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. Received: 10 November 1997 / Revised: 19 January 1998 / Accepted: 24 January 1998     

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。     

Separation of amino acids in plant tissue extracts by capillary zone electrophoresis with indirect UV detection using aromatic carboxylates as background electrolytes

Summary Amino acids in extracts of plant tissue were separated and detected by capillary zone electrophoresis (CZE) with indirect UV detection. Various aromatic carboxylates such as salicylate, benzoate, phthalate and trimellitate were investigated as background electrolytes (BGEs). A BGE of benzoate gave the best resolution and detector response. Amino acids were separated at a highly alkaline pH to charge amino acids negatively. Separation was achieved by the co-electroosmotic flow (Co-EOF) by the addition of the cationic surfactant myristyltrimethyl-ammonium bromide (MTAB) to the electrolyte. The condtions affecting the separation of amino acids, including electrolyte pH, concentrations of both benzoate and MTAB, were investigated and optimised. Separation of a mixture of 17 amino acids at pH 11.20 with indirect UV detection at 225 nm was achieved with a BGE of 10 mM benzoate containing 1.0 mM MTAB at pH of 11.20. Detection limits ranged between 10 and 50 μM. The proposed method was demonstrated by the determination of amino acids in extracts of Eucalypt leaves with direct injection of samples.     

On-column complexation and simultaneous separation of vanadium(IV) and vanadium(V) by capillary electrophoresis with direct UV detection

An on-column complexation method has been developed for the simultaneous determination of V(IV) and V(V). Vanadium species were chelated with aminopolycarboxylic acids to form anionic complexes which were separated by capillary zone electrophoresis (CZE) with direct UV detection. Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetric acid (DTPA), nitrilotriacetic acid (NTA), and N-2-hydroxyethylethlendiaminetriacetric acid (HEDTA) were investigated as both ligand and running electrolyte. Of the ligands studied the complexes of EDTA with V(IV) and V(V) resulted in the highest selectivity and UV response.The conditions used for on-column complexation and separation, including pH, and electrolyte ligand concentration, were examined to achieve reasonable separation selectivity and detection sensitivity. The optimum separation of the anionic forms of V(IV) and V(V) was obtained by use of CZE with UV detection at 185 nm and an electrolyte containing 5 mmol L(-1) EDTA at pH 4.0. Linear calibration plots were obtained in the concentration range10-300 micro mol L(-1); detection limits were 3 micro mol L(-1) for V(IV) and 1 micro mol L(-1) for V(V). The proposed method was demonstrated for the determination of vanadium in groundwater spiked with V(IV) and V(V).     

On the perspectives of capillary electrophoresis modes for the determination of morphine in human plasma without sample pretreatment

Screening and confirmation of drugs of abuse in body fluids are important for the medicinal treatment and form the legal basis of court judgments. A fast and precise identification of toxic substances is necessary. Morphine was determined in human plasma by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) using sample stacking mode. The electrophoretic separation was performed in an uncoated fused-silica capillary, 70 cm long to the detector, with an additional 10 cm to the cathode (75 microm i.d. and 360 microm o.d.). The UV absorbance detection was set at 190 nm. The electrophoretic buffers were prepared from 60 to 300 mm disodium tetraborate decahydrate, pH 10.5. Sodium dodecyl sulfate was added to the final solution in a concentration of 60 mm for MECC. All electrophoretic separations were carried out at 10 kV and the capillary temperature was ambient (25 degrees C). A linear calibration graph was obtained in the concentration range studied (50-5000 ng/mL). Several samples of drug-free plasma were checked for potential endogenous interference and the results showed no interference from the endogenous components, which co-migrated with morphine. As little as 50 ng/mL of morphine could be successfully analyzed by MECC in the concentration mode with acceptable precision. It is possible to determine morphine directly in plasma at therapeutic concentrations.     

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 on unmodified genetically coded amino acids

The separation of mixtures of free genetically coded amino acids by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) using UV and refractometric detection was studied. Mixtures of 16 and 14 amino acids were separated by CZE and MEKC, respectively.     

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.     

