Extension of the application range of UV-absorbing organic solvents in capillary electrophoresis by the use of a contactless conductivity detector.

A contactless conductivity detection (CCD) system is used for capillary zone electrophoresis (CZE) with non-aqueous solvents of the buffering background electrolyte, which exhibit strong UV absorbance below 230 nm. It is found that the CCD characteristics with such solvents (propylene carbonate, N,N-dimethylformamide and N.N-dimethylacetamide as examples) is the same as with aqueous solutions: the same signal and noise is obtained for a given electric conductance of the background electrolyte, independent of the kind of the solvent. Therefore CCD enables the extension of the application range to solvents with restricted use for common UV detection in CZE due to their unfavourable or even unfitting optical properties. The applicability of CCD is demonstrated by CZE of aliphatic ammonium compounds in these solvents.

New configuration in capillary isotachophoresis-capillary zone electrophoresis coupling.

The paper surveys possible configurations of a coupling capillary column operating in various electromigration modes. Special attention is given to capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) coupling and its description from the theoretical point of view. Computer simulations of separation are presented and compared with experiments. Further, we propose a new configuration of electrolyte systems in cITP-CZE coupling, which offers a possibility to perform complex analyses of micro- and macro-constituents in one run. The electrolyte system is verified by practical experiments for both anionic and cationic modes of analysis. The advantages and disadvantages of the new combination are discussed.

Effect of temperature on the separation of long DNA fragments in polymer solution.

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of lambda-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.

Electrokinetic chromatography with micelles, polymeric and monomeric additives with similar chemical functionality as pseudo-stationary phases.

A comparison is made of the retention properties of additives applied as positively charged pseudo-stationary phases for electrokinetic chromatography of neutral analytes. All additives have a quaternary ammonium as functional group. The polymeric additive [poly(N,N,N',N'-tetramethyl-N-trimethylenehexamethylenediammonium), Polybrene] has a concentration of 2% (w/w) in the background electrolyte (acetate, pH 5.2). Monomeric octyltrimethylammonium (OTMA) was used at a concentration below or above its critical micelle concentration (CMC) (140 mmol/l). At a concentration (259 mmol/l) above the CMC the system is that normally used for micellar electrokinetic chromatography with cationic micelles. However, even below the CMC, where OTMA is present as monomer, retention of the neutral analytes is observed as well. In all systems coating of the capillary wall with Polybrene establishes an electroosmotic flow directed towards the anode, counter-migrating to the electrophoretic movement of the additive. Based on the measurement of the mobility of the analytes (15 small, monofunctional aromatic compounds with different functional groups), their capacity factors, k(i), were determined in all systems. Low correlation of the k(i) values is observed between the particular systems, indicating their different selectivity at least for individual pairs of analytes. Based on the log k(i) values, a linear free energy relationship was applied to elucidate the main types of chemical interaction responsible for retention. As a result, cavity formation and n or pi electron interactions were found being significant for the micellar OTMA system, which agrees with findings described in the literature for other (cationic and anionic) micellar systems. For the polymeric system and for the monomeric OTMA system, the significant retention parameter is indicating n and pi electron interactions.

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.

Study on the aggregation of teicoplanin.

The aggregation of teicoplanin was studied in four solutions: 0.06 M phosphate buffers pH 4.3 and 6.3, both with and without 10% (v/v) of acetonitrile. Conductometry, capillary electrophoresis, cyclic voltammetry, and static light scattering were employed to determine the critical micelle concentration (CMC) of teicoplanin. Dynamic light scattering was used to give an additional information on the size and the size distribution of the particles formed. While the CMC was found in solutions without acetonitrile, attempts to detect any CMC failed in solutions containing acetonitrile. The results point out to different solvating mechanism in solutions with and without acetonitrile, leading to two different schemes of association.

Comparison of separation selectivity in capillary electrokinetic chromatography using a cationic linear polymeric pseudo-stationary phase or monomeric additives of similar structure.

