Capillary electrophoresis and capillary flow injection analysis with electrochemical detection

Measurements by capillary flow injection analysis (CFIA) and capillary electrophoresis (CE) in conjunction with electrochemical detection are described. The detection is based on an end-column electrode arrangement. Several novel electrodes, such as a spherical gold electrode and a dual-microdisk electrode, are presented and characterized regarding their analytical utility. In order to improve the selectivity of CFIA, dual-electrode and multiple-pulse detection are studied using couples of cyanometallates or metallocenes. Capillary electrophoretic experiments with amperometric detection are performed using 50 m i.d. capillaries without any electrical-field decoupler. The practicality and analytical characteristics of this detection strategy are illustrated for the separation of serotonin and some biological precursors and metabolites of neurotransmitter substances.     

Rapid inorganic ion analysis using quantitative microchip capillary electrophoresis

Rapid quantitative microchip capillary electrophoresis (CE) for online monitoring of drinking water enabling inorganic ion separation in less than 15 s is presented. Comparing cationic and anionic standards at different concentrations the analysis of cationic species resulted in non-linear calibration curves. We interpret this effect as a variation in the volume of the injected sample plug caused by changes of the electroosmotic flow (EOF) due to the strong interaction of bivalent cations with the glass surface. This explanation is supported by the observation of severe peak tailing. Conducting microchip CE analysis in a glass microchannel, optimized conditions are received for the cationic species K+, Na+, Ca2+, Mg2+ using a background electrolyte consisting of 30 mmol/L histidine and 2-(N-morpholino)ethanesulfonic acid, containing 0.5 mmol/L potassium chloride to reduce surface interaction and 4 mmol/L tartaric acid as a complexing agent resulting in a pH-value of 5.8. Applying reversed EOF co-migration for the anionic species Cl-, SO42- and HCO3- optimized separation occurs in a background electrolyte consisting of 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mmol/L HEPES sodium salt, containing 0.05 mmol/L CTAB (cetyltrimethylammonium bromide) resulting in a pH-value of 7.5. The detection limits are 20 micromol/L for the monovalent cationic and anionic species and 10 micromol/L for the divalent species. These values make the method very suitable for many applications including the analysis of abundant ions in tap water as demonstrated in this paper.     

Amperometric detection for capillary flow injection analysis

Ultrasmall-volume measurements of oxidizable compounds have been accomplished by coupling a capillary flow injection system with amperometric detection. Remarkably low (femtomole) mass detection limits result from the combination of nanoliter sample volume and the inherent sensitivity of the wall-jet detector. A substantial economy of reagent consumption and disposal accrues from the operation of the nl/min flow regime. Variables influencing the physical dispersion in the capillary flow injection system, including capillary length, sample volume or flow rate, are explored and optimized.     

High performance fourier transform ion cyclotron resonance mass spectrometric detection for capillary electrophoresis

We describe the current state of the on-line combination of capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS), and discuss aspects of the FTICR technique relevant to its use as a detection scheme for on-line separations. Aspects including sensitivity, mass resolution, duty cycle, and tandem mass spectrometric capabilities are discussed in the context of online separations with examples from the authors' laboratory.     

Determination of proteins by flow injection analysis coupled with the Rayleigh light scattering technique

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of protein concentrations. This method is based on the weak intensity of RLS of p-nitrohenzene-azo-3,6 disulfo-1-amino-8-naphthol-7-azo-benzene disodium salt (Amide Black-10B) which can be enhanced by addition of protein in weakly acidic solution. It has proved that the application of this method to quantify the proteins by using human serum albumin was available in real samples. In addition, this method is very sensitive (the determination limits are 0.11 μg/mL for human serum albumen (HSA) and 0.85 μg/mL for bovine serum albumen, BSA), simple, rapid and tolerance of most interfering substances. The FIA-RLS method was more stabile than the general RLS method and the average R.S.D. value of FIA-RLS less than general RLS. The effects of different interfering substances will be also examined. The amount of proteins in human serum sample was determined and the maximum relative error was no more than 3.00% as well as the recovery was between 94.9 and 105.9%.     

