Free solution capillary electrophoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with L- and D-Marfey's reagents

Separation of amino acid enantiomers and peptide isomers has been made possible through the use of Marfey's reagent and high-performance capillary electrophoresis (HPCE). Samples of amino acids and peptides were first derivatized with Marfey's reagent and subsequently analyzed by HPCE. Different modes of separation were investigated including free solution and micellar electrokinetic chromatography. The use of micellar electrokinetic chromatography in combination with L- and D-Marfey's reagent offered unequivocal means to confirm the presence of D-amino acid in an unknown sample. This method is also particularly useful for the analysis of peptide isomers.     

使用含离子涂层柱的毛细管电泳和毛细管电色谱的研究进展

 毛细管电色谱是近年发展起来的高效、高选择性的微分离技术。与一般的毛细管电泳和使用ODS反相填料的毛细管电色谱相比 ,含离子涂层柱的毛细管电泳和毛细管电色谱能提供较大且可控的电渗流 ,便于拓宽分离对象 ,优化分离条件。对使用含离子涂层柱的毛细管电泳和电色谱的特点、发展和应用状况进行了综述。     

毛细管电泳接触反应法测定血清中铁传递蛋白的浓度

建立了一种测定血清中铁传递蛋白的毛细管电泳接触反应方法，利用血清中铁传递蛋白计算化学结合铁的性质，用标准铁饮和铁传递蛋通过对铁准确定量，测定血清铁伟递蛋白的有浓度。     

高效毛细管电泳电化学检测器的研制

本文提出了一种用于高效毛细管电泳的新型安培电化学检测器设计，使用Ｎａｆｉｏｎ溶液制作的ＨＰＣＥ／ＥＤ接口，可有效地隔开两化学系统的干扰，且不引入附加体积，经对有机酚类化合物的胶束电动毛细管色谱分离与电化学检测知，该系统性能优良，对对苯二酚的检出限为３０ａｍｏｌ。     

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity.

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monitoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and appearance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.     

High-performance capillary electrophoresis for characterization of hapten—protein conjugates used for production of antibodies against soyasaponin I

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten—protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-- -galactoside; PNPG) coupled to KSTI. The PNPG—KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten—protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.     

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018–March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.     

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.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

对映异构体的高效毛细管电泳分离与测定

高效毛细管电泳是８０年代发展起来的一种高效、快速的新型分离技术，在对映异构体分离方面有着广泛的应用前景，本文介绍了这一新型分离技术用于对映体分离的基本原理，并列举了一些对映异构体分离的实例。     

基于毛细管电泳技术的高灵敏度蛋白质组学技术发展

近年来,蛋白质组学技术在样品前处理、分离技术和质谱检测技术方面获得了快速发展,已经可以实现在几小时内对上万种蛋白的同时定性和定量分析。然而,目前的主流蛋白质组学技术仍无法满足极微量生物样品,尤其是单细胞样品的组学分析需求。毛细管电泳分离技术具有峰宽窄、柱效高、样品用量少等优势,是与高分辨质谱在线联用的理想选择之一。该文评述了集成化和在线样品前处理以及主流的纳升液相色谱-质谱联用系统在高灵敏度蛋白质组学分析领域的发展现状和挑战,认为该领域的重要技术挑战之一在于目前的纳升液相色谱分离已经无法完全匹配现代高分辨质谱超过40 Hz的超高扫描速度,从而导致质谱使用效率的降低。针对上述技术挑战,该文重点探讨了毛细管电泳-质谱联用技术的独特技术优势和潜在发展机遇,主要包括：（1）面向微量酶解多肽样品的高柱效毛细管电泳分离。通过采用毛细管电色谱可以进一步改善毛细管电泳柱容量不足的局限;（2）面向高灵敏度分析的无鞘液/鞘液接口开发;（3）高效毛细管电泳分离与高扫描速度质谱检测的协同化使用。总之,我们预期毛细管电泳-质谱联用技术的进一步发展有望在针对单细胞等超微量生物学样品的蛋白质组学分析中获得更广泛的应用。     

Determination of vasoactive intestinal peptide in rat brain by high-performance capillary electrophoresis.

A high-performance capillary electrophoresis (HPCE) method for determining vasoactive intestinal peptide (VIP) in rat brain was developed. Cerebral cortex was first extracted by solid-phase extraction and purified by reversed-phase high-performance liquid chromatography. The VIP-rich fraction was further analysed by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography using a commercial HPCE instrument with UV detection. The identity of the peak of endogenous VIP was confirmed by performing multiple CZE analyses at different pH values. This HPCE method allows VIP to be detected and measured with good molecular specificity and could represent a reference method to validate data obtained by radioimmunoassay.     

