Determination of association constants between enantiomers of orciprenaline and methyl-beta-cyclodextrin as chiral selector by capillary zone electrophoresis using a partial filling technique

The principles for the determination of conditional association constants of enantiomers by capillary zone electrophoresis employing a partial filling technique (PFT) using methyl-beta-cyclodextrin as chiral selector is presented. Orciprenaline was used as a model compound. Partial filling is a separation technique, where different lengths of the chiral selector solution are introduced into the capillary to a final zone length shorter than the effective length of the capillary, prior to application of the solutes. Lengthening of the separation zone results in improving enantioresolution in addition to decreasing electrophoretic mobility of the enantiomers, because of longer interaction time between the solute and chiral selector. The degree of the reduction in electromobility depends on the affinity of the solute to the chiral selector, i.e. strength of the complex formed between the solute and cyclodextrin. The decrease in the electrophoretic mobility with increasing length of the separation zone is used for determination of the association constant. The association constants of the enantiomers of orciprenaline and the chiral selector were evaluated from the slope of the plot, observed electrophoretic mobility versus the ratio between the length of the separation zone and the effective length of the capillary. It was found that the association constants were independent of the chiral selector concentration. The mean values were 110 M(-1) and 160 M(-1) for respective enantiomer. Constants obtained by a conventional CE technique were in good agreement with those from the PFT experiments. The highest enantioselectivityy was obtained when about 50% of the solute was distributed to the selector phase.     

Simultaneous separation and enantioresolution of racemic local anesthetic drugs by capillary zone electrophoresis with Tween 20 and methyl-beta-cyclodextrin as selectors, employing a double plug technique

A new approach for simultaneous chiral and achiral separations by capillary zone electrophoresis is described. Two adjacent selector plugs, consisting of Tween 20 as an achiral and methyl-beta-cyclodextrin (CD) as a chiral selector, are employed and four related local anesthetics are used as model compounds. The principles of the partial filling technique, whereby the capillary is filled with the chiral selector solution followed by the micellar solution at different plug lengths and concentrations, prior to application of the solutes, was employed. During the run both capillary ends were dipped in a simple buffer, i.e., one without additives. The two separation media worked independently without any interaction. Separation of the solutes and their enantiomers was regulated by adjusting both the concentration and plug length (PL) of the micellar solution in the capillary, employing methyl beta-CD as chiral selector either at 38 or 76 mM. The solutes were separated on the basis of their affinity towards the micellar phase before they reached the methyl-beta-CD plug for enantioseparation. In the absence of the micellar plug, the enantiomers of prilocaine overlapped those of bupivacaine. The solutes and their enantiomers were completely separated by employing two adjacent plugs consisting of 100 mM Tween 20 solution (PL approximately 10 cm) and methyl-beta-CD solution at either 38 or 76 mM (PL approximately 30 cm).     

Evaluation of quail egg white riboflavin binding protein as a chiral selector in capillary electrophoresis by applying a modified partial filling technique.

A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on-line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol-coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 microM up to 900 microM and different plug lengths, while the running buffer pH (6.0), temperature (25 degrees C) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine.     

Di-n-amyl L-tartrate-boric acid complex chiral selector in situ synthesis and its application in chiral nonaqueous capillary electrophoresis

A chiral selector, di-n-amyl L-tartrate-boric acid complex, was in situ synthesized by the reaction of di-n-amyl L-tartrate with boric acid in a nonaqueous background electrolyte (BGE) using methanol as the medium. And a new method of chiral nonaqueous capillary electrophoresis (NACE) was developed with the complex as the chiral selector. It has been demonstrated that the chiral selector is suitable for the enantioseparation of some β-blockers and β-agonists in NACE. Some chiral analytes that could not be resolved in aqueous microemulsion electrokinetic chromatography (MEEKC) with the same chiral selector obtained baseline resolutions in the NACE system. The enantioseparation mechanism was considered to be ion-pair principle and the nonaqueous system was more favorable for the ion-pair formation which is quite useful for the chiral recognition. The addition of a proper concentration of triethylamine into the BGE to control the apparent pH (pH*) enhanced the enantiomeric discrimination. In order to achieve a good enantioseparation, the effects of di-n-amyl L-tartrate and boric acid concentration, triethylamine concentration, applied voltage, as well as capillary length were investigated. Under the optimum conditions, all of the tested chiral analytes including six β-blockers and five β-agonists were baseline resolved.     

