Simultaneous determination of dihydroxybenzene and phenylenediamine positional isomers using capillary zone electrophoresis coupled with amperometric detection

In general capillary zone electrophoresis (CZE) separation models, o‐, m‐, and p‐phenylenediamine isomers can be separated in a weak acidic running buffer for their pK_a values being 4.52, 5.64, 6.04, respectively, while o‐, m‐, and p‐dihydroxybenzene isomers can be separated in a weak basic buffer for their pK_a values being 9.40, 9.40 and 10.04, respectively. So, it is hard to find a suitable running buffer at a fixed pH in normal CZE for simultaneous separation of these two groups of positional isomers. In this paper, a novel method based on alternately running two different pH buffers in CZE coupled with amperometric detection (CZE‐AD) was designed to simultaneously determine these two groups of positional isomers. It is found that when two different pH running buffers were employed alternately under appropriate order and time, the six analytes could be separated perfectly in less than 20 min and the detection limits were as low as 10^–7 mol/L. Furthermore, the effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CZE–AD were investigated. Experimental results demonstrated that the introduced method was practical to simultaneously determine two groups of positional isomers with different pK_a and had some advantages of high sensitivity, good repeatability and small sample requirement.     

苯胺及其衍生物的毛细管电泳行为研究

采用紫外吸收检测，毛细管电泳分离，研究了九种苯胺及其衍生物在毛细管区带电泳(CZE)体系和胶束电动毛细管色谱(MECC)体系中的行为特征。讨论了缓冲溶液的浓度与pH、胶束浓度及混合胶束等在不同体系中对分离组分的影响，发现在CZE体系中，控制分离的主要因素是pKb值；在MECC体系中，控制分离的主要因素是溶质分子中碳原子数。建立了一种分离测定九种苯胺及其衍生物的高效毛细管电泳方法。     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.     

Protein mapping of peanut extract with capillary electrophoresis

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.     

Determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry

The determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) was investigated. Both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes of operation were studied. The optimal separation of four cobalamin species (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5′-deoxyadenosylcobalamin) and a potentially harmful corrinoid analogue (cobinamide dicyanide) was obtained using CZE at a pH of 2.5. Both 20 mM phosphate and 20 mM formate buffers were used with success, although the formate buffer provided improved resolution. The CZE-ICP-MS method was used to quantify cyanocobalamin in a vitamin supplement and the analytical results were in good agreement (±5%) with values obtained by ICP-MS for total Co levels. The solution detection limits for cobalamins using CZE-ICP-MS were approximately 50 ng/ml. MEKC was found to be useful for the screening of vitamin preparations because it provided a rapid means of distinguishing cyanocobalamin (the form most commonly used in vitamin preparations) from free cobalt. The separation of free cobalt and cyanocobalamin using MEKC was achieved in less than 10 min.     

Multivalent weak electrolytes - risky background electrolytes for capillary zone electrophoresis

Multivalent weak acids and bases are useful components of buffers in electrophoresis. The use of such buffers as background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is, however, risky due to the existence of unsafe regions in the analytical window of the separation. This contribution discusses the problems and shows that multivalent weak species in BGEs bring about the same effects as mixtures of two independent co-ions, i.e., the presence of two centers of symmetry in the electropherograms and the existence of a migrating system zone with a mobility in between these two centers of symmetry. The system zone deteriorates the analytical separation and detection of the analytes in its neighborhood. Illustrative experimental examples for both cationic and anionic CZE are shown and related discussion is given. Finally, some basic rules are formulated to avoid the preparation of risky BGEs.     

三种维生素的毛细管电泳和胶束电动毛细管色谱法分离与安培电化学检测

以自制毛细管电泳－电化学检测系统对ＶＣ,ＶＢ１和ＶＢ６的毛细管区带电泳和胶束电动色谱的分离与检测情况进行了初探。结果表明,在０．０１ｍｏｌ／ＬＮＨ３－ＮＨ４Ｃｌ介质中,检测电势定于５１０～５４０ｍＶ（对ＳＣＥ）时,三种维生素均有较好的ＣＺＥ图,对ＶＣ的分离效率达４６８８００块理论板。使用十二烷基硫酸钠时,分离效果欠佳。     

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.     

