Chiral capillary electrophoresis as predictor for separation of drug enantiomers in continuous flow zone electrophoresis

Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.     

Determination of the ionization constants of weak electrolytes by capillary zone electrophoresis

Capillary zone electrophoresis in untreated fused silica capillaries has proved suitable for the determination of the ionization constants of weak electrolytes. Several fundamental equations relating the electrophoretic mobilities of ionized solutes to hydronium ion concentrations in the running electrolyte have been verified experimentally. The observed dependence of the electrophoretic mobilities of weak bases and ampholytes on the pH of the electrolyte showed good agreement with predicted behavior. The pK_a values calculated from electrophoretic mobility data obtained by capillary zone electrophoresis were reasonably close to those reported in the literature.     

Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.     

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).     

赖氨酸的毛细管电泳特性及测定

研究了缓冲液pH和操作温度对赖氨酸毛细管区带电泳分离中运行电流、电渗迁移率、峰形等参数的影响,确定了赖氨酸电泳分离的最佳实验条件,建立了毛细管区带电泳测定赖氨酸的方法。线性范围为0．1－3．0g/L,检出限为0．03g/L。该法操作简便迅速,重复性好。用于赖氨酸发酵液中赖氨酸的含量测定,结果令人满意。     

Electrophoretic mobility of partially denatured DNA in a gel: qualitative and semiquantitative differences between bubbles and split ends

Partially melted DNA is known to exhibit an abrupt decrease of electrophoretic mobility in a gel. Although this is the main phenomenon exploited in TGGE/DGGE (temperature gradient gel electrophoresis/denaturing gradient gel electrophoresis), not much is known about the physical processes responsible for the blocking. While there is a commonly used formula for the reduced mobility based on the theory of branched polymers, it does not discriminate between denatured domains bounded on one (split end) or two sides (bubble). To better understand how the blocking occurs in both of these cases, a coarse-grained model of DNA gel electrophoresis is simulated using Langevin Dynamics. The simulations reveal that the low-field mobility is much more sensitive to denatured domains located at the ends of a DNA fragment. A denatured domain occurring at the center of a fragment indeed reduces the mobility, but at a much lower rate.     

Electropherogram of capillary zone electrophoresis with effective mobility axis as a transverse axis and its analytical utility. I. Transformation applying the hypothetical electroosmotic flow

For capillary zone electrophoresis, a new method of transformation from migration time to effective mobility was proposed, in which the mobility increase due to Joule heating and the relaxation effect of the potential gradient were eliminated successfully. The precision of the mobility evaluated by the proposed transformation was discussed in relation to the analysis of rare earth ions. By using the transformation, almost the same pherograms could be obtained even from the pherograms obtained originally at different applied voltages.     

Separation and characterization of sub-microm- and microm-sized particles by capillary zone electrophoresis

The analytical separation and characterization of particles in the size range of sub-microm and microm diameters by capillary zone electrophoresis (CZE) has been reviewed. The theoretical basis, on which the mobility can be interpreted to provide information regarding characteristics of particle surface, has shortly been presented. Particular emphasis was put on the model dependence of that interpretation and the need in most applications to forego the classical idealized model of spherical particles with "smooth" surfaces and to apply more realistic models, which take the "hairy" surface of real particles into account. Some highlights of the literature on the CZE of polystyrene latex microspheres, organic and inorganic colloids, lipoprotein particles, viruses, liposomes, biological membrane vesicles, and biological cells have been discussed. Also summarized are the reports on the particle size dependence of mobility and peak broadening in CZE and on electrophoretic behavior of rodlike particles and particle aggregates. Finally, the effects of neutral polymers in the background electrolyte on particle mobility and peak width are reviewed.     

Application of pressure in capillary zone electrophoresis to study the aggregation of chitosan 2-hydroxybutoxypropylcarbamate

It is demonstrated that the use of pressure extends the possibilities of capillary zone electrophoresis in studying aggregative states of substances, ensuring the detection of the presence of several types of aggregates with different electrophoretic mobilities. The electropherograms of chitosan 2-hydroxybutoxypropylcarbamate (CHBPC) in citrate solutions with pH 3.1, 4.5, and 5.8 indicate the dependence of aggregation on pH. A comparison of the data for CHBPC obtained by capillary zone electrophoresis, static light scattering, and scanning electron microscopy revealed a relationship between the electrophoretic mobility and sizes of aggregates, varying from 140 nm to several micrometers. The size of aggregates can be estimated by hydrodynamic contribution to their mobility. The effectiveness of the use of CHBPC for the dynamic modification of capillaries is shown.     

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis

Capillary zone electrophoresis of samples of recombinant human erythropoietin is performed. An in-house computer program is developed to compare the reliability of different migration parameters to assign the close migration bands of isoforms of erythropoietin. The migration time relative to the electroosmotic flow marker and the effective electrophoretic mobility are selected as the most accurate parameters. Percentages of correct assignment of bands higher than 99% are obtained with these parameters even when changes in operational factors are introduced. The chosen parameters have been applied to assign bands of isoforms in commercial samples of alpha- and beta-epoetin. The same capillary electrophoresis method has been applied to separate bands of isoforms of an erythropoietin analogue, darbepoetin alpha, the novel erythropoiesis-stimulating protein.     

