Detection and quantification of chitosan aggregates by pressure-assisted capillary zone electrophoresis

An approach to the detection and determination of chitosan aggregates in acetic acid solutions is proposed using pressure-assisted capillary zone electrophoresis. Processes of chitosan aggregation are studied depending on the composition of dispersion medium and storage time. The presence of several species of positively charged chitosan aggregates is revealed for the first time. Particle sizes in the range 20–2500 nm are determined by scanning electron microscopy and static and dynamic light scattering. The dependence of the shape of electropherograms on particle size distribution obtained under the same conditions is found. A trend to changing electrophoretic mobility depending on the size of the aggregate is observed, which enables the approximate evaluation of the polydispersity of chitosan solutions. Chitosan is used for the effective dynamic modification of capillaries, which does not require the introduction of a modifier into the background electrolyte.     

Stability study of somatropin by capillary zone electrophoresis

Human Growth Hormone (hGH) is a monomeric 22 kilo Dalton (kDa), 191 amino acid protein with an isoelectric point (pI) close to pH 5, produced in the anterior pituitary gland. High level production of somatropin (recombinant hGH) is done in Escherichia coli (E. coli) to meet the demand and to avoid possible Creutzfeldt-Jacob disease (CJD).The present study was initiated on the basis of results from post-marketing control of all somatropin preparations on the Norwegian market. The samples consisted of preparations presented as somatropin in solution, and some freeze-dried preparations were also included for comparison.The present study showed a significant degree of degradation of somatropin in solution. Deamidation increased over time for preparations in solution, as well as for freeze-dried preparations after dissolution. Preparations in solution showed high content of deamidated and cleaved forms. Freeze-dried preparations after dissolution and storage showed high content of deamidated forms, but low content of cleaved forms. Also, in one preparation, an unknown peak was detected in the electropherogram from capillary zone electrophoresis (CZE), eluting after the principal peak, in front of the Gln-18 somatropin peak.     

毛细管电泳电迁移扩散的定量研究

电迁移扩散是毛细管电泳中影响区带展宽的重要冈素之一。本文利用毛细管电泳电导检测装置对电迁移扩散进行了定量研究,提出一种评价电迁移扩散的方法。在该方法中引入一种描述电迁移扩散的物理量,它可通过几个易测的实验参数来计算。它不仅考虑到电迁移进样的歧视效应,而且考虑到样品浓度对电迁移扩散的影响。采用毛细管电泳中常用的两种缓冲液(磷酸和甲酸-组氨酸)对该方法进行了实验验证,结果表明它与理论预测符合良好。     

Application of capillary zone electrophoresis to the study of interactions betweenBacillus subtilis tryptophanyl-tRNA synthetase and tRNATrp

Summary The application of capillary zone electrophoresis to the study of interactions betweenBacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) and tRNA^Trp is described. Significant changes in peak shape of tRNA^Trp incubated with TrpRS indicated the occurrence of interactions between TrpRS and tRNA^Trp in pH 8.0 Tris-HCl buffer containing 0.1 mmol L⁻¹ EDTA and 1 mmol L⁻¹−5 mmol L⁻¹ mgCl₂. Addition of Mg²⁺ decreased the electrophoretic mobility of tRNA^Trp, which illustrated that conformation of tRNA^Trp depended on Mg²⁺. The dissociation constant of the TrpRS-tRNA^Trp complex was estimated to be 0.63 μmol L⁻¹ at 25°C in buffer solution.     

Stacking in capillary zone electrophoresis

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.     

Application of electrokinetic supercharging capillary zone electrophoresis to rare-earth ore samples

Capillary zone electrophoresis (CZE) using electrokinetic injection (EKI) with transient isotachophoresis, which was named “electrokinetic supercharging-CZE ” (EKS-CZE), was applied to model samples of rare-earth ores (xenotime and monazite) and a real sample of monazite ore, the abundance of the components being greatly different among samples. When simple EKI was applied, separation and detection of rare-earth ions with smaller mobilities than the major component became difficult with an increase of the content of the major component. In contrast, when EKS-CZE was applied, the minor components (Er, Tm, Yb) with contents less than 0.025% (rare-earth/total rare-earth) could be analyzed. The analytical results for minor components in monazite ore agreed with those obtained by isotachophoresis–particle-induced X-ray emission (ITP–PIXE) and inductively coupled plasma atomic emission spectrometry (ICP-AES) with errors less than 17%. The sample amount required for analysis was 9 μg which is 200-fold smaller than that used in ITP-PIXE analysis. Analytical sensitivity of EKS-CZE was comparable with that of ICP-AES.     

