Pavel Coufal

Optimization of the high-frequency contactless conductivity detector for capillary electrophoresis.

Two constructions of the high‐frequency contactless conductivity detector that are fitted to the specific demands of capillary zone electrophoresis are described. The axial arrangement of the electrodes of the conductivity cell with two cylindrical electrodes placed around the outer wall of the capillary column is used. We propose an equivalent electrical model of the axial contactless conductivity cell, which explains the features of its behavior including overshooting phenomena. We give the computer numerical solution of the model enabling simulation of real experimental runs. The role of many parameters can be evaluated in this way, such as the dimension of the separation channel, dimension of the electrodes, length of the gap between electrodes, influence of the shielding, etc. The conception of model allows its use for the optimization of the construction of the conductivity cell, either in the cylindrical format or in the microchip format. The ability of the high‐frequency contactless conductivity detector is demonstrated on separation of inorganic ions.

Methacrylate monolithic columns of 320 μm I.D. for capillary liquid chromatography

Monolithic capillary columns (320 microm I.D.) were prepared for capillary liquid chromatography (CLC) by radical polymerization of butylmethacrylate (BMA) and ethylenedimethacrylate (EDMA) in the presence of a porogen solvent containing propan-1-ol, butane-1,4-diol and water. The influence of the contents of the porogen solvent and EDMA in the polymerization mixture on the monolith porosity and column efficiency was investigated. The composition of the polymerization mixture was optimized to attain a minimum HETP of the order of tens of microm for test compounds with various polarities. The separation performance and selectivity of the most efficient monolithic column prepared was characterized by van Deemter curves, peak asymmetry factors and Walters hydrophobicity and silanol indices. It was demonstrated that the 320-microm I.D. monolithic column exhibited CLC separation performance similar to that observed for 100- and 150-microm I.D. monolithic columns reported in the literature; moreover, the 320-microm I.D. column was easier to operate in CLC and exhibited a higher sample loadability.

Optimization of background electrolytes for capillary electrophoresis I. Mathematical and computational model.

A mathematical and computational model is introduced for optimization of background electrolyte systems for capillary zone electrophoresis of anions. The model takes into account mono- or di- or trivalent ions and allows also for modeling of highly acidic or alkaline electrolytes, where a presence of hydrogen and hydroxide ions is significant. At maximum, the electrolyte can contain two co-anions and two counter-cations. The mathematical relations of the model are formulated to enable an easy algorithmization and programming in a computer language. The model assesses the composition of the background electrolyte in the analyte zone, which enables prediction of the parameters of the system that are experimentally available, like the transfer ratio, which is a measure of the sensitivity in the indirect photometric detection or the molar conductivity detection response, which expresses the sensitivity of the conductivity detection. Furthermore, the model also enables the evaluation of a tendency of the analyte to undergo electromigration dispersion and allows the optimization of the composition of the background electrolyte to reach a good sensitivity of detection while still having the dispersion properties in the acceptable range. Although the model presented is aimed towards the separation of anions, it can be straightforwardly rearranged to serve for simulation of electromigration of cationic analytes. The suitability of the model is checked by inspecting the behavior of a phosphate buffer for analysis of anions. It is shown that parameters of the phosphate buffer when used at neutral and alkaline pH values possess singularities that indicate a possible occurrence of system peaks. Moreover, if the mobility of any analyte of the sample is close to the mobilities of the system peaks, the indirect detector signals following the background electrolyte properties will be heavily amplified and distorted. When a specific detector sensitive on presence of the analyte were used, the signal would be almost lost due to the excessive dispersion of the peak.

Capillary electrophoresis of inorganic cations

The capillary electrophoresis (CE) methods of determination of inorganic cations are critically surveyed, with emphasis on the most recent works. The metal ion complexation is treated in detail, as well as the detection techniques. The advantages and drawbacks of CE compared to HPLC are briefly considered and typical examples of application to various matrices are given in a table.

Rapid capillary zone electrophoresis along short separation pathways and its use in some hyphenated systems: a critical review.