Selectivity in capillary electrokinetic separations

This review gives a survey of selectivity modes in capillary electrophoresis separations in pharmaceutical analysis and bioanalysis. Despite the high efficiencies of these separation techniques, good selectivity is required to allow quantitation or identification of a particular analyte. Selectivity in capillary electrophoresis is defined and described for different separation mechanisms, which are divided into two major areas: (i) capillary zone electrophoresis and (ii) electrokinetic chromatography. The first area describes aqueous (with or without organic modifiers) and nonaqueous modes. The second area discusses all capillary electrophoretic separation modes in which interaction with a (pseudo)stationary phase results in a change in migration rate of the analytes. These can be divided in micellar electrokinetic chromatography and capillary electrochromatography. The latter category can range from fully packed capillaries, via open-tubular coated capillaries to the addition of microparticles with multiple or single binding sites. Furthermore, an attempt is made to differentiate between methods in which molecular recognition plays a predominant role and methods in which the selectivity depends on overall differences in physicochemical properties between the analytes. The calculation of the resolution for the different separation modes and the requirements for qualitative and quantitative analysis are discussed. It is anticipated that selectivity tuning is easier in separation modes in which molecular recognition plays a role. However, sufficient attention needs to be paid to the efficiency of the system in that it not only affects resolution but also detectability of the analyte of interest.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

毛细管电泳法检测蜂蜜中残留的抗生素

 用毛细管电泳法对 5种常用的抗生素四环素 (TC) ,土霉素 (OTC) ,多西环素 (DOC) ,金霉素 (CTC)和氯霉素 (CP)进行了分离。研究了电泳缓冲液的 pH、不同有机试剂添加剂、温度等因素对抗生素分离的影响。实验结果表明 ,体积分数为 4%的N 甲基吗啡啉的加入可大大改善分离效果。在各自相应的浓度范围内 ,峰面积和样品浓度之间呈现良好的线性关系 ,重现性好。氯霉素的检测极限为 10 μg/L ,四环素、土霉素、多西环素为 2 0 μg/L ,金霉素为 40 μg/L(信噪比 >5 )。该方法成功地应用于蜂蜜中残留抗生素的测定 ,具有操作简单、快速方便、灵敏度及自动化程度高。     

High-performance capillary electrophoresis of meat, dairy, and cereal proteins

Food proteins play important roles in food functionality, nutrition, and human health. For these reasons, new analytical methods are continually being developed to separate and characterize these important proteins. High-performance capillary electrophoresis (HPCE) is one of the latest analytical methods to be applied to the separation of food proteins. This review covers methods and applications for the separation of three major groups of food proteins, meat, dairy, and cereal proteins.     

Chiral separation using capillary electromigration techniques based on ligand exchange principle

Over the last couple of decades, researchers have developed diverse chiral separation methods emerged from a few chiral separation principles. This review article is primarily focused on the application of chiral ligand-exchange (CLE) principle in capillary electromigration techniques, such as capillary electrophoresis (CE) and capillary electrochromatography (CEC). First, the most commonly used CLE-CZE separation mode by using different kinds of central ions, such as Cu(II), Zn(II), borate ion, and other metal ions, has been introduced. Meanwhile, several kinds of surfactants have been applied as the micelle-forming agents in the CLE micellar electrokinetic chromatography mode. The highlight of recent research of CLE-CEC is the exploitation of novel columns for chiral separation. Then, two kinds of capillary columns, packed capillary and monolithic capillary column, have been briefly described. Finally, the effective application of these chiral separation methods has been presented, including the application in life science and food analysis area.     

Simultaneous detection and quantitation of sodium, potassium calcium and magnesium in ocular lenses by high- performance capillary electrophoresis with indirect photometric detection

A high-performance capillary electrophoresis (HPCE) method which can be used to quantitatively determine Na⁺, K⁺, Ca²⁺ and Mg²⁺ simultaneously in ocular lenses has been developed. The proteins in the lens aqueous homogenates were precipitated by 10% trichloroacetic acid. The precipitated proteins were removed after a brief centrifugation, and the supernatant containing the cations was washed with ether and directly used for HPCE analysis. A 50 μm × 75 cm fused-silica capillary was used for separation and the detection wavelength was set at 214 nm. A 20-mM imidazole at pH 6.0 containing 0.1% hydroxypropyl methyl cellulose was used as background electrolyte. Sample solution was injected at 15 kV for 10 s, and the electrophoresis was carried out at 15 kV. All the cations can be separated and quantified from the peak areas within 9 min. The values obtained by this method were comparable with commonly used flame atomic absorption and flame atomic emission spectroscopy. It is demonstrated that this HPCE method can be used to quantify all the cation levels simultaneously within a short time even in a small single rat or mice lens.