Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.     

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.     

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.     

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.     

Separations of antifungal compounds in capillary electrophoresis with two anionic cyclodextrins

CD‐CZE methods were developed for complete stereoisomeric separations of a series of six γ‐lactam analogues, of which some were neutral, or cationic depending on the background electrolyte nature. The tested cyclodextrin was the versatile sulfobutylether‐ β‐CD, used either in a phosphate buffer using capillaries dynamically coated with polyethylene oxide or in a borate buffer using uncoated capillaries. Long‐end and short‐end modes and concentration variations of chiral selectors allowed finding conditions of complete separation of four out of the six derivatives (i.e., 1 , 2 , 3, and 4 ) in short run times, confirming their broad range of applications. To separate the two last compounds, the highly sulfated‐ γ‐CD was examined as chiral selector in acidic phosphate conditions. The enantiomers of the γ‐lactam analogues 5 and 6 were baseline resolved with 5.5 and 4%, respectively as concentration in the buffer.     

表面活性剂在高效毛细管电泳中的作用

表面活性剂作为缓冲液添加剂已广泛用于高效毛细管电泳中,综述了阴离子、阳离子、两性离子、非离子及手性等多种表面活性剂在离子、中性分子、手性化合物、多肽和蛋白质分离等方面的作用,介绍了其作用机理与改善高效毛细管电泳分离的原理。     

Enhancement of second-migrating enantiomer peak symmetry of basic drugs by using dual-cyclodextrin system in capillary electrophoresis

Today, chiral separations of cationic drugs by capillary electrophoresis are generally carried out by adding negatively charged cyclodextrins (CDs) to the running buffer while anionic or neutral drug separations require the use of dual-CD systems (mixtures of neutral and charged CDs). Chiral separation of some basic drugs (idazoxan, efaroxan, milnacipran) has been studied by using mixtures of sulfated-beta-CD (S-beta-CD) and hydroxypropyl-gamma-CD (HP-gamma-CD). The influence of the following parameters (nature and concentration of neutral CD, concentration of S-gamma-CD) on many separation factors (electrophoretic mobility, selectivity, efficiency, asymmetry factor, resolution) demonstrated that dual-CD systems are useful for chiral separation of basic drugs in order to improve the symmetry of the second-migrating enantiomer. Indeed, the neutral CD reduces the extent of electromigration dispersion by mobility tuning. Finally, the 0.5 mg/mL S-beta-CD/5 mg/mL HP-gamma-CD dual system has allowed the chiral separation of idazoxan, efaroxan and milnacipran enantiomers in less than 9 min.     

Estimation of the pH-independent binding constants of alanylphenylalanine and leucylphenylalanine stereoisomers with beta-cyclodextrin in the presence of urea

The separation of stereoisomers, particularly enantiomers, is important when their physiological activity differs. We have resolved the four stereoisomers each of alanylphenylalanine (Ala-Phe) and of leucylphenylalanine (Leu-Phe) by capillary electrophoresis using beta-cyclodextrin as a buffer additive and urea to enhance its solubility. A study of the influence of pH and beta-cyclodextrin concentration on the separations showed that weak inclusion complexes were formed between the dipeptides and chiral selector. It was found that pH could alter the migration order of enantiomers L-Ala-L-Phe and D-Ala-D-Phe, as well as L-Leu-L-Phe and D-Leu-D-Phe; however, there was no change in order for the other pairs of optical isomers. Electrophoretic mobility data were used to estimate the acid dissociation constants of the dipeptide isomers at pH < 7 with no chiral selector present. By varying the concentration of beta-cyclodextrin, the chiral selector, the binding constants of Ala-Phe and Leu-Phe optical isomers in their fully protonated and zwitterionic forms were estimated. For the four Ala-Phe stereoisomers, K = 42-66 M(-1) and 4-41 M(-1) for the cationic and zwitterionic forms, respectively. For the four Leu-Phe stereoisomers, K = 43-94 M(-1) and 1-28 M(-1) for the cationic and zwitterionic forms, respectively.     

Enantioselective separation of chiral vicinal diols in capillary electrophoresis using a mono‐6A‐aminoethylamino‐β‐cyclodextrin as a chiral selector

This paper describes an improved access to mono‐6^A‐aminoethylamino‐β‐CD (β‐CDen), a very efficient cationic chiral selector for CZE in the separation of eight chiral aromatic vicinal diols. The β‐CDen concentration has a strong influence on the efficiency of enantioseparation. The effects of the pH and concentration of the BGE, the capillary temperature, and the applied voltage on the resolution and separation selectivity have been studied. Excellent chiral resolution was achieved under the optimal conditions of β‐CDen 10 mM, pH 10, 200 mM borate buffer at 15 kV and 20°C within 20 min. Moreover, the developed method was successfully applied to the determination of the enantiomeric purity of the catalytic asymmetric dihydroxylation (AD) reaction products.     

