František Foret

Indirect photometric detection in capillary zone electrophoresis

Abstract 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.

Selection of the background electrolyte composition with respect to electromigration dispersion and detection of weakly absorbing substances in capillary zone electrophoresis

Abstract A characteristic feature of the migration of sample ions in zone electrophoresis is the formation of either fronting or tailing zones. This so-called electromigration dispersion can generally be suppressed by keeping the sample concentration more than two orders of magnitude below the concentration of the background electrolyte (BGE). In capillary zone electrophoresis the low sample concentration decreases the reliability of on-column UV absorbance detection, especially when substances having low molar absorption coefficients are to be detected. By the proper selection of the electrophoretic mobility of the background electrolyte co-ion, both electromigration dispersion and detection can be optimized. The migration behaviour of phenyllactic acid and four phenyl derivatives of acetic acid was studied in buffered electrolytes with different mobilities and concentrations of the BGE co-ions. For a better understanding of zone broadening, a simplified model of the electromigration together with some model calculations is presented.

Determination of halofuginone in feedstuffs by the combination of capillary isotachophoresis and capillary zone electrophoresis in a column-switching system.

A method has been developed for the determination of the coccidiocidic drug halofuginone in feedstuff concentrates which is based on the combination of capillary isotachophoresis and capillary zone electrophoresis in the column-switching mode. The high load capacity of the isotachophoretic step and high sensitivity of the zone electrophoretic step enabled analysis of up to 25 microliters of sample solution containing as little as 10(-8) M halofuginone with excellent reproducibility (R.S.D. about 1%). Attention was paid to the possibility of the existence of transient local isotachophoresis in the zone electrophoretic step, and experimental and theoretical methods of revealing zones migrating isotachophoretically in the background electrolyte were shown.

Recent devepolments in isotachopresis

Abstract This paper is summarizing the contributions to the analytical capillary isotachophoresis published during the period 1981–1984. It characterizes the present state of the method and covers theory, fundamental analytical aspects, instrumentation and applications. Special attention was payed to the fundamental analytical aspects, and a detailed discussion is given of the selection of electrolyte systems, stability of zones and separability of substances. The present commercial instrumentation is also briefly described.

Electric sample splitter for capillary zone electrophoresis

Abstract A method for reproducible sampling of small quantities of sample for high-performance capillary electrophoretic separations was developed. The method is based on the principle of the splitter. The sample migrates electrophoretically in two electrical circuits and the splitting ratio is given by the ratio of the corresponding electric currents. Very small aliquots of the sample in the form of short zones (pulses) can be introduced corresponding to direct sampling of nanolitre volumes.

Simple method for generation of dymanic pH gradient in capillary zone electrophoresis

Abstract A dynamic pH gradient extends the separation power of zone electrophoresis to mixtures of substances with widely differing p K values. A simple method for the generation of dynamic changes in the pH of the background electrolyte in the separation capillary is described. It is based on a controlled modification of the composition and, thus, of the pH of an electrolyte in the electrode chamber at the injection end of the separation capillary during the analysis. The higher the concentration of H + in this electrode chamber, the higher is the electromigration flow of H + into the capillary and the lower is the pH of the actual background electrolyte therein. The utilization of this principle is exemplified by the separation of eleven purine and pyrimidine bases with p K values ranging from 6.0 to 1.9.

Applicability of dynamic change of pH in the capillary zone electrophoresis of proteins.

A method to extend the separation power of CZE is described. The method is based on the separation of sample components at two different pH values during one separation run, and involves dynamic buffering of the pH inside a separation capillary by controlling the flow of H+ ions from the anodic electrode chamber. By changing the anolyte in the chamber, a dynamic pH step is generated, which proceeds rapidly along the capillary and establishes the required new pH value. The use of the method has been demonstrated by the cationic separation of a model mixture of proteins.

Selection of electrolyte systems in isotachophoresis

Abstract An attempt to formulate a strategy for the selection of electrolyte systems in capillary isotachophoresis is presented based on the discussion of (i) correct migration of zones, (ii) operational interval of pH, (iii) separability of substances and (iv) resolution of neighbouring species. The correct migration of zones is closely related to the buffering properties of the electrolyte systems. Such systems may be classified as buffered, buffer-free and non-buffered. Each of these systems covers a certain operational pH region which must coincide with the pH region required by the sample so as to ensure reasonable values of the effective mobilities of the sample species within the safe isotachophoretic region. Zone existence diagrams constructed for any of these systems may be advantageously used to reveal both the domain of substances that give correct stable zones and the operational pH region of the system. The problem of separability of substances may be solved by using both the mobility data and pH in the respective zones. The resolution of neighbouring species in a given electrolyte system may be estimated by a graphical procedure involving the mobility curves of these species and the respective existence line of the zone existence diagram.