Enantiomer separation by nonaqueous and aqueous capillary electrochromatography on cyclodextrin stationary phases

Native beta- and gamma-cyclodextrin bound to silica (ChiraDex-beta and ChiraDex-gamma) were packed into capillaries and used for enantiomer separation by capillary electrochromatography (CEC) under aqueous and nonaqueous conditions. Negatively charged analytes (dansyl-amino acids) were resolved into their enantiomers by nonaqueous CEC (NA-CEC). The addition of a small amount of water to the nonaqueous mobile phase enhanced the enantioselectivity but increased the elution time. The choice of the background electrolyte (BGE) determined the direction of the electroosmotic flow (EOF). With 2-(N-morpholino) ethanesulfonic acid (MES) or triethylammonium acetate (TEAA) as BGE an inverse EOF (anodic EOF) was observed while with phosphate a cathodic EOF was found. The apparent pH (pH*), the concentration of the BGE, and the nature of the mobile phase strongly influenced the elution time, the theoretical plate number and the chiral separation factor of racemic analytes.     

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.     

Hydrogen-bond effect and ion-pair association in the separation of neutral calix[4]pyrroles by nonaqueous capillary electrophoresis

A study on the migration behavior of four neutral macrocyclic compounds calix[4]pyrroles (C4Ps) by nonaqueous capillary electrophoresis (NACE) with tetrabutylammonium halide salts as background electrolyte (BGE) is described. Systematic studies were carried out with different BGE in acetonitrile (ACN) to elucidate the involved phenomena. The effect of BGE concentration on analytes effective mobilities mu eff,i was studied. Rough values of analytes absolute mobilities mu infinity,i were obtained by extrapolation of effective mobilities to zero ionic strength of BGE. Stokes radius of C4Ps in the form of C4PF(-) and C4PCl(-) was calculated. The complex constants of C4Ps with F(-) anion and Cl(-) anion and ion-pair association constants of each C4PF(-) and C4PCl(-) with tetrabutylammonium cations was evaluated and confronted. Hydrogen bonding effect is prerequisite in the separation; meanwhile ion-pair association which is intensified by steric hindrance effect plays an important role.     

Analysis of Calix[4]arenes Using Nonaqueous Capillary Electrophoresis

In nonaqueous capillary electrophoresis (NACE), an organic solvent is used in place of an aqueous medium as the background solution to improve the solubility and selectivity for hydrophobic analytes. In this study, we employed NACE with UV detection for the analysis of eight calix[4]arenes. We examined the influence of several parameters—the buffer composition, the nonaqueous solvent‘s composition and proportion, and the concentration of the electrolyte of the nonaqueous buffer—on the efficiency of the electrophoretic separation. The separation was achieved through the analyte's different effective mobility via different degrees of deprotonation on the phenolic OH groups of the calix[4]arene. This deprotonation can further affect the analyte's ability to form a complex with the metal ion. The optimized background electrolyte (BGE), comprising a mixture of N‐methylformamide/acetonitrile (30:70, v/v) and 100 mM AcOH/20 mM NH₄OAc, provided rapid (<11 min) separation of the calix[4]arenes with good resolution. The relative standard deviations of the migration times for the eight analytes were all less than 1%. Within the calibration concentration range, the coefficients of determination (R²) were all greater than 0.9914. Thus, the present study demonstrated NACE can provide adequate separation for the analysis of calix[4]arenes.     

Nonaqueous capillary electrophoresis with alcoholic background electrolytes: separation efficiency under high electrical field strengths

The effect of high voltage on capillary electrophoresis (CE) separations of anionic analytes in nonaqueous separation media was investigated. Methanol, ethanol, 1-propanol, and 1-butanol were tested as background electrolyte (BGE) solvents. Experiments were carried out with a laboratory-built CE instrument suitable for high-voltage separations. Potentials up to 60 kV were applied with reversed polarity to generate unusually high field strengths (e.g. 2000 Vcm-1) and so achieve fast and efficient separations. Highest separation efficiencies were obtained with propanol as BGE solvent, and the dependency of the efficiency on the separation voltage was more or less linear. With the other alcohols, separation efficiency decreased or remained roughly constant with increasing absolute voltage. The separation efficiencies are discussed in terms of longitudinal diffusion, Joule heating, and analyte interaction with the capillary wall. Capillary preconditioning had a varied effect on the separations in the different BGEs as the BGE and the conditioning process affected the electroosmotic flow (EOF) velocity and direction.     

