Peaks in capillary zone electrophoresis: fact or fiction

Non-steady-state electrophoretic processes can be estimated by a repeated application of a steady-state model based on the electroneutrality equation, the modified version of Ohm's law, and the mass balances of the co- and counterions. With such a mathematical model, all parameters in sample zones in capillary zone electrophoresis (CZE) can be calculated. The relationships between the calculated parameters for sample zones in CZE, such as the pH, concentrations of co- and counterions, and the ratio E1m1/E2m2 versus the mobilities of both anionic and cationic analytes can be visualized in a SystChart, a set of eight panels, for a given background electrolyte (BGE). All properties of a zone, such as the fronting/tailing character and the question of peaks/dips can be read from such a SystChart. Applying n coions, n-1 discontinuities are present in such a SystChart, indicating the presence of system peaks applying that BGE. For BGEs with one coion, no system peaks (discontinuities) exist at moderate pH values. SystCharts calculated for BGEs with a low pH do show discontinuities, however, which indicates that system peaks are present in electropherograms applying BGEs at low pH. Experimentally, it is shown that system peaks are indeed present in electropherograms applying BGEs with one coion at low pH and the mobilities of the system peaks generally increase with decreasing pH. Hydrogen ions seem to act as a second coionic species. Of course, these system peaks are only visible in the UV signal if the BGE has UV-absorbing properties.     

The preparation of background electrolytes in capillary zone electrophoresis: golden rules and pitfalls

In this article the methodology of the design of suitable background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is described. The principal aspects of the role of a BGE in CZE are discussed with respect to an appropiate migration behavior of analytes, including the transport of the electric current, the buffering of pH, the Joule heat, the electro-endosmotic flow (EOF) and the principal migration and detection modes. The impact of the composition of the BGE upon migration and detection is discussed. It is shown that the total concentration of the BGE is a principal factor and the adjustment of migrating analyte zones according to the Kohlrausch regulating function (KRF) is the principal effect in most of the sample stacking techniques. The number of co-ions and their properties are of key importance for peak shapes of the analyte peaks and for the existence of system zones. The detection of UV-transparent analytes may advanteously be done in the indirect UV mode, by using UV-absorbing co-ions, however, both peaks and dips may be expected in the UV trace in case of multiple co-ionic BGEs. Properties of BGEs can be predicted applying mathematical models and it is shown that with SystCharts, predictions can be given concerning the existence of system zones, detection modes and the peak shapes of analytes for a given BGE. Practical examples of methodological considerations are given in the design of suitable BGEs for four principal combinations of migration and detection modes. The properties of the BGEs selected are exemplified with experimental results. Golden rules are summarized for the preparation of suitable BGEs in CZE.     

System peaks in capillary zone electrophoresis of anions with negative voltage polarity and counter-electroosmotic flow

The system peaks that often appear on electropherograms in anion separation by CE with indirect spectrophotometric detection, negative voltage polarity and cathodic EOF are studied. The system peaks are shown to correspond to the zones with the changed concentration of the BGE constituents; they appear while the zone of each analyte anion passes through the outlet end of the capillary and are transported to the detector by EOF. An equation is suggested for predicting migration times of the system peaks with an error of 1%. The ratios of the system peak area to the analyte peak area are found to amount to 20%. It is shown that it is possible to avoid overlapping of the system peaks and analyte peaks by controlling the EOF velocity owing to hydrodynamic pressure. Using the mathematical simulation of CE shows that the system peaks and baseline shift can result from changing the transference numbers of the BGE ions and analyte ions at the capillary edge. The cases when the system peak may be incorrectly identified as the peak of analyte ion are considered. In order to avoid such errors, some practical recommendations are given.     

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.     

Modeling the zeta potential of silica capillaries in relation to the background electrolyte composition

A theoretical relation between the zeta potential of silica capillaries and the composition of the background electrolyte (BGE) is presented in order to be used in capillary zone electrophoresis (CZE). This relation is derived on the basis of the Poisson-Boltzmann equation and considering the equilibrium dissociation of silanol groups at the capillary wall as the mechanism of charge generation. The resulting model involves the relevant physicochemical parameters of the BGE-capillary interface. Special attention is paid to the characterization of the BGE, which can be either salt or/and buffer solutions. The model is successfully applied to electroosmotic flow (EOF) experimental data of different aqueous solutions, covering a wide range of pH and ionic strength. Numerical predictions are also presented showing the capability of the model to quantify the EOF, the control of which is relevant to improve analyte separation performance in CZE.     

