Simulation of the effects of complex- formation equilibria in electrophoresis: II. experimental verification

The complete mathematical model of electromigration in systems with complexation agents introduced in the Part I of this article (V. Hru?ka et al., Eletrophoresis, 2012, 33, this issue), which was implemented into our simulation program Simul 5, was verified experimentally. Three different chiral selector (CS) systems differing in the type of the CS, the magnitude of the complexation constants as well as in the experimental conditions were selected for verification. The experiments and simulations were performed at various concentrations of the CSs in order to discuss the influence of the concentration of the CS on the separation. The simulated and experimental electropherograms show very good agreement in the position, shape and amplitude of the analyte peaks. The new Simul 5 Complex offers a deep insight into electrophoretical separations that take place in systems containing complexing agents, for example into enantiomer separations. Using Simul 5 Complex we were able to predict and explain the significant electromigration dispersion of analyte peaks. It was clarified that the electromigration dispersion in these systems results directly from complexation. The new Simul 5 Complex was also shown to be a useful and powerful tool for the prediction of the results of enantioseparations.     

Effect of urea on analyte complexation by 2,6‐dimethyl‐β‐CD in peptide enantioseparations by CE

The aim of the present study was the investigation of the effect of urea on analyte complexation in CD‐mediated separations of peptide enantiomers by CE in the pH range of about 2–5. pH‐independent complexation and mobility parameters in the absence and presence of 2 M urea were obtained by three‐dimensional, non‐linear curve fitting of the effective analyte mobility as a function of pH and heptakis‐(2,6‐di‐O‐methyl)‐β‐CD concentration. Urea led to decreased binding strength of the CD towards the protonated and neutral analyte enantiomers as well as to decreased mobilities of the free analytes. In contrast, mobilities of the fully protonated enantiomer–CD complexes as well as the pK_a values of the free and complexed analytes increased. The effect of urea on separation efficiency varied with pH and CD concentration. In the case of Ala‐Tyr and Ala‐Phe, separations improved in the presence of urea at pH 2.2. In contrast, separations were impaired by urea at pH 3.8 and low concentrations of the CD. Decreased separation efficiency was noted for Asp‐PheOMe and Glu‐PheNH₂ at low CD concentrations when urea was added but separations improved at higher CD concentrations over the entire pH range studied. The effect of urea on analyte complexation appeared to be primarily non‐stereoselective. Furthermore, the pH‐dependent reversal of the enantiomer migration order observed for Ala‐Tyr and Ala‐Phe can be rationalized by the complexation and mobility parameters.     

Systematic investigations of different capillary electrophoretic techniques for separation of methylquinolines

The migration behaviour of isoquinoline, quinoline, and methyl derivatives of quinoline in different capillary electrophoretic modes has been systematically investigated. Optimised separation conditions were established by varying the key parameters (solvent, pH, temperature, surfactant concentration, core phase) for aqueous and non‐aqueous capillary zone electrophoresis (NACE), micellar electrokinetic chromatography (MEKC) with anionic or non‐ionic micelles (SDS, Brij 35), and microemulsion electrokinetic chromatography (MEEKC) with charged or uncharged microemulsion droplets. A separation of all quinolines could be achieved by MEEKC with charged droplets, by MEKC or by formamide‐based NACE. Comparing the separations with respect to separation selectivity, substantial changes in migration order could be observed between the different techniques. Regarding separation efficiency, the number of theoretical plates and limits of detection (LOD) have been compared. The best LODs were achieved using SDS as surfactant in MEKC, followed by MEEKC.     