Chiral separation with gradient elution isotachophoresis for future in situ extraterrestrial analysis

The first results of chiral separations with the gradient elution isotachophoresis method are presented. As previously described, citrate is used in the run buffer as the leading ion and borate in the sample buffer as the terminating ion. Modulation of parameters such as electrolyte pH, pressure scan rate, chiral selector concentration, combinations of CD or the percentage of ampholytes provides an easy optimization of the separations. To perform fluorescent detection 5-carboxyfluorescein succinimidyl ester and two fluorogenic-labeling agents, fluorescamine (Fluram) and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, are used to label amino acids. With the 5-carboxyfluorescein amino acids, chiral separations are easily obtained using a neutral CD ((2-hydroxypropyl)-beta-CD) at a low concentration (2 mmol/L). With Fluram amino acids, the situation is more complicated due to the formation of diastereoisomers and due to weak interactions with the different CDs used. The use of the 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde-labeling agent solves the problems observed with the Fluram agent while retaining the fluorogenic properties. These first results demonstrate the simplicity and the feasibility of gradient elution isotachophoresis for chiral separations.     

Rapid separation of pharmaceutical enantiomers using electrokinetic chromatography with a novel chiral microemulsion

A novel oil-in-water microemulsion incorporating the chiral surfactant dodecoxycarbonylvaline (DDCV) was used to achieve the rapid enantiomeric separation of pharmaceutical drugs by electrokinetic chromatography (EKC). Incorporation of DDCV into a microemulsion resulted in an elution range more than double that provided the micellar form of the surfactant aggregate. Interestingly, for the same compounds the enantioselectivity provided by the chiral DDCV microemulsions ranged from 1.06-1.30 for the neutral and cationic drugs, which was slightly higher than that provided by chiral DDCV micelles. The use of a low surface tension oil (ethyl acetate) permitted a much lower concentration of chiral surfactant to be employed; this, together with the use of a zwitterionic buffer (ACES) resulted in a very low conductivity microemulsion that allowed a higher separation voltage to be utilized, resulting in rapid enantiomeric separations (< 8 min.). Mobility matching of the buffer cation(s) was used to improve peak shape and efficiencies. In our limited survey of the phase diagram, the optimum composition of the microemulsion buffer was 1.0% (w/v) DDCV (30 mM), 0.5% (v/v) ethyl acetate, 1.2% (v/v) 1-butanol and 50 mM ACES buffer at pH 7.     

Enantioseparation of 3,4-dihydroxyphenylalanine and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid by capillary electrophoresis using cyclodextrins

The enantiomeric separations of 3,4-dihydroxyphenylalanine (dopa) and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid (carbidopa) by capillary electrophoresis were studied using several native, neutral and anionic cyclodextrins as chiral additives and uncoated fused-silica capillaries. The effect of the type and concentration of the cyclodextrin added to 20 mM phosphate buffer (pH 2.5) on enantioseparation and migration times was studied. A high resolution value of 15.63 was obtained for dopa enantiomers with a buffer containing 20 mM single isomer, heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin. The enantiomers of carbidopa were separated using 20 mM carboxymethyl-beta-cyclodextrin as a chiral resolving agent. Both methods allowed the determination of 0.1% of the D-enantiomer (second migrating) in the presence of the L-enantiomer (first migrating) of dopa and carbidopa with a good precision. These methods also gave good results in terms of precision for both peak area, migration time, linearity and accuracy.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

Contemporary chiral simulators for capillary zone electrophoresis

For separation of enantiomers in presence of a chiral selector, data obtained with the 1D dynamic simulators SIMUL5complex and GENTRANS are compared to data predicted by PeakMaster 6, a recently released generalized model of the linear theory of electromigration. Four electrophoretic systems with stereoisomers of weak bases were investigated. They deal with the estimation of input data for complexation together with the elucidation of the origin of observed system peaks, the interference of analyte and system peak migration, the change of enantiomer migration order as function of the selector concentration and the inversion of analyte migration direction in presence of a multiply negatively charged selector. For all systems, data predicted with PeakMaster 6 are in agreement with those of the dynamic simulators and simulation data compare well with experimental data that were monitored with setups featuring conductivity and/or UV absorbance detection along the capillary. SIMUL5complex and GENTRANS provide the full dynamics of any buffer and sample arrangement and require very long execution time intervals. PeakMaster 6 is restricted to conventional CZE, is based on an approximate solution of the transport equations, provides data for realistic experimental conditions within seconds and represents a practical tool for an experimentalist.     

Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4 x 10(-4) cm2 V(-1) s(-1) The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte pKa values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.     

Not only pH. Specific buffer effects in biological systems

The aim of this work is to overview the specific effect of pH buffers in biological systems. The pH of a buffer solution changes only slightly when a small amount of a strong acid or bases is added to it. This is widely accepted and applied both in chemical and in biological (i.e. enzyme catalysis) systems. Here we show some examples – spanning from pH measurements, enzyme activities, electrophoretic mobilities, antibody aggregation, protein thermal stability – that demonstrate additional roles of buffers. They not only set pH, but also address specific ion effects, in terms of Hofmeister series, when strong electrolytes are also added. From the experimental data referred to some charged biological moieties it emerges that different buffers, at the same nominal pH, can specifically adsorb at the charged surface. Buffer specific adsorption modifies several molecular and macroscopic properties amongst which electrophoretic mobilities, and hence effective surface charges, are particularly significant. More importantly, buffers' weak electrolytes, even at low concentration, are found to compete for the adsorption at the charged surfaces with strong electrolytes, thus modulating Hofmeister effects.     

Separation of enantiomers by capillary electrophoresis using pentosan polysulfate

Pentosan polysulfate, a semisynthetic polysaccharide, was employed as a chiral run buffer additive in capillary electrophoresis. Twenty-eight racemic analytes were resolved. The separations were successful only at low pH when the analytes were significantly protonated. This suggests that ionic interactions were the dominant associative interactions between the anionic pentosan polysulfate and the positively charged analytes. Compared to other linear, carbohydrate-based chiral selectors (i.e., chondroitin sulfates, heparin and dextran sulfate) pentosan polysulfate has some characteristics common of anionic polysaccharides; yet it has several differences in its structure and properties which account for its unusual enantioselectivity. The effects of pH, concentration of phosphate buffer, concentration of pentosan polysulfate and the type and concentration of organic modifier on the enantiomeric separations were investigated. The optimization of these separations were dependent on the nature of the analytes and could be achieved by the proper choice of experimental conditions.     

Investigation of the Enantioseparation of Basic Drugs with Erythromycin Lactobionate as a Chiral Selector in CE

The macrocyclic antibiotics including ansamycins, glycopeptides, aminoglycosides and polypeptides have been demonstrated to exhibit powerful enantioselectivity towards numerous chiral compounds. By comparison with the four classes of antibiotics, macrolides are another type of macrocyclic antibiotics. In this study erythromycin lactobionate belonging to the group of macrolides is first used as a chiral selector in CE for the enantiomeric separations of basic drugs. As observed, erythromycin lactobionate allowed excellent separation of the enantiomers of N,N-dimethyl-3-(2-methoxyphenoxy)-3-propylamine, propranolol and duloxetine, as well as partial enantioresolution of primaquine, chloroquine and nefopam. In addition, erythromycin lactobionate possesses advantages such as high solubility and low viscosity in the solvent and very weak UV absorption. Among several experimental factors including buffer pH, BGE and erythromycin lactobionate concentrations, capillary temperature and applied voltage, we found that the enantioseparations of basic drugs above-mentioned strongly depended on the pH of BGE and the concentration of the chiral additive. The optimum pH was in the neutral or weak basic region but varied among drugs. An erythromycin lactobionate concentration of about 10% (w/v) and a low capillary temperature of 16 °C were recommended for the practical analysis.     

Electrolysis phenomena in electrophoresis

We present a new theoretical approach for calculating changes in the physico-chemical properties of BGEs for measurements by CZE due to the electrolysis in electrode vials (vessels). Electrolysis is an inevitable phenomenon in any measurement in CZE. Water electrolysis, which occurs in most measurements, can significantly alter the composition of the BGE in electrode vials and in the separation capillary and has a negative influence on the robustness and quality of separations. The ability to predict changes in the composition of the BGE is important for evaluation of the suitability of the BGEs for repeating electrophoretic runs. We compared theoretically calculated changes in the physico-chemical properties (pH, conductivity) with those measured using pH-microelectrode and contactless conductivity detection of the BGE after the electrophoretic run. We confirmed the validity of our theoretical approach with a common BGE composed of acid-base pair, where one constituent is fully dissociated while the second constituent is dissociated by only half, and with Good's buffer. As predicted by theoretical approach, the changes in the physico-chemical properties of the Good's buffer after the electrophoretic run were several times lower than in the case of a common BGE composed of a weak acid – strong base pair.