Jl Jozef Beckers

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.

Determination of absolute mobilities, pK values and separation numbers by capillary zone electrophoresis : effective mobility as a parameter for screening

Migration times or apparent mobilities can never be used for the identification of ionic species in capillary zone electrophoresis if an electroosmotic flow (EOF) is present, because the velocity of this flow varies considerably with the “state” of the capillary. From the migration times of the EOF and the ionic species, the effective mobilities can be calculated. These effective mobilities are nearly independent of the concentrations of the sample ionic species. Although a large excess of one of the sample components can cause different values of the calculated effective mobility, they are reproducible if the matrix has a constant composition and in this way effective mobilities can be used for screening purposes. In the determination of effective mobilities the use of a “true” EOF marker is extremely important. If effective mobilites are measured in two different electroyte systems at different pH values, at which the degrees of dissociation differ sufficiently, the absolute ionic mobilities and pK values of ionic species can be calculated. Values obtained in this way, for mobility and pK were compared with data obtained isotachophoretically, showing good agreement. Theoretically, the separation number in zone electrophoresis, defined as the number of components that can be separated within a unit of mobility, varies widely with the mobilities of the ionic species and the EOF. Experimentally obtained values of the separation number are significantly lower than the calculated values owing to the method of injection, temperature effects during analysis and amount of sample. For low-molecular-weight ionic species separations are possible if the effective mobilities differ by about one unit for cations and 0.2–0.3 for anions. A negative wall charge (at higher pHs) diminishes the separation number of cations considerably, especially on applying small diameter capillaries, owing to attractive forces between the wall and analytes.

Quantitative analysis in capillary zone electrophoresis with conductivity and indirect UV detection

An interesting point in quantitative capillary zone electrophoresis, when applying conductivity detection or indirect UV detection with non-UV absorbing components, is the existence of a relationship between effective mobilities and peak area, independent of the kind of ionic species. This relationship is theoretically considered for fully ionized monovalent ions resulting in a linear relationship, passing through the origin, between temporal peak area and the product of a correction factor (dependent only on the effective mobilities of the ionic species) and migration time for an equimolar sample composition. A good correlation between theory and practice could be established by applying experimental measured data.

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.

Isotachophoresis : The qualitative separation of cation mixtures

Abstract The formulae needed for computations on buffered systems are given. Using these formulae, a computer program was evaluated and calculations were correlated with the results of some experiments. The possibility of the simultaneous separation of cations depends on the differences between the effective mobilities, which can be affected by the choice of pH, counter-ion and solvent. The qualitative simultaneous separation of cations was studied in eight electrolyte systems using methanol and water as solvents. In each system, simultaneous separations of 7–10 cations were possible, which could be increased by combining electrolyte systems.

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.

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.

UV detection in capillary zone electrophoresis Peaks or dips - that is the question

Abstract Applying capillary zone electrophoresis with UV detection, both UV-absorbing and UV-transparent components can be present in electropherograms as negative peaks (dips) or as positive peaks. Starting from Kohlrauschs regulation function, derived for fully ionized monovalent ionic constituents and under the assumption that the molar absorptivities of the UV-absorbing components are identical, eight different cases can be distinguished and in several cases components can occur both as peaks or as dips depending on their mobilities and those of the co- ions of the system. Applying background electrolytes containing two co-ions, system peaks are present, with a mobility that is between the mobilities of the two co-ions and determined by the concentration ratio of these two co-ions. In the background electrolytes studied, containing the co-ions potassium and histidine, UV-transparent sample components with a mobility higher than that of the system peak migrate as a positive peak, whereas UV-transparent components with lower mobilities migrate as negative peaks. System peaks themselves can also be peaks or dips depending on the sample composition. Sample peaks in the vicinity of system peaks interact with the system peaks through which both sample and system peaks are enlarged and quantitative properties are lost. Similar phenomena can be measured for anions in background electrolytes containing the co-ions phenylacetate and acetate, indicating that these phenomena are probably not associated with adsorption phenomena of cations on the fused-silica surface.

Isotachophoresis with two leading ions and migration behaviour in capillary zone electrophoresis: II. Migration behaviour in capillary zone electrophoresis

Abstract During a zone electrophoretic analysis, components can migrate in the isotachophoretic mode. If, for example in anionic separation in capillary zone electrophoresis an anion with a high effective mobility is present in the sample at a very high concentration, it migrates forwards into the background electrolyte, separates from the other components and forms an isotachophoretic system with two leading ions together with the anion of the background electrolyte. Some of the sample components will therefore migrate in the isotachophoretic mode for the greater part of the analysis time. Because in the isotachophoretic mode the zone lengths are constant, very small sample zone lengths will give extremely high plate numbers. Of course, migration times will vary strongly depending on the composition of the sample.

Effect of sample stacking on resolution, calibration graphs and pH in capillary zone electrophoresis

For the determination of components present in samples at very low concentrations, large injection volumes have to be applied in order to introduce a detectable amount of the analytes in capillary zone electrophoresis (CZE). To obtain a good resolution, the sample analytes have to be concentrated in narrow bands and therefore sample stacking is often applied. Sample stacking can lead to an increase in the electroosmotic flow and extra peak broadening during the analysis, through which the gain in resolution will be lost. Further, the presence of different electrolytes in the capillary can cause pH shifts. In this paper a model is given for the calculation of migration times of components applying sample stacking, and the effects of sample stacking in CZE on resolution, calibration graphs and pH are discussed