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.

Peak deformation in cationic analysis caused by system zones.

In electrophoretic processes, often zones migrate through the separation compartment, with a composition different from that of the background electrolyte (BGE) but which do not contain, however, any component of the sample mixture. These zones migrate with a mobility mainly determined by the composition of the BGE and are called system zones (SZs). If these SZs are visible in electropherograms they are called system peaks (SPs). If sample components have a mobility close to that of a SZ, the separation process can be disturbed and the sample peak shapes are deformed. SZs can appear applying BGEs containing more co-ionic species or if BGEs are used at high or low pH. Recently, the existence of SZs has been described applying BGEs containing weak multivalent anionic species. In this paper, the diverse kinds of system zones, are discussed for cationic systems and the effect of invisible SZs on separations is shown. As an example of a weak multivalent cation, the behavior of the divalent cation histamine is studied, which can be used as co-ion in BGEs for the separation of cations in the indirect UV mode. Applying BGEs containing histamine, SZs are visible in the electropherograms and there existence could also be established theoretically by the use of SystCharts. A mathematical model for the calculation of the mobility of SZs is verified and it has been shown that an unsafe region with a mobility window of msp +/- 10% can be indicated, for the separation of fully ionized sample components.

Success and failure with phthalate buffers in capillary zone electrophoresis.

Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).

Why robust background electrolytes containing multivalent ionic species can fail in capillary zone electrophoresis.

In this paper it is demonstrated that system peaks are induced by multivalent weak ionic species in background electrolytes. Their existence is derived from SystCharts and from Peak Shape Diagrams and the theory is confirmed experimentally. If analytes are present in a sample, with mobilities approximately equal to the mobility of a system peak, they interact, resulting in a strong increase of electromigration dispersion. This leads to strong peak broadening, peak deformation and a loss of resolution. Typical background electrolytes containing multivalent ionic species, e.g. phosphate and phthalate buffers, often reported to be robust electrolytes, are therefore not always universally applicable and can fail for the application of specific analytes. This paper reports a systematic study of the above phenomena and shows both theoretical and experimental results for background electrolytes containing phosphoric acid and phthalic acid.

Sample stacking in capillary zone electrophoresis: principles, advantages and limitations.

The principles of stacking procedures are described and their properties are discussed, including the fundamentals of the behavior of zone boundaries and the consequences of the self-correcting properties of boundaries in moving boundary electrophoresis, isotachophoresis, and zone electrophoresis. Further, the diverse possibilities of stacking procedures and the unavoidable destacking are described, and several examples of practically applied stacking procedures are given, besides many references to applications. Some limitations in the use of stacking procedures are discussed. The paper is arranged in such a way that it can serve both as an introduction into the field and as a reference overview.

Window optimization in isotachophoresis superimposed on capillary zone electrophoresis.

A sample stacking procedure to which a specific combination of electrolyte solutions is applied is isotachophoresis (ITP) superimposed on capillary zone electrophoresis (CZE), a so-called ITP/CZE system. In ITP/CZE some components migrate in an ITP fashion on top of a background electrolyte, and the other analytes migrate in a zone electrophoretic manner. For such a system, the leading electrolyte consists of a mixture of an ionic species, L1, of high mobility (the leading ion of the ITP system), an ionic species, L2, of low mobility (the coions of the CZE system), and a buffering counter-ionic species, whereas the terminating solution only contains the ionic species L2 and the buffering counterions. The zones of the components migrating in the ITP/CZE mode are sharp owing to the self-correcting properties of the zones and the concentrations of the L1 ions of the system. Mobility windows can be calculated, indicating which ions can migrate in the ITP/CZE mode. In this article mobility windows are calculated by applying both strong and weak acids as L1 and L2 ions and it appears that mobility windows can be optimized by chosing different ratios of L1 and L2 as well as different pH values. It is possible to construct very narrow mobility windows, and thereby choose which component of a sample solution can be concentrated, and to what concentration, in a very selective way. The big advantage of ITP/CZE compared with applications such as transient ITP and transient stacking is that the stacked sample ionic species migrate in the ITP mode during the whole experiment; furthermore, they do not destack. Experimentally obtained electropherograms validate the calculated mobility windows for the ITP/CZE mode.

