Determination of phenolic constituents in citrus samples by on-line coupling of a flow system with capillary electrophoresis

Phenolic compounds extracted from different citrus were determined. Calibration, extraction, elution, and introduction into the sample vial was carried out automatically by a continuous flow system (CFS) coupled to capillary electrophoresis (CE) equipment via a programmable arm. The only manual operation was the centrifugation of the sample to remove the pulp. The supernatant solutions were introduced into the CFS-CE system. A C-18 minicolumn coupled into the CFS was used to perform cleanup of the samples. The analytes were eluted from the minicolumn using methanol. Quantitative analysis was carried out by the standard addition method. The method presented allows a fast, quantitative, and reproducible determination of six main phenolic compounds in citrus samples, with precision in the range of 3.0-6.5%, expressed as relative standard deviations.     

An Automated Hydrodynamically Mediated Technique for Preparation of Calibration Solutions via Capillary Electrophoresis System as a Promising Alternative to Manual Pipetting

In this paper, a novel procedure for preparing calibration solutions for capillary electrophoresis (CE)-based quantitative analysis is proposed. Our approach, named the automated hydrodynamically mediated technique (AHMT), uses a capillary and a pressure system to deliver the expected amount of working solution and diluent directly to a sample vial. As a result, calibration solutions are prepared automatically inside the CE instrument, without any or with minimal manual operation. Two different modes were tested: forward and reverse, differing in the direction of hydrodynamic flow. The calibration curves obtained for a model mixture of analytes using AHMT were thorough compared to the standard procedure based on manual pipetting. The results were consistent, though the volume of obtained calibration solutions and the potential risk of random errors were significantly minimized by AHMT. Its effectiveness was further enhanced by the application of SCIEX^® nanoVials, reducing the actual volume of calibration solutions down to 10 μL.     

Direct multiparametric determination of anions in soil samples by integrating on-line automated extraction/filtering with capillary electrophoresis

An integrated continuous flow-capillary electrophoresis for the determination of soluble anions in soil samples is presented. A filtration probe coupled with the flow system, which is located before the capillary electrophoresis instrument, was designed to simplify sample pretreatment and to perform clean-up of aqueous soil suspensions. Only the manual weighing of the samples is needed. The extraction process for soil samples was optimized. The clear filtrate containing the soluble anions from soil was then passed to the capillary electrophoresis equipment by a home-made programmable arm. Chloride, sulfate, nitrite and nitrate were determined in soil samples at μg g^–1 level and the results compared to manual reference methods. The precision expressed as relative standard deviation was in the range of ± 1.6 to ± 2.5% for the set of analytes determined. The procedure is up to 4 times faster than the competitive manual methods.     

Determination of pesticides in waters by automatic on-line solid-phase extraction-capillary electrophoresis

The separation of seven pesticides by micellar electrokinetic capillary chromatography in spiked water samples is described, allowing the analysis of pesticides mixtures down to a concentration of 50 microg l(-1) in less than 13 min. Calibration, pre-concentration, elution and injection into the sample vial was carried out automatically by a continuous flow system (CFS) coupled to a capillary electrophoresis system via a programmable arm. The whole system was electronically coupled by a micro-processor and completely controlled by a computer. A C18 solid-phase mini-column was used for the pre-concentration, allowing a 12-fold enrichment (as an average value) of the pesticides from fortified water samples. Under the optimal extraction conditions, recoveries between 90 and 114% for most of the pesticides were obtained.     

Direct multiparametric determination of anions in soil samples by integrating on-line automated extraction/filtering with capillary electrophoresis

An integrated continuous flow-capillary electrophoresis for the determination of soluble anions in soil samples is presented. A filtration probe coupled with the flow system, which is located before the capillary electrophoresis instrument, was designed to simplify sample pretreatment and to perform clean-up of aqueous soil suspensions. Only the manual weighing of the samples is needed. The extraction process for soil samples was optimized. The clear filtrate containing the soluble anions from soil was then passed to the capillary electrophoresis equipment by a home-made programmable arm. Chloride, sulfate, nitrite and nitrate were determined in soil samples at μg g^–1 level and the results compared to manual reference methods. The precision expressed as relative standard deviation was in the range of ± 1.6 to ± 2.5% for the set of analytes determined. The procedure is up to 4 times faster than the competitive manual methods. Received: 29 July 1997 / Revised: 10 September 1997 / Accepted: 13 September 1997     

The Development of a Sheathless Interface for Capillary Electrophoresis Electrospray Ionization Mass Spectrometry Using a Cellulose Acetate Cast Capillary