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.     

Calibrationless quantitative analysis by indirect UV absorbance detection in capillary zone electrophoresis: the concept of the conversion factor

A new, fast and efficient procedure is described for the simultaneous quantitative analysis of various non-UV absorbing species in a sample, by capillary zone electrophoresis with indirect UV absorbance detection. The procedure is based on the concept of the conversion factor (CF). The CF of an analyte is defined as the ratio of the measured temporal peak area and the product of its migration time and transfer ratio (TR). Thus defined, the CF is of general validity for all analytes separated and detected in a given background electrolyte (BGE), since it has the same value for the same amounts of various analytes. If a sample is enriched with a known concentration of a standard component and analyzed by CZE, the CF of this standard component can be calculated and then the concentrations of all other analytes can be determined, without the use of any calibration graph. The individual TRs can be determined a priori from tabulated ionic mobilities and pK values of the analytes and of the constituents of the BGE or, for strong analytes, by using experimental data from the electropherogram of the analysis itself. The practical procedure of the analysis includes enrichment of the sample with a known quantity of a suitable standard and a single CZE run of the resulting mixture. The injected volume does not need to be known and thus the procedure also eliminates the injection error. The proposed procedure has been verified experimentally and reproducible and accurate values were obtained by using four different CZE apparatus for the analyses of standard mixtures of cations in three different BGEs.     

Eigenmobilities in background electrolytes for capillary zone electrophoresis: III. Linear theory of electromigration

A mathematical model of capillary zone electrophoresis (CZE) based on the conception of eigenmobilities, which are the eigenvalues of a matrix M tied to the linearized governing equations is presented. The model considers CZE systems, where constituents, either analytes or components of the background electrolyte (BGE), are weak electrolytes--acids, bases, or ampholytes. There is no restriction on the number of components nor on the valence of the constituents nor on pH of the BGE. An electrophoretic system with N constituents has N eigenmobilities. In most BGEs one or two eigenmobilities are very close to zero so their corresponding eigenzones move very slowly. However, there are BGEs where no eigenmobility is close to zero. The mathematical model further provides: the transfer ratio, the molar conductivity detection response, and the relative velocity slope. This allows the assessment of the indirect detection, conductivity detection and peak broadening (distortion) due to electromigration dispersion. Also, we present a spectral decomposition of the matrix M to matrices allowing the assessment of the amplitudes of system eigenpeaks (system peaks). Our model predicted the existence of BGEs having no stationary injection zone (or water zone, gap, peak, dip). A common practice of using the injection zone as a marker of the electroosmotic flow must fail in such electrolytes.     

Chiral Separation by Capillary Zone Electrophoresis Used Cyclodextrins and Their Derivatives as Chiral Selector

Capillary zone electrophoresis (CZE) is a very pronising analytical technique for the optical isomer resolution of the compounds studied. The drawbacks of the techniques such as HPLC [1] were sophisticated stationary phases and/or the relatively high quantity of the chiral agent in the mobile phase, which do not exist in CZE. The capillary electrophoresis (CE) method can offer advantages on lower consumption of analyte and background electrolyte (BGE), shorter analysis time, and higher efficiencies [2-3]     

逍遥丸的毛细管电泳指纹图谱的建立

采用毛细管区带电泳法建立了逍遥丸(Xiaoyao Pill, XYP)的毛细管电泳指纹图谱(CEFP)。运用正方形优化法,以色谱指纹图谱分离量指数(RF)为优化的目标函数,对建立指纹图谱的实验条件进行了优化,确定了最佳背景电解质(BGE)溶液50 mmol/L硼砂-50 mmol/L磷酸氢二钠-150 mmol/L磷酸二氢钠-50 mmol/L碳酸氢钠(1:1:1:5, v/v/v/v; pH 7.40)、紫外检测波长228 nm、运行电压12 kV、重力进样25 s (高度14 cm)的分离检测条件。采用未涂层石英毛细管(70 cm×75 μm,有效分离长度57 cm)分离,以咖啡酸色谱峰为参照,确定13批逍遥丸样品的21个共有指纹峰。通过聚类分析确定用其中10批样品生成对照CEFP,以此为标准用系统指纹定量法鉴别13批逍遥丸的质量,结果显示: S3号样品的化学成分数量和分布比例不合格,S10和S12号样品含量明显偏高,其余批次质量均合格。所建立的正方形优化法操作简便,适用于中药的毛细管区带电泳BGE的选择;所建立的逍遥丸CEFP具有较好的精密度和重现性,可以为逍遥丸的质量控制提供新的参考。     

