毛细管电泳柱上安培检测装置的研制

介绍了一种新的毛细管电泳柱上安培检测装置。以碳纤维微电极为工作电极,在自组装的ACS－2000毛细管电泳仪上,测定了三种苯二酚的异构体：邻苯二酚、对苯二酚、间苯二酚,在20min内达到了基线分离。     

新型富集仪的研制及其实验教学应用

报道了新型实验教学仪器——"毛细管电泳富集仪"的研制及实验教学开发应用。对该仪器的工作原理、仪器性能、工作参数以及实际富集效果和教学应用进行了详细的说明和讨论,对于影响样品富集效果的各个因素进行了探讨。实验结果表明,该仪器的实际富集因子可达560倍。采用研制的毛细管电泳富集仪对原有实验进行深度开发,深化和丰富了仪器分析实验的实验教学内容。     

Fraction collector for capillary zone electrophoresis

An instrument is described which is capable of collecting fractions from a capillary zone electrophoresis apparatus. The fraction collector is characterized in terms of discretely collecting the separated components of a multi-component sample. In addition, the fraction collector permits the study of the effect of capillary zone electrophoresis on the biological activity of alpha-chymotrypsin.     

Rapid determination of short chain carnitines in human plasma by electrospray ionisation-ion trap mass spectrometry using capillary electrophoresis instrument as sampler

A capillary electrophoresis apparatus was used as sampler for flow injection analysis (FIA) in tandem mass spectrometry of L-carnitine and its acetyl- and propionyl-metabolites in human plasma. The capillary electrophoresis instrument was coupled to the ion trap mass spectrometer by an electrospray ionization coaxial sheath liquid interface. The electrophoresis capillary introduced the sample directly into the source by applying a prolonged sample injection. The use of the capillary electrophoresis apparatus miniaturised the FIA procedure, substantially reducing the quantities of solvents and samples used, and allowed rapid automated sequential analyses. The method was optimised and validated using a dialyzed human plasma matrix. The plasma samples were analysed after a simple, rapid deproteinisation procedure with acetonitrile and diluted 70 times before direct injection into the mass spectrometer for product ion scan MS/MS analysis in positive ionisation. The total analysis time was 5 min, including capillary preconditioning and acquisition time (3 min). The method was sensitive, allowing the determination of L-, L-acetyl- and L-propionyl-carnitines at 140, 14 and 3.6 nM concentrations (injected values) corresponding to lower limit of quantitation values in plasma of 10, 1 and 0.25 microM, respectively. The method was processed for full validation and applied to the analysis of L-carnitine and its short chain derivatives in human plasma samples.     

Home-made temperature-control unit for the Waters Quanta 4000 capillary electrophoresis system

A home-made temperature control unit has been developed for a commercial capillary electrophoresis apparatus, the Waters Quanta 4000. The effect of the unit on the absolute migration times and peak areas of nine β-blockers is described. It was found that this homemade temperature control unit considerably (≥ 100 %) improved the repeatability of the separation.     

Capillary electrophoretic determination of S-carboxymethyl-L-cysteine and its major metabolites in human urine: feasibility investigation using on-column detection of non-derivatized solutes in capillaries with minimal electroosmosis

The capillary electrophoretic analysis of S-carboxymethyl-L-cysteine and some of its metabolites in human urine is reported using (i) on-column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE-100 (Bio-Rad), featuring coated fused silica capillaries of 25 microns i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 microns i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.     

Capillary electrophoresis-chemiluminescence detection system equipped with a consecutive sample-injection device.

We have developed a consecutive sample-injection device for capillary electrophoresis, which comprises one four-way cock, two syringe pumps, and an interface part taking advantage of two three-way Teflon joints. Sample introduction into the capillary is made hydrodynamically by pressure, caused by the flow of the sample solution at the tip of the capillary inlet. We combined the injection device with a capillary electrophoresis-chemiluminescence detection system. A mixture solution of N-(4-aminobutyl)-N-ethylisoluminol, isoluminol isothiocyanate, and luminol was analyzed as a model sample by the present system. The sample solution was consecutively injected and detected with about a 230 s interval. The present capillary electrophoresis-chemiluminescence detection system with the consecutive sample injection device features easy and rapid operation, an inexpensive apparatus, high sensitivity, as well as consecutive analysis.     