The fabrication of a novel sheathless interface for capillary electrophoresis–electrospray–mass spectrometry (CE–ESI–MS) is described. A programmable CO₂ laser was used to ablate small channels in the walls of a polyimide capillary near the terminus. Subsequent exposure of the channel region to a cellulose acetate solution followed by drying resulted in the formation of an electrically conductive semi-permeable membrane. Application of an appropriate voltage to the reservoir resulted in the simultaneous establishment of an electrical connection for CE and ESI. Interface viability was demonstrated by conducting a CE separation of a peptide mixture, with detection accomplished via positive ion mode ESI–MS. For the peptide Val-Tyr-Val, a limit of detection of 0.1 femtomole (S/N 3) was achieved using single reaction monitoring. Attributes of the interface include structural robustness, ease of fabrication, minimal interface dead volume, and the ability to alter post-separation analyte ionization status by use of appropriate buffers in the interface reservoir.     

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.     

High-performance liquid chromatographic,capillary electrophoretic and capillary electrophoretic-electrospray ionisation mass spectrometric analysis of selected alkaloid groups

Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described. The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures. The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids. The described buffer system is also applicable in the combination of CE with electrospray ionisation mass spectrometry. This analytical technique allowed the separation and identification of components of standard indole alkaloid mixture as well as crude extracts of R. serpentina roots, R. serpentina cell suspension cultures and cortex of Aspidosperma quebracho-blanco. The influence of buffer composition and analyte structures on separation is discussed.     

Capillary electrophoretic determination of thiosulfate,sulfide and sulfite using in-capillary derivatization with iodine

A new capillary electrophoresis (CE) method was developed for the rapid, simple and selective determination of thiosulfate, sulfide and sulfite species. The proposed method is based on the in-capillary derivatization of separated sulfur anions by mixing their zones with the iodine zone during the electrophoretic migration and direct UV detection of iodide formed. The optimal conditions for the separation and derivatization reaction were established by varying electrolyte pH, electrolyte counter-ion, concentration of iodine, and applied voltage. The optimized separations were carried out in 20 mmol/L Tris-chloride electrolyte (pH 8.5) using direct UV detection at 214 nm. All three sulfur species were well resolved in less than 4 min. The method gives repeatability comparable or even better than this obtained for sulfur anions using standard CE technique. The proposed CE system was applied to the monitoring of sulfur anions in spent fixing solutions during the electrolytic oxidation.     

The importance of capillary electrophoresis,capillary electrochromatography,and ion chromatography in separations of inorganic ions

The importance of capillary electrophoresis (CE), capillary electrochromatography (CEC), and ion chromatography (IC) in inorganic ion analyses is outlined. Methods for improving the reliability of the CE measurements are briefly described. Selectivity optimization in CE analyses of inorganic cations and anions is discussed. Using the Peakmaster program, CE system peaks (system zones, eigenmobilites) and some important CE parameters, such as effective mobilities, electromigration dispersion, indirect UV, and direct conductivity signals, are predicted and compared with experimental analyses.     

Use of phosphobetaine-type zwitterionic surfactant for the determination of alkali and alkaline earth metal ions and ammonium ion in human saliva by capillary electrophoresis

With conventional capillary electrophoresis (CE), it was difficult to directly analyze samples containing proteins as a result of the irreversible adsorption of proteins onto the inner surface of the capillary column. This difficulty, however, was completely overcome by adding N-dodecylphosphocholine (DPC, a phosphobetaine-type zwitterionic surfactant) to the background electrolyte (BGE). DPC made two essential contributions to the determination of common inorganic cations in the protein-containing samples: protein adsorption onto the capillary walls was completely avoided, and the resolution of the analyte cations was essentially improved. The optimal BGE for analysis of biological samples was found to be 5 mM DPC, 5 mM copper(II) acetate/10 mM ethylenediamine (pH 8). The detection limits (signal-to-noise ratio =3 and UV at 215 nm) of sodium, potassium, calcium, magnesium, and ammonium ions were 25, 31, 24, 45, and 60 micro M, respectively. These five species of the common inorganic cations in human saliva samples were detected successfully within 2 min by the proposed system with direct sample injection.     

Coupling of biological sample handling and capillary electrophoresis.

