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.     

Field-amplified sample injection and in-capillary derivatization for sensitivity improvement of the electrophoretic determination of histamine

The feasibility of the combination of field-amplified sample injection (FASI) and in-capillary derivatization was explored for improving sensitivity of histamine in capillary electrophoresis (CE). Naphthalene-2,3-dicarboxaldehyde (NDA) was used as derivatization reagent. The reagent and sample was introduced by tandem mode. The derivatization was accomplished by at-inlet mode with standing time of 1.5 min. The combination of FASI and in-capillary derivatization was successfully achieved with about 400-fold concentration sensitivity enhancement compared to pre-capillary derivatization at the same set-up. The detection limit of concentration for histamine reached 1.25 x 10(-11) M by CE and fluorescence detection with S/N = 3. Parameters affecting FASI and in-capillary derivatization process including sample matrix, buffer concentration and reagent injection amount, were investigated.     

Sensitivity enhancement by on-line preconcentration and in-capillary derivatization for the electrophoretic determination of amino acids

This study describes an application of on-line preconcentration by large-volume stacking in combination with in-capillary derivatization for enhancing spectrophotometric detection sensitivity in capillary electrophoresis. The method is illustrated by an example dealing with the determination of amino acids with 1,2-naphthoquinone-4-sulfonate as a labelling agent. Samples are dissolved in water in order to create a stacking process based on differences in the conductivity between this medium and a concentrated running buffer. The in-capillary derivatization is accomplished following a sandwich procedure in which the sample is inserted between two segments of reagent. Amino acid derivatives are obtained and separated in a fused-silica capillary with a sodium borate electrolyte buffer using 2-propanol as an organic modifier. The method is applied to the analysis of amino acids in pharmaceutical and feed samples. A good concordance between the predicted values and those obtained with the standard method is observed, with overall quantification error below 5%. The proposed procedure allows the detection limits sensitivity to be enhanced in 1000-fold with respect to conventional precapillary derivatization.     

Capillary electrophoresis determination of biogenic amines by field-amplified sample stacking and in-capillary derivatization

A sensitive CE method for determining biogenic amines in wines based on in-capillary derivatization with 1,2-naphthoquinone-4-sulfonate is presented. In this method, reagent and buffer solutions are introduced hydrodynamically into the capillary whereas the sample is injected electrokinetically, thus, allowing a selective preconcentration of the analytes by field-amplified sample stacking. Amines are labeled inside the capillary using a zone-passing derivatization approach in mixed tandem mode. The most relevant variables influencing on the derivatization and separation as well as significant interactions have been evaluated using experimental design. Multi-criteria decision making is utilized for the simultaneous optimization of interacting variables through overall desirability response surfaces. The validation of the method has proven an excellent separation performance and accuracy for the determination of biogenic amines such as histamine, tryptamine, phenylethylamine, tyramine, agmatine, ethanolamine, serotonin, cadaverine, and putrescine in red wines. Detection limits range from 0.02 mg/L for ethanolamine to 0.91 mg/L for serotonin. The RSDs for migration time and peak area are around 1.2 and 6.2%, respectively. Red wines from different Spanish regions have been analyzed using the proposed method.     

Capillary electrophoretic determination of ammonia using headspace single-drop microextraction

A new method involving headspace single-drop microextraction (SDME) and capillary electrophoresis (CE) is developed for the preconcentration and determination of ammonia (as dissolved NH₃ and ammonium ion). An aqueous microdrop (5 μL) containing 1 mmol/L H₃PO₄ and 0.5 mmol/L KH₂PO₄ (as internal standard) was used as the acceptor phase. Common experimental parameters (sample and acceptor phase pH, extraction temperature, extraction time) affecting the extraction efficiency were investigated. Proposed SDME-CE method provided about 14-fold enrichment in about 20 min. The calibration curve was linear for concentrations of NH₄⁺ in the range from 5 to 100 μmol/L (R² = 0.996). The LOD (S / N = 3) was estimated to be 1.5 μmol/L of NH₄⁺. Such detection sensitivity is high enough for ammonia determination in common environmental and biological samples. Finally, headspace SDME was applied to determine ammonia in human blood, seawater and milk samples with spiked recoveries in the range of 96-107%.     