色氨酸、半胱氨酸和酪氨酸的高效毛细管电泳分析

用自组装的高效毛细管电泳安培检测装置,对具有常规电活性的色氨酸,半胱氨酸和酪氨酸进行了分离分析条件的研究,采用重力进样方式,进样高度25cm,进样时间15S,在分离毛细管长70cm,内径25um,的电泳装置上,10mmol/L K2HPO4-H3PO4缓冲液（PH10．5）,20KV分离高压,＋1．05V（vs SCE)检测电位的条件下,对酸解毛发中得到的混合氨基酸中的色氨酸,半胱氨酸和酪氨酸进行了分离测定,结果令人满意。     

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.     

Very fast electrophoretic separation on commercial instruments using a combination of two capillaries with different internal diameters

A capillary formed by connecting a 9.7 cm‐long separation capillary with id 25 μm with an auxiliary 22.9 cm‐long capillary with id 100 μm (coupled capillary) was tested for electrophoretic separation at high electric field intensities. The coupled capillary was placed in the cassette of a standard electrophoresis apparatus. It was used in the short‐end injection mode for separation of a mixture of dopamine, noradrenaline, and adrenaline in a BGE of 20 mM citric acid/NaOH, pH 3.2. An intensity of 2.7 kV/cm was attained in the separation part of the capillary at a separation voltage of 30 kV, which is 2.9 times more than maximum intensity value attainable in a capillary with the same length with uniform id. At these high electric field intensities, the migration times of the tested neurotransmitters had values of 12.3–13.3 s and the attained separation efficiency was between 2350 and 2760 plates/s. It is thus demonstrated that an effective separation instrument ‐ a coupled capillary ‐ can be used for very rapid separation in combination with standard, commercially available instrumentation.     

Simultaneous chromatography and electrophoresis: two-dimensional planar separations

Single-dimension separations are routinely coupled in series to achieve two-dimensional separations, yet little has been done to simultaneously exploit multiple dimensions during separation. In this work, simultaneous chromatography and electrophoresis is introduced and evaluated for its potential to achieve two-dimensional separations. In simultaneous chromatography and electrophoresis, chromatography occurs via capillary action while an orthogonal electric field concurrently promotes electrophoresis in a second dimension. A novel apparatus with a dual solvent reservoir was designed to apply the concurrent electric field. Various compounds were used to characterize the apparatus and technique, i.e., vitamins, amino acids, and dyes. Improved separation is reported with equivalent analysis times in comparison to planar chromatography alone. The feasibility of simultaneously employing chromatography and electrophoresis in two dimensions is discussed.

Rational approach to quantitative sodium dodecyl sulfate capillary gel electrophoresis of monoclonal antibodies

Sodium dodecyl sulfate capillary gel electrophoresis has been used to separate and quantify murine monoclonal antibodies. The method uses a murine IgG, whose subclass differs from the analyte antibody, as an internal reference. The internal reference is chosen based on knowing that mouse IgG1 can be separated from mouse IgG2a or IgG2b. Good intra- and inter-day reproducibility [relative standard deviation (RSD)<2%] of peak-area ratio has been obtained. A calibration curve also demonstrates high linearity (R2=0.9999) of response for the analyte. The described method is highly suitable for accurate determination of the antibody concentration even if a capillary electrophoresis apparatus is unable to provide good injection reproducibility.     

An economic approach for construction of a multiplexed capillary electrophoresis system

We describe in this report an economic approach to construct a multiplexed capillary electrophoresis (CE) system with optic-fiber UV detection. To demonstrate the feasibility of this approach, a seven-lane CE setup is built. The system has a background noise of ∼10⁻⁵ absorbance unit, a limit of detection of 3-4 μM for uracil at 254 nm, and a linear dynamic range of 2.5 orders of magnitude. The apparatus consists of three major components: a UV light source, a photodiode array (PDA) and optical module assembly, and a computer. Compared to the recently reported 96-lane capillary electrophoresis system with UV-vis absorbance detection, the cost of goods is reduced by more than 10-fold, and the noise level is improved by 2 to 3 folds. Parallel CE separations of a protein mixture have been performed on this apparatus, and reproducible and high resolution separation results have been obtained. Detailed construction and characterization of the system have also been discussed.     