The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) requires efficient sample preparation (i.e., concentration and clean-up) procedures to remove interfering solutes (endogenous/exogenous and/or low-/high-molecular-mass), (in)organic salts and particulate matter. The sample preparation modules can be coupled with CE either off-line (manual), at-line (robotic interface), on-line (coupling via a transfer line) or in-line (complete integration between sample preparation and separation system). Sample preparation systems reported in the literature are based on chromatographic, electrophoretic or membrane-based procedures. The combination of automated sample preparation and CE is especially useful if complex samples have to be analyzed and helps to improve both selectivity and sensitivity. In this review, the different modes of solid-phase (micro-) extraction will be discussed and an overview of the potential of chromatographic, electrophoretic (e.g., isotachophoresis, sample stacking) and membrane-based procedures will be given.     

Identification of kappa and lambda chains of the major immunoglobulin G subclasses by capillary zone electrophoresis

Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains. Various analytical conditions and CE modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC) have been thoroughly studied. CZE was found to be the most convenient way to separate IgG subclasses. Three of the human IgG subclasses were resolved using uncoated fused-silica and 50 mM phosphate, pH = 9.3, as operating buffer at 20 kV and detection at 214 nm. IgG1kappa was completely separated from IgG2kappa and IgG3kappa, whereas IgG2kappa co-migrated with IgG4kappa, which is the minor IgG subclass. Under the same conditions IgG4lambda was completely separated from IgG1lambda, IgG2lambda and IgG3lambda, enabling the identification of the various lambda chains. The developed CE method is rapid and can be applied to the identification of the major immunoglobulin G subclasses in respect to their variable kappa and lambda chains.     

Separation of the positional isomers of phthalic acids in hydroorganic solvents using capillary electrophoresis

The positional isomers of phthalic acids (ortho-, meta-, and para-) and benzoic acid could be completely separated by capillary electrophoresis (CE). A simple CE method employing direct detection in mixed methanol/water buffers is presented. The effect of the electrolyte buffer system, including pH, buffer concentration, and organic solvent on the electrophoretic mobility of the analytes, is investigated. The electroosmotic flow is reversed using cationic surfactant and cetyltrimethylammonium bromide so that anions are separated under the co-EOF mode. The resolution of the analytes and selectivity could be improved by the adjustment of the methanol content. Ion association with the surfactant in methanol/water buffer is discussed. The validity of the method in terms of sensitivity, reproducibility, and linearity is also reported. The text was submitted by the authors in English.     

Analysis of serotonin in brain microdialysates using capillary electrophoresis and native laser-induced fluorescence detection

Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.     

Spherical molecularly imprinted polymer particles: a promising tool for molecular recognition in capillary electrokinetic separations

Spherical molecularly imprinted polymer particles obtained via precipitation polymerization, were introduced as a pseudostationary phase in capillary electrophoresis (CE) to study molecular recognition. Analyses were performed via a partial filling technique using (+)-ephedrine-imprinted microspheres (100-200 nm) which were polymerized from methacrylic acid and 1,1,1-Tris(hydroxymethyl)propanetrimethacrylate using acetonitrile as the solvent. The influence of pH and the modifier content on the separation was investigated. A 0.1% w/v suspension in an aqueous 10 mM phosphate buffer (pH 2.5 with 40% acetonitrile) was hydrodynamically injected into the CE system (80% of the effective capillary length) and led to full baseline separation of racemic ephedrine within 10 min.     

Coupling of a large-size capillary column with an electrospray mass spectrometer. A reliable and sensitive sheath flow capillary electrophoresis-mass spectrometry interface

The concept of interfacing a large-size column for capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS) for robust and automatic CE-MS operation is reported. Both standard ionspray interface and microionspray interface have been modified to operate in a sheath flow pattern to overcome the common stability problem in CE-MS coupling. To make the interface sensitive, a step-down stainless steel tube with smaller inner diameter and tapered tip was incorporated onto a larger tube embracing the CE column via cold soldering. The devices were evaluated for quantitative analysis of nucleotides at femtomole level and stable analytical performance in peptide profiling.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine

A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed.     

Simultaneous analysis of plural samples in a CE-CL detector possessing micro-space area for reaction/detection.

Plural samples were simultaneously analyzed in a capillary electrophoresis (CE)-chemiluminescence (CL) detector system, taking advantage of a micro-space area for reaction/detection at the tip of the capillary. The CL reaction of 1,10-phenanthroline and hydrogen peroxide was adopted and a Cu(II) sample was used as a model. Three different length capillaries were inserted into a flow-type CL detection cell made of a Teflon tube. Three samples migrated in the corresponding capillaries at the same time and mixed with the CL reagent at the tip of capillary to produce CL. The simultaneous analysis of plural samples in the present system supported the possibility that a real sample could be determined more easily, rapidly, and precisely with the calibration curve.