Capillary electrophoretic determination of cathepsin D activity using Oregon Green-labeled hemoglobin.

Cathepsin D is an aspartyl protease of lysosomal origin and functions in a variety of roles including protein turnover, catabolism of peptide hormones, antigen processing and presentation, and neoplastic disease. In breast cancer, the level of cathepsin D has been linked to metastasis and prognosis for survivability. Many of these studies concerning the role of cathepsin D in cancer have used immunological detection methods to determine the level of enzyme. These indirect methods to assess the cathepsin D level may not reflect enzyme activity accurately. The significance of cathepsin D to physiological and pathophysiological processes suggests that rapid and sensitive methods for determining cathepsin D activity would contribute to a more complete assessment of this enzyme in its various roles. This work describes a procedure to determine cathepsin D activity based on hydrolysis of fluorescently labeled hemoglobin and employs capillary electrophoresis to separate and measure the products of reaction. A single major cleavage product, representing the first 32 residues of the hemoglobin alpha-chain, appeared after a very short incubation time (less than 10 min) and was used to determine activity. The procedure described here requires very small sample volumes, has a low detection limit (approximately 10(-9) M) and thus represents an additional approach to determine cathepsin D activity in biological samples.     

Enzymatic in-capillary derivatization for glucose determination by electrophoresis with spectrophotometric detection

The following paper compares several procedures of in-capillary bienzymatic derivatization with regard to glucose determination with the use of glucose oxidase and horseradish peroxidase. The procedures discussed below include continuous contact in the capillary, plug-plug injection, and sequential injection with incubation in the capillary inlet. The reaction of hydrogen peroxide catalyzed by peroxidase was performed using two different substrates. The best results were achieved for nicotinamide adenine dinucleotide, reduced disodium salt (NADH) acting both as a chromogenic reagent and a substrate for peroxidase, while the method employed was sequential injection and incubation at the capillary inlet. The LOD was estimated to be 25 nM with a linear response up to 0.1 microM.     

Field-amplified sample injection and in-capillary derivatization for capillary electrophoretic analysis of metal ions in local wines

In-capillary derivatization and field-amplified sample injection (FASI) coupled to capillary zone electrophoresis (CZE) was evaluated for the analysis of metals (Co(II), Cu(II), Ni(II), and Fe(II)) using 2-(5-Nitro-2-Pyridylazo)-5-(N-Propyl-N-Sulfopropylamino)Phenol (Nitro-PAPS) as the derivatizing agent. For FASI, the optimum conditions were water as sample solvent, 1 s hydrodynamic injection (0.1 psi) of a water plug, 5 s of electrokinetic introduction (10 kV) of the sample. The in-capillary derivatization was successfully achieved with zone-passing strategy in order tandem injection of Nitro-PAPS reagent (0.5 psi, 7 s), a small water plug (0.1 psi, 1 s), and metal ion introduction (10 kV, 5 s). The solution of 45 mmol L^− 1 borate pH 9.7 and 1.0 × 10^− 5 mol L^− 1 Nitro-PAPS containing 20% acetonitrile was used as the running buffer. The limit of detection obtained by the proposed method was lower than those from pre-capillary derivatization about 3–28 times. The recovery of the method was comparable to pre-capillary derivatization method. In-capillary derivatization-FASI-CZE was applied to analysis of metals in wine samples. The results were compared with those obtained by CZE with pre-capillary derivatization method and atomic absorption spectrometry (AAS).     

Capillary electrophoretic determination of the protease Savinase in cultivation broth.

The highly basic washing enzyme Savinase and various analogues were analysed by micellar electrokinetic chromatography (MEKC) and electrophoresis. Broth samples were withdrawn during the cultivation of Savinase by recombinant microorganisms. Savinase peak areas obtained by MEKC-electrophoretic analysis were normalized with respect to migration time and compared with traditional enzyme activity measurements. The electropherograms indicated thermal degradation of the Savinase molecule at high field strengths. Baseline separation of Savinase and two analogues was achieved.     