Application of capillary zone electrophoresis to the analysis and to a stability study of nitrite and nitrate in saliva

The applicability of capillary zone electrophoresis for the determinations of nitrite and nitrate was studied. Using direct UV detection the limit of detection values of the analytes were 0.14 and 0.21 microg/mL, respectively. The developed method was found to be useful to directly determine nitrite, nitrate and thiocyanate in saliva. It was found that adjusting the pH of the sample to 11 and storing the saliva at 4 degrees C was adequate to make constant the nitrite/nitrate ratio in saliva samples at least 7 days.     

Hemoglobin A2 quantification by capillary zone electrophoresis

Hemoglobin A2 (HbA2) comprises about 2.2% of the total hemoglobin in the erythrocytes. The separation and quantitation of this minor hemoglobin by capillary electrophoresis (CE) using an arginine Tris buffer is described. Some of the variables affecting the accuracy and precision of HbA2 quantification are investigated. Furthermore, the quantification of this hemoglobin by CE is compared to that of a microcolumn chromatography method. The CE method is better suited than the microcolumn method for measuring HbA2 in the sickle cell trait.     

Analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis

The ability of capillary zone electrophoresis in the development of analytical methods devoted to the quality control of the thiol drug penicillamine is shown. Using 50 mM phosphate running buffer (pH 2.5), good quantitations of underivatized penicillamine and its disulfide were achieved; detection at 200 nm allowed checking the presence of the disulfide impurity in pharmaceuticals. The use of 1,1-[ethenylidenebis(sulfonyl)]bis-benzene as a thiol specific reagent resulted in an increased sensitivity for the quantitation of D-penicillamine (limit of detection at 200 nm wavelength was 1.5 microM). Introducing beta-cyclodextrin as chiral selector in the running buffer, enantioseparation of D-L-penicillamine was obtained; for this purpose (+)-camphor-10-sulfonic acid, a chiral ion-pairing reagent, was found to be an essential additive in obtaining a baseline separation. The resulting enantioseparative system was validated in order to evaluate the presence of the toxic L-penicillamine enantiomer in pharmaceutical samples.     

The study of polyoxometalates formation using capillary zone electrophoresis

The formation process of polyoxometalates [PMo₁₂O₄₀]^3? and [PMo_{12 ? x}V_xO₄₀]^?3?x has been studied in aqueous solutions of 0.1 M malonate buffer at pH 2.8–3.0 using CZE. Two different approaches, pre‐capillary and in‐capillary, were examined and compared. In precapillary mode, the reaction mixture of the reactants and reaction products was injected into the capillary followed by the separation procedure. In in‐capillary mode, the sequential input of the reagents and running electrolyte into the capillary and the species separation occurs simultaneously. The optimal parameters of in‐capillary separation were established as functions of applied voltage and the length of the intermediate buffer zone between the reagents in the capillary. As a result the best‐compromise conditions for the separation of the mixtures containing the reactants, intermediates, and reaction products, in order to achieve the best efficiency, symmetry, and peak areas, were achieved at ?18 kV and the input parameter of 900 mbar·s. It was also shown that in‐capillary mode is more informative than pre‐capillary mode for studying the complex compound formation process.     

System zones in capillary zone electrophoresis

When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.     

Separation of nicotine metabolites by capillary zone electrophoresis and capillary zone electrophoresis/mass spectrometry

The use of capillary zone electrophoresis (CZE) and capillary zone electrophoresis/mass spectrometry (CZE/MS) has been demonstrated, in principle, for the separation of nicotine and nicotine metabolites. The buffer system developed for separation and detection by CZE/UV was modified for use in CZE/MS analysis. Several of the metabolites are isobaric and tandem mass spectrometric (MS/MS) techniques have been used to differentiate such analytes.     

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.     