Steady-state models in electrophoresis: from isotachoporesis to capillary zone electrophoresis.

Although all electrophoretic techniques are closely allied and controlled by the same rules, we often distinguish between steady-state and dynamic models in the modeling of electrophoretic processes, whereby steady-state models are applied for isotachophoresis (ITP) and dynamic models are applied for other electrophoretic processes, wherein a steady-state is not reached. This paper shows how, starting from a mathematical model for the steady-state in ITP, mathematical models can be derived for several modifications of ITP and that even nonsteady-state processes can be estimated by a repeated application of a steady-state model. In this way all parameters in sample zones in capillary zone electrophoresis (CZE), through which temporal electropherograms can be simulated, can be calculated. Realistic simulations can be obtained for zone electrophoretic processes, for which the electrodispersive character is the dominating peak broadening mechanism, and simulated electropherograms resemble measured electropherograms concerning migration times, peak shapes, fronting or tailing character and the question of peaks and dips.

Determination of propionate in bread using capillary zone electrophoresis.

A method for the determination of propionate in bread is described. The propionate was extracted from the bread with a repeated extraction procedure and measured using capillary zone electrophoresis in the indirect UV mode applying a background electrolyte of 0.005 M Tris adjusted at pH 4.6 by adding benzoic acid. Using laboratory-baked bread containing known amounts of sodium propionate, recoveries of ca. 95% could be established, validating the method.

Determination of aminoglycoside antibiotics in pharmaceuticals by capillary zone electrophoresis with indirect UV detection coupled with micellar electrokinetic capillary chromatography.

Aminoglycoside antibiotics can be determined by capillary zone electrophoresis (CZE) with indirect UV detection in the anionic mode with a reversed electroosmotic flow (EOF) by addition of FC 135 to the background electrolyte. The effective mobilities of thirteen aminoglycoside antibiotics were determined as a function of pH. Applying CZE with indirect UV detection in the anionic mode and reversed EOF coupled with micellar electrokinetic capillary chromatography with the cationic surfactant cetyltrimethylammonium bromide, both neutral and charged antibiotics can be determined in combined pharmaceuticals. As an example, neomycin and hydrocortisone were determined in Otosporin eardrops.

Determination of sulphonamides in pork meat extracts by capillary zone electrophoresis.

For sixteen sulphonamides the effective mobility was measured as a function of pH and from the effective mobilities determined in two different electrolyte systems the pK value and mobility at infinite dilution were calculated. A pH of 7.0 was found to be the optimum pH for the separation for both standard mixtures and mixtures of sulphonamides dissolved in pork meat extracts. For the determination of the sulphonamides in pork meat only a very simple pretreatment consisting of extraction with acetonitrile and centrifugation is suitable, as the matrix effects at pH 7.0 do not affect the separation. Calibration graphs for five sulphonamides were constructed, and regression coefficients of at least 0.999 were obtained. The limit of detection for the method varies from 2 to 9 ppm for a pressure injection time of 10 s (injection volume ca. 18 nl) using a Polymicro Technology capillary of length 116.45 cm, distance between injection and detection 109.75 cm and I.D. 50 microns.

Comparison of isotachophoresis, capillary zone electrophoresis and high-performance liquid chromatography for the determination of salbutamol, terbutaline sulphate and fenoterol hydrobromide in pharmaceutical dosage forms.

Reversed-phase high-performance liquid chromatography (RP-HPLC), isotachophoresis (ITP) and capillary zone electrophoresis (CZE) were applied to the determination of salbutamol, terbutaline sulphate and fenoterol hydrobromide in commercially available pharmaceutical dosage forms. The comparison showed that especially with the use of ITP, high concentrations of other charged sample components can disturb the separation process. If special attention is paid to ensure a complete separation, all methods give comparable results. For the regression lines of the calibration graphs, regression coefficients of at least ca. 0.999 and nearly zero intercepts are obtained with relative standard deviations of ca. 1-2% for peak area or zone lengths. By applying the different techniques, often different components of the sample matrix can be detected, i.e., a more complete impression of the sample composition can be obtained by using all the three techniques.