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.     

Brush-like copolymer as a physically adsorbed coating for protein separation by capillary electrophoresis

A brush-like copolymer consisting of poly(ethylene glycol) methyl ether methacrylate and N,N-dimethylacrylamide (PEGMA-DMA) was synthesized and used as a novel static physically adsorbed coating for protein separation by capillary electrophoresis for the first time, in order to stabilize electroosmotic flow (EOF) and suppress adsorption of proteins onto the capillary wall. Very stable and low EOF was obtained in PEGMA-DMA-coated capillary at pH 2.2-7.8. The effects of molar ratio of PEGMA to DMA, copolymer molecular mass, and pH on the separation of basic proteins were discussed. A comparative study of bare capillary with PEGMA-DMA-coated capillary for protein separation was also performed. The basic proteins could be well separated in PEGMA-DMA-coated capillary over the investigated pH range of 2.8-6.8 with good repeatability and high separation efficiency because the copolymer coating combines good protein-resistant property of PEG side chains with excellent coating ability of PDMA-contained backbone. Finally, the coating was successfully applied to the fast separation of other protein samples, such as protein mixture and egg white, which reveals that it is a potential coating for further proteomics analysis.     

Novel approach for the rapid determination of water-soluble organic acids in wine by co-electroosmotic flow capillary zone electrophoresis

An innovative protocol for the fast analysis of some organic acids in red wine by co-electroosmotic capillary zone electrophoresis and indirect UV detection using hexadimethrine bromide (HDB) as coating agent was proposed. The adsorption of HDB onto the capillary wall provided a stable electroosmotic flow and separation of small anions was carried out using background electrolytes containing no polymer additive. Low RSD% values (<3.6%) in terms of migration times and effective mobilities were obtained from the analysis of a mixture of nitrate and nitrite and of a mixture of organic acids. An experimental design approach was used to investigate the effects of temperature, separation voltage, and percentage of methanol added to the running buffer solution on the separation of the analytes. A faster method allowing the separation of the organic acids involved in the malolactic fermentation of wine was developed. Using a running electrolyte consisting of 35% (v/v) methanol in a solution of 22 mM benzoic acid at pH 6.10 adjusted with 1.0 M TRIS-base buffer, the separation of tartaric, malic, succinic, acetic, and lactic acids was feasible in less than 210 s. Application of the method to the quantification of the above-mentioned organic acids in Italian red wine samples is reported.     

New physically adsorbed polymer coating for reproducible separations of basic and acidic proteins by capillary electrophoresis

In this work, a new physically adsorbed coating for capillary electrophoresis (CE) is presented. The coating is based on a N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EPyM) copolymer synthesized in our laboratory. The capillary coating is simple and easy to obtain as only requires flushing the capillary with a polymer aqueous solution for 2 min. It is shown that by using these coated capillaries the electrostatic adsorption of a group of basic proteins onto the capillary wall is significantly reduced allowing their analysis by CE. Moreover, the DMA-EPyM coating provides reproducible separations of the basic proteins with RSD values for migration times lower than 0.75% for the same day (n = 5) and lower than 3.90% for three different days (n = 15). Interestingly, the electrical charge of the coated capillary wall can be modulated by varying the pH of the running buffer which makes possible the analysis of basic and acidic proteins in the same capillary. The usefulness of this coating is further demonstrated via the reproducible separation of whey (i.e. acidic) proteins from raw milk. The coating protocol should be compatible with both CE in microchips and CE-MS of different types of proteins.     

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.     

Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.     