The contemporary research in natural sciences and technologies and its applications to a wide range of human activities are characterized by two primary features: (a) The immense volume of concrete findings is growing more and more rapidly. (b) The greatest development takes place at the boundaries among scientific disciplines, e.g., at the boundaries between mathematics and physics, chemistry, and biology. Consequently, successful solutions of research problems require teamwork, with intense and continuous cooperation among the team members specialized in various fields. To attain this, the specialists in individual disciplines must possess a sufficient overview and understanding of the theoretical and experimental approaches used in the cooperating fields and of the characteristics of all the results contributed. Analytical chemistry is a very good example of this situation. It is an applied discipline that widely uses the results of not only the general chemistry but also of other fields, primarily mathematics, informatics, and physics, and progressively more those of biology, imitating or directly utilizing the processes occurring in living organisms. On the other hand, most scientific and technological projects cannot be solved without analytical data on the identity and amounts of a great variety of substances present in various materials and on the distribution of these substances in space and time. Therefore, the analytical field is very wide and its boundaries with other disciplines are quite diffuse. Analytical chemists are, or should be, members of very many research teams and should continuously cooperate with the other team members who, on the other hand, should sufficiently understand the possibilities and limitations of analytical procedures, the character of the analytical data, and their reliability. The requirements on the amount of analytical data and on the quality of analytical information are rapidly increasing. The most common analytical tasks involve: (a) Identification and sufficiently selective, sensitive, and reliable simultaneous determinations of the components of large sets of very similar substances (e.g., in biochemistry, biology, and medicine); (b) Monitoring of rapid changes in the presence and concentration of a component or several components in complex matrices (e.g., in biology, theoretical and practical medicine, or environmental monitoring, or in studies of reaction mechanisms and kinetics in many fields); (c) Determinations of spatial distribution of substances in various matrices (e.g., in material sciences, solid-phase physics, geology, etc.); (d) Identification and determination of ultratrace amounts of various analytes (e.g., in physics, electronics, or material sciences); (e) Simple, rapid and inexpensive analyses of very large numbers of samples (e.g., in practical medicine, environmental monitoring, or food industry); (f) Completion at a satisfactory technical level, possibly with extensive miniaturization and automation, and with acceptable financial costs. Analytical chemistry employs a very wide range of experimental approaches and their combinations to meet all these requirements. Among the most important ones are the uses of high-performance separations (primarily gas and liquid chromatography or capillary electrophoresis), hyphenated with various measuring techniques (mostly mass spectrometry and other spectrometric measurements) and many more complex combinations. This review concentrates on the very young and highly promising area of capillary electrophoresis along short separation pathways, because it has so far been covered by the literature much less than the classical fields of high-performance separations, because it is developing rapidly and makes it possible to carry out rapid, reliable, relatively technically simple, and not very expensive analyses of very complex samples, primarily in the fields of biology, medicine, and environmental studies. Chem. Rev. 2009, 109, 4487–4499 4487

Monolithic columns based on a poly(styrene-divinylbenzene-methacrylic acid) copolymer for capillary liquid chromatography of small organic molecules.

A very simple and readily performed method is described for the preparation of poly(styrene-divinylbenzene-methacrylic acid) monolithic columns for capillary liquid chromatography. The effect of the methacrylic acid content on the morphological and chromatographic properties has been investigated. Methacrylic acid is shown to be essential for isocratic separations of small organic analytes by capillary liquid chromatography. Column efficiencies of about 28,000 theoretical plates/m have been obtained for all the test compounds. The batch-to-batch and run-to-run repeatability of the retention times is better than 1.5%.

Effect of association with sulfate on the electrophoretic mobility of polyarginine and polylysine.

Domains rich in cationic amino acids are ubiquitous in peptides with the ability to cross cell membranes, which is likely related to the binding of such polypeptides to anionic groups on the membrane surface. To shed more light on these interactions, we investigated specific interactions between basic amino acids and oligopeptides thereof and anions by means of electrophoretic experiments and molecular dynamics simulations. To this end, we measured the electrophoretic mobilities of arginine, lysine, tetraarginine, and tetralysine in sodium chloride and sodium sulfate electrolytes as a function of ionic strength. The mobility was found to be consistently lower in sodium sulfate than in sodium chloride at the same ionic strength. The decrease in mobility in sodium sulfate was greater for tetraarginine than for tetralysine and was larger for tetrapeptides compared to the corresponding free amino acids. On the basis of molecular dynamics simulations and Bjerrum theory, we rationalize these results in terms of enhanced association between the amino acid side chains and sulfate. Simulations also predict a greater affinity of sulfate to the guanidinium side chain groups of arginine than to the ammonium groups of lysine, as the planar guanidinium geometry allows simultaneous strong hydrogen bonding to two sulfate oxygens. We show that the sulfate binding to arginine, but not to lysine, is cooperative. These results are consistent with the greater decrease in the mobility of arginine compared to that of lysine upon addition of sulfate salt. The nonspecific mobility retardation by sulfate is ascribed to its electrostatic interaction with the cationic amino acid side chain groups.

Separation of haloacetic acids in water by capillary zone electrophoresis with direct UV detection and contactless conductivity detection

The separation of haloacetic acids (HAAs) in water by capillary zone electrophoresis with direct UV and contactless conductivity detection was investigated using phosphate, citrate, and borate buffers, and the experimental data were compared to simulation data predicted by a computational program known as PeakMaster. Good agreement between the experimental data and simulation data predicted by PeakMaster was found. Using the phosphate buffer or the citrate buffer and electrokinetic injection it was possible to quantitate HAAs at 0.1 ppm levels in water.

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinsons disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO4 and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min.

Separation and quantification of ropinirole and some impurities using capillary liquid chromatography

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinsons disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary liquid chromatography (CLC) was used for the separation and quantification of ropinirole and its five related impurities, potentially formed during its synthesis. A simultaneous optimization of three mobile phase parameters, i.e., pH, buffer concentration and acetonitrile content was performed employing an experimental design approach which proved a powerful tool in method development. The retention factors of the investigated substances in different mobile phases were determined. Baseline resolution of the six substances on a C18 reversed stationary phase was attained using a mobile phase with an optimized composition [acetonitrile-8.7 mM 2-(N-morpholino)ethanesulfonic acid adjusted to pH 6.0 (55:45, v/v)]. It was shown that CLC, operated in the isocratic mode under the mobile phase flow-rate of 4 microl/min, can determine the level of these impurities, down to a level of 0.06% of the main component within 25 min.