三角形法和四面体法优化选择毛细管区带电泳背景电解质

建立了两种高效、快速的毛细管区带电泳背景电解质(BGE)的优化方法三角形优化法和四面体优化法。以色谱指纹图谱指数F和色谱指纹图谱相对指数Fr作为评价毛细管电泳分析系统的目标函数,以雪莲药材水提取液为样品,考察一定浓度的硼砂、硼酸、磷酸氢二钠和磷酸二氢钠溶液按三角形优化法和四面体优化法构成背景电解质时对样品的分离情况,通过添加有机改性剂和调节pH进行再优化。用三角形法优化出以50 mmol/L硼砂-含3%乙腈的150 mmol/L磷酸二氢钠(体积比为1∶1)作为BGE时分离效果最佳,用四面体法优化出以50 mmol/L硼砂-150 mmol/L磷酸二氢钠-200 mmol/L硼酸(体积比为1∶1∶2,用0.1 mol/L氢氧化钠调pH 8.55)作为BGE时分离效果最佳,分别获得28个和25个电泳峰。所建立的方法操作简捷,适用于中药材水提取液或醇提取液的毛细管区带电泳BGE的选择。     

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Quantification of buserelin in a pharmaceutical product by multiple-injection CZE

An efficient and rapid separation method based on reversed-polarity multiple-injection CZE (MICZE), has been developed for the quantification of buserelin in a pharmaceutical product. The determinations were performed by serially injecting five standard solutions of buserelin (50-300 microg/mL) and one reference analyte into a Polybrene-coated capillary. All the samples contained goserelin, an analog peptide to buserelin, as internal standard (IS). Immediately after pressure injection, the applied sample plugs were subjected to electrophoresis for 2 min at -25 kV. Consequently, each sample plug became isolated from its neighboring plugs by the BGE, composed of 100 mM phosphate-triethanolamine buffer at pH 3.0 containing 10% v/v ACN. During separation the individual sample components migrated at similar velocities and as distinct zones through the capillary giving 24 peaks, 12 from the analyte and the IS and 12 from the sample matrix. The buserelin content of the pharmaceutical product was determined to be 0.94 +/- 0.05 mg/mL, which is only a slight deviation from the declared concentration (1 mg/mL).     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.     

Reproducible and efficient separation of aggregatable zein proteins by CZE using a volatile background electrolyte

Zein proteins are a complex mixture of polypetides that belong to the alcohol-soluble storage proteins group (prolamines) in corn. These proteins constitute about 50-60% of the total endosperm protein and are classified in different groups on the basis of differences in their solubility and sequence. Among them, zein proteins are considered the majority group showing a high tendency to aggregate what makes their analysis by any analytical method very difficult. Thus, CZE of these proteins requires the use of very complex BGEs noncompatible with online ESI-MS analysis. The aim of this work was to find a new BGE for the CZE separation of zein protein fully compatible with ESI-MS while providing further light on the complex CZE separation of aggregatable proteins. Thus, it is demonstrated in this work that efficient and reproducible CZE separations of zein proteins can be achieved by using a BGE composed of water, ACN, formic acid and ammonium hydroxide. Besides, it is shown that zein analysis is significantly improved by including the effect of an ammonium gradient during their separation. It is experimentally verified that the ammonium gradient can easily be achieved in CZE by either working with a sample zone with a low concentration of ammonium and a BGE with a high concentration, or conversely, working with a sample zone with high ammonium concentration and a BGE with low concentration of ammonium, giving rise in both cases to a significant improvement in the CZE separation of these proteins. It is demonstrated that this procedure can give rise to efficiency improvements of up to 20-fold in the CZE separation of zein proteins. Under optimized conditions, 20 proteins could be separated with average efficiencies higher than 400 000 theoretical plates/m. Some possible explanations of this effect are discussed including stacking, protein-capillary wall adsorption, protein solubility and protein-salt interactions.     

