Separation of the unique proteins of wheat protein fractions by capillary electrophoresis

Summary The wheat maturation process was monitored by high-performance capillary electrophoresis. The different protein components of the albumin, globulin, gliadin and glutenin fractions from the Osborne extraction procedure were analysed. The wheat sample was a Hungarian winter wheat, cultivar Martonvásári 23. The protein fractions were analysed by capillary zone electrophoresis using a low pH phosphate buffer containing a polymeric additive and organic modifiers. The albumins and gliadins as well as glutenins showed a characteristic pattern of development during the maturation process. For these fractions the development occurred at different stages of maturation. The formation of protein fractions of wheat at different stages of maturation—and thus the entire maturation process—could be well characterised by high-performance capillary electrophoresis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September, 1–3, 1999     

Prediction of wheat dough W and P/L inflation test parameters by capillary zone electrophoresis of a protein extract followed by multivariate regression

A procedure for the evaluation of the wheat flour hardness, based on capillary electrophoresis of a protein extract in an isoelectric acidic buffer, was developed. The 13 flour samples were extracted twice, and two injections of each extract were made. Separations were performed in a background electrolyte (BGE) containing 40 mM aspartic acid, 6 M urea, and 0.5% hydroxyethylcellulose at 60 degrees C. Using the normalized and corrected areas of 79 peaks and peak groups, a partial least squares regression (PLS1) model was able to predict the flour strength or dough deformation work (W) and the dough tenacity/extensibility ratio (P/L) (Alveograph parameters) with an average relative standard deviation in the predictions of +/- 3% and +/-8%, respectively. These values amounted to a +/- 6-8% and +/- 11% with multiple linear regression (MLR) and PLS1 models constructed by measuring only 12 peaks and peak group areas on the electropherograms.     

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.     

Use of zwitterionic buffer additives to improve the separation of proteins in capillary zone electrophoresis

In CZE, the adsorption of the proteins on the capillary wall is a common problem. This paper describes the simple method of utilizing zwitterionic buffer additives to improve the separation of proteins in untreated fused silica capillaries at neutral pH. Three kinds of zwitterion are evaluated in the separation of acidic, neutral, and basic proteins, including their effect on protein efficiency, mobility, separation, and resolution; the difference between the effects of the different additives are also highlighted. The method has proved to be a possible means of reducing protein adsorption, especially for basic proteins.     

Approaches to optimisation of precision in capillary electrophoresis

Summary A study has been performed to obtain insight into the relative importance of critical factors affecting the repeatability of hydrodynamic injections in CE. Precision was measured for repeated analysis of a test mixture containing two acidic compounds.The use of an internal standard was clearly shown to improve precision especially when peak area precision was poor. It is suggested that precision is maximised by employing a combination of a constant temperature, an appropriate electrolyte system, an internal standard, long injection times, and high sample concentrations. Other factors are discussed, but are classified as having only a minor impact.     

A covalent modified hydrophilic capillary for enhanced capillary electrophoresis of biopolymers

δ-Gluconolactone was covalently coupled to aminopropyl derivatized capillary,which created hydrophilic brushes on the inner wall of the capillary.The coated capillary was shown to generate a stable electroosmotic flow(EOF) in the investigated pH range of 2.0-9.0 and to suppress effectively the adsorption of proteins.And it enabled separation of some biopolymer mixtures including basic proteins,DNA and tryptic digested bovine serum albumin(BSA) within 15 min with efficiencies up to 450,000 plates/m.The in...     

Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.     