Partial filling technique in capillary electrophoresis for the separation of phenolic isomers with sulfonatocalix[4]arene as a selector

p-Sulfonatocalix[4]arene was used as a selector in capillary electrophoresis to separate phenolic positional isomers. To avoid the detection interference caused by the high UV absorption of calixarene, the partial filling technique was applied. The operation variables, including buffer, separation voltage, the concentration of the selector and the plug length of the selector zone, were systematically optimized. The detection limits of mass were in the range of 0.07-0.28 pg. Molecular modeling was used to explain the interaction between calixarene and phenolic isomers.     

Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis

A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.     

Chiral Separation by Capillary Zone Electrophoresis Used Cyclodextrins and Their Derivatives as Chiral Selector

Capillary zone electrophoresis (CZE) is a very pronising analytical technique for the optical isomer resolution of the compounds studied. The drawbacks of the techniques such as HPLC [1] were sophisticated stationary phases and/or the relatively high quantity of the chiral agent in the mobile phase, which do not exist in CZE. The capillary electrophoresis (CE) method can offer advantages on lower consumption of analyte and background electrolyte (BGE), shorter analysis time, and higher efficiencies [2-3]     

Predefine resolution of enantiomers in partial filling capillary electrophoresis and two discontinuous function plugs coupling in‐capillary

In common partial filling CE (PF‐CE), the capillary contains the selectors plug between the injection and detector end to avoid the selector going into the detector zone. To expand this method, we propose a mode of two discontinuous function plugs coupling in‐capillary, named as plug–plug PF‐CE (ppPF‐CE). Initially, we present the method to predefine the effective length of chiral selector to meet the requirement of enantiomers' resolution, which could avoid some experimental procedures. With α‐CD as a chiral selector, a satisfactory resolution of enantiomers d,l ‐tryptophan and d,l ‐tyrosine was obtained with a partial filling α‐CD plug of optimal length and concentration. Subsequently, a second plug containing hydroxypropyl methylcellulose, organic solvents (acetonitrile and methanol), anionic and cationic surfactants (SDS and CTAB), and different concentrations of sodium phosphate buffer was inserted after the selector plug. Effects of plug–plug filling on enantiomers' migration and resolution are discussed. The ppPF‐CE might be a new flexible mode for CE application.     

Simultaneous stereoselective analysis of tramadol and its main phase I metabolites by on-line capillary zone electrophoresis-electrospray ionization mass spectrometry

On-line combination of partial filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the simultaneous enantioseparation of tramadol and its main phase I metabolites. The partial filling technique was efficient at avoiding MS contamination by the chiral selector. Different experimental factors were investigated, including the chiral selector nature and concentration, plug length as well as the separation temperature. The best enantioseparation of the investigated compounds was achieved with a coated polyvinyl alcohol capillary and a 40 mM ammonium acetate buffer, pH 4.0, adding sulfobutyl ether beta-cyclodextrin (2.5 mg/ml) as the chiral selector. The charged cyclodextrin not only allowed enantioseparation of tramadol and its metabolites, but also improved the selectivity of compounds with the same molecular mass. Finally, CE-electrospray ionisation-MS was successfully applied to the stereoselective analysis of tramadol and its main metabolites in plasma after a simple liquid-liquid extraction.     