Advances in detection of inorganic ions in capillary electrophoresis

Recent advances in capillary electrophoresis methodology to improve the detection sensitivity for inorganic ions are briefly reviewed. Specifically discussed are current developments of nonabsorbance-based CE detectors and on-capillary enrichment techniques as well as some possible future perspectives.     

Electrokinetic injection in capillary electrophoresis and its application to the analysis of inorganic compounds

In capillary electrophoresis, electrokinetic injection is a highly controversial sampling technique. It is a simple mode of sample introduction which is suitable for on-line preconcentration of the analytes, but its precision and accuracy are more strongly affected by experimental conditions compared to hydrodynamic injection. In the first part of this paper the features of electrokinetic and hydrodynamic injections are compared, followed by a detailed discussion on the different biases of electrokinetic injection and on how to reduce them. Finally, applications of the electrokinetic injection are reviewed with special emphasis on the analysis of inorganic compounds.     

An automated method of on-line extraction coupled with flow injection and capillary electrophoresis for phytochemical analysis

In this study, an automated system for phytochemical analysis was successfully fabricated for the first time in our laboratory. The system included on-line decocting, filtering, cooling, sample introducing, separation, and detection, which greatly simplified the sample preparation and shortened the analysis time. Samples from the decoction extract were drawn every 5 min through an on-line filter and a condenser pipe to the sample loop from which 20-μL samples were injected into the running buffer and transported into a split-flow interface coupling the flow injection and capillary electrophoresis systems. The separation of glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8) and +10 kV voltage. Calibration curves showed good linearity with correlation coefficients (R) more than 0.9991. The intra-day repeatabilities (n = 5, expressed as relative standard deviation) of the proposed system, obtained using GTA and GA standards, were 1.1% and 0.8% for migration time and 0.7% and 0.9% for peak area, respectively. The mean recoveries of GTA and GA in the off-line extract of Glycyrrhiza uralensis Fisch root were better than 99.0%. The limits of detection (signal-to-noise ratio = 3) of the proposed method were 6.2 μg/mL and 6.9 μg/mL for GTA and GA, respectively. The dynamic changes of GTA and GA on the decoction time were obtained during the on-line decoction process of Glycyrrhiza uralensis Fisch root.     

Comparison of ion chromatography and capillary electrophoresis for the determination of inorganic ions

The techniques of ion chromatography and capillary electrophoresis are compared as analytical methods for the determination of inorganic anions and cations. Comparison is made in the areas of stage of development, separation efficiency, separation selectivity, analytical performance parameters, method development procedures, applications, strenghts and weaknesses, and future directions. It is shown that the two techniques are complementary rather than competitive, especially with regard to their separation selectivities and the type of applications to which they are most suited.     

Determination of verapamil hydrochloride with 12-tungstophosphoric acid by resonance Rayleigh scattering method coupled to flow injection system

A flow injection analysis (FIA) method coupled to resonance Rayleigh scattering (RRS) detection for the determination of verapamil hydrochloride (VP) was proposed. In pH 1.0 acidic medium, 12-tungstophosphoric acid (TP) reacted with VP to form an ion-association complex, which resulted in a significant enhancement of RRS intensity. The maximum scattering peak was located at 293 nm. RRS intensity was proportional to the concentration of VP in the range of 0.017-13.0 μg mL⁻¹, and the detection limit (3σ) was 5.1 ng mL⁻¹. The proposed method exhibited satisfactory reproducibility with a relative standard derivation (R.S.D.) of 2.1% for 11 successive determinations of 5.0 μg mL⁻¹ VP. Therefore, a novel method for the determination of VP by FIA-RRS was developed. The optimum reaction conditions and the parameters of the FIA operation such as flow rate, injection volume, reactor length, and so on had been optimized in this paper. The present method had been applied to the determination of VP in serum samples and pharmaceuticals with satisfactory results. The maximal sample throughput in the optimized system was 80 h⁻¹.     

