Isotachophoretic Migration Research Articles

Front Cover: Mathematical model of electromigration allowing the deviation from electroneutrality

DOI: 10.1002/elps.202000207 The cover picture shows the graphical output of the mathematical model describing electromigration in nanodimensions. It depicts isotachophoretic migration in a nanochannel, the diameter of which is 50 nm. You can see in color the simulated pH of the charged silica gel layer at the inner channel wall and in the adjacent liquid solution. The solution is composed of weak multivalent electrolytes.

Computer simulation of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers with a free or immobilized neutral chiral selector

A detailed computer simulation study of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers for cases with the neutral selector added to the leader, immobilized to the capillary wall or support, or partially present in the separation column is presented. The electrophoretic transport of the analytes from the sampling compartment into the separation medium with the selector, the formation of a transient mixed zone, the separation dynamics of the stereoisomers with a free or immobilized selector, the dependence of the leader pH, the ionic mobility of norpseudoephedrine, the complexation constant and selector immobilization on steady-state plateau zone properties, and zone changes occurring during the transition from the chiral environment into a selector free leader are thereby visualized in a hitherto unexplored way. For the case with the selector dissolved in the leading electrolyte, simulation data are compared to those observed in experimental setups with coated fused-silica capillaries that feature minimized electroosmosis and zone detection with conductivity and absorbance detectors.

Study of the mechanism of acetonitrile stacking and its application for directly combining liquid-phase microextraction with micellar electrokinetic chromatography

Acetonitrile stacking is an online concentration method that is distinctive due to its inclusion of a high proportion of organic solvent in sample matrices. We previously designed a universal methodology for the combination of liquid-phase microextraction (LPME) and capillary electrophoresis (CE) using acetonitrile stacking and micellar electrokinetic chromatography (MEKC) mode, thereby achieving large-volume injection of the diluted LPME extractant and the online concentration. In this report, the methodology was extended to the analysis of highly substituted hydrophobic chlorophenols in wines using diethyl carbonate as the extractant. Additionally, the mechanism of acetonitrile stacking was studied. The results indicated that the combination of LPME and MEKC exhibited good analytical performance: with ∼40-fold concentration by LPME, a 20-cm (33% of the total length) sample plug injection of an eight-fold dilution of diethyl carbonate with the organic solvent-saline solution produced enrichments higher by a factor of 260–791. Limits of qualification ranged from 5.5 to 16.0ng/mL. Acceptable reproducibilities of lower than 1.8% for migration time and 8.6% for peak areas were obtained. A dual stacking mechanism of acetonitrile stacking was revealed, involving transient isotachophoresis plus pH-junction stacking. The latter was associated with a pH shift induced by the presence of acetonitrile. The pseudo-stationary phase (Brij-35) played an important role in reducing the CE running time by weakening the isotachophoretic migration of the analyte ions following Cl− ions. The combination of acetonitrile stacking and nonionic micelle-based MEKC appears to be a perfect match for introducing water-immiscible LPME extractants into an aqueous CE system and can thus significantly expand the application of LPME-CE in green analytical chemistry.

Isotachophoresis with counterflow in an open capillary: Computer simulation and experimental validation

The purpose of applying a countercurrent flow to isotachophoretic migration is to increase the effective separation channel length during ITP. However, severe dispersion induced by applying a counterflow can be detrimental to ITP. This paper uses numerical simulations in a 2D axisymmetric domain to investigate the dispersion caused by a parabolic counterflow in open-capillary ITP. Counterflow in these simulations was generated by applying a back pressure to stop the isotachophoretic stack, i.e., forming stationary ITP zones. It is found that dispersion is strongly related to analyte molecular diffusivity: R-phycoerythrin, due to its small diffusivity, showed ~20-fold increase in zone width in stationary counterflow ITP, compared to ITP in the absence of counterflow, while fluorescein only had ~10% increase in zone width under similar operating conditions. Applying the Taylor-Aris dispersion formula in counterflow ITP simulations provided only a rough estimate of the dispersion, e.g., overestimation of analyte zone widths. Experiments on counterflow ITP were conducted in a silica capillary that was covalently and dynamically coated to exclude electroosmosis effect. The counterflow was generated by adjusting the relative height of the fluids in the two reservoirs at the capillary ends. Good qualitative agreement between simulations and experiments was found.