毛细管电泳迁移时间重现性影响因素的探讨

以电泳介质为研究对象,在毛细管区带电泳(CZE)和胶束电动毛细管色谱(MECC或MEKC)两种电泳模式下,探讨了电泳 介质中各组分浓度、冲洗程序、电泳介质在实验中发生的变化对溶质迁移时间重现性的影响。根据描述缓冲液中各组分 浓度与电渗流迁移时间和溶质迁移时间的关系式,证明利用电导值可准确地表征电泳缓冲液的浓度;通过控制缓冲液的电 导值可提高迁移时间的重现性。运行缓冲液pH值的变化影响毛细管壁上的硅羟基的电离,因此需选择合理的冲洗程序使硅 羟基的电离达到平衡,以提高迁移时间的重现性。毛细管入口端缓冲液是影响溶质迁移行为的主要因素,其在电泳中发生 的变化是影响电泳重现性的重要原因。目前在实验中可通过提高运行缓冲液的更换频率来保证迁移时间的重现性。     

Ultramicro enzyme assays in a capillary electrophoretic system.

This paper describes an ultramicro method for achieving enzyme assays. Enzyme saturating concentrations of substrate, coenzyme when appropriate, and running buffer were mixed and used to fill a deactivated fused-silica capillary in a capillary zone electrophoresis apparatus. The enzyme glucose-6-phosphate dehydrogenase was injected by either electrophoresis or siphoning and mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced nicotinamide adenine dinucleotide phosphate, to the detector where it was detected at 340 nm. Under constant potential, the transport velocity of enzyme and the product was generally different. This caused product to be separated from the enzyme after it was formed. Because product formation was much faster than the rate of enzyme-product separation, product accumulated. The amount of accumulated product was inversely related to operating potential. In the extreme case, the operating potential was zero. Zero potential assays were generally carried out by electrophoresing the enzyme partially through the capillary and then switching to zero potential. This capillary was left at zero potential for several minutes to allow additional product to accumulate. After this additional amplification step, potential was again applied and the product transported to the detector. Product formed under constant potential appears as a broad peak with a flat plateau. When the voltage is switched to zero at intermediate migration distance, a peak will be observed on top of this plateau. Either the eight of the plateau or the area of the peak may be used to determine enzyme concentration. The lower limit of detection was 4.6.10(-17) mol of glucose-6-phosphate dehydrogenase.     

Continuous fractionation of enantiomer pairs in free solution using an electrophoretic analog of simulated moving bed chromatography

Continuous fractionation of the left and right enantiomers of Piperoxan was performed in free solution in a vortex-stabilized electrophoresis apparatus. Sulfated beta-cyclodextrin was used as the chiral selector. A capillary electrophoresis (CE) study of the separation of Piperoxan enantiomers was carried out in order to find the buffer conditions that produce the maximum peak separation time between the two enantiomers and the optimal chiral selector concentration. These peak separation times were then used to calculate the electrophoretic mobilities of the enantiomer-ligand complexes. The difference in electrophoretic mobilities, when used in a preliminary model of the enantiomer separation, indicated that, by imposing a fluid flow opposite the direction of electromigration, it would be possible to force the fast and slow enantiomers to move in opposite directions within the vortex-stabilized apparatus. Using the predictions of the preliminary separation model, the vortex stabilized electrophoresis apparatus was configured with a feed port at the center of the chamber axis and offtake ports near the cathode and anode. This allowed for continuous operation of the apparatus. Continuous fractionations were completed at throughputs of 1.5 and 4.0 mg/h with both offtakes showing greater than 99% enantiomeric purity at 4.0 mg/h using CE. Fractionation was achieved at a throughput of 10 mg/h, but while the slow enantiomer was recovered with greater than 99% purity, only 96% enantiomeric purity of the fast stereoisomer was achieved. The loss of resolution at higher volumetric throughputs supports our hypothesis that a mobility-dependent "window" of operation exists in which two solutes can be completely separated.     

Determination of 2,4-D and Dicamba in food crops by MEKC

Summary The determination of 2,4-D (2,4-dichlorophenoxyacetic acid) and Dicamba (2-methoxy-3,6-dichlorobenzoic acid) residues in sugar cane, rice and corn was performed by a supercritical fluid extraction (SFE) method using CO₂/acetone as extraction mix and an SFE apparatus developed in our laboratory. The extracts were cleaned up after extraction by both liquid-liquid partition and a Florisil column. Micellar electrokinetic capillary chromatography (MEKC) coupled with ultraviolet on-column detection was used for the analysis of these pesticides. The detection limits were improved by the preparation of a special detection cell with an increased pathlength that gave detection limits of ca. 0.6 pg for 2,4-D and Dicamba. Our results demonstrated that capillary electrophoresis can be a powerful new analytical tool for pesticide residue analysis.     