Semi-permanent surfactant coatings for inorganic anion analysis in capillary electrophoresis

Capillary electrophoretic separations of inorganic anions are performed using a capillary coated with a mixture of the cationic surfactant didodecyldimethylammonium bromide (DDAB) and the zwitterionic surfactant 1,2-dilauroyl-sn-phosphatidylcholine (DLPC). These double-chained surfactants form semi-permanent coatings on the capillary wall, which allows the excess surfactant to be removed from the buffer prior to separation. Interactions between surfactant aggregates in the buffer and analyte anions are thus eliminated. The electroosmotic flow (EOF) can be altered from fully reversed (100% DDAB) to near zero (100% DLPC) using different ratios of DDAB and DLPC. Controlling the EOF allows for improved resolution of the anions while maintaining a rapid, co-EOF separation, free from analyte-surfactant additive interactions.     

Automated two-dimensional separation flow system with electrochemical preconcentration, stripping, capillary electrophoresis and contactless conductivity detection for trace metal ion analysis

This paper describes the automation of a fully electrochemical system for preconcentration, cleanup, separation and detection, comprising the hyphenation of a thin layer electrochemical flow cell with CE coupled with contactless conductivity detection (CE-C?D). Traces of heavy metal ions were extracted from the pulsed-flowing sample and accumulated on a glassy carbon working electrode by electroreduction for some minutes. Anodic stripping of the accumulated metals was synchronized with hydrodynamic injection into the capillary. The effect of the angle of the slant polished tip of the CE capillary and its orientation against the working electrode in the electrochemical preconcentration (EPC) flow cell and of the accumulation time were studied, aiming at maximum CE-C?D signal enhancement. After 6 min of EPC, enhancement factors close to 50 times were obtained for thallium, lead, cadmium and copper ions, and about 16 for zinc ions. Limits of detection below 25 nmol/L were estimated for all target analytes but zinc. A second separation dimension was added to the CE separation capabilities by staircase scanning of the potentiostatic deposition and/or stripping potentials of metal ions, as implemented with the EPC-CE-C?D flow system. A matrix exchange between the deposition and stripping steps, highly valuable for sample cleanup, can be straightforwardly programmed with the multi-pumping flow management system. The automated simultaneous determination of the traces of five accumulable heavy metals together with four non-accumulated alkaline and alkaline earth metals in a single run was demonstrated, to highlight the potentiality of the system.     

Fluorescence detection with an immersed flow cell in capillary electrophoresis

An immersed flowcell has been developed for fluorescence detection in capillary electrophoresis (CE) by modifying an ordinary HPLC spectrofluorimetric detector flowcell. The minimum detectability obtained for riboflavine was about 4 fmol. The use of the flowcell to measure a fluorescence spectrum by the stopped-flow method of shutting down the DC high voltage during a wavelength scan, has also been achieved successfully.     

共振瑞利散射光谱法测定异烟肼

基于在PH=1.5的CL缓冲溶液中异烟肼的加入使曙红Y的共振瑞利散射信号增强,建立了一种异烟肼测定方法.异烟肼浓度在0.02～4.2μg/mL范围内与体系散射强度的增强呈线性关系,检出限为0.01851μg/mL.该方法用于异烟肼片剂的测定,结果满意.     

Comparison between capillary electrophoresis and ion-chromatography for analysis of inorganic anions

Separation and detection of some selected inorganic anions with capillary electrophoresis are shown. The anions are separated in microbore capillaries (25 m* 0.2 m) and detected with a UV-detector. Results are compared with the method of ion exchange chromatography. In consideration of the most important physical and chemical parameters an easy kind of computer simulation for such electropherograms was developed.To get optimal results of separation and UV-detection in capillary electrophoresis some parameters of the device HPE 100, i.e. loading time in the electrokinetic sample injection mode and the running voltage are varied. The behaviour of absorption in the UV region of the chosen anions as well as the influence of pH values in retention behaviour are investigated. There is a simple way to calculate the electrophoretic mobilities from known retention times. Approximate limits of detection for all anions and for each technique are given.     