寡糖的毛细管电泳分析

多种寡糖经α－萘胺衍生化后，用硼砂作为电泳介质，实现了高效毛细管电泳分离。比较了毛细管区带电泳和胶束毛细管电动色谱分离寡糖α－萘胺衍生物的电泳行为，对影响分离度的诸因素进行了考察，选择了最佳分离条件。     

Determination of heterocyclic compounds by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) based on sodium cholate (NaCh) and sodium dodecyl sulphate (SDS) was developed for the determination of aromatic amino acids and heterocyclic legume constituents. The influence of temperature, voltage, micellar system, pH, zwitterion and modifier concentrations in the buffer on migration times, peak areas, resolution and number of theoretical plates was investigated. This MECC method makes possible the sensitive determination of the individual compounds with detection limits in the picomole range. Up to 300 000 theoretical plates per metre of capillary were obtained together with satisfactory linearity and repeatability of the NaCh method. The applicability of MECC to samples prepared from plant material, following a fast and simple technique of isolation, purification and group separation, is illustrated by selected examples.     

Measurement of desmosine and isodesmosine by capillary zone electrophoresis

Desmosine (DES) and isodesmosine (IDE) were separated and quantitated by a simple and sensitive capillary zone electrophoretic (CZE) method, using hydrostatic injection and direct UV detection at 254 or 185 nm. Two different electrophoretic mobilities for the two isoforms were observed in 90 mM phosphoric acid pH 2.2. The presence of a mixture of amino acids in the sample did not affect the separation of DES and IDE. The method was successfully used to quantitate the amounts of DES and IDE in elastin hydrolysates.     

Recent advances in capillary electrophoresis of proteins, peptides and amino acids

The current status of high-performance capillary electrophoresis as an analytical separation method for proteins, peptides and amino acids is assessed. Recent advances in suppressing the effects of electroosmotic flow and irreversible adsorption of proteins at the capillary wall are reviewed, together with procedures for optimal separations of peptides and amino acids. The detection aspects emphasize the role of laser-induced fluorescence and capillary electrophoresis/mass spectrometry in high-sensitivity measurements.     

Open tubular capillary electrochromatography of underivatized amino acids using Rh(III) tetrakis(phenoxyphenyl)porphyrinate as wall modifier

The separation of 17 “common” underivatized amino acids was attempted by open tubular capillary electrochromatography (OT-CEC) in fused-silica capillaries coated with Rh(III) tetrakis(phenoxyphenyl)porphyrinate (Rh(III)TPP(m-OPh)₄OAc) using sodium phosphate and Tris–phosphate buffers as background electrolytes (BGEs). The OT-CEC separation of amino acids was compared with that obtained by capillary zone electrophoresis in bare fused-silica capillaries using the same BGEs. The amino acids were not derivatized and the UV-absorption detection was set at 200 nm. Depending on the experimental conditions at least 15 amino acids were separated. The best separations were obtained in a Rh(III)TPP(m-OPh)₄OAc-coated capillary in 50 mM Tris–100 mM phosphate buffer at pH 2.25. Separation of the critical triplet Val–Ile–Leu was always at least indicated being better at higher BGE concentrations. Regarding the sensitivity of the method, lower concentration limits of detection (LODs) in the coated capillary were obtained for Thr, Gly, Tyr, and Val; the other amino acids exhibited lower LODs in the uncoated capillary. The separation of acidic amino acids was not achieved.     

Postcolumn reactor using a laser-drilled capillary for light-emitting diode-induced fluorescence detection in CE

This study investigated a novel postcolumn reactor for fluorescence detection in CE. A laser-drilled capillary, with an aperture made by laser ablation, was used for mixing derivatization reagents with the analytes separated by CZE. The derivatization reagents, o-phthaldialdehyde (OPA), and 2-mercaptoethanol, were introduced into the capillary through the aperture and reacted with the analytes after CZE separation. High voltages were applied to both the inlet reservoir and the reservoir filled with the derivatization reagents. Thus, the flow rate of the derivatization reagents was controlled by the electric potential that was applied to the reservoir of the derivatization reagents. A UV light-emitting diode was used as an excitation light source for the fluorescence detection of OPA derivatives. A commercially available tee connector was compared with the laser-drilled capillary. The results implied that the dead volume of the laser-drilled capillary was less than that of the tee connector, since the laser-drilled capillary suppressed band broadening more efficiently. The LODs for amino acids were determined to be approximately 5 microM. The method was applied to the determination of amino acids in a Japanese beverage.     