Evaluation of balhimycin as a chiral selector for enantioresolution by capillary electrophoresis

The glycopeptide antibiotic balhimycin and its haloanalogue bromobalhimycin were evaluated as chiral selectors for enantioresolution by capillary electrophoresis. In order (i) to eliminate the adsorption of the glycopeptide antibiotics on the capillary wall, (ii) to shorten the separation time and (iii) to improve the detection sensitivity, a combined approach of the dynamic surface coating technique, the co-electroosmotic flow electrophoresis technique and the partial filling technique was employed for the enantioresolution of 16 acidic racemates. The effect of experimental parameters (plug length of the partial filling solution containing the chiral selector, selector concentration and buffer pH) on enantiorecognition was investigated. Furthermore, the enantiorecognition ability imparted by balhimycin, bromobalhimycin and vancomycin were compared. For most tested compounds, the highest enantiorecognition was obtained with balhimycin as chiral selector. Only in the case of the enantioresolution of tiaprofenic acid, vancomycin showed a superior enantiorecognition.     

Electrolyte and additive effects on enantiomer separation of peptides by nonaqueous ion-pair capillary electrophoresis using tert.-butylcarbamoylquinine as chiral counterion

Nonaqueous ion-pair capillary electrophoresis separations of N-protected (all-R)/(all-S) alanine peptide enantiomers with up to six amino acid residues using tert.-butylcarbamoylquinine as selector and employing the partial filling technique are presented. The effects of various conditional parameters on separation were studied, namely chemical nature of the capillary wall, solvent composition of the background electrolyte (BGE), acid-base-ratio (equivalent to apparent pH), ionic strength and selector concentration. The influence of the solvent composition (methanol-ethanol ratios) on resolution turned out to be rather complex. The separation of the peptide enantiomers was strongly altered by small changes in pH and ionic strength. An increase of the selector concentration was found to offer an easy way for enhancing enantioselectivity, although some drawbacks, e.g., elongation of run times, have to be considered. A method was developed that allowed the separation of N-3,5-dinitrobenzoyl oligoalanine enantiomers containing 1-6 amino acid residues in one run. Like in a recent high-performance liquid chromatography (HPLC) study, separation selectivity thereby decreased from 1.541 (Ala), 1.340 (Ala(2)), 1.054 (Ala(3)), 1.029 (Ala(4)), 1.024 (Ala(5)) to 1.020 (Ala(6)). In addition, all four stereoisomers of N-2,4-dinitrophenyl- and N-3,5-dinitrobenzyloxycarbonyl-protected alanylalanine could be baseline-resolved.     

Enantioseparation by CE with vancomycin as chiral selector: Improving the separation performance by dynamic coating of the capillary with poly(dimethylacrylamide)

An approach for improving the separation performance of the enantioseparation by CE with vancomycin as chiral selector is described. In the present method, a solution of poly(dimethylacrylamide) (PDMA) was used for dynamic coating of the capillary wall to minimize the adsorption of vancomycin onto the capillary wall, and to depress the EOF. Compared with the bare fused-silica capillaries and the capillaries coated with the polycationic polymer hexadimethrine bromide (HDB), the PDMA-coated capillary displayed the best separation performance. The resulting coating could withstand hundreds of runs without losing its function. Moreover, a partial filling technique was applied to avoid interference in detection caused by the presence of vancomycin in the buffer. The separation time was shortened when a short-end-injection technique was applied. Several parameters such as buffer pH, vancomycin concentration and plug length of the vancomycin solution for the separation were optimized. Under the optimal conditions, all tested enantiomers, including FMOC amino acids derivatives, ketoprofen and fenoprofen, were baseline-separated in less than 4.2 min.     

Use of chiral amino acid ester‐based ionic liquids as chiral selectors in CE

In this study, the applicability of a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester‐based CILs were synthesized and used as additives in the BGE in order to evaluate their chiral recognition ability. The performance of these CILs as the sole chiral selectors was evaluated by using 1,1′‐binaphthyl‐2,2‐diylhydrogenphosphate (BNP) as the analyte and by comparing the resolution values. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation, the anion type of the CIL and its concentration, and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral‐recognition ability of the CIL. Baseline separation of BNP within 13 min was achieved by using a BGE of 100 mM Tris/10 mM sodium tetraboratedecahydrate (pH 8) and a chiral selector of 60 mM l ‐alanine tert butyl ester lactate. The run‐to‐run and batch‐to‐batch reproducibilities were also evaluated by computing the %RSD values of the EOF and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all %RSD values were below 1%.     