End-column electrochemical detection for inorganic and organic species in high-voltage capillary electrophoresis

The electronics and construction for an end-column ultramicroelectrode (3–10 μm) detection system that permits the use of medium-sized capillaries (25 μm I.D.) without appreciable effects from the potential field at the end of the capillary. Normal peak-to-peak noise over 10 s was 0.01–0.1 pA. The background noise observed for a 200 × μm carbon-fiber electrode placed either 180 μm within a 25-μm capillary or at a point 500 μm away from the capillary was essentially the same. A study of detector response as a function of the position of the electrode has shown that accurate location of the electrode is important for sensitive and reproducible detection. These studies also showed that differences between the density of the electrolyte existing the capillary and the electrolyte in the detection cell could cause anomalous electrode response depending on the location of the electrode relative to the end of the capillary. Application of a carbon fiber or an Hg film electrode gave detection limits (twice the peak-to-peak noise over 10 s) of 2 · 10⁻⁸ mol/l for Pb²⁺, 1 · 10^{− 5} mol/l for NO₂⁻ and 5 · 10⁻¹⁰ mol/l for catechol.     

Electroosmotic flow reversal for the determination of inorganic anions by capillary electrophoresis with methanol-water buffers

Manipulation of the electroosmotic flow (EOF) is essential for achieving optimized separations of small anions by capillary electrophoresis (CE). In this work, efficient suppression or reversal of EOF is achieved upon addition of small amounts of the cationic surfactants, cetyltrimethylammonium bromide (CTAB) or didodecyldimethylammonium bromide (DDAB) to the electrophoretic buffer. Highly stable and reversed EOF are achieved using the surfactants in the presence of up to 50% MeOH. In aqueous and low methanol containing solutions (up to 30%, v/v) surface aggregation of the surfactants at the capillary wall occurs at a concentration below the critical micelle concentration (CMC). The impact of MeOH on reversed EOF is predominantly a function of the diminished zeta potential of the silica, and to a lesser extent on the CMC in the bulk solution of the surfactant. Fast baseline separation and selectivity changes for small inorganic anions are observed when mixed aqueous-organic buffers are employed. Changes in EOF, micellar properties of the surfactant and selectivity for inorganic anions upon addition of various percent of methanol are also discussed.     

The determination of alendronate ion and certain inorganic ions using capillary electrophoresis

A procedure for simultaneous determination of alendronate, chloride, phosphite, phosphate, and methylsulphonate anions is proposed. These ions are the main admixtures in synthetic alendronic acid, and their content should be controlled when producing pharmaceutical preparations of alendronic acid. The developed method makes it possible to determine all the indicated ions over a period of 4 min. Excellent efficiency (200000–500000 theoretical plates) makes it possible to determine ions even with a high content of one of the components. The detection limits are 0.4−2.3 mg/l. The developed method has been successfully applied for the analysis of methanesulfonic acid regenerated from the reaction mixture.     

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.     

Application of hot platinum microelectrodes for determination of flavonoids in flow injection analysis and capillary electrophoresis

The determination of quercetin and rutin by flow injection analysis (FIA) and capillary electrophoresis (CE) using electrochemical detection was described. These flavonoids were determined at normal (unheated) and hot platinum microelectrodes using cyclic voltammetry. When quercetin or rutin is reaching the platinum electrode, a change of the current in the region of the platinum oxide formation is observed. Integration of the current changes in this in this region creates analytical signals in the form of peaks. An increase of temperature to about 76 ?C in a small zone adjacent to the microelectrode causes an increase of the analytical signal by more than 6 times under FIA conditions. This method enables the use of hot microelectrodes as detectors in HPLC or CE. In CE the improvement of the analytical signal at hot microelectrodes is smaller than in FIA and increase only 1.3–3.4 times. Heated microelectrodes were used for analysis of the flavonoids in natural samples of the plant (extract of sea buckthorn) and a pharmaceutical preparation (Cerutin).