Conductivity detection in capillary zone electrophoresis: inspection by PeakMaster

A simple rule stating that the signal in conductivity detection in capillary zone electrophoresis is proportional to the difference between the analyte mobility and mobility of the background electrolyte (BGE) co-ion is valid only for systems with fully ionized electrolytes. In zone electrophoresis systems with weak electrolytes both conductivity signal and electromigration dispersion of analyte peaks depend on the conductivity and pH effects. This allows optimization of the composition of BGEs to give a good conductivity signal of analytes while still keeping electromigration dispersion near zero, regardless of the injected amount of sample. The demands to achieve minimum electromigration dispersion and high sensitivity in conductivity detection can be accomplished at the same time. PeakMaster software is used for inspection of BGEs commonly used for separation of sugars (carbohydrates, saccharides) at highly alkaline pH. It is shown that the terms direct and indirect conductivity detection are misleading and should not be used.     

The principles of migration and dispersion in capillary zone electrophoresis in nonaqueous solvents

Nonaqueous solvents are interesting media for capillary zone electrophoresis as they can affect all relevant parameters governing the separation of sample zones. However, for a rational planning of the working conditions and an appropriate interpretation of the results obtained, the basic principles of ion migration and zone dispersion must be understood. Many solvent induced effects need to be carefully considered and recognized before full exploitation of nonaqueous solvents can take place. It is the goal of this overview to present the fundamental physicochemical aspects of capillary zone electrophoresis in nonaqueous solvent systems. Therefore, the detailed discussion is related to the effect of organic solvents on electrophoretic mobilities (based on the theory of conductance), acid-base dissociation behavior (based on the transfer activity coefficient and medium effect), pH, separation efficiency (with regard to mobility and diffusion coefficient in dilute solutions), resolution, and electroosmotic flow.     

Determination of ionization constants of saccharides by capillary zone electrophoresis with amperometric detection

Summary A method based on a linear model enabling the efficient determination of the ionization constants (K _a) of saccharides by capillary zone electrophoresis with amperometric detection has been demonstrated. TheK _a values obtained from the plots of the reciprocal effective mobility against the inverse concentration of sodium hydroxide were in agreement with literature values.     

非水毛细管电泳应用新进展

非水毛细管电泳(NACE)已经被广泛用于药物、环境和生物等领域。由于有机溶剂种类繁多,它们的物理和化学性质各不相同,因此可以针对被分析物的性质及检测方法的不同,选择不同的有机溶剂用于NACE分离,从而拓宽了毛细管区带电泳(CZE)的应用范围。本综述根据近年来NACE在分析领域的应用,对NACE的优势、检测方法、富集方式以及在实际样品中的应用等方面进行了总结,并对其今后的发展进行了展望。     

New method for standardization of electropherograms obtained in capillary zone electrophoresis

A new method for standardization of electropherograms obtained by capillary zone electrophoresis was proposed, where the migration time axis was replaced by the effective mobility axis. The mobility increase due to temperature increase by Joule heating and the relaxation effect of the potential gradient were eliminated successfully by introducing a temperature coefficient for mobility expression and a delay time, respectively. The precision of the mobility evaluated by the proposed conversion methods was evaluated for a model sample. By using the conversion method, almost the same electropherograms could be obtained even from the electropherograms originally obtained by using different hardware conditions.     

Chitosan as cationic polyelectrolyte for the modification of electroosmotic flow and its utilization for the separation of inorganic anions by capillary zone electrophoresis.

Cationic polyelectrolyte of chitosan was used for the reversal of electroosmotic flow in capillary zone electrophoresis. The chitosan was dissolved in acetic acid solution, and stable electroosmotic flow was obtained at the chitosan concentrations between 50 and 300 microg/mL. Separation of inorganic anions was carried out using the dynamically coated capillary by capillary zone electrophoresis. Nine kinds of anions were separated and detected with the capillary. The electrophoretic mobility of the analyte anions decreased with increasing concentrations of chitosan in the migrating solution through ion-ion interaction, but the migration order of the analyte anions was not changed in the concentration range of the chitosan examined. The signal shape for the analyte anions was developed by using field-enhanced sample stacking with 10 mM sodium sulfate.     

Experimental observation of light-induced solitary waves of analyte bands in capillary electrophoresis.

The phenomenon of electrophoresis in free solution has been studied theoretically down to the molecular level for decades. In addition, intermolecular photo-induced proton transfer reactions, which occur in a wide class of molecules (phenols and aminoarenes) as well as proteins (green fluorescent protein), were also studied extensively. However, the study of the effect of light-induced electrophoretic mobility changes of the analytes in electrophoresis was begun only recently. In the present work, capillary zone electrophoresis was chosen as the environment to measure the magnitude of these electrophoretic mobility shifts induced by light. Background electrolytes (running electrolytes) with high refractive indices were developed, allowing the capillary to work like an optical fiber. The experimental conditions for obtaining stable coupling and guided laser light along the liquid core are discussed. Experimental evidence of band compression is observed, leading to a solitary wave behavior of the analyte band (2-naphthol). These solitary waves result from competition between thermal diffusion (dispersion mechanism) and a nonlinear (band compression) effect due to the combined electrophoresis phenomenon and absorption of guided light by the molecules of the band (which are subjected to a "reversible intermolecular proton transfer reaction" as one of their decay routes). The possibilities of applying this effect to different methods and techniques are also discussed.     

The application of spline wavelet transform to the determination of electroosmotic mobility in capillary electrophoresis

The rule of current change was studied during capillary electrophoresis (CE) separation process while the conductivity of the sample solution was different from that of the buffer. Using a quadratic spline wavelet of compact support, the wavelet transforms (WTs) of capillary electrophoretic currents were performed. The time corresponding to the maximum of WT coefficients was chosen as the time of current inflection to calculate electroosmotic mobility. The proposed method was suitable for different CE modes, including capillary zone electrophoresis, nonaqueous CE and micellar electrokinetic chromatography. Compared with the neutral marker method, the relative errors of the developed method for the determination of electroosmotic mobility were all below 2.5%.