The retention properties in electrically driven systems with monomeric additives were compared to an electrokinetic chromatographic system with a linear, charged polymer of similar chemical structure (all additives are quaternary tetraalkyl ammonium ions). The monomeric additives were tetramethylammonium (TMA), tetraethylammonium (TEA) and dimethylpyrrolidinium (DMP), respectively, the polymeric additive was poly(diallyldimethyl)ammonium (PDADMA). The additive concentration in the background electrolyte was 2 and 4% (w/w). The retention characteristics were based on the apparent mobilities of 10 non-charged analytes with different chemical functionality, which were transported by the anodic electroosmotic flow in the dynamically coated capillary, and retained by the counter-flowing cationic additives. From these data capacity factors were derived, which ranged up to 0.8. Association constants were calculated, and were found between 10 and 170. Roughly, the association constants increased for a given analyte in the sequence TMA

Diffusion coefficient and capacity factor in capillary electrokinetic chromatography with replaceable charged polymeric pseudophase.

Apparent diffusion coefficients, Dapp,i, were determined in solutions with a polycationic additive -- poly(diallyidimethylammonium) -- acting as a pseudostationary phase for electrokinetic chromatography. They were determined for six small neutral analytes at five concentrations of the polymeric additive (between 0 and 4% w/w) by a stopped migration method. The apparent diffusion coefficients decrease with increasing polymer concentration only within 40% maximum, an effect that cannot be associated with the macroscopic viscosity of the polymer solution (which increases by a factor of 10). The change of the apparent diffusion coefficients is related to the interaction of the neutral analyte molecules with the polyelectrolyte chain. Applying the model of analyte partitioning between "free" solution and polymer, capacity factors and partition constants were derived from the slope of the 1/Dapp,i vs. polymer concentration curves. Partition constants determined by this method (ranging between 40 and 170) agree with those obtained by electrokinetic chromatography.

Capillary electrokinetic chromatography with charged linear polymers as a nonmicellar pseudostationary phase: determination of capacity factors and characterization by solvation parameters.

A permanent polycation, polydiallyldimethylammonium (PDADMA), is applied as a linear, polymeric, replaceable, and nonmicellar pseudostationary phase for the separation of neutral analytes by capillary electrokinetic chromatography. It is shown that this polymer used in the background electrolyte is able to separate the analytes even if it does not form micelles under the given conditions. The most favorable aspect for practical use lays in the simple replacement of the separation media after each run, thus generating highly reproducible conditions. To determine the capacity factors of the analytes, a new method, based on an isotachophoretic regime, has been introduced for the measurement of the electrophoretic mobility of the polymeric pseudo-stationary phase. The capacity factors in the separation system, derived from the mobilities of the polymer, the electroosmotic flow, and the mobilities of 15 individual aromatic analytes, range between 0.3 and 1.2 for the given separation media (aqueous solution of acetate buffer, pH 5.2, with 4% w/w PDADMA). The type of interaction in the pseudochromatographic system was clarified from solvation parameters based on the linear free energy relationship model. It was found that pi and n electron interactions and hydrogen-bond basicity of the polymer, as compared with the aqueous bulk phase, are the main cause of retention of the analytes.

Dispersive phenomena in electromigration separation methods.

A review on dispersive effects and on peak broadening in electromigration separation methods (capillary electrophoresis and electrochromatography) is presented, mainly covering papers published between the beginning of 1997 and the beginning of 2000. Most attention is drawn to work dealing with nonlinear effects that cause anomalous electromigration dispersion in electrolyte systems with two or multiple coions. Further, topics cover the comparison of electroosmotic and pressure-driven modes in electrochromatography, dispersive effects due to nonhomogeneous velocity fields in packed electrochromatography columns, to nonuniform electroosmotic flow, to sorption of analytes (mainly proteins) at the column wall or the stationary phase, and due to the influence of the nonideal column geometry like coiling or irregularities in shape.

Capillary electrokinetic chromatography with polyethyleneimine as replaceable cationic pseudostationary phase. Influence of methanol and acetonitrile on separation selectivity.