Capillary electrophoretic determination of inorganic selenium species

The performance of pyromellitic electrolyte for capillary zone electrophoresis of inorganic selenium species in the presence of selected common anions with indirect UV detection was investigated. The separation was achieved with pyromellitic electrolyte at pH 8.8 and hexamethonium hydroxide as the electroosmotic flow modifier. Obtained detection limits of 0.17 microg ml(-1) for Se(VI) and 0.29 microg ml(-1) for Se(IV) were improved by a factor of 5-7 in comparison with chromate electrolyte, which has been mainly employed for selenium analysis. Good resolution for nitrate-Se(VI) peaks were obtained.     

Capillary electrophoretic analysis of carbohydrates derivatized by in-capillary condensation with 1-phenyl-3-methyl-5-pyrazolone

Our previous papers on capillary electrophoresis (CE) have shown that samples can be derivatized in a capillary and the derivatives can be analyzed immediately after derivatization, provided that the derivatization reaction is so rapid as to complete in seconds. The present paper presents extended application of in-capillary derivatization to a much slower reaction such as the condensation of reducing carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) which requires 30 min at 70 degrees C in pre-column derivatization by manual operation. It was necessary to first drive the introduced plugs of sample and reagent solutions to put them together at the entrance of the heated portion of a capillary, then to allow the superimposed plugs to react for a relevant period. We showed how to determine the introduction times of the sample and the reagent solutions as well as intermediate running buffer, the voltages to be applied for plug driving and product analysis, and the duration of voltage application, all of which are important for effective in-capillary derivatization. An example of the analysis of maltooligosaccharides by this technique is presented. It was shown that maltooligosaccharides were quantitatively derivatized with PMP in 35 min at 57 degrees C, and the derivatives could be analyzed in ca. 15 min by CE immediately after derivatization. Separation was satisfactory in 200 mM borate buffer, pH 8.2 containing sodium dodecyl sulfate to a concentration of 200 mM. Although the theoretical plate number, and accordingly the resolution, were significantly lower than the corresponding values in pre-capillary derivatization, reasonable reproducibility was ensured for both migration time (RSD 3.5% on average) and peak area (RSD less than 3%) under the optimized conditions. It is notable that sample amount could be lowered to the 10 fmol level, in contrast to the 10 pmol level in pre-capillary derivatization. In addition, since the technique employed here (the modified at-inlet technique of in-capillary derivatization) is easily automated, the established system will be highly beneficial for routine analysis of carbohydrates. Analysis by this technique was also shown to be useful for kinetic study of the derivatization reaction.     

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.     

一种基于二喹啉甲酸- Cu+显色反应的毛细管电泳检测蛋白质的新方法

采用毛细管电泳法和蛋白质显色反应-二喹啉甲酸(BCA)法,结合微波辅助反应,在60 mmol/L硼酸盐缓冲液(pH 9.5)中,实现了对蛋白质的快速毛细管电泳分析检测。同时以β-环糊精为包合添加剂,实现了BCA-Cu+复合物和游离BCA的分离,从而在波长200 nm处以测定特征生成的BCA-Cu+复合物来间接检测蛋白质,其峰强度比直接检测蛋白质自身吸收的峰强度提高了2个数量级。对于转铁蛋白、蓖麻毒素,其线性范围分别为2～200 mg/L和2～100 mg/L,检出限分别为0.33和0.37 mg/L。将该方法成功地应用于第一届蓖麻毒素国际实验室间比对测试的部分样品,含量测定结果满意。     

Capillary electrophoretic determination of dithiocarbamates and ethyl xanthate

A simple and sensitive capillary electrophoretic method was developed for the separation and determination of sodium methyldithiocarbamate (metham), manganese ethylenebisdithiocarbamate (maneb) and ethyl xanthate in boric acid buffer by direct UV absorbance detection at lambda = 254 nm. The separation is dependent on pH and nature of the buffer. The detection limits (S/N = 3) are 1.7 x 10(-6) mol/L for ethyl xanthate, 1.3 x 10(-6) mol/L for metham and 2.1 x 10(-6) mol/L for maneb. The method has been successfully applied to the analysis of wheat samples spiked with maneb and in a commercial sample.     