开管毛细管电色谱分析3种解热镇痛药物

针对生物体液样品开展药物的绿色高效毛细管电泳分离分析具有重要的研究意义。该研究以3种解热镇痛药（4-氨基安替比林、氨基比林及非那西汀）为研究对象,以嵌段聚合物为涂层,建立了药物的开管毛细管电色谱（OT-CEC）分析新策略。首先,采用活性/可控自由基可逆加成-断裂链转移聚合方法,合成制备得到了两亲性嵌段聚合物-聚（苯乙烯-甲基丙烯酸缩水甘油酯）（P（St-GMA））,并将其涂覆到毛细管内壁;其次,通过考察影响OT-CEC分离效率的关键因素,包括嵌段聚合物的聚合时间、涂覆毛细管嵌段聚合物的浓度、电泳运行缓冲液的种类和pH值、有机溶剂添加剂等,优化了3种解热镇痛药物的OT-CEC分离条件;最终发现,不需添加任何有机溶剂及表面活性剂,仅采用50.0 mmol/L乙酸钠-乙酸（pH 5.7）作为OT-CEC的缓冲溶液,就能实现3种解热镇痛药物的基线分离。在8.0~2.5×10³ μmol/L范围内,分析物峰面积与其对应的浓度呈现良好的线性关系,相关系数（R²）均大于0.995,检出限为1.0~2.5 μmol/L。结果表明：P（St-GMA）在溶液中自组装所形成的类表面活性剂胶团结构增强了两亲性嵌段聚合物与解热镇痛药物之间的相互作用,显著提升了解热镇痛药物的OT-CEC分离效率。该工作不仅为制备新型聚合物及调控嵌段聚合物的自组装行为提供了研究思路,也展示了两亲性嵌段聚合物在药物的绿色OT-CEC分析中的实际应用潜力。     

Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis

To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis.     

Separation and determination of carnosine-related peptides using capillary electrophoresis with laser-induced fluorescence detection

A capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the separation and detection of carnosine-related peptides (carnosine, anserine, and homocarnosine). A sensitive and fluorogenic regent, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) was selected as a precapillary labeling reagent for imidazole dipeptides to form isoindole derivatives. The optimized molar ratio between CBQCA and peptide was found to be 75:1, and 50 mmol/L borate buffer (pH 9.2) was used for the derivatization in order to achieve good efficiency. Three imidazole dipeptides were baseline-separated within 20 min by using 112 mmol/L sodium borate (pH 10.4-10.8) as running buffer. Concentration detection limits (signal-to-noise ratios) for carnosine, anserine, and homocarnosine were 4.73, 4.37, and 3.94 nmol/L, respectively. This method has been applied to the analysis of human cerebrospinal fluid (CSF) and meat dry powder of pig and sheep. Recoveries were in the range of 82.9-104.8% for homocarnosine in CSF. For carnosine and anserine, the recoveries are 98.3% and 80.2% in meat dry powder of pig and 111.2% and 112.8% in meat dry powder of sheep, respectively.     

Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection

A dynamic coating capillary electrophoresis coupled with a simplified on-line chemiluminescence detection system was designed and evaluated. In the proposed system, poly-vinylpyrrolidone was used as dynamic coating substance in the separation buffer to reduce the unwanted protein non-specific adsorption, which was first applied in capillary electrophoresis coupling with on-line chemiluminescence detection. In order to avoid complex processing, an ordinary plastic cuvette was modified as a three-way joint. The chemiluminescence reaction conditions and capillary electrophoresis separation conditions were investigated in detail. The results showed that the coated capillary can be injected protein samples at least 30 times continuously with good repeatability. Under optimal conditions, the chemiluminescence relative intensity was linear with the concentration of hemoglobin in the range of 4-1850 μg mL(-1) and the detection limit was 2.0 μg mL(-1) (S/N=3). The relative standard deviation of migration times and peak heights for 40 μg mL(-1) hemoglobin were 2.5% and 4.1% (n=11) respectively. Interference of matrix effects was overcome by the calibration according to standard addition methods. Afterwards, the method was validated successfully and was applied to detect the concentration of hemoglobin in the serum of haemolytic patients.     

Nanoparticle‐based capillary electroseparation of proteins in polymer capillaries under physiological conditions

Totally porous lipid‐based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas^®, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle‐based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single‐amino‐acid‐substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one‐step procedure based on self‐assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid‐based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary‐based method facilitates future electrochromatography of proteins on polymer‐based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.     

Field-amplified on-line sample stacking for determination of carnosine-related peptides by capillary electrophoresis

An on-line sample stacking method, namely field-amplified sample injection, has been developed for the separation and determination of carnosine, anserine, and homocarnosine by capillary electrophoresis. Using electrokinetic injection, about 130- to 160-fold improvement of sensitivity was achieved without loss of separation efficiency when compared to conventional sample injection. For conventional injection, the samples were dissolved in running buffer and then hydrodynamically injected for 10 s (3.45 kPa). Various parameters affecting separation and sample stacking were optimized. Under optimum conditions, linear responses were obtained over two orders of magnitude and the detection limits (defined as S/N = 3) of carnosine, anserine, and homocarnosine were 1.5 x 10(-8) to 1.6 x 10(-8) mol/L.     

