by using central composite design (CCD). The best separation of four flavonoids could be obtained using 50 mM borate buffer (pH 10.0) containing 22% acetontrile as modifier, while separation voltage was 15 kV and temperature was at 25 degrees C. The method developed is accurate, simple and reproducible, which could be used for quality control of Epimedium and its medical preparations.     

Evaluation of coelectroosmotic capillary zone electrophoresis for the rapid analysis of twelve aromatic sulphonate compounds

Summary Coelectroosmotic capillary zone electrophoresis (CZE) has been investigated as a means of rapid analysis of twelve aromatic sulphonate compounds. The main factors affecting reversal of electroosmotic flow (EOF)—type of osmotic modifier and concentration-were studied. Two types of osmotic modifier, an alkylammonium salt (cetyltrimethylammonium bromide, CTAB) and a cationic polyelectrolyte (hexadimentrine bromide, HDB) were investigated. The composition of the running buffers was optimized according to the characteristics of each osmotic modifier. A concentration of HDB as low as 0.0001% (w/v) was used successfully to provide a stable and reversed EOF.     

On-line sequential preconcentration of inorganic anions by anion-selective exhaustive injection and base-stacking in capillary zone electrophoresis

A sequential electrostacking method based on anion-selective exhaustive injection (ASEI) and base-stacking (BS) is presented for the preconcentration and determination of inorganic anions by capillary zone electrophoresis (CZE) in this paper. Tetradecyltrimethylammonium bromide as an electroosmotic flow (EOF) modifier was added into the buffer to suppress EOF of the capillary. Firstly, a water plug was hydrodynamically injected into the capillary. During ASEI under negative high voltage, the sample anions migrated quickly towards the boundary between the water plug and buffer in the capillary. Then an alkaline zone was injected electrokinetically to concentrate the anions further. With the sequential electrostacking method, the preconcentration factor of (0.8-1.3) x 10(5) was obtained compared with the conventionally electrokinetic injection and the relative standard deviation of peak area was 3.3-5.3% (n = 5). The detection limits of ASEI-BS-CZE for six inorganic anions were 6-14 ng/L. The proposed method has been adopted to analyze six anions in cigarette samples successfully.     

Capillary zone electrophoresis of albumin-depleted human serum using a linear polyacrylamide-coated capillary: separation of serum alpha-and beta-globulins into individual components

The separation of human serum globulins into individual components was investigated by capillary zone electrophoresis (CZE) using a linear polyacrylamide-coated capillary at pH 7.4. Prior to CZE analysis of globulin components present in serum, it was found that it was necessary to remove albumin. Preparation of albumin-depleted human serum with a HiTrap Blue column allowed the detection of alpha- and beta-globulin components as a series of peaks. Almost all the peaks, both narrow and broad, observed in CZE analysis could be assigned to six globulin components (alpha1-acid-glycoprotein, alpha1 -antitrypsin, haptoglobin, alpha2-macroglobulin, Gc-globulin, and transferrin) by using the technique of antibody-based indirect detection. The CZE results, obtained from serum preparations from three healthy adults and six patients, showed that the CZE system might be capable of detecting qualitative differences among individuals with regard to individual globulin 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 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.     

Fenofibrate and fenofibric acid analysis by capillary electrophoresis

A capillary electrophoresis method has been developed to measure fenofibrate in capsules based on micellar electrokinetic capillary chromatography with detection at 280 nm using a borate buffer containing sodium dodecyl sulfate (SDS). However, the metabolite of this drug (fenofibric acid) in serum and whole blood was analyzed by capillary zone electrophoresis (CZE) in a borate-carbonate buffer using acetonitrile stacking. The analysis is rapid, < 7 min with no interferences. Incubation of fenofibrate in whole blood caused hydrolysis of the ester bond with the release of fenofibric acid.     

