Rapid isoelectric focusing of proteins in hydrolytically stable capillaries.

Three new types of capillary coatings for capillary isoelectric focusing that avoid siloxane chemistry, resulting in hydrolytically stable coatings, are described and tested: phenyl-silica, acrylamide-reacted vinyl-silica, and pure PTFE. Capillaries of these three types were compared using standard proteins and a biological mixture of proteins similar to what might be encountered in actual use. Of these, the acrylamide-coated capillary produced the highest-quality results. In contrast to capillaries prepared using siloxane reactions, the capillaries described herein exhibited greatly enhanced stability at high pH.     

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography (CEC) is achieved with open-tubular capillaries (o-CEC), with packed capillaries (p-CEC) or with monolithic capillaries. In o-CEC, capillaries are coated with a thin film containing cyclodextrin derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as chiral selectors. In p-CEC, typical chiral HPLC stationary phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins, glycoproteins, macrocyclic antibiotics, quinine-derived and 'Pirkle' selectors, polyacrylamides and molecularly imprinted polymers are used as chiral selectors. Chiral monolithic stationary phases prepared by in situ polymerization into the capillary were also developed for electrochromatographic enantiomer separation.     

毛细管柱表面鸡卵清蛋白快速改性并用于等电聚焦电泳分离蛋白质

开发了一种利用鸡卵清蛋白改性毛细管柱内表面的快速方法。改性后的毛细管柱可避免蛋白质样品的吸附作用，在较温和的条件下，柱性能有很好的稳定性，可适用于等电聚焦电泳分离生物样品的需要。     

Simultaneous operation of plural separation modes in capillary electrophoresis with a chemiluminescence detector possessing a micro-space area for reaction/detection

Analysis of alpha-amino acids, proteins, and phenolic compounds was simultaneously performed using three capillaries in capillary electrophoresis with chemiluminescence detection, taking advantage of the micro-space area for reaction/detection at the tip of the capillary. The three capillaries included usual, polymer-containing, and sodium docley sulfate (SDS)-containing migration buffers for separation. The eluted samples from the capillaries, which were inserted into the chemiluminescence detection cell, were mixed with chemiluminescence reagent at the tips of the capillaries to generate visible light. The specific micro-space area for reaction/detection at the tips of the capillaries enabled the simultaneous operation of the three separation modes in the present system.     

Liposome Capillary Electrophoresis of Peptides and Proteins

Liposome capillary electrophoresis (LCE) using unilamellar liposomes composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a suspended pseudo stationary phase has been investigated for its capability at separating peptides and proteins in bare fused-silica capillaries. The study has explored different strategies for allowing the liposome suspension to act as a disperse pseudo stationary phase with the ability of modulating selectivity, resolution and separation performance of peptides and proteins in bare-fused silica capillaries. Such strategies comprise the use of capillaries either partially or totally filled with the liposome suspension, whereas the electrolyte solution is liposome-free, or the incorporation of the liposomes into the buffer solution employed for rinsing the capillary and as the background electrolyte. Three synthetic peptides of similar amino acid sequence and four basic standard proteins have been employed as test analytes. Varying the volume of the liposome suspension introduced in the capillary promoted differentiated variations in the migration velocity of the three peptides reflecting their selective interactions with the liposomes. Efficient separation of basic proteins was obtained at pH 7.4 in a bare fused-silica capillary with the electrolyte solution containing 60 μM POPC.     

Surface initiated polymerization of a cationic monomer on inner surfaces of silica capillaries: Analyte separation by capillary electrophoresis versus polyelectrolyte behavior

[2‐(Methacryloyl)oxyethyl]trimethylammonium chloride was successfully polymerized by surface‐initiated atom transfer radical polymerization method on the inner surface of fused‐silica capillaries resulting in a covalently bound poly([2‐(methacryloyl)oxyethyl]trimethylammonium chloride) coating. The coated capillaries provided in capillary electrophoresis an excellent run‐to‐run repeatability, capillary‐to‐capillary and day‐to‐day reproducibility. The capillaries worked reliably over 1 month with EOF repeatability below 0.5%. The positively charged coated capillaries were successfully applied to the capillary electrophoretic separation of three standard proteins and five β‐blockers with the separation efficiencies ranging from 132 000 to 303 000 plates/m, and from 82 000 to 189 000 plates/m, respectively. In addition, challenging high‐ and low‐density lipoprotein particles could be separated. The hydrodynamic sizes of free polymer chains in buffers used in the capillary electrophoretic experiments were measured for the characterization of the coatings.     

