Enantiomer separations by nonaqueous capillary electrophoresis using octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The newest member of the single-isomer isomer sulfated cyclodextrin family, octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma-CD) was used for the first time as a resolving agent for the nonaqueous capillary electrophoretic separation of the enantiomers of 26 weak base pharmaceuticals in an acidic methanol background electrolyte. The solubility limit of ODAS-gamma-CD at room temperature proved to be 55 mM in this background electrolyte, which afforded good, fast enantiomer separations for most of the basic drugs tested. For all the bases studied, the effective mobilities and separation selectivities were found to follow the predictions of the charged resolving agent migration model of electrophoretic enantiomer separations. The effective mobilities of the weakly binding weak bases remained cationic throughout the entire 0 to 45 mM ODAS-gamma-CD concentration range; separation selectivities increased as the ODAS-gamma-CD concentration was increased. The effective mobilities of the moderately binding weak bases became anionic in the 2.5 to 45 mM ODAS-gamma-CD concentration range; separation selectivities first increased as the effective mobilities approached zero, then decreased again as the ODAS-gamma-CD concentration was increased further. The effective mobilities of the strongly binding weak bases became anionic in the 0 to 2.5 mM ODAS-gamma-CD concentration range; separation selectivities decreased as the ODAS-gamma-CD concentration was increased above 2.5 mM.     

A family of single-isomer,sulfated gamma-cyclodextrin chiral resolving agents for capillary electrophoresis: octa(6-O-sulfo)-gamma-cyclodextrin

The second member of the single-isomer, sulfated gamma-cyclodextrin family, the sodium salt of octa(6-O-sulfo)-gamma-cyclodextrin (OS) has been synthesized, characterized and used to separate the enantiomers of nonelectrolyte, acidic, basic, and ampholytic analytes by capillary electrophoresis in acidic aqueous background electrolytes. The anionic effective mobilities of the nonelectrolyte and anionic analytes increased with increasing concentration of OS. The effective mobilities of strongly complexing cationic analytes became anionic with very low OS concentrations and passed local anionic effective mobility maxima as the OS concentration, and along with it, the ionic strength, of the background electrolyte increased. The effective mobilities of the weakly binding cationic analytes became only slightly anionic at high OS concentration values and did not show the local anionic effective mobility maxima. For nonelectrolyte analytes, separation selectivities decreased with increasing OS concentration. For cationic analytes, separation selectivities were highest where the effective mobilities of the less mobile enantiomers approached zero. OS proved to be a broadly applicable chiral resolving agent.     

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.     

Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis

Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.     

The use of a bivalent counter anion to control the effective mobility of the hydrogen ion constituent in cationic isotachophoresis

The use of a bivalent counter anion for cationic isotachophoresis was studied both theoretically and experimentally. A mathematical model was proposed and solved to give the effective mobility of the hydrogen ion constituent, uH,H, in the respective electrolyte systems. The theoretical values agreed well with the experimental data in a series of electrolyte systems. The dependence of uH,H on such parameters as pH of the leading electrolyte, ionic mobilities of leading and counter ions, and dissociation constants was calculated and discussed. A bivalent counter anion will prove useful for the separation of low mobility cations and weak bases, as shown for the separation of substituted anilines in a succinate electrolyte system.     

Influence of a soluble anionic polymer on the electrophoresis of proteins

The influence of a soluble anionic polymer on electrophoresis of proteins was studied in relation to the nonspecific ionic effect of an affinophore on application to affinophoresis. Zone electrophoresis of proteins was carried out in agarose gel in the presence of succinyl-poly-L-lysine (degree of polymerization, 120) by using three electrophoresis buffers differing in ionic strength (0.06, 0.12 and 0.18) and pH (7.0 and 7.9). Proteins migrated as distinct single bands even in the presence of the polymer. The mobility of cationic proteins towards the cathode was first decreased and then increased towards the anode as the polymer concentration increased, while that of anionic proteins was not affected. The dependence of the apparent mobility changes of the proteins on the concentration of the polymer was treated quantitatively in the same way as affinity electrophoresis. The extent of the ionic interaction between a cationic protein and the polymer could be estimated as an apparent dissociation constant. It greatly depended on the ionic strength of the electrophoresis buffer. Except for the extremely cationic proteins such as lysozyme, the ionic interaction with up to 0.1 mM of the polymer could be practically suppressed by the use of 0.1 M sodium phosphate buffer (pH 7.0).     