An extraction technique for analytical sample preparation in aqueous solution based on controlling dispersion of ionic surfactant assemblies in isotachophoretic migration

An extraction technique for analytical sample preparation in aqueous solution has been developed based on controlling dispersion of ionic surfactant assemblies. An extraction technique was realized based on controlling dispersion of the ionic surfactant assemblies in their isotachophoretic migration during the extraction by arranging the solutions of leading electrolyte, ionic surfactant and terminating electrolyte in order and applying voltage. Potential of the technique for analytical sample preparation in aqueous solution has been demonstrated by extracting a model sample of four alkylphenones, which were analyzed by HPLC after the extraction. The extraction showed concentration effects on all the four alkylphenones of butyrophenone, valerophenone, hexanophenone and heptanophenone in the model sample. The enrichment factors were 5.29, 7.70, 7.25 and 7.49 for the four alkylphenones of butyrophenone, valerophenone, hexanophenone and heptanophenone, respectively. Linear relationship was obtained with all the four alkylphenones between their chromatographic peak areas before and after the extraction in the range of concentration from 0.05ppm to 1.5ppm. The RSD of the chromatographic peak areas in triplicate extractions was 7.97%, 3.75%, 2.91% and 4.45% for butyrophenone, valerophenone, hexanophenone and heptanophenone, respectively. Advantages of the extraction technique developed include ease of operation, low reagent cost, no consumption of organic solvents and no requirement for additional phase separation.

Autofocusing and ESI‐MS analysis of protein digests in a miniaturized multicompartment electrolyzer

Free-solution IEF of protein digests was studied in a newly introduced MicroRotofortrade mark multicompartment electrolyzer. The fractionation was performed in a cylindrical separation chamber divided into ten compartments with or without the addition of carrier ampholytes. In the case of autofocusing mode of operation, the tryptic digest itself served as the mixture of ampholytes leading to the separation of the peptides with well-defined pI's. The focusing process was monitored visually using colored pI markers. The resulting fractions from both modes of the separation were analyzed by CE and electrospray-TOF mass spectrometer using electrospray tips microfabricated in polyimide. Additional experiments, aiming at visualization of the mass flux within the focusing compartments were performed using isotachophoretic migration of color cationic tracers. The study considered the autofocusing of both the peptides with well-defined narrow pI's as well as those showing negligible net charge in a broader pH range. Although not all peptides in the protein digests have well-defined pI's the autofocusing process can preseparate many of them leading to higher S/N in the ESI-MS signals and improved protein sequence coverage.

On‐line sample concentration techniques in capillary electrophoresis: Velocity gradient techniques and sample concentration techniques for biomolecules

Methods with a high sensitivity and high separation efficiency are goals in analytical separation techniques. On-line sample concentration techniques in capillary electrophoresis (CE) separations have rapidly grown in popularity over the past few years because they achieve this goal. This review describes the methodology and theory associated with a number of different techniques, including electrokinetic and chromatographic methods. For small molecules, several on-line concentration methods based on velocity gradient techniques are described, in which the electrophoretic velocities of the analyte molecules are manipulated by field amplification, sweeping, and isotachophoretic migration, resulting in the on-line concentration of the analyte zones. In addition, the on-line concentration methods for macromolecules are described, since the techniques used for macromolecules (DNAs and proteins), are different from those for small molecules, with respect to either mechanism or methodology. Recent studies relating to this topic are also discussed, including electrophoretic and chromatographic techniques on capillary or microchip.