Nonaqueous CE using contactless conductivity detection and ionic liquids as BGEs in ACN

N,N'-Alkylmethylimidazolium cations have been separated in NACE when one of the N,N'-dialkylimidazolium salts (ionic liquids (ILs)) was used as an electrolyte additive to the organic solvent separation medium. The separated species were 1-methyl-, 1-ethyl-, 1-butyl-, 1-octyl-, 1-decyl-3-methylimidazolium and N-butyl-3-methylpyridinium cations and BGE composed of 1-ethyl-3-methylimidazolium ethylsulfate or 1-butyl-3-methylimidazolium trifluoroacetate [BMIm][FAcO] (A6; B2) diluted in ACN. It was demonstrated that contactless conductivity detection (CCD) may be applied to monitoring the separation process in nonaqueous separation media, allowing to use the UV light-absorbing imidazolium-based electrolyte additives. There could be marked three concentration regions of added ILs; at first ionic strength of BGE below 1-2 mM, and then the actual electrophoretic mobility of analytes rises from 0. At concentrations above 1-2 mM, the added IL facilitated separation. In concentration region of 1-20 mM, the actual electrophoretic mobility of analyzed imidazolium cations was increasing with decrease in separation medium ionic strength. At higher concentrations of BGE (above 30 mM), the conductivity of the separation media became too high for this detector. Some organic dyes were also successfully separated and detected by contactless conductivity detector in a 20 mM A6 separation electrolyte in ACN.     

Enantioseparation of venlafaxine andO-desmethylvenlafaxine by capillary electrophoresis with mixed cyclodextrins

Summary Capillary electrophoresis (CE) has been successfully applied to the separation of the enantiomers of venlafaxine (Vx) and its main active metabolite,O-desmethylvenlafaxine (ODV), by use of a mixture of two cyclodextrins (CDs) added to the background electrolyte (BGE). The use of carboxymethylated β-cyclodextrin (CMB) enabled the enantioseparation of both Vx and ODV, although separation of all four enantiomers was not achieved. Addition of a second cyclodextrin (α-CD) to the BGE resulted in the simultaneous resolution of the enantiomers of both compounds. In this system, α-CD enabled discrimination between Vx and ODV whereas CMB enabled the enantioseparation. Resolution was strongly influenced by the ratio of the concentrations of the two complexing agents. Baseline resolution of Vx and ODV was achieved in an uncoated capillary. Addition of organic modifiers to the BGE had a substantial effect on the interaction of the analytes with the α-CD.     

Comparison of aqueous and nonaqueous carrier electrolytes for the separation of penicillin V and related substances by capillary electrophoresis with UV and mass spectrometric detection

A method for the determination of penicillin V together with its impurities and by-products formed during biosynthesis, using capillary electrophoresis (CE) with UV and electrospray-mass spectrometric (ESI-MS) detection is presented. Aqueous and nonaqueous electrolytes containing 20 mM ammonium acetate were investigated to determine their suitability for the separation of these analytes. These carrier electrolytes were optimized with respect to the pH and the solvent/s used (water, methanol, acetonitrile, ethanol and isopropanol) and it was shown that although the nonaqueous electrolytes offered unique separation selectivities, the best results in terms of selectivity and sensitivity were obtained for the aqueous system. Finally, the applicability of this method for the analysis of a mixture representative of a real fermentation broth was demonstrated using an aqueous carrier electrolyte with both UV and ESI-MS detection.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Analysis of the lipophilic peptaibol alamethicin by nonaqueous capillary electrophoresis-electrospray ionization-mass spectrometry

The microheterogeneous peptaibol alamethicin F30 isolated from the culture broth of Trichoderma viride was analyzed by nonaqueous CE-electrospray-MS using an IT and a TOF mass analyzer. Compared to aqueous buffers, higher separation selectivity was observed for methanolic BGE allowing the detection of more minor components. The low electrophoretic mobility observed for neutral analytes under nonaqueous conditions may be explained by ion-dipole interactions between the peptide analytes and electrolyte ions. The amino acid sequences of the individual components were derived from MS(n) using the doubly or triply charged pseudomolecular ions as well as characteristic fragments as precursor ions. The exchange of Ala by alpha-aminoisobutyric acid (Aib) which is frequently observed for peptaibols was detected for several components. Additional variations included the exchange of Gln to Glu, and the loss of the C-terminal amino alcohol or of the first six amino acids from the N-terminus with concomitant formation of pyroglutamyl residues. In most cases comigration of the Aib peptaibols with the respective Ala component was observed as the mass difference of 14 Da as the result of the amino acid exchange was not sufficient to translate into an electrophoretic separation under the conditions applied. However, proper selection of the precursor ions allowed the unequivocal analysis of the components. Additional TOF-MS measurements were performed in order to resolve the ammonium adducts from comigrating compounds (i.e., Aib-Ala exchange) and to confirm the amino acid composition of the individual components. Except for neutral compounds migrating close to the EOF the mass accuracy was better than 4 ppm for the doubly charged pseudomolecular ions and better than 2 ppm for triply charged ions.     