Identification of peaks in capillary zone electrophoresis based on actual mobilities.

A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.     

Influence of methanol as a buffer additive on the mobilities of organic cations in capillary electrophoresis

The mobilities of a series of aromatic ammonium ions, ranging in charge from +1 to + 3, were investigated by capillary electrophoresis using buffers consisting of 0-75% v/v methanol. This is an extension of our previous studies involving anion mobility in methanol-water media [1]. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. For all of the buffer compositions studied, the ionic strength effect increased with increasing cation charge, and varied as a function of solvent 1/eta epsilon (1/2) as predicted by the electrophoretic term within the Pitts' equation. In the presence of methanol, the ionic strength effects became more dramatic. The absolute mobilities of the cations were altered by the addition of methanol to the electrophoretic media. For example, at 75% MeOH, a migration order reversal was observed between the + 2 and + 3 ammonium ions. These solvent-induced selectivity changes are attributed to dielectric friction. As predicted by the Hubbard-Onsager dielectric friction model, dielectric friction increased with increasing methanol content and with increasing analyte charge. Further, the changes in cation mobility correlated to the changes in solvent relaxation time (tau), epsilon and eta. Although not predicted by the Hubbard-Onsager theory, the + 3 ammonium ion experienced more dielectric friction than the - 3 sulfonate and - 3 carboxylate investigated previously [1]. This apparent failure of the Hubbard-Onsager model results from its continuum nature, whereby ion-solvent interactions are not taken into account.     

Dual-opposite injection electrokinetic chromatography for the unbiased, simultaneous separation of cationic and anionic compounds

The concept of dual opposite injection in capillary electrophoresis (DOI-CE) for the simultaneous separation, under conditions of suppressed electroosmotic flow, of anionic and cationic compounds with no bias in resolution and analysis time, is extended to a higher pH range in a zone electrophoresis mode (DOI-CZE). A new DOI-CE separation mode based on electrokinetic chromatography is also introduced (DOI-EKC). Whereas conventional CZE and DOI-CZE are limited to the separation of charged compounds with different electrophoretic mobilities, DOI-EKC is shown to be capable of separating compounds with the same or similar electrophoretic mobilities. In contrast to conventional EKC with charged pseudostationary phases that often interact too strongly with analytes of opposite charge, the neutral pseudostationary phases appropriate for DOI-EKC are simultaneously compatible with anionic and cationic compounds. This work describes two buffer additives that dynamically suppress electroosmotic flow (EOF) at a higher pH (6.5) than in a previous study (4.4), thus allowing DOI-CZE of several pharmaceutical bases and weakly acidic positional isomers. Several DOI-EKC systems based on nonionic (10 lauryl ether, Brij 35) or zwitterionic (SB-12, CAS U) micelles, or nonionic vesicles (Brij 30) are examined using a six-component test mixture that is difficult to separate by CZE or DOI-CZE. The effect of electromigration dispersion on peak shape and efficiency, and the effect of surfactant concentration on retention, selectivity, and efficiency are described.     