New method to determine stability constants of lanthanide-EDTA complexes by isotachophoresis

The zone reduction in the isotachophoretic migration of several lanthanide-EDTA complexes at low pH was investigated. The typical leading electrolyte was 20 m M HCl buffered by triglycine at pH 3.1 and the terminating electrolyte was 20 m M ammonia solution buffered by acetic acid at pH 4.8. It was observed that a good linear relationship exists between the stability constants and the zone reduction due to the bleeding effect (liberating free metal ions). This relationship is the basis of a new method for the evaluation of the stability constants of the lanthanide complexes.

Computer simulation of electrophoretic transport. IV. Anionic isotacophoresis of ampholytes using a common component which migrates faster than the leading component.

Our computer simulation procedure was applied to an anionic isotachophoretic system of ampholytes in which a component having a migration velocity faster than the leading component of the system was added to the catholyte. After a certain time of electrophoresis depending on the initial condition, isotachophoretic migration resulted even in the presence of the faster component in the catholyte. The isotachophoretic concentration of the ampholytes and the potential gradient were lower in the presence than those in the absence of the faster component. The lower potential gradient is preferable from the standpoint of Joule's heat so that the proposed isotachophoretic system seems to be useful for slab gel isotachophoretic experiments where thinning of the gel occurrs in the region of high potential gradient.

Some practical aspects of utilizing the on-line combination of isotachophoresis and capillary zone electrophoresis

The on-line combination of isotachophoresis (ITP) and zone electrophoresis is a very effective tool for increasing the separation capability and sensitivity of capillary zone electrophoresis (CZE). Its most effective version is performed in column coupling instrumentation and is characterized by isotachophoresis in the first capillary serving as the efficient preseparation and concentration stage followed by on-line transfer of the sample cut into the second capillary where analytical zone electrophoresis proceeds as the second stage. The on-line transfer of the sample from the first capillary into the second is always accompanied by the segments of some additional amounts of the leader and/or terminator from the ITP step. This results in transient survival of isotachophoretic migration during the second stage. Hence the separation process during the second stage can be characterized as the destacking of analytes followed by zone electrophoretic separation. In this paper, both a theoretical and an experimental study is presented showing that destacking of analytes is a selective process, which affects strongly the most important final analytical parameters of the detected zones, namely the detection time and zone variance, and thus it makes the simple use of detection data for qualitative analysis misleading. It was shown that both the detection time and variance of a zone of an analyte depend strongly on the amount of the accompanying segments of the leader/terminator transferred and on the actual type of electrolyte system selected from the generally possible types. These types are (i) L-S-L, where the leading electrolyte (L) from ITP stage serves as the background electrolyte (BGE) during CZE (S = sample), (ii) T-S-T, where the terminating electrolyte (T) from the ITP stage serves as the BGE during CZE, and (iii) BGE-S-BGE, where L, T and BGE are mutually different. Explicit equations were derived enabling one to predict migration time and zone variance for actual working conditions, and, for model systems, the theoretical data were calculated and verified experimentally. Further, it is shown that the systems T-S-T and L-S-L are user friendly and a simple standardization procedure was proposed, allowing correct qualitative evaluation of the analytical data in these systems. Finally, a theory is presented predicting the existence of anomalous variances of zones in the record of analysis in BGE-S-BGE systems and its experimental verification is given.

Electromigration in systems with additives in background electrolytes: II. Ionic admixture

A theoretical model is presented for isotachophoretic migration in systems where both the leading and terminating electrolytes contain another common component (admixture) ( e.g., a difficult to remove impurity). The effective mobility of the admixture is lower than that of the leading ion and higher than that of the terminating ion. The model system includes a further two samples. The mathematical simulation of the separation dynamics of this system was obtained by solution of basic physico-chemical laws. This facilitated a comprehensive formulation of the principles of the steady state and calculations of the parameters of all zones. Based on the theoretical model, a method for the determination of admixture concentrations in the leading and terminating electrolytes from isotachophoretic measurements and accurate determination of the limiting mobilities of the separated compounds from experimental data affected by the presence of the admixture are described.