Simul 6: A fast dynamic simulator of electromigration

Simul 6 is a 1D dynamic simulator of electromigration based on the mathematical model of electromigration in free solutions. The model consists of continuity equations for the movement of electrolytes in a separation channel, acid–base equilibria of weak electrolytes, and the electroneutrality condition. It accounts for any number of multivalent electrolytes or ampholytes and provides a complete picture about dynamics of electromigration and diffusion in the separation channel. The equations are solved numerically using software means which allow for parallelization and multithreaded computation. Simul 6 has a user-friendly graphical interface. It is typically used for inspection of system peaks (zones) in electrophoresis, stacking and preconcentrating analytes, optimization of separation conditions, method development in either capillary zone electrophoresis, isotachophoresis, and isoelectric focusing. Simul 6 is the successor of Simul 5, and has been launched as a free software available for download at https://simul6.app/ .     

Capillary electrophoretic separations of proteins using carrier ampholytes

Capillary separations of proteins using carrier ampholytes are performed between an anolyte and a catholyte of same pH (pH 3). Depending upon the concentration of carrier ampholytes used, two different separation processes take place. At a 10% concentration, the high-resolution separation of six model proteins is achieved, which can be described as a transient capillary isoelectric focusing (cIEF) system moving isotachophoretically. The isotachophoretic (ITP) behaviour of the system is evidenced by the influence of the catholyte concentration on the separation. The separation is neither pure cIEF nor pure cITP and the migration order of the proteins results from the influence of both their isolelectric points and their mobilities.     

Strategies in two‐dimensional liquid chromatographic separation of complex polymer systems

Complex synthetic polymer systems as for example copolymers exhibit distributions in at least two of the three basic molecular characteristics which are molar mass, chemical structure/composition and molecular architecture. Size exclusion chromatography (SEC) separates macromolecules according to their size in solution which simultaneously depends on all molecular characteristics. Therefore, multi‐dimensional liquid chromatographic techniques are to be applied to independently assess all different distributions present in the sample. So far, two‐dimensional separations have been attempted. In the first dimension separation column, selected liquid chromatographic mechanisms are intentionally combined to suppress effects of all but one molecular characteristic. Consequently, polymer species are separated exclusively or at least predominantly according to one single parameter. In the second dimension separation column, macromolecules are separated according to another molecular characteristic. In this contribution the methods are briefly reviewed in which effect of polymer molar mass on polymer retention is suppressed. The resulting ”one parameter separation systems” can be on‐line or off‐line connected to another separation system such as SEC to provide more detailed characterization of complex polymers. Besides, selected procedures for the re‐concentration of diluted polymer solutions are concisely treated. These may be utilized for increasing the concentration of sample(s) leaving the first dimension separation column. Eventually, some arrangements for controlled sample re‐introduction into the second dimension separation column are outlined.     

Simulation of the effects of complex- formation equilibria in electrophoresis: I. mathematical model

Simul 5 Complex is a one-dimensional dynamic simulation software designed for electrophoresis, and it is based on a numerical solution of the governing equations, which include electromigration, diffusion and acid-base equilibria. A new mathematical model has been derived and implemented that extends the simulation capabilities of the program by complexation equilibria. The simulation can be set up with any number of constituents (analytes), which are complexed by one complex-forming agent (ligand). The complexation stoichiometry is 1:1, which is typical for systems containing cyclodextrins as the ligand. Both the analytes and the ligand can have multiple dissociation states. Simul 5 Complex with the complexation mode runs under Windows and can be freely downloaded from our web page http://natur.cuni.cz/gas. The article has two separate parts. Here, the mathematical model is derived and tested by simulating the published results obtained by several methods used for the determination of complexation equilibrium constants: affinity capillary electrophoresis, vacancy affinity capillary electrophoresis, Hummel-Dreyer method, vacancy peak method, frontal analysis, and frontal analysis continuous capillary electrophoresis. In the second part of the paper, the agreement of the simulated and the experimental data is shown and discussed.     