Determination of berberine and strychnine in medicinal plants and herbal preparations by pressurized liquid extraction with capillary zone electrophoresis.

A simple and rapid method for the determination of berberine and strychnine in medicinal plants and herbal preparations for regulatory purposes using a home-made pressurized liquid extraction (PLE) system with capillary zone electrophoresis (CZE) using ultraviolet detection at 254 nm was developed. The effects of pH, concentration of buffer, and organic modifiers in the electrophoretic separation were investigated. The buffer used for CZE contained 50 mM ammonium acetate, pH 3.1. The effect of temperature on the extraction efficiency of strychnine in medicinal plants by PLE was demonstrated. Comparable or higher extraction efficiency was achieved with PLE for strychnine in medicinal plants and berberine in herbal preparations compared to soxhlet extraction. The effect of matrix interference in medicinal plants and herbal preparations containing a number of medicinal plants samples using CZE was investigated by standard additional experiments. The reproducibility of the method using PLE with CZE was found to vary between 2.4 and 10.7% (n = 5/6) for different types of samples on different days.     

Determination of ciprofloxacin and its impurities by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been elaborated for separation, identification and determination of ciprofloxacin and its impurities. The separation, phosphate buffer pH 6.0 was supplemented with 0.075 M pentane-1-sulfonic acid sodium salt. The elaborated method was validated. The selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision, and accuracy of capillary zone electrophoresis were evaluated. The results obtained by CZE were also compared with those obtained by liquid chromatography. Regarding the validation results the CE method fulfils the current European Pharmacopoeia (Eur. Ph.) requirements. The evaluated CE method could be applicable to the analysis of different medicinal products containing ciprofloxacin.     

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.     

Stacking in capillary zone electrophoresis

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.     

卤代乙酸及其结构相近化合物的高效毛细管电泳分离

氟、氯、溴等卤代乙酸是结构非常相近的离子型化合物,对它们的分离测定比较困难。用高效毛细管电泳法在碱性或酸性缓冲液条件下可将它们分离。在酸性缓冲液条件下,可提高有机酸分离的选择性。较低的操作电压有利于提高阴离子的分离度,而改变温度对分离度的影响不大。     

Analysis of galactoglucomannans from spruce wood by capillary electrophoresis

The aim of this study was to setup a method for detection and quantification of monosaccharide components in technical galactoglucomannas (T-GGM) from spruce wood using capillary zone electrophoresis (CZE). CZE technique was optimised regarding borate buffer concentrations, EOF modifier application, and system pH. Aqueous solution of T-GGM was chemically hydrolysed by sulphuric acid, in an autoclave. In this way obtained monosaccharides were derivatized with 4-amino benzoic acid ethyl ester via reductive amination using sodium cyanoborohydride. The results of the optimisation procedure showed that the borate buffers at lowest concentrations (100 and 200 mM) with acetonitrile addition as EOF modifier gave the optimal measurement results, as it showed sufficient separation at relatively short migration times. The amounts of single monosaccharide components in the T-GGM samples obtained by the optimised CZE procedure were practically the same in comparison to the results of the well established HPLC-anion exchange chromatography. On the basis of this research, it was concluded that the capillary zone electrophoresis is an efficient analytical procedure for the characterisation of galactoglucomannans derived from softwoods.     

Separation of phospholipids by capillary zone electrophoresis with indirect ultraviolet detection

A simple method for separation of different anionic and zwitterionic phospholipid classes by capillary zone electrophoresis (CZE), using indirect UV detection with adenosine monophosphate (AMP) as background electrolyte and the UV-absorbing additive, was successfully developed in this study. The separation conditions including apparent pH (pH*) of running buffer, concentration of AMP, organic solvent, applied voltage and capillary temperature were systematically optimized. The application of this method to human blood sample was also briefly examined.     

New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis

Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused-silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated.     

Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries.