Bidirectional isotachophoresis: I. Verification of bidirectional isotachophoresis and simultaneous determination of anionic and cationic components

Bidirectional isotachophoretic migration was confirmed by the direct measurement of the pH profiles and the boundary velocities of the separated zones. The anolyte used was 10 m M HCl—β-alanine (pH 3.6) and the catholyte was 10 m M KOH-acetic acid (pH 4.8). It was shown that bidirectional isotachophoresis could be achieved with an electrolyte system consisting of a leading electrolyte for an anionic analysis and one for a cationic analysis. The combination was not arbitrary but the pH difference between the anolyte and the catholyte was restricted to keep the effective mobility of the terminating ion not too small. The simultaneous separation and determination of anions and cations in a test mixture were demonstrated on the basis of the time-based zone length measured by the use of a dual detection system.

Trace analysis of proteins by capillary zone electrophoresis with on-column transient isotachophoretic preconcentration.

The qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co-ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co-ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface-coated fused-silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 microL was injected into a 75 microns ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10(-7)-10(-8) M, with detection limits of approximately 10(-9) M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.

On-column transient and coupled column isotachophoretic preconcentration of protein samples in capillary zone electrophoresis

Two strategies for the isotachophoretic preconcentration of samples were evaluated using a standard protein mixture as an example. In the first, on-column transient isotachophoretic migration permits the injection of relatively large volumes of sample into a commercial instrument. Depending on the composition, 30–50% of the column length can be filled with the sample while maintaining good resolution of the sample components. When proper electrolyte compositions are selected, the conventional single-column instrument can be used for an isotachophoretic sample preconcentration of 50-fold or more without any modification. In the second strategy, a coupled-column system was examined which, in principle, provides a higher degree of freedom in the selection of capillary zone electrophoretic running conditions and the possibility of injection of higher sample volumes. The gain in detection level is at least a factor of 1000 in the coupled-column arrangement; however, the instrumentation is more complicated.

Some problems encountered in the selection of electrolyte systems in isotachophoresis

AbstractA theory is described which gives a new algorithm for the selection of suitable electrolyte systems based on assuming (i) the zone stability as its correct isotachophoretic migration; (ii) the separability as the establishment of a fixed migration order of the zones of individual substances. The theory is based on data of the typeūi,j, i. e., effective mobility of substance i in the j‐th zone. For checking the stability of zones, the use of the zone existence diagrams is demonstrated, where the groups of substances giving stable zones are easily characterized, with special attention to anionic analyses with OH− as the terminator. For checking the separability of substances, the use of the sequence contours in an existence diagram is demonstrated with respect to the zone order as well as to the formation of stable mixed zones. Experiments are given where anionic isotachophoresis of some amino acids with OH− as the terminator is shown, and the formation of stable mixed zones of substances with mutually different effective mobilities is demonstrated.

Zone order in isotachophoresis : The concept of the zone existence diagram and its use in cationic systems

The migration behaviour of zones of strong and weak bases in cationic isotachophoresis is considered. Based on the protolytic effects on the effective mobilities, the criteria decisive for the migration mode and zone order are formulated and the concept of the zone existence diagram is introduced, where zone pH vs. effective mobility is plotted for u BH and K BH as parameters. From such a diagram (for a given leading electrolyte—terminator system) one may predict whether a given base will or will not migrate in stack and what the migration mode and zone order of a given pair of bases are. Based on this diagram, a general discussion is given of possible migration modes of a pair of bases, distinguishing (i) classical isotachophoretic migration, (ii) migration with inversion of effective mobilities or pH, (iii) steady-state mixed zone migration and (iv) enforced migration. Further, a new view of the formation and migration of the steady-state mixed zones is introduced and discussed. It is shown that if a steady-state mixed zone may be formed in the system then the character of the isotachopherogram is strongly affected by the ratio of the substances sampled. Based on the above-mentioned knowledge, a detailed classification is given of zone patterns potentially formed by a two-component sample of cathodically migrating bases in an isotachophoretic electrolyte system. It is shown that the elaborated principles may also be used in multi-component cationic analysis and the necessary criteria are given in the form of explicit mathematical equations.