Development of a capillary electrophoresis-77 K luminescence detection system for online spectral identification

We have demonstrated that capillary electrophoresis (CE) can be easily interfaced with 77 K luminescence spectroscopy (LS) for separation and online spectral identification of structurally similar analytes. This novel CE-LS apparatus consists of a regular CE system, instrumentation for LS and a specially designed capillary Dewar. When the separating molecules traverse into the cryostat detection window, liquid nitrogen is added, freezing the separating analyte zones within the capillary. At low temperature, detection limits are improved via signal averaging and the inherent increase in quantum yield at 77 K. We present the first application of the CE-LS system to structural isomers (2,3- and 3,4-methylenedioxymethamphetamine) and stereoisomers (trans- and cis-resveratrol). With this approach, the CE-LS interfacing provides a sensitive, accurate, rapid, simple and economic methodology for analytical chemistry.     

A new sequential injection analysis‐capillary electrophoresis system with amperometric detection

A novel and fully automated sequential injection analysis manifold coupled to a capillary electrophoresis apparatus with amperometric detection, is described. The sequential injection manifold was isolated from the high voltage by inserting an air plug into the circuit. Small buffer reservoirs were used to avoid the need to pump fresh buffer to the interface during the electrophoretic separation. No decoupling device was used to mitigate the interference from the high voltage electric field, instead the potential shift induced by the separation voltage, was accounted for. The new hydrodynamic injection method presented is based on the overpressure created in the circuit when a pinch valve is closed for a predetermined time. The injection method yields RSD values of peak height and area below 2.55 and 1.82%, respectively, at different durations of valve closure (n = 5). The capillary and working electrode alignment was achieved by adapting a commercial available capillary union. When the electrode was replaced, the alignment method proved to be very reliable, yielding RSD values of peak height and area lower than 2.64 and 2.08%, respectively (n = 8). Using this system with a gold microelectrode, dopamine, and epinephrine could be quantified within the concentration range of 1–500 μM and detected at a concentration of 0.3 μM. The methods here presented could be applied for the development of new capillary electrophoresis systems with amperometric detection and/or to the design of fully automated systems for online process monitoring purposes.     

Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with on-line isotachophoretic sample pretreatment.

Analytical capabilities of capillary zone electrophoresis (CZE) with on-line coupled capillary isotachophoresis (ITP) sample pretreatment in the column-coupling capillary electrophoresis equipment to separate and determine enantiomers present in multicomponent ionic matrices were studied. Tryptophan was used as a model analyte in the ITP-capillary zone electrophoresis experiments performed in this context while a 90-component model mixture of UV-light absorbing organic anions and urine served as multicomponent sample matrices. Various working modes in which the on-line coupled capillary isotachophoresis-capillary zone electrophoresis combination in the column-coupling separation system can operate were employed in the anionic regime of the separation with direct injections of the samples. Advantages and limitations of these working modes in the separations of enantiomers present in model and urine matrices were assessed. Experiments with model mixtures of tryptophan enantiomers revealed that the two were resolved in the capillary zone electrophoresis stage with the aid of alpha-cyclodextrin also when their concentration ratio in the sample was 1:200 while the concentration of L(-)-tryptophan was 25 nmol/l. The limits of detection for the enantiomers were at approximately 10 nmol/l (approximately 1.5 ng/ml) concentrations for a 220 nm detection wavelength of the UV detector employed in the capillary zone electrophoresis stage and for a 30 microliters sample load. A high sample load capacity of the on-line coupled capillary isotachophoresis stage was effective in separating the samples corresponding to 3-6 microliters volumes of undiluted urine. The results from the runs with urine samples showed that only the capillary isotachophoresis-capillary zone electrophoresis combination with a post-column on-line coupled capillary isotachophoresis sample clean-up (responsible for a removal of more than 99% of the sample anionic constituents migrating in the on-line coupled capillary isotachophoresis stack and detectable in the capillary zone electrophoresis stage) provided a universal alternative for the detection and quantitation of the model analyte (L(-)-tryptophan).