Numerous efforts have been made to separate proteins by capillary zone electrophoresis (CZE). The most common optimization techniques are changing the pH of the running buffer, coating the capillary surface with a hydrophilic polymer, or using additives in the sample solution. Surface coatings and solution additives can reduce the adsorption of the protein onto the capillary surface, but they diminish the separation efficiency and the resolution of CZE. This paper reports the successful separation of proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide. Under these conditions, model proteins and proteins in human serum have been determined by CZE. It is shown that the results from CZE are compatible with those of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

A chip-type thin-layer electrochemical cell coupled with capillary electrophoresis for online separation of electrode reaction products

A coupling technique of thin-layer electrolysis with high-performance capillary electrophoresis/UV–vis technique(EC/HPCE/UV–vis) is developed for online separation and determination of electrode reaction products. A chip-type thin-layer electrolytic (CTE) cell was designed and fabricated, which contains a capillary channel and a background electrolyte reservoir, allowing rapid electrolysis, direct sampling and online electrophoretic separation. This chip-type setup was characterized based on an electrophoresis expression of Nernst equation that was applied to the redox equilibrium of o-tolidine at different potentials. The utility of the method was demonstrated by separating and determining the electro-oxidation products of quercetin in different pH media. Two main products were always found in the studied time, potential and pH ranges. The variety of products increased not only with increasing potential but also with increasing pH value, and in total, at least 13 products were observed in the electropherograms. This work illustrates a novel example of capillary electrophoresis used online with thin-layer electrolysis to separate and detect electrode reaction products.     

Study on the systematic optimization of capillary electrophoresis using a controlled weighted centroid variable size simplex algorithm

The systematic optimization of capillary electrophoretic separations using a dynamic scouting optimum method-controlled weighted centroid variable size simplex algorithm is described. The factors affecting the efficiency of the separation are simultaneously considered during the optimization procedures. The established optimization method is applied to amino acid separation by capillary zone electrophoresis (CZE) with on-column indirect UV detection and to the separation of local anesthetics by micellar electrokinetic chromatography (MECC) with on-column UV detection. The optimization procedures include the pH and the background absorption electrolyte (BGAE) concentrations together with the applied voltage in the CZE separation of amino acids. The pH, the SDS concentrations together with the percentage of methanol are considered in the MECC separation of local anesthetics. Two methods, i.e., the Long Coefficient and Uniform Design Table, are used to define the start vertexes during the optimization procedure and similar final experimental conditions for the separations are achieved. Thirteen native amino acids are baseline separated by CZE and 4 local anesthetics are satisfactorily separated by MECC.     

不同缓冲体系对毛细管电泳法分离15种核苷类化合物效果的比较

对毛细管电泳法分离15种核苷类化合物所用的不同缓冲液体系进行了系统比较,确定不同模式毛细管电泳法分析多种核苷类化合物的最适合背景缓冲液体系(BGE)。分别以四硼酸钠、磷酸氢二钠、乙酸钠、碳酸氢钠、乙酸铵和乙二胺(DEA)为背景电解质,对毛细管区带电泳(CZE)、毛细管电泳-电喷雾飞行时间质谱(CE-ESI-TOF/MS)以及胶束电动毛细管电泳(MEKC)3种模式进行比较,并对其中几种优势缓冲体系进行了优化。结果表明,CZE模式下使用四硼酸钠和磷酸氢二钠缓冲体系无法同时分离15种核苷类化合物,因此只适用于分析核苷类化合物数量较少的样品。而使用含有2%丙酮的300 mmol/L DEA能完全分开15种核苷类化合物,且分辨率和峰形良好。MEKC模式下,以25 mmol/L磷酸氢二钠(添加70 mmol/L十二烷基磺酸钠(SDS))为缓冲盐的分离结果最佳,并且此方法能成功应用于海洋生物海葵中核苷类化合物的分离。CE-ESI-TOF/MS分析中,以20 mmol/L乙酸铵(pH 10.0)为背景电解质,正离子模式检测,15种核苷类化合物的质谱信号均良好,检测灵敏度明显优于文献中报道的使用DEA缓冲体系的结果。本研究阐明了不同缓冲体系对15种核苷类化合物分离的适用性,为毛细管电泳技术在复杂基质中多种核苷类化合物的分离方法中的应用奠定了基础。