Analysis of aromatic acids by nonaqueous capillary electrophoresis with ionic‐liquid electrolytes

The separation of six kinds of aromatic acids by CZE with 1‐ethyl‐3‐methylimidazolium chloride (EMIMCl) and 1‐ethyl‐3‐methylimidazolium hydrogen sulfate (EMIMHSO₄)_, two kinds of ionic liquids (ILs) as background electrolytes, and acetonitrile as solvent were investigated. The six kinds of aromatic acids can be separated under positive voltage with low IL concentration with either of the two ILs and separation with EMIMHSO₄ is better in consideration of peak shapes and separation efficiency. But the migration order is different when the IL is different. Under negative voltage with high IL concentration, the six analytes can be separated with EMIMCl as background electrolytes and the migration order of the analytes is opposite to those with low concentration of EMIMCl as background electrolyte. The separations are based on the combination effects of heteroconjugation between the anions and cations in the ILs and the analytes, of which the heteroconjugation between the anions in the ILs and the analytes plays a dominant role. The heteroconjugation between the anions of the ILs and analytes is proton sensitive and only a very small amount of proticsolvents added into the electrolyte solution can harm the separation. When EMIMCl concentration is high, the heteroconjugation between the IL anions and the proton in the analytes make the effective mobility of the analytes much higher than the EOF and their migration direction reversed. Finally, the six aromatic acids in water samples were analyzed by nonaqueous CE with low concentration of EMIMHSO₄ as background electrolytes with satisfactory results.     

Dynamic high-resolution computer simulation of electrophoretic enantiomer separations with neutral cyclodextrins as chiral selectors

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.     

Separation of anions by ion chromatography-capillary electrophoresis

Capillary electrophoresis (CE) with a water-soluble ion-exchange polymer in the background electrolyte is very efficient for the separation of organic and inorganic anions because the ion-exchange selectivity, as well as differences in electrophoretic mobility, can be used for separating sample ions. Poly(diallyldimethylammonium chloride) (PDDAC) was employed for this purpose. A very stable electroosmotic flow was obtained between pH 2.3 and 8.5 due to the strong adsorption of PDDAC onto the capillary wall. The effect of ion exchange on the migration of sample anions and their separation was controlled by varying the concentration of PDDAC, the concentration and the type of salt used in the CE background electrolyte. Addition of organic solvent (e.g., acetonitrile) could also modify the sample migration and the separation. Baseline separations were obtained for anions with very similar mobilities, such as bromide and iodide, naphthalenesulfonates, and bi- and tricarboxylic acids. Typical separation efficiencies were between 195,000 and 429,000 theoretical plates per meter. Ten replicate separations gave an average RSD of 1.0% for migration times of the sample anions studied. Excellent separations were obtained for a variety of samples, including a separation of 17 inorganic and organic anions in less than 6 min.     

Nonaqueous media for separation of nonionic organic compounds by capillary electrophoresis

For the separation of neutral compounds by micellar electrokinetic chromatography, separations are usually carried out in predominantly aqueous solution in order to preserve the charged micelle necessary for the separation. We now show that polycyclic aromatic hydrocarbon (PAH) compounds can be separated efficiently by capillary electrophoresis in pure methanol or in aqueous-organic mixtures containing a high percentage of methanol. Sodium tetradecyl sulfate was the preferred surfactant. The effects of pH, solvent composition, surfactant structure, and surfactant concentration on the separations were studied. Reproducible migration times and linear calibration plots were obtained.     

Optimization of separation and migration behavior of cephalosporins in capillary zone electrophoresis