Multi-purpose capillary electrophoresis system with concentration gradient detection

A robust, inexpensive and versatile capillary electrophoresis (CE) system for routine and rapid analysis is reported, which consists of a rugged cartridge holding a 20-mum i.d. 15-cm long capillary, and an inexpensive, universal and sensitive concentration gradient detector. The design of the cartridge simplifies the sample introduction process and makes it possible to perform many separation modes, including moving boundary capillary electrophoresis (MBCE), capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF), on the same system. This arrangement provides more information about a sample's components since analytes can be separated by different modes performed on the same CE system. The detector only consists of a low-power HeNe laser, or laser diode, and a photodiode position sensor. Amino acids and proteins of 10(-6)-10(-3)M concentration can be separated by different capillary electrophoretic modes, and detected directly by the detector. The universal detector shows particularly good sensitivity when applied to CE separation modes having self-concentration and focusing effects. Femtomoles of proteins were separated and detected with CIEF. In addition, a short and narrow capillary allows use of high electrical fields which facilitate rapid separations. Four amino acids at millimolar concentrations were fully separated and detected in less than 80 sec by the MBCE mode when a high electric field was applied. The physical size of the whole system is much smaller than that of conventional CE instruments with UV absorbance or fluorescence detector.     

Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen L-amino acid standards were labelled with NBD-F at 60 degrees C for 1 min, and separated in a buffer system containing 20 mM borate, 25 mM sodium cholate, 10 mM Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.     

Sample introduction and separation in capillary electrophoresis, and combination with mass spectrometric detection

Capillary-electrophoresis methods are attracting interest owing to the ability to yield rapid high-resolution separations, but many aspects, such as sample injection, separation conditions and detection, need further development. Effects related to sample injection and buffer composition have been investigated. Automated methods for electromigration injection of nl-size sample volumes are shown to give a precision of approximately +/-1%. Problems encountered with manual injection procedures have been examined by an electric field reversal technique. The effect of buffer pH on capillary zone-electrophoresis (CZE) separations can be attributed to changes in electro-osmotic flow velocities and to changes in the isoelectric points of analytes. The interfacing of capillary electrophoresis with mass spectrometry is described and demonstrated for a range of conditions, with a quaternary phosphonium salt mixture. Separations obtained by CZE and capillary isotachophoresis are compared and the relative advantages of the two techniques discussed.     

Separation and Determination of Four Amino Acids by Micellar Electrokinetic Capillary Chromatography

The most methods used to determine amino acids developed in the past were via pre- or post-column derivatization of the analytes to produce a fluorescent reagent in order for detection of them by measuring the fluorescence. The detection of underivatized amino acids is also accomplished by indirect methods. In this work, micellar electrokinetic capillary chromatography(MECC) based on sodium dodecyl sulphate (SDS) was developed for the direct determination of the four amino acids, histidine,tyrosine, tryptophan and phenylalanine, by using UV-detector. The apparatus used was Model 3850 capillary electrophoresis system (ISCO,USA) with a 60cm 0.05mm I.D.fused-silica capillary,where was a detection window at a position 35 cm from the injection end of the capillary. And the detection performed by on-column measurement of ultraviolet absorption at 210nm. SP4600 integrator was used for the data acquisition and processing.     

Capillary electrophoretic separation of organophosphonic acids using borate esterification and direct UV detection

One major challenge in the analysis of small ions by capillary zone electrophoresis (CZE) is detection. The most common commercially available detector for CZE is based on UV absorbance. For many small molecules, however, little UV absorbance occurs above 210 nm, limiting the usefulness of this detection method. Carbohydrates, alcohols and amides have been separated and directly detected by complexation with sodium borate. It has been shown that these complexes have UV absorbances in the range of 220 to 280 nm, whereas the analytes alone are UV transparent at energies less than 200 nm. Separation and direct detection of organophosphonic acids using sodium borate as both a buffer and derivatization agent is demonstrated. Detection limits on the order of nanograms are reported with separations that exhibit 10 000 to 1 740 000 theoretical plates. The ultraviolet, infrared, nuclear magnetic resonance and mass spectra of various borate/phosphonic acid esters are explored.     

粒子群算法优化中药化学成分的毛细管电泳分离条件

建立了一种基于粒子群优化算法的毛细管电泳条件辅助优化方法。以丹参为研究对象,将改良的色谱指数方程用于评价酚酸类成分的电泳分离性能,用粒子群优化算法对分离条件进行全局寻优,获得最佳的区带电泳分离条件(5.0 mmol/L硼砂,18.5 mmol/L磷酸二氢钠,6.1%乙腈,运行电压18.2 kV)。为进一步改善分离,在所获优化条件下添加50.0 mmol/L SDS,在胶束电动毛细管色谱分离模式下使酚酸类成分(原儿茶醛、丹参素、丹酚酸B等)得到更好分离。本方法准确可靠,可推广应用于其他复杂化学体系的毛细管电泳分离条件优化。