Capillary zone electrophoresis of alpha 1-acid glycoprotein fragments from trypsin and endoglycosidase digestions

Capillary zone electrophoresis with fused-silica tubes having hydrophilic coating on the inner walls was evaluated in the separation of peptide and glycopeptide fragments from trypsin digestion of alpha 1-acid glycoprotein. Submapping of glycosylated and nonglycosylated tryptic fragments of the glycoprotein by capillary electrophoresis was facilitated by selective isolation of the glycopeptides on concanavalin A silica-based stationary phases prior to the electrophoretic run. In addition, the electrophoretic map and submaps of the whole tryptic digest and its concanavalin A fractions, respectively, allowed the elucidation of the microheterogeneity of the glycoprotein. Also, capillary zone electrophoresis proved suitable for the mapping of the oligosaccharide chains cleaved from the glycoproteins by endoglycosidase digestion. The oligosaccharides cleaved from human and bovine alpha 1-acid glycoprotein were analyzed after derivatization with 2-aminopyridine, which allowed their sensitive detection by on column UV absorption. The separation was best achieved when 0.1 M phosphate solution, pH 5.0, containing 50 mM tetrabutylammonium bromide was used as the running electrolyte. The effect of the organic salt on separation was attributed to ion-pair formation and/or hydrophobic interaction.     

Increased selectivity for electrochromatography by dynamic ion exchange

The separation of compounds with similar mobilities is expect to be difficult with capillary zone electrophoresis. Increased selectivity is shown for compounds of this type when other modes of separation are added to the system. A capillary coated with a hydrophobic stationary phase is shown to be a dynamic ion exchanger when a quaternary ammonium compound is added to the running buffer. Compounds are shown to have a decrease in retention when the concentration of the buffer ions is increased. The effect of adding an organic modifier and the influence of the concentration of the surface active reagent are also studied.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecylsilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecysilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

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.     

β‐cyclodextrin‐ionic liquid polymer based dynamically coating for simultaneous determination of tetracyclines by capillary electrophoresis

Tetracyclines are a group of broad spectrum antibiotics widely used in animal husbandry to prevent and treat diseases. However, the improper use of tetracyclines may result in the presence of their residues in animal tissues or waste. Recently, great attention has been drawn towards the green solvents ionic liquids. Ionic liquids have been employed as a coating material to modify the electroosmotic flow in capillary electrophoresis. In this study, a functionalized ionic liquid, mono‐6‐deoxy‐6‐(3‐methylimidazolium)‐β‐cyclodextrin tosylate, was synthesized and used for the simultaneous separation and quantification of tetracyclines by capillary electrophoresis. Good separation efficiency could be achieved due to the multiple functions of β‐cyclodextrin derived ionic liquid, including the electrostatic interaction, the hydrogen bonding, and the cavity structure in β‐cyclodextrin ionic liquid which can entrap the tetracyclines to form inclusion complex. After optimization, baseline separation achieved in 25 min with the running buffer consisted of 10 mmol/L, pH 7.2 phosphate buffer and 20 mmol/L β‐cyclodextrin ionic liquid. The satisfied result demonstrated that the β‐cyclodextrin ionic liquid is an ideal background electrolyte modifier in the separation of tetracyclines with high stability and good reproducibility. And it is an effective strategy to design and synthesize specific ILs as additive applied in separation.     

Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis

Polyelectrolytes are widely used in capillary electrophoresis as coating agents of silica capillaries to prevent adsorption phenomena and improve the repeatability of peptide and protein analysis. A systematic study of the coating experimental conditions has been carried out to optimize coating stability and performance. The main experimental parameters studied were the type and concentration of polyelectrolytes used in several monolayer and multilayer coatings, the ionic strength of coating and stabilizing solutions, and the procedures used for coating and capillary storage. Electroosmotic flow magnitude, direction and repeatability were used to monitor coating stability. Coating ability to limit adsorption was investigated by monitoring variations of migration times, time-corrected peak areas and separation efficiency of test peptides. Capillary-to-capillary and batch-to-batch reproducibility was also studied. In addition, the separation performance of polyelectrolyte coatings were compared to those obtained with bare silica capillaries.