毛细管电泳法对铝胁迫下小麦根部有机酸的直接测定

在进行植物抗铝逆性研究中,从植物的培养液中发现从根部分泌出的有机酸与铝结合从而实现无毒化是其抗铝逆性机理的重要依据,但是其分析需繁杂的前处理过程。而本文用毛细管电泳法直接测定了铝胁迫下培育的小麦根中的铝和有机酸,确认了主要积累的是苹果酸和柠檬酸。发现随着提高培养液中的铝浓度,根部的铝量也相应增加。同时,虽然根中的柠檬酸量无明显变化,但苹果酸被诱导增加。在pH7．8的Na3PO4缓冲溶液中加入表面活性剂溴化十六烷基三甲铵作为电泳溶液体系,紫外检测波长214nm,可以有效地分离检测以阴离子形式存在的上述有机酸。探讨了植物抗铝胁迫研究中简便易行的植物根部有机酸的直接测定方法。     

Influence of temperature control in capillary electrophoresis

We have investigated the influence of capillary temperature on migration time and peak area and have evaluated different cooling systems. It was found that for applied voltages below 15 kV (i.e. those most frequently used) temperature control effectively improves peak area reproducibility but has less effect on migration time.     

An evaluation of the use of capillary electrophoresis to monitor trace drug residues following the manufacture of pharmaceuticals

Summary The novel use of capillary electrophoresis to the important and developing area of monitoring possible drug residues on pharmaceutical manufacturing equipment is reported. The CE method is applicable to a wide range of basic drugs with sensitivity as low as 25ng/ml (equivalent to 8×10^–8 M). This sensitivity is equivalent to that obtainable for HPLC for the drugs tested and is obtained by employing a combination of a wider bore capillary with low wavelength UV detection. Preliminary evaluation of the method performance shows acceptable precision, linearity, sensitivity and accuracy. Features of the method compared to HPLC include simplicity, ease of method transfer, reductions in analysis set-up time, and reduced costs of solvents and columns.     

Simultaneous separation of nitrate, nitrite and ammonium by capillary electrophoresis

Summary A capillary electrophoretic method for the simultaneous separation of nitrate, nitrite and ammonium has been developed. Direct (NO₃ ⁻, NO₂ ⁻) and indirect (NH₄ ⁺) UV detection at 214 nm in conjunction with electromigration sampling from both ends of the capillary was used. Two electrolyte systems based on imidazole-sulfate (pH 3.8) and copper(II)-ethylenediamine-chloride (pH 8.0) were investigated. Optimisation of the experimental parameters such as electrolyte concentration, pH, nature of the counter-ion, was studied. The method permits excellent separation of three nitrogen species in only 4 min. The analytical performance of both electrolyte systems is compared in terms of migration time and peak area repeatability and detectability. Alkaline electrolyte shows a better overall analytical performance.     

Bioanalytical applications of fast capillary electrophoresis

Capillary electrophoresis is unique among liquid-phase separations in its utility for fast separations. Development of technology such as optical-gating, flow-gating, and microfabrication has allowed separations on the millisecond time scale to be developed. The fast separation times place great demands on the detector systems, frequently requiring detection limits below 1 amol to be practical. The development of such fast separations has opened many new applications not previously feasible for separations-based methods. This has included real time chemical monitoring, detection of short-lived species such as protein conformers or non-covalent complexes, and rapid multi-dimensional separations. Other applications currently being developed include high-throughput assays for clinical laboratories or screening combinatorial libraries. This review covers recent developments in the instrumentation for fast CE and some of the applications.     

Purity control of oxytetracycline by capillary electrophoresis

The applicability of capillary electrophoresis for the purity control of oxytetracycline (OTC) was investigated. OTC is a broad-spectrum antibiotic belonging to the group of the tetracyclines. Several related substances can be present due to fermentation or degradation, such as 4-epioxytetracycline, -apooxytetracycline, β-apooxytetracycline, anhydrooxytet racycline, 2-acetyl-2-decarboxamidooxytetracycline, tetracycline and 4-epitetracycline. Using fused-silica capillaries, the influence of buffer type, buffer pH and buffer concentration were investigated. In all cases 1 mM EDTA was added to prevent metal-ion complexation. The influence of the buffer counter-ion type was examined. Consequently, some instrumental parameters were changed such as capillary length and diameter as well as capillary temperature and applied voltage. The following method is finally proposed: fused-silica capillary, l (effective length) = 38 cm, L (total length) = 44 cm, 50 μm I.D.; buffer, sodium carbonate 20 mM-EDTA 1 mM, pH 11.25; voltage, 10 kV; temperature, 10°C. Linearity, limit of detection and limit of quantitation were determined as well as the relative standard deviations for all the analytes involved. This method is less selective then existing liquid chromatographic methods but it may be used as a complementary tool in purity control and stability studies.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