Discrete non-UV-absorbing anionic spacers for isotachophoretic separations at high pH

In the same way as glutathione S-transferase could be identified in isotachopherograms through its ligand-binding ability, so can other enzymes be identified after binding, for example, a specific inhibitor or substrate of the enzyme. We have also achieved identification of adenylate kinase in a commercial preparation in this way. This is useful information, if one is considering a further purification step, especially if preparative isotachophoresis is being considered. Adenylate kinase only just migrates in front of the terminator (forced isotachophoresis) in the electrolyte system C. However, after binding of a nucleotide inhibitor, the net negative charge is increased greatly, leading to a large increase in net mobility. So great is this increase, that the complex can be found in the electrolyte system B. However, the alkaline conditions for anionic separations prove to lead to a dissociation of complexes with substrates such as AMP or inhibitor ANS. They are, however, stable in acid media. This is one of the great advantages of isotachophoresis. The operating systems can be manipulated over a wide range to suit other prerequisites of the analysis. Thus, the enzyme itself, and complexes, could be suitably analysed in the cationic system D. A general rule of isotachophoresis is that the pH of the operating system should be at least 1 unit removed from the isoelectric point of the protein under consideration. However, isotachophoretic migration is then dependent on the mobility of the terminator. Immunoglobulins

Migration behaviour of the hydrogen ion and its role in isotachophoresis of cations

A theoretical and experimental study has been made of the behaviour of hydrogen ions under the conditions of cationic isotachophoresis. It is shown that, for successful cationic isotachophoresis, the electrolyte conditions should be such that H + migrates in an enforced isotachophoretic manner as terminator and its front boundary shows self-sharpening. A mathematical description of the migration of H + as the terminating zone is elaborated, giving the criteria decisive for the character of the terminating zone front boundary depending on the composition of the leading electrolyte and on the properties of the cationic substances separated (strong and weak bases). It has been found theoretically that, for a given set of cations to be separated and counter anion in the leading electrolyte, the concentration of the leading electrolyte must be higher than a certain critical value to obtain stable isotachophoretic migration. Model experiments with different counter-anion systems (acetic acid—acetatate and formic acid—formate) at various concentrations have given good agreement with the theory. Illustrative examples of cationic isotachophoresis of some amino acids and dipeptides are described. The model described enables both the prediction of migration behaviour of cationic substances and the selection of suitable electrolyte systems in order to obtain stable isotachophoretic migration of these substances.

Isotachophoresis of Kinetically labile complexes

This paper deals both theoretically and experimentally withe the behaviour of kinetically labile anionic complexes under the conditions of isotachophoretic migration. It has been shown that in an isotachophoretic zone of an anio nic metal—ligand complex, owing to the dissociation equilibrium free metal cations and free ligand anions are present, which, in proportion to the signs and values of their mobilities, migrate out of the zone of the complex. The migrating zone of the complex, leaving behind a trace of the free metal cation, appears as a bleeding zone and decomposes slowly in the course of the migration. A mathematical description of the decomposing zone is elaborated, giving the decomposition rate of the complex depending on the conditional complex stability constant, operational parameters of the column and the composition of the leading electrolyte. It has been demonstrated that in both main cases of mutual movement of the complex and the free ligand (the ligand overtakes the complex or lags behind it) the decomposition of the complex during the migration can be negligible under properly selected operating conditions (mainly the composition of the leading electrolyte), and the zone will appear isotachophoretically stable and can be utilized for quantitative analysis. Model experiments with NTA and EDTA complexes agree with the theory. The model described makes it possible both to predict the isotachophoretic behaviour of anionic complexes under the given operating conditions and to select these conditions so that the stability of the zones of the complexes under investigation may be influenced selectivity in order to obtain the required separation.