Electrokinetic separation of peptides and proteins using a polyvinylamine-coated capillary with UV and ESI-MS detection

In order to accomplish the analysis of peptides and proteins by capillary electrophoresis, Lupamin, a high-molecular-weight linear polyvinylamine (PVAm) polymer, was introduced to modify the inner wall of fused-silica capillaries by physical absorption. Thanks to the high density of positively charged amino groups in Lupamin under acidic conditions, not only is a strong reversed electroosmotic flow generated in the coated capillary but the adsorption of analytes on the inner wall of the capillary is also efficiently eliminated. It has been demonstrated that the Lupamin-coated capillary can be used to advantage for the rapid analysis of amino acids, peptides, and proteins with good resolution and peak shape by capillary electrophoresis. In order to evaluate the basic feature of a Lupamin-coated capillary, electroosmotic flows generated by a Lupamin coating layer under different conditions including pH, coating time, concentration, and the composition of electrolytes on Lupamin-coated and uncoated capillaries were investigated. Furthermore, electrospray ionization-mass spectrometry (ESI-MS) detection was carried out for the analysis of amino acids and peptides.     

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long‐term stability of fused‐silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three‐protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused‐silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life‐time.     

金纳米微粒修饰毛细管电泳分离蛋白质的研究

报道了金纳米微粒(Au NP)修饰毛细管电泳分离蛋白质的方法.采用物理吸附法将Au NP修饰在熔融石英毛细管内表面,制备成Au NP修饰毛细管.探讨了修饰剂Au NP的浓度对电渗流及蛋白质分离的影响.结果表明,Au NP修饰的毛细管能有效地抑制电渗流及蛋白质在毛细管内壁上的吸附,提高分离效率.在优化的实验条件下,实现了...     

High-performance capillary electrophoresis of hydrophobic membrane proteins

Hydrophobic membrane proteins, extrinsic and intrinsic ones, were separated by high-performance capillary zone electrophoresis (HPCZE) and high-performance capillary isotachophoresis (HPCITP). In the case of HPCZE with both coated and uncoated quartz capillaries the addition of 7 M urea to the separation buffers was necessary to achieve reproducible results. In the HPCITP experiments PTFE capillaries were used. When spacers were used, e.g., ampholytes, additional splitting of peaks was observed. The splitting was caused by the microheterogeneity of the investigated proteins, which are differently glycosylated and/or phosphorylated.     

毛细管电泳涂层柱技术的进展

毛细管电泳涂层柱是解决蛋白质在毛细管壁吸附的最有效的方法。较为系统地综述了毛细管电泳涂层柱的几种制作方法,指出了毛细管电泳涂层柱（包括毛细管电色谱柱）的发展趋势,３９篇。     

Bonded dimethylacrylamide as a permanent coating for capillary electrophoresis

A method for coating capillaries for capillary electrophoresis with chemically bonded polydimethylacrylamide has been developed, and the properties of the capillaries have been evaluated. The coated capillaries provided high separation efficiency, 12 x 10(5) theoretical plates/m was obtained for cytochrome c. The electroosmotic flow at pH 8.0 was 10 x 10(-10) to 6 x 10(-10) m2 V(-1) s(-1). The coated capillaries were quite stable at high pH. At least 150 runs could be done at pH 10 without appreciable performance deterioration. The excellent performance of the coated capillaries was illustrated by separation of basic proteins, acidic proteins, 9-fluorenylmethyl chloroformate-derivatized neurotransmitter amino acids, peptide reference mixtures and peptides digested from a bacteria protein.     

预聚合法制备聚丙烯酰胺毛细管凝胶柱

提出了一种温和条件下制备交联聚丙烯酸胺毛细管凝胶电泳柱的新方法──预聚合法.讨论了制备过程中的问题,获得了凝胶组成、毛细管内径及长度等不同的交联聚丙烯酸胺毛细管凝胶电泳柱.采用激光回射干涉检测技术,实现了蛋白质标准样品的毛细管凝胶电泳在柱分离检测.     

Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.     

Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.     

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C⁴D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused‐silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused‐silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.     