High-resolution computer simulation of electrophoretic mobilization in isoelectric focusing

Cationic and anionic electrophoretic mobilization for focusing of hemoglobins (Hb's) in the presence of 100 carrier ampholytes covering a pI range of 6.00-7.98 was studied by computer simulation at a constant current density of 300 A/m(2). Electropherograms that would be produced by whole column imaging and by single detectors placed at different locations along the focusing column are presented. Upon mobilization, peak heights of the Hb zones decrease, but the zones retain a relatively sharp constant profile and are migrating at a constant velocity. A further peak decrease occurs during readjustment at the locations of the original buffer/column interfaces, indicating that detection sensitivity is the lowest at these locations. An anionic carrier ampholyte mobility smaller than that of its cationic species produces a cathodic drift which is smaller than the transport rate used for electrophoretic mobilization. Compared to the case with equal mobilities of carrier ampholyte species, a small increase (decrease) is predicted for the cationic (anionic) mobilization rate within the focusing column. Simulation data suggest that electrophoretic mobilization after focusing and focusing with concurrent electrophoretic mobilization are comparable isotachophoretic processes that occur when there is an uninterrupted flux of an ion through the focusing column. Cathodic drift caused by unequal mobilities of the species of carrier ampholytes, electrophoretic mobilization, and decomposition occurring at the pH gradient edges are related electrophoretic processes.     

Estimation of the possibility of internal solid-phase reactions to occur in some oxide systems

We calculated the energy of defect formation and the mobility of defects in cubic ZrO₂-based solid solutions and in nickel and cadmium oxides in a view of developing methods for controlling internal solidphase reacitons in an inert matrix, these reactions being of considerable importance for manufacturing matrix/inclusion composites. The calculations were carried out for an ionic model of chemical bond using GULP software. The frequency factors in the Arrhenius temperature funcitons of diffusion coefficients were estimated for various cationic and anionic defects using circular vibration frequencies of hopping atoms. A frequency factor was estimated as the mean vibration frequency obtained from lattice dynamic calculations also using GULP software followed by the Fourier-transform analysis of the projection of the trajectory of an atom on the direction of transition into an activated state. The results were used to estimate the excess anion concentrations necessary for reaching equal cationic and anionic mobilities at various temperatures; from these estimations, it was inferred that, of the systems under consideration, an internal solid-phase reaction is expected to occur only for NiO.     

Controlling the electrophoretic mobility of single-walled carbon nanotubes: a comparison of theory and experiment

The electrophoretic mobilities of single-walled carbon nanotubes (SWNTs) in agarose gels subjected to negatively charged covalent functionalization and noncovalent anionic surfactant adsorption are compared using a simplified hydrodynamic model. Net charges are calculated on the basis of estimated friction coefficients for cylindrical rodlike particles. The effects of functionalization with negatively charged 4-hydroxybenzene diazonium and anionic sodium cholate are quantified and compared with model predictions. The adsorption of Na+ counterions into the nonionic surfactant layer adsorbed on SWNTs (Triton-X-405) is shown to induce a positive charge and reverse the mobility under select conditions. This effect has not been identified or quantified for nanoparticle systems and may be important in the processing of these systems.     

Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

A hydrophilic polymer, poly(vinylpyrrolidone) (PVP), was employed for suppressing the electroosmotic flow (EOF). A capillary was filled with aqueous PVP solution for coating the capillary wall with PVP; the PVP solution was then replaced by a migration buffer solution containing no PVP. Three types of PVP with different molecular weights were examined. The EOF was suppressed more effectively as the molecular weight of PVP increased. The EOF in the coated capillary was approximately 10-fold smaller than that of a bare capillary and was constant in the pH range of 6-8. The suppressed EOF was stable even when no PVP was added to the migration buffer. However, the EOF increased significantly when sodium dodecyl sulfate was added into the migration buffer. The method was applied for determining the electrophoretic mobilities of inorganic anions that have negative electrophoretic mobilities larger than the electroosmotic mobility of the bare capillary. A novel method for determining the electrophoretic mobilities was proposed based on the linear relationship between electric current and electrophoretic mobility. The electrophoretic mobility was proportional to the electric current. Therefore, the intercept of the regression equation represents the electrophoretic mobility at room temperature. The electrophoretic mobilities were in good agreement with the absolute electrophoretic mobilities.     