Identification and determination of aesculin and aesculetin in ash barks by capillary zone electrophoresis

Summary A capillary zone electrophoresis method for identification and determination of aesculin and aesculetin has been established using borate-phosphate buffer containing 30% ethanol with on-column UV detection. A detailed investigation of the influence of changes in borate concentration, pH, applied voltage, temperature and organic modifier was then carried out. For both aesculin and aesculetin, a linear plot of migration time (MT) against borate concentration was obtained, and ln[measured peak area (MA)] and lnMT both gave linear plots against ln(applied voltage) with correlation coefficient r>0.999, which also resulted in a linear correlation between MA and MT (r≥0.9998) under varied voltage. Ethanol as organic modifier to the background electrolytes helped in separating aesculin and aesculetin from other components in ash barks. The reproducibility with relative standard deviation in MT and in normalized peak area(NA) and linearity based on NA against concentration were evaluated. Finally, the method was successfully applied to monitor the quality of different ash barks and to compare the effect of sample preparation on content of bioactive components in ash bark. Results indicate that CZE promises to be applicable to quality control of traditional Chinese medicines containing aesculin and aesculetin.     

Capillary zone electrophoresis with electroosmotic flow controlled by external radial electric field.

A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.     

Capillary electrophoresis of seed 2S albumins from Lupinus species

Two modes of capillary electrophoresis (CE)--free-solution capillary zone electrophoresis (CZE) and sodium dodecyl sulfate capillary electrophoresis (SDS-CE) using a non-gel sieving matrix--have been developed for comparative analysis of low-molecular-mass 2S albumin isoforms from lupins. The albumin fraction and 2S albumins were separated in uncoated fused-silica capillary by CZE with 0.02 M phosphate buffer, pH 7.3, containing the sodium salt of phytic acid. The use of phytic acid (0.025 M) as buffer modifier and ion-pairing agent improved migration reproducibility, peak shape and separation efficiency. The reduced 2S albumins were separated by SDS-CE using a high concentration (0.3-0.5 M) mixture of tris(hydroxymethyl)aminomethane and borate buffers in uncoated fused-silica capillary. Of the various polymers used as non-gel sieving matrix, SDS-CE with a 10% dextran solution was found to be suitable for separation of 2S albumin polypeptides with molecular masses of 4,000-7,000 and 8,000-11,000. The addition of glycerol or ethylene glycol to the SDS separating buffer improved the resolution of polypeptides. The examined Lupinus species showed species-specific CZE and SDS-CE migration profiles of the 2S albumins.     

Simultaneous determination of anthraquinones in Rhubarb by pressurized liquid extraction and capillary zone electrophoresis

Rhubarb, a well-known Chinese herbal medicine, is also used in Europe and other places of the world. Anthraquinones derivatives are thought to be the major active components. A pressurized liquid extraction (PLE) and capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of five anthraquinones including aloe-emodin, emodin, chrysophanol, physcion, and rhein in Rhubarb. The effects of the experimental variables on PLE and CZE have been optimized. The optimum conditions of PLE were: solvent, methanol; temperature, 140 degrees C; particle size, 0.13-0.2 mm; static extraction time, 5 min; pressure, 1500 psi; and one extraction. The best separation of the five anthraquinones could be obtained using 50 mM borate buffer (pH 8.2) containing 25% isopropyl alcohol and 25% acetontrile as modifier, while the separation voltage was 25 kV and the temperature was at 20 degrees C. The method developed is accurate, simple, and reproducible, and could be used for quality control of Rhubarb and its medical preparations.     

Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum

Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di-, mono-, and asialo-Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 microm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40 degrees C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo- to pentasialo-Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-Tf isoform related to the area of tetrasialo-Tf in 50 healthy donors (24 males and 26 females; aged 25-50 years) was 3.15 +/- 0.76% (mean +/- SD). The comparison between CDT values obtained by this CZE procedure and the "Axis-Shield %CDT" kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.     

A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.     