Eigenmobilities in background electrolytes for CZE. V. Intensity (amplitudes) of system peaks

We present a mathematical model of CZE based on the concept of eigenmobilities - the eigenvalues of matrix M tied to the linearized governing equations of electromigration, and the spectral decomposition of matrix M into matrices of amplitudes P(j). Any peak in an electropherogram, regardless of whether it is an analyte peak or a system peak (system zone), is matched with its matrix P(j). This enables calculation of the peak parameters, such as the transfer ratio and the molar conductivity detection response (which give the indirect detection signal and the conductivity detection signal, respectively), when the initial disturbance caused by the injection of the sample is known. We also introduce new quantities, such as the generalized transfer ratio and the conductivity response of system zones, and show how the amplitude (intensity, area) of the analyte peaks and the system peaks can be calculated. We offer a free software, PeakMaster (http://www.natur.cuni.cz/gas), which yields this information in a user-friendly way.     

Experimental assessment of electromigration properties of background electrolytes in capillary zone electrophoresis

Electromigration dispersion (EMD) properties of background electrolytes (BGEs) used in capillary zone electrophoresis (CZE) are of key importance for the success of an analysis. The knowledge of these properties may serve well for the prediction of the asymmetry of peaks of analytes, for the prediction of unsafe regions where a strong interference of system zones may be expected, and for the selection of optimum conditions where the analytes of interest may give sharp and practically symmetric peaks. Present theories enable one to calculate and predict EMD properties of many BGEs but there is also a lot of BGEs that are beyond the present theoretical models as far as their composition and equilibria involved are considered. This contribution brings a method for assessment of EMD properties of any BGE from easily accessible experimental data. The method proposed is illustrated by model examples both for cationic and anionic separations. Imidazole acetate, histamine acetate, and histidine acetate served as model BGEs for cationic separations; as the model BGE for anionic separations, Tris-borate and sodium-borate BGEs have been selected since these buffers are frequently used and borate is well-known for its complexing equilibria in aqueous solutions.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

Electromigration peak dispersion of isotachophoretic protein zones during capillary zone electrophoresis

The relationships between electromigration dispersion (EMD) and on-line isotachophoresis-capillary zone electrophoresis (ITP-CZE) are described for several basic model proteins and interleukin-6 (rhIL-6). During CZE separation of the highly concentrated analyte zones which were generated during the initial ITP step EMD evolves from intrinsic differences in conductivity between the focused ITP zones and the leading electrolyte. Nearly triangular peaks with a sharp front and diffuse rear side were observed. An electromigration dispersion factor (F_EMD) was introduced to measure peak asymmetry. EMD of individual peaks was shown to increase with the absolute amount of the respective analyte injected and with analyte mobility. Good linearity was observed when F_EMD was plotted against protein mobility (r > 0.95). The slope of the graphs describing this relationship increased with the amount of analyte injected. The influence of EMD on the separation efficiency of neighboring peaks appeared to be less pronounced than expected. Consecutive release from the ITP-stack during transition from ITP to CZE might be an explanation for this observation.     

On-line column coupled isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: Varenicline and its metabolite in human urine

A new highly advanced analytical approach, based on two-dimensional column coupled CE (ITP-CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed, evaluated and applied in biomedical field in the present work. Capillary isotachophoresis (ITP) coupled on-line with capillary zone electrophoresis (CZE) used in hydrodynamically closed separation system was favorable for increasing the sample load capacity, increasing the analyte concentration, and removing the deteriorative highly conductive major matrix constituents. These factors considerably reduced the concentration limits of detection (cLOD) and external sample preparation (comparing to single column CZE), and, by that, provided favorable conditions for the mass spectrometry (enhanced signal to noise ratio, reproducibility of measurements, working life of MS). Here, the CZE–ESI combination provided more effective interfacing than ITP–ESI resulting in both a higher obtainable intensity of MS detection signal of the analyte as well as reproducibility of measurements of the analyte’s peak area. The optimized ITP-CZE–ESI-QqQ method was successfully evaluated as for its performance parameters (LOD, LOQ, linearity, precision, recovery/accuracy) and applied for the direct identification and ultratrace (pg mL⁻¹) determination of varenicline and, in addition, identification of its targeted metabolite, 2-hydroxy-varenicline, in unpretreated/diluted human urine. This application example demonstrated the real analytical potential of this new analytical approach and, at the same time, served as currently the most effective routine clinical method for varenicline.