Capillary electrophoretic determination of oxalate in amniotic fluid

A capillary electrophoretic (CE) assay for oxalate has been applied to the quantitative determination of free oxalate in amniotic fluid. Indirect absorbance detection of oxalate is accomplished with a chromate-based background electrolyte modified with ethylenediaminetetraacetic acid (EDTA). Detection interference due to the presence of high levels (≈4 mg/ml) of inorganic chloride is eliminated through a direct sample clean-up procedure based on cation (Ag⁺-form) resins. Separation interference from amniotic fluid proteins is prevented through the use of a simple aqueous-based dilution procedure. This method for the determination of oxalate in amniotic fluid provides precision of ≈5% relative standard deviation (RSD). Within-day precisions for the oxalate response and migration time are better than 3% RSD and 1% RSD, respectively. Between-day precisions for the oxalate response and migration time are better than 6% RSD and 3% RSD, respectively. The analytical recovery of oxalate (1000 ng/ml) spiked into amniotic fluid was better than 96%. The limit of detection (LOD) for the method is ≈100 ng/ml oxalate. This method also shows promising results for the determination of oxalate in human blood plasma samples.     

Capillary electrophoretic determination of dithiocarbamates and ethyl xanthate

A simple and sensitive capillary electrophoretic method was developed for the separation and determination of sodium methyldithiocarbamate (metham), manganese ethylenebisdithiocarbamate (maneb) and ethyl xanthate in boric acid buffer by direct UV absorbance detection at λ = 254 nm. The separation is dependent on pH and nature of the buffer. The detection limits (S/N = 3) are 1.7 × 10^–6 mol/L for ethyl xanthate, 1.3 × 10^–6 mol/L for metham and 2.1 × 10^–6 mol/L for maneb. The method has been successfully applied to the analysis of wheat samples spiked with maneb and in a commercial sample.     

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.     

Capillary electrophoretic determination of mercury compounds in different matrixes

Summary Capillary electrophoresis has been used to separate inorganic (Hg²⁺) and organic (methyl-, ethyl-, and phenylmercury) mercury compounds as their cysteine complexes. The optimized electrophoretic separation was performed in fused-silica capillary tubing at 25 kV with 25mm sodium borate buffer (pH 9.3). Identification and quantification of the mercury species at mg L⁻¹ levels was achieved by use of UV detection at 200 nm. The relative standard deviation (n=10) ranged from 0.38 to 0.51% for migration times and from 0.43 to 2.94% for corrected peak areas. Good recovery (>90%) was obtained for all four mercury species in surface waters, and for inorganic mercury and methylmercury in five- to tenfold diluted biofluids (urine, saliva, and cerebrospinal fluid). TheLOQ values obtained were too high to be useful for determination of mercury species in real samples. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL?1 for each zopiclone enantiomer, 0.8-1.6 μg mL?1 for 2-amino-5-chloropyridine and 0.4-0.8 μg mL?1 for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.     

Capillary electrophoretic determination of thiosulfate and its oxidation products

Capillary electrophoresis (CE) was developed for the rapid and simple determination of thiosulfate and its oxidation products such as common polythionates, sulfite and sulfate. Direct and indirect UV detection techniques were investigated. The optimized separations of UV absorbing S2O3(2-), S4O6(2-), S5O6(2-) and S6O6(2-) anions were carried out in 5 mmol l(-1) (NH4)2SO4, 5 mmol l(-1) KH2PO4 electrolyte at pH 5.0, with direct UV detection at 214 nm. All analytes were well resolved in less than 4 min. Analysis of S2O3(2-), S4O6(2-), SO4(2-) and SO3(2-) ions can be performed in 5 mmol l(-1) H2CrO4, 1 mmol l(-1) hexamethonium hydroxide electrolyte neutralized with triethanolamine to pH 8.0, using indirect UV detection at 254 nm. However, the detection sensitivity for tetrathionate was poor. Other polythionates can not be detected at all because of their high absorbance even at 254 nm. The developed CE method was applied for the monitoring of sulfur species in spent fixing solutions during the electrolytic oxidation.