A new dynamic computer model permitting the combined simulation of the temporal behavior of electroosmosis and electrophoresis under constant voltage or current conditions and in a capillary which exhibits a pH-dependent surface charge has been constructed and applied to the description of capillary zone electrophoresis, isotachophoresis, and isoelectric focusing with electroosmotic zone displacement. Electroosmosis is calculated via use of a normalized wall titration curve (mobility vs pH). Two approaches employed for normalization of the experimentally determined wall titration data are discussed, one that considers the electroosmotic mobility to be inversely proportional to the square root of the ionic strength (method based on the Gouy-Chapman theory with the counterion layer thickness being equal to the Debye-Hückel length) and one that assumes the double-layer thickness to be the sum of a compact layer of fixed charges and the Debye-Hückel thickness and the existence of a wall adsorption equilibrium of the buffer cation other than the proton (method described by Salomon, K.; et al. J. Chromatogr. 1991, 559, 69). The first approach is shown to overestimate the magnitude of electroosmosis, whereas, with the more complex dependence between the electroosmotic mobility and ionic strength, qualitative agreement between experimental and simulation data is obtained. Using one set of electroosmosis input data, the new model is shown to provide detailed insight into the dynamics of electroosmosis in typical discontinuous buffer systems employed in capillary zone electrophoresis (in which the sample matrix provides the discontinuity), in capillary isotachophoresis, and in capillary isoelectric focusing.

Software is available, which simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. These dynamic models are based upon equations derived from the transport concepts such as electromigration, diffusion, electroosmosis and imposed hydrodynamic buffer flow that are applied to user-specified initial distributions of analytes and electrolytes. They are able to predict the evolution of electrolyte systems together with associated properties such as pH and conductivity profiles and are as such the most versatile tool to explore the fundamentals of electrokinetic separations and analyses. In addition to revealing the detailed mechanisms of fundamental phenomena that occur in electrophoretic separations, dynamic simulations are useful for educational purposes. This review includes a list of current high-resolution simulators, information on how a simulation is performed, simulation examples for zone electrophoresis, ITP, IEF and EKC and a comprehensive discussion of the applications and achievements.

GENTRANS, a comprehensive one-dimensional dynamic simulator for electrophoretic separations and transport, was extended for handling electrokinetic chiral separations with a neutral ligand. The code can be employed to study the 1:1 interaction of monovalent weak and strong acids and bases with a single monovalent weak or strong acid or base additive, including a neutral cyclodextrin, under real experimental conditions. It is a tool to investigate the dynamics of chiral separations and to provide insight into the buffer systems used in chiral capillary zone electrophoresis (CZE) and chiral isotachophoresis. Analyte stacking across conductivity and buffer additive gradients, changes of additive concentration, buffer component concentration, pH, and conductivity across migrating sample zones and peaks, and the formation and migration of system peaks can thereby be investigated in a hitherto inaccessible way. For model systems with charged weak bases and neutral modified β-cyclodextrins at acidic pH, for which complexation constants, ionic mobilities, and mobilities of selector-analyte complexes have been determined by CZE, simulated and experimentally determined electropherograms and isotachopherograms are shown to be in good agreement. Simulation data reveal that CZE separations of cationic enantiomers performed in phosphate buffers at low pH occur behind a fast cationic migrating system peak that has a small impact on the buffer composition under which enantiomeric separation takes place.

electrophoresis simulation model comprising the determined pH and ionic strength dependent wall titration data as input for calculation of electroosmosis is shown to provide CZE and CITP electropherograms that qualitatively agree well with those obtained experimentally. In contrast to FS and glass, electroosmosis at any pH in Ž PMMA is too weak to perform bidirectional i.e., simultaneous cationic and . anionic CZE and CITP analyses with a detector placed toward the cathodic capillary end. Furthermore, the same is true for the performance of CIEF with electroosmotic zone displacement. Imposed flow is demonstrated to provide the required net buffer flow.

Effects originating from the variability of the sample matrix can be efficiently eliminated when the separation conditions are selected so that compounds of like charge with high concentration referred to as macrocomponents are embodied into the system of transient isotachophoresis. For stacking and separation of anionic trace analytes in biological samples, the presence of chloride is shown to be important to balance out effects of other macrocomponents that act against isotachophoretic stacking. Having acetoacetate, malate, citrate, and some drug metabolites in untreated human serum samples, the stacking mechanism of these compounds in an electrolyte system comprising 5 mM mandelic acid and epsilon -aminocaproic acid, pH 3.8, is explained. Analytes are monitored by indirect UV-absorption detection. Attention is paid to the minimum chloride concentration required with respect to the concentration ratio of phosphate (stacker) and lactate (destacker) present in the sample so as to ensure both stacking and separation of trace analytes. Insight into the separation process is given both with computer simulations and experiments. For selected analytes, the effect of chloride concentration on quantitative evaluation, sensitivity and limit of detection is demonstrated as well. Moreover, the applicability of the mobility window between phosphate and lactate for an additional group of metabolites is sketched.

The separation of methadone enantiomers by cationic capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) having (2-hydroxypropyl)-beta-cyclodextrin (OHP-beta-CD) as chiral selector in the leading electrolyte is described. Sodium acetate/acetic acid (pH between 4 and 5) served as leading electrolyte (catholyte) and acetic acid as terminator (anolyte). Complete separation of the enantiomers was obtained by CITP in a 50 microm internal diameter (ID) fused-silica capillary and in a 500 microm ID Teflon capillary. In the first approach, enantiomeric separation could be monitored via UV absorbance detection at low wavelength. With the second instrumental setup, an additional conductivity sensor permitted the visualization of the enantiomeric separation and the characterization of the buffer system employed. A 10 mM sodium acetate/acetic acid leading buffer of pH 4.3, containing 5 mM OHP-beta-CD, was found to provide best enantiomeric separation and was thus chosen for RITP. With RITP processing of a few mg of racemic methadone, partial separation of methadone enantiomers was obtained. R-(-)-methadone and S-(+)-methadone were found to be significantly (up to about 80%) enriched at the front and back side, respectively, of the isotachophoretic zone. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary zone electrophoresis (CZE) and circular dichroism spectroscopy. CZE was found to represent a simple and efficient method for the determination of the enantiomeric excess, whereas the latter technology was noted to be the superior approach for properly characterizing fractions that contain similar amounts of the two enantiomers. Furthermore, chiral RITP and analysis of the collected fractions by circular dichroism spectroscopy is shown to be potentially useful for identification of single enantiomers in absence of pure chiral standards.

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This paper presents principle and first results of a novel competitive binding immunoassay for monitoring of theophylline in human serum. The assay is based upon short time incubation of a mixture of antiserum, containing the antibody raised against theophylline, fluorescein labelled theophylline (tracer) and serum prior to injection of a few nanoliter of this mixture onto a fused-silica capillary for subsequent separation and analysis of free tracer and the antibody-tracer-complex by micellar electrokinetic capillary chromatography with laser induced fluorescence detection. Quantitation based upon multi-level calibration using the height of the peak produced by the free tracer is shown to provide theophylline serum levels which are in agreement with those obtained by a commercial fluorescence polarization immunoassay and with those determined by micellar elektrokinetic capillary chromatography with direct serum injection and on-column UV absorption detection.