Experimental verification of a predicted, hitherto unseen separation selectivity pattern in the nonaqueous capillary electrophoretic separation of weak base enantiomers by octakis (2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of cationic enantiomers with single-isomer multivalent anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (mu0(RCD)/mu0(SCD)), and parameter a, the complexation-induced alteration of the analyte's mobility (mu0(RCD)/mu0). In addition to the previously observed discontinuity in separation selectivity that occurs as mu(eff) of the less mobile enantiomer changes from cationic to anionic, a new feature, a separation selectivity maximum was predicted to occur in the resolving agent concentration range where both enantiomers migrate cationically provided that (i) K(RCD)/K(SCD) <1 and mu0(RCD)/mu0(SCD) >1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) > 1, or (ii) K(RCD)/K(SCD) >1 and mu0(RCD)/mu0(SCD) <1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) <1. This hitherto unseen separation selectivity pattern was experimentally verified during the nonaqueous capillary electrophoretic separation of the enantiomers of four weak base analytes in acidic methanol background electrolytes with octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gammaCD) as resolving agent.     

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.     

The effect of surfactants on the dissociation constants of phenothiazine derivatives Alkalimetric determination of diethazine and chlorpromazine

The effect of a cationic, an anionic and a non-ionic surfactant on the acid-base equilibria of the phenothiazine derivatives, diethazine hydrochloride and chlorpromazine hydrochloride, has been studied. It has been found that the presence of cationic and non-ionic surfactants strongly enhances the dissociation of the two derivatives, whereas the anionic surfactant decreases the dissociation constant. These effects are in agreement with a theory based on a pseudophase, ion-exchange model of micelles. From the dissociation-constant values as a function of the surfactant concentration, the binding constants for diethazine and chlorpromazine with the surfactants Septonex and sodium dodecylsulphate have been calculated. The ability of cationic surfactants to solubilize the free bases of the phenothiazine derivatives and to increase their dissociation constants has been utilized to develop a new method for alkalimetric determination of the derivatives in a micellar medium. The method has been applied to determination of the content of the active component in pharmaceutical preparations.     

Size-based characterization of nanometric cationic maghemite particles using capillary zone electrophoresis

Size-sorted maghemite (gamma-Fe(2)O(3)) particle populations of number mean solid diameters ranging from 6 to 10 nm were suspended and directly characterized in their stabilizing acidic, citrated or basic aqueous media using CZE coupled with UV detection. Analytical conditions were optimized in order to ensure reliable mobility measurements of these ferrofluids in their anionic and cationic forms. Particular interest has been paid to the investigation of the positively charged ferrofluids since cationic colloids have received little attention so far. A strategy for capillary wall modification was chosen in order to prevent particle adsorption while preserving high analytical performances. The influence of experimental conditions such as particle volume fraction, injection volume, electric field strength and electrolyte nature on electrophoretic profiles and measured electrophoretic mobilities was evaluated. A size-dependent electrophoretic mobility was demonstrated and discussed in terms of the ratio of the particle radius to Debye length with reference to existing models (Henry, etc.). Although these nanometric particle distributions lie in a very narrow size range, partial separation was obtained with selectivity varying as a function of electrolyte ionic strength.     

Smooth model surfaces from lignin derivatives. II. Adsorption of polyelectrolytes and PECs monitored by QCM-D

For the first time to the knowledge of the authors, well-defined and stable lignin model surfaces have been utilized as substrates in polyelectrolyte adsorption studies. The adsorption of polyallylamine (PAH), poly(acrylic acid) (PAA), and polyelectrolyte complexes (PECs) was monitored using quartz crystal microgravimetry with dissipation (QCM-D). The PECs were prepared by mixing PAH and PAA at different ratios and sequences, creating both cationic and anionic PECs with different charge levels. The adsorption experiments were performed in 1 and 10 mM sodium chloride solutions at pH 5 and 7.5. The highest adsorption of PAH and cationic PECs was found at pH 7.5, where the slightly negatively charged nature of the lignin substrate is more pronounced, governing electrostatic attraction of oppositely charged polymeric substances. An increase in the adsorption was further found when the electrolyte concentration was increased. In comparison, both PAA and the anionic PEC showed remarkably high adsorption to the lignin model film. The adsorption of PAA was further studied on silica and was found to be relatively low even at high electrolyte concentrations. This indicated that the high PAA adsorption on the lignin films was not induced by a decreased solubility of the anionic polyelectrolyte. The high levels of adsorption on lignin model surfaces found both for PAA and the anionic PAA-PAH polyelectrolyte complex points to the presence of strong nonionic interactions in these systems.     