Electroosmotic flow changes due to interactions of background electrolyte counter-ions with polyethyleneimine coating in capillary zone electrophoresis of proteins

The properties and behavior of polyethyleneimine (PEI) covalently coated capillaries with respect to different background electrolytes used in capillary zone electrophoresis (CZE) are described. The coating stability and changes of inner surface charge in the capillary were followed by measurement of electroosmotic flow (EOF). Interest was focused mainly on conjugate bases of carboxylic acids as anionic background electrolyte components (acetate, citrate, malate, malonate, tartrate, and succinate). An interesting phenomenon was observed in PEI-coated capillaries: The direction (and the magnitude) of EOF depends on the composition of the background electrolyte and at a certain pH it can undergo reversible change. Ionic complex formation was suggested as a hypothesis to explain this behavior. With this knowledge, the PEI-coated capillary was used for the separation of basic proteins in the above-mentioned background electrolytes. A standard protein mixture of cytochrome c, ribonuclease A, and lysozyme at a concentration of 0.25 mg/mL each was chosen as model sample.     

Use of eosin as a fluorophore in capillary electrophoresis with laser detection.

Eosin has been used to generate the background signal for indirect fluorimetric detection of inorganic and organic ions, simultaneously separated by capillary zone electrophoresis (CZE). This reagent provides constant fluorescence over the pH range of 5-10 and is compatible with the excitation by an argon ion laser at 488 nm with emission at 520 nm. The use of esosine as fluorophore, H3BO3, and Na2B4O7 as electrolyte and diethylentriamine as modifier of the electroosmotic flow in CZE were optimised. The analytical potential of the studied buffer was tested on a group of 12 anions, used as model compounds. Both, hydrodynamic and electrokinetic injection mode were optimised. The detection limits determined by the last injection mode, were in the range 0.008-0.037 mg l(-1). By using this method, the quantitation of the common anions in tap and mineral water has been carried out successfully.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

Capillary electrophoretic determination of the component monosaccharides in hemicelluloses

This study describes the precolumn derivatization of carbohydrates with p-aminobenzoic acid and their efficient separation as borate complexes by means of capillary zone electrophoresis, using a capillary tube of fused silica containing 150 mmol/L borate buffer, pH 10.0, as carrier. On-column UV-monitoring at 285 nm allowed the detection of aldoses and uronic acids in the lower femtomole range. Reproducible quantification of carbohydrates was achieved at least in the concentration range of 0.1–10 mmol/L by the relative peak area method, using cinnamic acid as internal standard. The method was successfully applied to the determination of the monosaccharide composition of both commercially obtained xylans as well as of hemicelluloses obtained by hydrothermal degradation of biomass.     

Identification and determination of effective components in Euphrasia regelii by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method has been developed for simultaneous determination of eukovoside, cinnamic acid and ferulic acid in Euphrasia regelii for the first time. The electrophoresis buffer was 20 mmol/L sodium borate containing 10% (v/v) methanol (pH 8.50). The effects of concentration of borate and electrolyte pH on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients 0.9995-0.9998) between the peak area of each analyte and the concentration. The levels of analytes in the different parts of Euphrasia regelii were easily determined with recoveries ranging from 95.5 to 104.2%.     

Determination of adenosine deaminase activity in human erythrocytes by on-column capillary isotachophoresis-capillary zone electrophoresis in the presence of electroosmotic flow

Transient capillary isotachophoresis (CITP)-capillary zone electrophoresis (CZE) in presence of electroosmotic flow (EOF) was utilized for the measurement of adenosine deaminase activity in human erythrocytes. Phosphates, dominant anions of the sample matrix, were used as leading ions for transient isotachophoresis, and borates (0.3 M, pH 10) were used as terminating ions and background electrolyte for CZE. Final experimental conditions made it possible to inject 70% of the total capillary volume (1.45 microL) with the sample. Enzymatic conversion products (inosine and hypoxanthine), present in the sample in the low-micromolar range, were determined using optimized conditions. The limit of detection was 28 nM using UV detection at 202 nm. The presented data shows that CITP-CZE can be performed in uncoated capillaries in the presence of strong EOF.