毛细管电泳在火炸药研究中的应用

近年来,毛细管电泳以其高效、快速、微量的特点,在火炸药分析领域被广泛应用并迅速发展。本文从毛细管区带电泳、胶束电动毛细管色谱、毛细管电色谱、芯片毛细管电泳、微乳毛细管电动色谱这五种毛细管电泳的模式出发,结合本实验室的最新研究进展,综述毛细管电泳在火炸药研究中的应用。     

Application of capillary isotachophoresis and capillary zone electrophoresis to the determination of inorganic ions in food and feed samples

The purpose of this review is to summarise critically the possibilities of capillary isotachophoresis and capillary zone electrophoresis for the determination of inorganic ions in food and feed samples. This article covers papers published since 1977.     

Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis

Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.     

System zones in capillary zone electrophoresis.

This paper brings an overview of system zones (SZs) in capillary zone electrophoresis (CZE) and their effects upon the migration of zones of analytes. It is shown that the formation and migration of SZs is an inherent feature of CZE, and that it depends predominantly on the composition of an actual background electrolyte (BGE). One can distinguish between stationary SZs and migrating SZs. Stationary SZs, which move due to the electroosmotic flow only, are induced in any BGE by sample injection. Migrating SZs may be induced by a sample injection in BGEs which show at least one of the following features: (i) BGE contains two or more co-ions, (ii) BGE has low or high pH whereby H+ or OH- act as the second co-ion, and (iii) BGE contains multivalent weak acids or bases. SZs do not contain any analyte and show always BGE-like composition. They contain components of the BGE only and the concentrations of these components are different from their values in the original BGE. Providing that some of the ionic components of the BGE are visible by the detector, the migrating SZs can be detected and they are present as system peaks/dips in the electropherogram. It is shown that a migrating SZ may be characterized by its mobility, and examples are given how this mobility can depend on the composition of the BGE. Further, the effects of the migrating SZs (either visible or not visible by the detector) upon the zones of analytes are presented and the typical disturbances of the peaks (extra broadening, zig-zag form, schizophrenic behavior) are exemplified and discussed. Finally, some conclusions are presented how to cope with the SZs in practice. The proposed procedure is based on the theoretical predictions and/or measurements of the mobilities of SZs and on the so-called unsafe region. Then, such operational conditions should be selected where the unsafe region is outside of the required analytical window.     

A new approach to dynamic deactivation in capillary zone electrophoresis

This paper describes a simple means of obtaining high resolution separations of basic proteins at a pH below their pl. Small amounts of a cationic fluorosurfactant are added to the running buffer. A positively charged wall is thereby obtained which will repel positively charged proteins. The particular chemistry of fluorosurfactants is believed to enhance the efficiency of the deactivation. Examples are presented of the separation of some model proteins, including a human insulin-like growth factor (IGF-I), and a misfolded by-product of the growth factor.     

Indirect photometric detection in capillary zone electrophoresis

An indirect photometric detection method is described which is based on the use of an absorbing co-ion as the principal component of the background electrolyte. The zones of non-absorbing ionic species are revealed by changes in light absorption due to charge displacement of the absorbing co-ion. Theoretical considerations are given for selecting a suitable absorbing co-ion to achieve a high sensitivity of detection.The role of electromigration dispersion is illustrated by experiments and the effects of the differences in the effective mobilities of sample ions and that of the absorbing co-ion are discussed. The highest sensitivity can be achieved for sample ions having an effective mobility close to the mobility of the absorbing co-ion. In such a case, the concentration of the sample component in its migrating zone can be high while electromigration dispersion is still negligible. The useful dynamic range of the detection is then limited by the linearity and noise of the detector, the former parameter being given mostly by the shape of the on-column detection cell. The best sensitivities can be obtained in low-concentration background electrolytes containing a co-ion with high absorption at a given detection wavelength.It is shown that indirect photometric detection can be useful for detecting substances that have no optical absorption in the UV and/or visible region, provided that the composition of the background electrolyte is selected correctly.     

Split-flow injector for capillary zone electrophoresis

A simple construction of a split-flow injector eliminating some common problems connected with the use of such devices is described. It consists of a low-pressure pump, an injection valve and a delivery tube in which the separating capillary inlet is fixed. The sample is injected without moving the separating capillary inlet and without interrupting the applied voltage. The grounded electrophoretic electrode is close to the injection valve so that all metal parts of the injector are kept at a sufficiently low potential. Minimum length and small internal diameter of delivery tube minimizes additional sample zone broadening. The effects of some experimental parameters, such as the position of the separation capillary inlet with respect to the background solution flow direction and background solution flow-rate are experimentally studied. The injector was tested primarily for the electrokinetic injection.