Position and intensity of system (eigen) peaks in capillary zone electrophoresis

The intensity of system (or eigen) peaks encountered in capillary zone electrophoresis (CZE) can be predicted by considering mass balances for each of the analyte constituents and each of the constituents in the background electrolyte (BGE). As a result of coherence, in each zone the proportions in which the constituent concentrations vary are fixed; they are determined by the composition of the BGE and the nature of the analyte constituent (if present) and described as eigenvectors of a transport matrix. Considering the effect of an injection, the mass balances for all constituents can be satisfied only via the intensity of each zone. This leads to an n-equations, n-unknowns problem, with the intensities as the unknowns and the mass balances as equations.The latter can be easily solved to obtain the intensities of the zones, of analytes as well as of system peaks. In this work the approach has been applied to CZE systems with two co-ions in the BGE, and experimental results have been compared to the predictions obtained from the model. Agreement was seen to be reasonable, but the quantitative comparison often failed, probably due to experimental difficulties.     

Identification of peaks in capillary zone electrophoresis based on actual mobilities.

A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.     

Theory of system zones in capillary zone electrophoresis

In the last years, it has been shown that the formation and migration of system zones is an inherent feature of capillary zone electrophoresis (CZE) and that it depends predominantly on the composition of an actual background electrolyte (BGE). In most of the currently used BGEs, the SZs are invisible by the UV absorbance detection system, however, the comigration of SZs with the zones of analytes deteriorates the analytical performance of CZE and may be fatal for its utilization. Therefore, the theoretical predictions of the existence and migration of SZs is of key importance for the expediency of CZE. This is a review of the theoretical treatments of SZs which reveals the origin and the properties of SZs and shows how to cope with them. Also, a table of some typical BGEs is presented where the existence and mobilities of SZs are given.     

Determination of ebrotidine metabolites in overlapping peaks from capillary zone electrophoresis using chemometric methods

This paper illustrates the possibilities of chemometric methods in the resolution and quantification of various compounds in overlapping peaks from capillary electrophoresis. Ebrotidine and most of its metabolites were efficiently separated by capillary zone electrophoresis (CZE) in a fused-silica capillary. However, the procedure was not suitable for the physical separation of the three less ionizable metabolites, which comigrated and overlapped with the electroosmotic flow signal. Multivariate curve resolution based on an alternating least squares procedure was used for their mathematical resolution. For such a purpose, data obtained in the CZE system with a diode array detector, which consisted of UV spectra registered over time, were analyzed. The ebrotidine metabolites were successfully resolved and quantified in synthetic mixtures and urine samples.     

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.     

Prediction of the separation of phenols by capillary zone electrophoresis

The effect of pH and ionic strength on the migration of neutral acids in capillary zone electrophoresis (CZE) has been studied for several phenols. The mobilities of the phenols and the efficiency of the capillary have been related to the studied factors. The mobility can be related to the pH of the running buffer through the mobility of the phenolate ion, and the conditional acidity pK value of the phenol at the working ionic strength. This allows prediction of the migration of the phenol, solely from its pK_a^′ value (literature pK_a corrected for the ionic strength of the solution) and mobility of the anion, which can be easily calculated from the mobility at a basic pH value and the pK_a^′ value. Combination of the predicted mobility with the efficiency allows estimation of the resolution of the consecutive peaks obtained for a mixture of phenols. This method has been tested for two groups of phenols of environmental interest.     

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.     

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.     

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.     

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis

Poor sensitivity and low phase ratio are the main drawbacks of open tubular capillary electrochromatography (OTCEC). The poor sensitivity results from the use of narrow bore size capillary, whereas the low phase ratio, which limits the separation capability, is caused by the limited surface area of conventional capillary. Two strategies may be useful to overcome these disadvantages. First, an extended light path (ELP) capillary, which has a bubble cell at the detection point, is used to improve the sensitivity. Secondly, an etched capillary of a 1,000-fold increased surface area is used to enhance the phase ratio. In this work, use of an ELP capillary and an etched capillary in OTCEC was evaluated with a chiral stationary phase of avidin prepared with the physical adsorption method. With a 20 microm I.D. ELP capillary with a 150 microm bubble cell, the peak height was enhanced by 4-10-fold and the corrected peak area was increased by 12-fold relative to a 20 microm I.D. conventional capillary. However, the peak efficiency and resolution decreased noticeably. The phase ratio on the etched capillary was slightly enhanced, by a factor of 1.64 relative to an unetched capillary. Consequently, the separation capability was slightly improved. The increase in the phase ratio was much lower than that expected from the increase in surface area, the reason for which is probably the reduced density of surface silanol group and the generation of nitrogen-containing groups due to the etching process.     

On-line multichannel Raman spectroscopic detection system for capillary zone electrophoresis

An on-line multichannel Raman spectroscopic detection system for capillary zone electrophoresis using a charge-coupled device as the detector is described. Resonant, near-resonant and non-resonant excitation Raman spectroscopies are employed. The 400 cm-1 spectral window provides adequate information to identify resolved and unresolved compounds. The use of analyte velocity reduction to allow increased data acquisition times is described. With near-resonant enhancement, the technique is shown to allow detection of 500 attomoles of methyl orange from a 5-nl injection volume, corresponding to 1 x 10(-7) M.     

Artificial neural networks for quantification in unresolved capillary electrophoresis peaks

The application of the combination of experimental design (ED) and artificial neural networks (ANNs) for the quantification of overlapped peaks in capillary zone electrophoresis is described. When the total separation cannot be achieved by separation techniques, the use of ED-ANN can be a suitable approach. The unstability of EOF causes peak shift that has to be corrected in order to apply ED-ANN methods. In this work, normalization procedure of electropherograms with consequent application of ANNs for quantification purpose was developed. Both, spectra and electropherograms can be used as multivariate data. In general, both kinds of data were found to be suitable for unresolved peaks quantification by ED-ANN approach.     

Stacking and discontinuous buffers in capillary zone electrophoresis

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.     

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.     

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).     

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.     

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.     

Polypyrrole-coated capillaries for capillary zone electrophoresis

Fused-silica capillaries are permanently coated by silanization with 3-{[3-(N-pyrrole)-2-hydroxypropyl]amino}propyltriethoxysilane followed by oxidative polymerization of the pyrrole moieties with iron (III) or peroxodisulfate in the presence of chloride, perchlorate, or dextransulfate as anions. This approach allows to modulate the EOF in its magnitude as well as in its direction. With the small anions chloride and perchlorate, the EOF is reversed below pH 5 while with the large dextransulfate polyanions (DS) the EOF is relatively constant over the pH range from 2.5 to 9.4. This can be of advantage at low pH, at which the EOF of uncoated capillaries is close to zero. Application for separation of some herbicides is shown. The lifetime of PP-modified capillaries is satisfactory: the decrease in EOF is less than 3% during 80 analyses (160 min) and less than 5% over three months of storage. The reproducibility of capillary modification is about 5% (RSD of EOF).     

Fraction collector for capillary zone electrophoresis

An instrument is described which is capable of collecting fractions from a capillary zone electrophoresis apparatus. The fraction collector is characterized in terms of discretely collecting the separated components of a multi-component sample. In addition, the fraction collector permits the study of the effect of capillary zone electrophoresis on the biological activity of alpha-chymotrypsin.     

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.