The effect of methanol and acetonitrile, respectively, on the separation of neutral compounds (benzyl alcohol, phenols) is investigated in electrokinetic chromatographic (EKC) systems consisting of polyethyleneimine (PEI) as charged, polymeric, replaceable pseudostationary phase. The separation systems consist of a buffer solution (2-morpholinoethanesulfonic acid, pH 7.0, 20 mM) containing 0.3-0.9% (w/v) PEI as additive and a varying percentage of methanol (0-50%, v/v) or acetonitrile (0-30%, v/v). EKC is carried out in fused-silica capillaries [47.0 cm (effective length 40.3 cm) x 100 microns I.D.]. They are dynamically coated with PEI, resulting in an electroosmotic flow directed towards the anode. The neutral analytes are migrating with the electroosmotic flow, and are retarded by the electrically driven counterflow of PEI. Separation of the analytes follows in the sequence benzyl alcohol, phenol, resorcinol, pyrogallol, reflecting the increasing hydrogen bond acidity and polarity (polarizibility) of the solutes. However, addition of methanol or acetonitrile causes a drastic loss of resolution, whereby the relative retention of the separands (related to benzyl alcohol) indicates a decrease of retardation upon addition of the organic solvents.

Separation of neutral compounds by capillary electrokinetic chromatography using polyethyleneimine as replaceable cationic pseudostationary phase.

Polyethyleneimine (PEI, molecular weight 6 x 10(5) - 1 x 10(6)) is applied as a positively charged pseudostationary phase for electrokinetic chromatography (EKC) of uncharged mono- and oligophenols. EKC is carried out in PEI-coated fused-silica capillaries (with electroosmotic flow directed towards the anode) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 7.0, 20 mM) with PEI added to the solution in concentrations up to 0.70% w/v. The pseudostationary phase leads to a retardation of the solutes mainly according to the number (and the position) of the OH-groups of the separands, and is not influenced significantly by methyl groups. For 0.70% w/v PEI solution, for instance, the relative retention, rho, has values between 0.33 and 0.53. For the systems with the highest resolution of the separands (0.25-0.30% PEI) 190,000 plates per meter are observed. The results indicate that the separation selectivity is mainly caused by ion-dipole interactions between the OH-groups of the solutes and the pseudostationary phase.

Size-based separation of polyelectrolytes by capillary zone electrophoresis: migration regimes and selectivity of poly(styrenesulphonates) in solutions of derivatized cellulose.

The migration regimes and the separation selectivity of linear poly(styrenesulfonates) (PSS) were investigated in solutions of linear polymeric hydroxyethylcellulose (HEC). Variable parameters were the number of PSS monomer units (in the range of 25-6520), HEC concentration (below and above the entanglement threshold), molecular mass of HEC (between 35900 and 438000 Da) and electric field strength (from 190 to 1140 V/cm). Besides those regimes which are known for migration of polyelectrolytes in physical networks (Ogston sieving, reptation without and with orientation), the observed separation below the entanglement threshold indicates an effect of solute-chain interactions similar to that observed for the separation of double-stranded DNA. From the dependence of the separation selectivity on HEC concentration and molecular weight it was found that both parameters increase the separation. The selectivity of the solutions of higher concentrated low molecular HEC was compared to lower concentrated high molecular HEC, which has a similar bulk viscosity, and therefore obeys comparable peak dispersion due to thermal broadening. At high field strengths (preferably used in capillary electrophoresis) the former gave better separation only for shorter PSS molecules. For separation of longer PSS molecules, only high molecular HEC is applicable due to the deformation of the low molecular matrix by large analyte molecules.

Peak broadening in capillary zone electrophoresis.

A review on peak broadening in capillary zone electrophoresis in free solutions is given which covers a selection of the literature published on this topic over the period mainly between 1992 and the beginning of 1997 (consisting of 71 publications). The contributions to peak dispersion from extracolumn effects (e.g. due to the finite length of the injection zone, or the aperture of the detector), from longitudinal diffusion, Joule heating, electromigration dispersion (concentration overload), a different path length of the solute ions, wall adsorption, laminar flow and the (longitudinally) homogeneous or nonhomogeneous electroosmotic flow are described. The latter may also occur when a longitudinally nonhomogeneous radial electric field is applied. Peak dispersion is depicted either by the plate-height model, or the concentration of the solute as a function of space and time is calculated either analytically or numerically by solving the equation of continuity with appropriate initial and boundary conditions and possibly completed by equations governing further quantities.

Influence of transference number on migration and deformation of concentration boundaries in capillary zone electrophoresis.