Preparation of an iminodiacetic acid-modified capillary and its performance in capillary liquid chromatography and immobilized metal chelate affinity capillary electrophoresis

We prepared iminodiacetic acid (IDA)-modified and Cu(II)-IDA-modified capillaries through polymerization of N-(vinylbenzylimino) diacetic acid. The fundamental performance of these capillaries was examined in capillary liquid chromatography (LC) and immobilized metal chelate affinity capillary electrophoresis (IMACE). Copper(II), cobalt(II), and hematin were detected at different retention times by means of capillary LC with a chemiluminescence detector, during which the IDA-modified capillary was used. The difference in the retention times was attributed to the difference in the interaction between metal ions or complex and IDA moieties on the inner wall of the capillary. In addition, human serum albumin (HSA) and human serum gamma-globulin (HgammaG) were separated and detected using IMACE with an absorption detector, during which the Cu(II)-IDA-modified capillary was used. The separation of HSA and HgammaG was achieved through the interaction between proteins and Cu(II) chelate moieties on the inner wall of this capillary.     

超支化聚合物化学键合毛细管电泳柱的制备及其对蛋白质的分离行为

分别合成了以三羟甲基丙烷和季戊四醇为核的超支化聚(胺-酯),并对其进行了红外测定、羟值测定、粘度测定等表征。采用化学键合方法将其涂于毛细管内壁,并测定涂层柱的电渗流以及对碱性蛋白质的分离能力,结果表明,涂层柱能有效地抑制碱性蛋白质在毛细管内壁上的吸附,大大降低电渗流;以三羟甲基丙烷为核的超支化聚(胺-酯)涂层柱的塔板数达105/m,而以季戊四醇为核的超支化聚(胺-酯)涂层柱的分离柱效更高,塔板数达107/m。实验结果表明这两类涂层柱都具有较好的分离效果和稳定性。     

Fast and easy coating for capillary electrophoresis based on a physically adsorbed cationic copolymer

In this work, a new copolymer synthesized in our laboratory is used as physically adsorbed coating for capillary electrophoresis (CE). The copolymer is composed of ethylpyrrolidine methacrylate (EPyM) and methylmethacrylate (MMA). The capillary coating is easily obtained by simply flushing into the tubing an EPyM/MMA solution. It is demonstrated that the composition of the EPyM/MMA copolymer together with the selection of the background electrolyte (BGE) and pH allow tailoring the direction and magnitude of the electroosmotic flow (EOF) in CE. It is also shown that the EOF obtained for the EPyM/MMA-coated capillaries was reproducible in all cases independently on pH or polymer composition. Thus, RSD values lower than 1.9% (n=5) for the same capillary and day were obtained for the migration time, while the repeatability interdays (n=5) was observed to provide RSD values lower than 0.5%. The stability of the coating procedure was also tested between capillaries (n=3) obtaining RSD values lower than 0.6%. It is demonstrated with several examples that the use of EPyM/MMA coatings in CE can drastically reduce the analysis time and/or to improve the resolution of the separations. It is shown that EPyM/MMA-coated capillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. Also, EPyM/MMA-coated capillaries provide a faster separation of samples containing simultaneously positive and negative analytes. Moreover, it is demonstrated that the use of EPyM/MMA-coated capillaries can incorporate an additional chromatographic-like interaction with nucleosides that highly improves the separation of this group of solutes.     

Capillary electrophoresis of proteins in dextran-coated columns

A simple coating technique by using uncross-linked dextran has been developed for fused-silica capillaries to be used in capillary electrophoresis of basic proteins. The capillaries were first silanized with a heterobifunctional silane (gamma-aminopropyltriethoxylsilane), which served as a coupling agent between the capillary inner wall and the polysaccharide coating. Dextran of high molecular mass (about 70 kDa) was activated with 1,1'-carbonyldiimidazole. Then the activated dextran was coupled to the primary amino groups that were anchored onto the inner wall of the silanized capillaries. The residual reactive groups on the dextran were further substituted by neutral functions in a coupling reaction with excess ethanolamine. By using dimethyl sulfoxide (DMSO) rather than aqueous buffer as the reaction medium, the extent of substitution was improved by minimizing the residual reactive groups at the surface. Since they are ionogenic, the electrosmotic flow in the system is relatively low. The chemically bound dextran coating showed good reproducibility and stability. In electrophoretic experiments basic proteins were separated with high efficiency by use of the dextran-coated fused-silica capillary columns. The main advantage of the method described here is that both polysaccharide activation and amine-coupling reactions were carried out under mild conditions at room temperature without catalysts. For this reason, the method is recommended to coat the inner wall of microfluidic separation channels which would not tolerate a harsh treatment.