The influences of buffer pH, buffer concentration and buffer electrolyte on the migration behavior and separation of 12 cephalosporin antibiotics in capillary zone electrophoresis using three different types of buffer electrolyte, including phosphate, citrate, and 2-(N-morpholino)ethanesulfonate (MES), were investigated. The results indicate that, although buffer pH is a crucial parameter, buffer concentration also plays an important role in the separation of cephalosporins, particularly when cefuroxime and cefazolin, cephalexin and cefaclor, or cefotaxime and cephapirin are present as analytes at the same time. The electrophoretic mobility of cephalosporins and electroosmotic mobility measured in citrate and MES buffers are remarkably different from those measured in phosphate buffer. With citrate buffer, optimum buffer concentration is confined to a small range (35-40 mM), whereas buffer concentrations up to 300 mM can be used with MES buffer. Complete separations of 12 cephalosporins could be satisfactorily achieved with these three buffers under various optimum conditions. However, the separability of 12 cephalosporins with citrate or MES buffer is better than that with phosphate buffer. As a consequence of a greater electrophoretic mobility of cephalosporins than the electroosmotic mobility with citrate buffer at pH below about 5, some cephalosporins are not detectable. The cloudiness of the peak identification and of the magnitudes of the electrophoretic mobility of cefotaxime and cefuroxime reported previously are clarified. In addition, the pKa values of cephradine, cephalexin, cefaclor, and cephapirin attributed to the deprotonation of either an amino group or a pyridinium group are reported, and the migration behavior of these cephalosporins in the pH range studied is quantitatively described.     

Enantiomeric Separation of Epinephrine and Salbutamol by Micellar Electrokinetic Chromatography Using β-Cyclodextrin as Chiral Additive

Introduction The development of chiral substances, especially inthe pharmaceutical field, places increasing demands onanalytical methods for the separation of these kinds ofisomers and the chiral purity control of drugs in phar-macokinetic studies. As the enantiomers of epinephrineand salbutamol have different pharmacological andtoxicological characteristics, separation and quantitationof the single enantiomers are required. Analytical methods used so far for the enantiomerseparation inclu…     

Continuous dielectrophoretic separation of particles in a spiral microchannel

Particle separation is a fundamental operation in the areas of biology and physical chemistry. A variety of force fields have been used to separate particles in microfluidic devices, among which electric field may be the most popular one due to its general applicability and adaptability. So far, however, electrophoresis‐based separations have been limited primarily to batchwise processes. Dielectrophoresis (DEP)‐based separations require in‐channel micro‐electrodes or micro‐insulators to produce electric field gradients. This article introduces a novel particle separation technique in DC electrokinetic flow through a planar double‐spiral microchannel. The continuous separation arises from the cross‐stream dielectrophoretic motion of particles induced by the non‐uniform electric field inherent to curved channels. Specifically, particles are focused by DEP to one sidewall of the first spiral, and then dielectrophoretically deflected toward the other sidewall of the second spiral at a particle‐dependent rate, leading to focused particle streams along different flow paths. This DEP‐based particle separation technique is demonstrated in an asymmetric double‐spiral microchannel by continuously separating a mixture of 5/10 μm particles and 3/5 μm particles.     

Performance implications of chemical mobilization after microchannel IEF

Chemical mobilization following IEF enables single‐point detection of an ideally stationary equilibrium electrophoresis mode. Despite prior studies exploring optimization of chemical mobilization conditions and recent insight from numerical simulations, understanding of both chemical mobilization mechanisms and the implications of mobilization on IEF analytical performance remains limited. In this study, we utilize full‐field imaging of microchannel IEF to assess the performance of a range of canonical chemical mobilization schemes. We empirically demonstrate and characterize key areas where limited understanding of performance implications exists, including: the effects of mobilization solution pH and ion concentration, differences between ionic and zwitterionic mobilization, and diffusion as a source of zone broadening. We utilize Simul5 simulations to gain insight into the sources of the measured performance differences. Measurements of the location, linearity, and slope of the IEF pH gradient (via fluorescent pH markers imaged before and during mobilization) as well as mobilization‐associated broadening of focused analytes were performed to quantify performance and determine the dominant sources of variability. Our results suggest that nonuniform broadening of the pH gradient and changes in the pH gradient linearity stem from conductivity nonuniformities in the separation channel and not diffusion‐associated band broadening during mobilization.     