Separation of chiral drugs with β-CD phosphate by capillary electrophoresis

In capillary electrophoresis (CE) chiral separation is accomplished by adding suitableselector in the running electrolyte"'. The type of selector is of primary importance forachieving successful resolution. Selector concentration has considerable influence aswell3'. Charged cyclodextrins were first introduced for chiral separation of aminoacidss and used for enantioseparation of drugs by Terabe group'. Many kinds ofcharged CDs are now commercially available7-11. The charged CD commonly use…     

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.     

Quantification of buserelin in a pharmaceutical product by multiple-injection CZE

An efficient and rapid separation method based on reversed-polarity multiple-injection CZE (MICZE), has been developed for the quantification of buserelin in a pharmaceutical product. The determinations were performed by serially injecting five standard solutions of buserelin (50-300 microg/mL) and one reference analyte into a Polybrene-coated capillary. All the samples contained goserelin, an analog peptide to buserelin, as internal standard (IS). Immediately after pressure injection, the applied sample plugs were subjected to electrophoresis for 2 min at -25 kV. Consequently, each sample plug became isolated from its neighboring plugs by the BGE, composed of 100 mM phosphate-triethanolamine buffer at pH 3.0 containing 10% v/v ACN. During separation the individual sample components migrated at similar velocities and as distinct zones through the capillary giving 24 peaks, 12 from the analyte and the IS and 12 from the sample matrix. The buserelin content of the pharmaceutical product was determined to be 0.94 +/- 0.05 mg/mL, which is only a slight deviation from the declared concentration (1 mg/mL).     

Simultaneous contactless conductivity detection and UV detection for the study of separation of tamsulosin enantiomers in discontinuous electrolyte systems by CE

This work shows the potential of using discontinuous electrolyte systems for the separation of tamsulosin enantiomers by CE. Sulfated beta-cyclodextrin was used as a chiral selector. In acidic electrolytes, sulfated beta-cyclodextrin migrates as an anion and the analyte (tamsulosin) migrates as a cation. Due to this, four experimental arrangements were proposed. These arrangements differ in composition of electrolytes in the inlet compartment, in the capillary and in the outlet compartment. The separation of tamsulosin enantiomers in acetate buffers with sodium and Tris counterions was studied. Simultaneous contactless conductivity detection and UV detection were used for the study of the separation mechanism in these systems. Mobilities of sulfated beta-cyclodextrin were used for the calculation of the time when the analyte migrates through the BGE zone with the selector. The simulation program Simul 4.0 was used for the calculations of the concentration profiles of the electrolyte components dependent on the time of the separation. The mechanism of enantioseparation in these arrangements was suggested.     

Direct injection of seawater for the analysis of nitroaromatic explosives and their degradation products by micellar electrokinetic chromatography

Practical considerations for the injection and separation of nitroaromatic explosives in seawater sample matrices are discussed. The use of high surfactant concentrations and long electrokinetic injections allows for improved detection limits. Sensitivity was enhanced by two mechanisms, improved stacking at the detector-side of the sample plug and desorption of analyte from the capillary wall by surfactant-containing BGE from the inlet side of the sample plug. Calculated limits of detection (S/N = 3) for analytes prepared in pure seawater were 70–800 ppb with injection times varying from 5 to 100 s.     

Modeling the zeta potential of silica capillaries in relation to the background electrolyte composition

A theoretical relation between the zeta potential of silica capillaries and the composition of the background electrolyte (BGE) is presented in order to be used in capillary zone electrophoresis (CZE). This relation is derived on the basis of the Poisson-Boltzmann equation and considering the equilibrium dissociation of silanol groups at the capillary wall as the mechanism of charge generation. The resulting model involves the relevant physicochemical parameters of the BGE-capillary interface. Special attention is paid to the characterization of the BGE, which can be either salt or/and buffer solutions. The model is successfully applied to electroosmotic flow (EOF) experimental data of different aqueous solutions, covering a wide range of pH and ionic strength. Numerical predictions are also presented showing the capability of the model to quantify the EOF, the control of which is relevant to improve analyte separation performance in CZE.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.