Analysis of pyrimidine derivatives by capillary electrophoresis

Summary A capillary electrophoretic method has been developed for the determination of the main product as well as of by-products in technical samples of substituted pyrimidines. Both zone electrophoresis and micellar electrokinetic chromatography have been used for the separation employing electrolytes consisting of borate buffers (pH 9 to 9.4) with or without sodium dodecylsulfate. Optimization of separation selectivity could be achieved by addition of up to 20% 2-propanol or methanol to the carrier electrolyte. Quantification by internal standards resulted in relative standard deviations between 0.2 and 0.8%. By-products could be analyzed down to levels of 0.1% in technical samples.

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Analyse von Pyrimidinderivaten mitteles Kapillarelektrophorese

Zusammenfassung Für die Bestimmung von Haupt- und Nebensubstanzen in technischen Proben von substituierten Pyrimidinen wurde ein kapillarelektrophoretisches Analysenverfahren entwickelt. Sowohl Zonenelektrophorese als auch mizellare elektrokinetische Chromatographie mit Trägerelektrolyten bestehend aus Boratpuffern (pH 9 bis 9.4) mit oder ohne Natriumdodecylsulfat wurden für die Trennung eingesetzt. Eine Optimierung der Trennselektivität war durch die Zugabe von bis zu 20% 2-Propanol oder Methanol zum Trägerelektrolyten möglich. Quantifizierung mittels interner Standards ergab relative Standardabweichungen zwischen 0.2 und 0.8%. Nebenprodukte konnten in technischen Proben bis zu Gehalten von 0.1% analysiert werden.

     

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R_f), which provided information about the binding strength and the overall charge of the protein‐ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.     

Nonaqueous and aqueous capillary electrophoresis of synthetic polymers

In this work, the use of capillary electrophoresis (CE) to analyze synthetic polymers is reviewed including works published till February 2004. The revised works have been classified depending on the CE mode (e.g., free solution capillary electrophoresis, capillary gel electrophoresis, etc.) and type of buffer (i.e., nonaqueous, aqueous and hydro-organic background electrolytes) employed to separate synthetic macromolecules. Advantages and drawbacks of these different separation procedures for polymer analysis are discussed. Also, physicochemical studies of complex polymer systems by CE are reviewed, including drug release studies, synthetic polyampholytes, dendrimers, fullerenes, carbon nanotubes and associative copolymers.     

The use of a short-end injection procedure to achieve improved performance in capillary electrophoresis

Summary Minimum capillary lengths on commercial instruments are fixed and cannot be decreased further. To effectively reduce the capillary length used for separation the sample can be injected from the end of the capillary nearest the detector. This procedure is known as a short-end injection and can reduces analysis times by at least two-thirds compared to conventional injections. The time reduction benefits are shown in rapid separations of basic drugs, drug-related impurities and chiral compounds. Short-end injections, in combination with both increased electrolyte strength and reduced voltage are an effective approach to reducing the detrimental impact of high sample solution ionic strength. They can also lead to improved resolution by increasing stacking effects and reducing peak tailing. Peak area and migration time precision obtained are shown to be equivalent to those obtained for conventional injection procedures. It is concluded that short-end injections should be considered for routine operation as they are a useful means of reducing analysis time, increasing sensitivity, decreasing buffer depletion effects. They also allow use of higher electrolyte strengths which can improve resolution and reduce peak tailing, and can overcome significant problems which occur when analysing samples containing high salt contents.     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。