Capillary zone electrophoresis of histidine-containing compounds

Capillary zone electrophoresis has been tested for the separation of angiotensins, cationic heptapeptides and model histidine derivatives. Good separation efficiencies are seen for peptides and model compounds with negative to small positive net charges. For net charge greater than +2, addition of putrescine to pH 6 buffer greatly suppresses ion exchange at anionic sites on fused silica. When operating at pH values where histidine groups are neutral, addition of Zn2+ allows separations based on metal, rather than proton, binding. Separation efficiencies and relative migration times are dependent on capillary length when ion-exchange behavior occurs.     

Synthesis, analytical characterization and initial capillary electrophoretic use in an acidic background electrolyte of a new, single-isomer chiral resolving agent: heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)-β-cyclodextrin

The sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)cyclomaltoheptaose (HAMS), the first single-isomer sulfated β-CD that carries the sulfo group exclusively at the C2 position, has been synthesized. The purity of each synthetic intermediate and of the final product was determined by hydrophilic interaction (HILIC) and reversed-phase HPLC. The structural identity of each intermediate and of the final product was verified by 1-D and 2-D NMR spectroscopy and MALDI-TOF MS. HAMS was used for the capillary electrophoretic separation of the enantiomers of a set of non-ionic and weak base analytes in pH 2.5 background electrolytes. Rapid separations with satisfactory peak resolution values were obtained for most enantiomers using low concentrations of HAMS. The effective mobilities and separation selectivities were dependent on the concentration of HAMS according to the predictions of the charged resolving agent migration model. The separation selectivities observed with HAMS, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose and heptakis(2-O-methyl-3,6-di-O-sulfo)cyclomaltoheptaose were different for some of the analytes studied in detail.     

Nucleoside monophosphates recognition using macrocyclic polyamine bonded phase in capillary electrochromatography

An open-tubular wall-coated macrocyclic polyamine capillary column (70 cm x 75 microm ID) with 50 cm effective length for the separation of nucleoside monophosphates is described. Some parameters with respect to concentration, pH, composition of the buffer, and voltage in order to optimize the separation were studied. The coated capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. Baseline separations were achieved for the eight nucleotides in less than 26 min using a background electrolyte consisting of H(3)PO(4)-NaH(2)PO(4) (30 mM, pH 3.10), an applied voltage of -15 kV, and detection at 254 nm. The macrocyclic polyamine on the capillary wall introduced anion coordination for the interaction with the analytes, the strength of which could be moderated by the type and concentration of the competing ion used in the background electrolyte (BGE). With a low concentration of the competing ion (phosphate ion), the migration behavior followed that obtained in the electrophoretic system. Increasing the concentration of the competing ion resulted in a faster migration and more complete elution of the analyte. The method established was also employed for the analysis of nucleotides in mushrooms. Aqueous extracts of mushrooms from different species and various extraction methods were injected directly for the analysis. Uridine 5'-monophosphate, guanosine 5'-monophosphate, adenosine 5'-monophosphate, and cytidine 5'-monophosphate, were found in the sample tested.     

Separation of lidocaine and its metabolites by capillary electrophoresis using volatile aqueous and nonaqueous electrolyte systems

The separation of the basic drug lidocaine and six of its metabolites has been investigated both by using volatile aqueous electrolyte system, at low pH and by employing non-aqueous electrolyte systems. In aqueous systems, the best separation of the compounds under the investigated conditions was achieved by using the electrolyte 60 mM trifluoroacetic acid (TFA)/triethylamine (TEA) at pH 2.5 containing 15% methanol. With this electrolyte, all seven compounds were well separated with high efficiency and migration time repeatability. The separations with bare fused-silica capillaries and polyacrylamide-coated capillaries were compared with higher separation efficiency with the latter. On the other hand, near baseline separation of all the seven compounds was also obtained by employing the non-aqueous electrolyte, 40 mM ammonium acetate in methanol and TFA (99:1, v/v), with comparable migration time repeatability but lower separation efficiency relative to the aqueous system.