The change of the position and shape of a concentration boundary existing in the background electrolyte in capillary zone electrophoresis is treated theoretically. Such boundaries may occur when the ionic concentration, c, of the sample differs from that of the background electrolyte. Both mathematical treatment and numerical simulation lead to the result that when an (initially symmetrical) concentration boundary migrates in the capillary it becomes nonsymmetrical. The reason for this is the nonsymmetry of the function, which is obtained as the difference of the transference number (which is a function depending on c) and its linear approximation at the average value of the concentration. Numerical simulations for capillary zone electrophoresis with LiI or potassium benzorate as a hypothetical background electrolyte show the formation of asymmetrical plugs for samples of more diluted electrolyte solutions.

Dynamics of peak dispersion in capillary zone electrophoresis including wall adsorption: II. Exact analysis of unsteady linear adsorptive dispersion.

An exact analysis of the unsteady axial dispersion of an analyte, undergoing a linear adsorption at the column wall in capillary zone electrophoresis, is presented. A system of partial differential equations--in which the radial coordinate is one of the independent variables--is taken as a model for linear wall adsorption. It is shown that the dispersion is a sum of two terms, one which depends linearly on time and whose exact form is generally known, and a non-linear one. The most interesting result of this work is that it derives another system of differential equations, which this nonlinear term is to satisfy. It makes it possible to present a closed formula for the asymptotic value of the nonlinear term, i.e., its limit for large time. Its behavior for times close to zero is also studied.

Contribution of capillary coiling to zone dispersion in capillary zone electrophoresis.

A mathematical model of the deformation of the analyte zone in capillary zone electrophoresis (CZE) due to capillary coiling is presented and the influence of capillary coiling on the distribution of electric current density inside the capillary is described. The model gives a quantitative description of the potential contribution of capillary coiling to the total zone dispersion in CZE. The zone broadening caused by capillary coiling is calculated as the difference of migration distances of the particles migrating at the inner circumference of the capillary coil and those migrating at the outer circumference. This difference is shown to be directly proportional to the capillary diameter and to the number of capillary coils, i.e. indirectly proportional to the radius of the capillary coils into which the capillary of the given length is coiled. The contribution of capillary coiling to the total zone dispersion is compared with the contribution of the longitudinal diffusion of low and high molecular mass analytes and bioparticles. It is shown that, especially in the case of CZE separation of macromolecules and particles, capillary coiling can significantly decrease the separation efficiency. For that reason the small radius coiling of the capillary column in the CZE apparatuses should be avoided.

Dynamics of peak dispersion in capillary zone electrophoresis including wall adsorption I. Theoretical model and results of simulation.

A mathematical model is described for the simulation of peak profiles in capillary zone electrophoresis taking wall adsorption into account. It is based on such physico-chemical relations as mass balance equations, sorption rate equations and appropriate boundary conditions. The numerical solution of the model is carried out, allowing the depiction of the concentration profile of the sample in the capillary as a result of the dynamics of the processes involved, which depend on a number of input parameters: rate constants, adsorption isotherms, column dimensions, field strength etc. Four cases are discussed in detail, namely those with either slow or fast adsorption kinetics and linear or nonlinear isotherms.

Simulated quantitative and qualitative isotachophoretic indices of 73 amino acids and peptides in the pH range 6.4-10.

Qualitative and quantitative isotachophoretic indices of 73 amino acids, dipeptides and tripeptides were simulated under 24 leading electrolyte conditions covering the pH range 6.4-10. The RE values and time-based zone lengths are tabulated together with the absolute mobility (m0) and pKa values used. The leading electrolyte used was 10 mM HCl and the pH buffers were imidazole, tris(hydroxymethylamino)methane, 2-amino-2-methyl-1,3-propanediol and ethanolamine. The simulated indices will be useful in the assessment of the separability and determination of the listed and related compounds.

Determination of limiting ionic mobilities and dissociation constants of some local anaesthetics.

The limiting ionic mobilities and thermodynamic acid dissociation constants were calculated from isotachophoretic experiments for the local anaesthetics procaine, tetracaine, lidocaine, trimecaine, bupivacaine, cinchocaine, diperodone, diocaine, cocaine, psicaine-neu, tropacocaine, amylocaine, beta-eucaine and leucinocaine. The pH values at which the local anaesthetics with very similar limiting ionic mobilities can be isotachophoretically separated were determined from simulated mobility curves. The measuring apparatus employed a high-frequency contactless conductivity detector.