Quantitative analysis of cation binding to the adenosine nucleotides using the variable ionic strength method: validation of the Debye-Hückel-Onsager theory of electrophoresis in the absence of counterion binding

The free solution mobilities of the adenosine nucleotides 5'-adenosine triphosphate (ATP), 5'-adenosine diphosphate (ADP), 5'-adenosine monophosphate (AMP), and 3'-5'-cyclic AMP (cAMP) have been measured in diethylmalonate buffers containing a wide variety of monovalent cations. The mobilities of all nucleotides increase gradually with the increase in intrinsic conductivity of the cation in the BGE. However, at a given conductivity, the mobilities observed for ATP, ADP, and AMP in BGEs containing alkali metal ions and other cations are lower than these observed in BGEs containing tetraalkylammonium ions. Since the mobility of cAMP is independent of the cation in the BGE, the results suggest that the relatively low mobilities observed for ATP, ADP, and AMP in BGEs containing cations other than a tetraalkylammonium ion are due to cation binding, reducing the effective net charge of the nucleotide and thereby reducing the observed mobility. To measure the binding quantitatively, the mobilities of the nucleotides were measured as a function of ionic strength. The mobilities of ATP, ADP, and AMP decrease nonlinearly with the square root of ionic strength (I(1/2)) in BGEs containing an alkali metal ion or Tris(+). By contrast, the mobilities decrease linearly with I(1/2) in BGEs containing a nonbinding quaternary ammonium ion, as expected from Debye-Hückel-Onsager (DHO) theory. The mobility of cAMP, a nonbinding analyte, decreases linearly with I(1/2), regardless of the cation in the BGE. Hence, a nonlinear decrease of the mobility of an analyte with I(1/2) appears to be a hallmark of counterion binding. The curved mobility profiles observed for ATP, ADP, and AMP in BGEs containing an alkali metal ion or Tris(+) were analyzed by nonlinear curve fitting, using difference mobility profiles to correct for the effect of the physical properties of BGE on the observed mobilities. The calculated apparent dissociation constants range from 22 to 344 mM, depending on the particular cation-nucleotide pair. Similar values have been obtained by other investigators, using different methods. Interestingly, Tris(+) and Li(+) bind to the adenosine nucleotides with approximately equal affinities, suggesting that positively charged Tris(+) buffer ions can compete with alkali metal ions in Tris-buffered solutions.     

Electrophoresis of a biocolloid covered with a cation-absorptive membrane

The electrophoretic behavior of a biocolloid covered with a charged membrane is theoretically analyzed in the present study. Here, the influences of nonuniformly distributed fixed groups, absorption of cations by fixed original functional groups, variation in dielectric constant in the electrophoretic system, and ionic sizes are considered. The results of numerical simulation suggest that a larger absolute value of the electrophoric mobility of biocolloids could be generated by larger membrane electricity. The absolute value of the electrophoric mobility for the nonlinear distribution of the fixed groups is larger than that for the linear distribution of the fixed groups. The absolute value of the electrophoric mobility increases with (1) the concentration of total fixed groups, (2) the cation-absorption equilibrium constant, (3) the nonuniform feature index for functional-groups distribution, (4) the dielectric constants of the inner uncharged membrane zone for only mobile cationic charge and for both mobile cationic and anionic charge, and (5) the effective size of the cations. An increase in the absolute value of the electrophoric mobility can also be resulted from a decrease in the following parameters: (1) the friction coefficient of the biocolloidal membrane phase, (2) the membrane thickness, (3) the dielectric constant of space for all charge and of outer uncharged membrane zone, (4) the effective sizes of anions and fixed groups, and (5) the number of cations and the fixed original functional groups involved in the formation of a cation-functional group complex.