Mt Mariëtte Ackermans

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.

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.

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

Determination of some drugs by micellar electrokinetic capillary chromatography : the pseudo-effective mobility as parameter for screening

Abstract In contrast to capillary zone electrophoresis, micellar electrokinetic capillary chromatography can be applied to the determination of compounds that are unchanged and almost insoluble in water. As a screening parameter, the pseudo-effective mobility is to be preferred to the capacity factor k ′ because it can be calculated if t MC is unknown, and because it gives a better indication of whether components can be separated or not. Special attention should be paid, however, to the composition of the sample solution. The use of organic solvents to dissolve the sample can influence the separation enormously. Calibration graphs were constructed for some drugs and as an example dapsone in tablets was determined.

Isotachophoresis with electroosmotic flow: open versus closed systems

If isotachophoretic (ITP) experiments are carried out in open systems, an electroosmotic flow (EOF) will act on the ITP systems. In ITP experiments with EOF in open systems four modes can be distinguished, viz., anionic, cationic, reversed anionic and reversed modes. The applicability of these modes depends strongly on the velocity of the EOF. Examples of separations are given showing some typical features of these modes. Detailed consideration of a mathematical model for ITP with EOF showed that this model is identical with that of ITP without EOF.

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.

Isotachophoresis in open systems : Problems in quantitative analysis

So far, isotachophoretic (ITP) analyses have been carried out in commercially available or laboratory-made ITP instruments, generally in closed systems. At present several instruments are commercially available, originally designed for capillary zone electrophoresis with open capillaries, and these instruments can also be used for ITP. If ITP experiments are carried out using such apparatus, however, an electroosmotic flow (EOF) will act on the ITP system. The velocity of the EOF strongly varies with the choice of the leading electrolyte and terminating electrolyte and also the composition of the sample. Hence the reproducibility in quantitative analyses is a serious problem. For quantitative experiments at least an internal standard must be used to correct for undesirable fluctuations in the EOF and irreproducible injections. Better results can be obtained by effectively suppressing the EOF by using additives such as methylhydroxyethylcellulose. Results of quantitative experiments using the Beckman P/ACE System 2000 HPCE are presented, showing some of the problems in quantitative analyses with ITP in open capillaries.