Analysis of neutral nitromusks in incenses by capillary electrophoresis in organic solvents and gas chromatography-mass spectrometry

Nitromusks used as fragrances in a variety of personal-care products, cleansers, and domestic deodorants, including incense sticks, are neutral nitro aromatic compounds; some of these have been reported as photosensitizers. In this work, their analysis was performed by capillary electrophoresis (CE) in a methanol-based background electrolyte (BGE). In particular, a 10 mM solution of citric acid in methanol was used; under these conditions the strong suppression of the electroosmotic flow favored the use of negatively charged surfactants as additives for the anodic migration of the studied analytes. To this end, sodium taurodeoxycholate (TDC) was supplemented at high concentration (190 mM) to the organic background electrolyte (BGE), showing strong indication of the ability to give micelle-like aggregates. Since nitromusks are characterized by the presence of a nitroaromatic ring with low charge density, their association with TDC aggregates can be ascribed to donor-acceptor interactions. Separation of musk xylene, musk ketone, and the banned musk moskene and musk ambrette was obtained under full nonaqueous BGE; the addition of relatively small water percentages (15% v/v) was found to be useful in improving the separation of pairs of adjacent peaks. Under optimized conditions (190 mM sodium TDC in methanol-water, 85-15 v/v containing citric acid 10 mM) the system was applied to the analysis of nitromusks in incense sticks extracted with methanol. The results were compared with those obtained by the analysis of the same samples using gas chromatography with mass detector. The expected different selectivity of separation obtained using the two techniques can be useful in the unambiguous determination of the analytes; furthermore, a substantial accord of the preliminary quantitative results achieved with the two methods was assumed as the confirmation of the potential reliability of CE performed with high percentage of organic solvent.     

Tungstate as complex-forming reagent facilitating separation of selected polyphenols by capillary electrophoresis and its comparison with borate

A novel electrophoretic BGE containing tungstate as complex-forming reagent is suitable for the separation of polyphenols. Similar to molybdate-containing BGE reported earlier (cf. M. Polásek, et al.., Talanta 2006, 69, 192) addition of tungstate to BGE affects significantly migration of compounds/ligands with vicinal -OH groups due to the formation of negatively charged complexes involving W(VI) as central ion. Baseline separation of mixtures of flavonoids (apigenin, luteolin, hyperoside, quercetin, and rutin) and phenolic acids (chlorogenic and p-coumaric acid) was achieved within 15 min with optimized BGE of pH 7.4 containing 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES), 2.2 mM tungstate, and 25% v/v of methanol. The separation was performed in a 75 cm (effective length 42 cm)x 75 microm id uncoated fused-silica capillary at 30 kV with spectrophotometric detection at 275 nm. The calibration curves were rectilinear for 25-175 microg/mL of all analytes (cinnamic acid as the internal standard). The LODs ranged from 1.8 to 6 microg/mL for all analytes except for chlorogenic acid. Intraday precision (n = 6) of migration times (RSD < or = 1.2%) and peak areas (RSD < or = 5.6%) was evaluated. The tungstate-based BGEs can be alternatively utilized for the analysis of polyphenols at considerably lower pH than with conventional alkaline borate-based BGEs.     

Nonaqueous capillary electrophoresis of dextromethorphan and its metabolites

This study deals with the nonaqueous capillary electrophoretic separation of dextromethorphan and its metabolites using a methanolic background electrolyte. The optimization of separation conditions was performed in terms of the resolution of dextromethorphan and dextrorphan and the effect of separation temperature, voltage, and the characteristics of the background electrolyte were studied. Complete separation of all analytes was achieved in 40 mM ammonium acetate dissolved in methanol. Hydrodynamic injection was performed at 3 kPa for 4 s. The separation voltage was 20 kV accompanied by a low electric current. The ultraviolet detection was performed at 214 nm, the temperature of the capillary was 25°C. These conditions enabled the separation of four analytes plus the internal standard within 9 min. Further, the developed method was validated in terms of linearity, sensitivity, and repeatability. Rat liver perfusate samples were subjected to the nonaqueous capillary electrophoretic method to illustrate its applicability.     

Separation of iota-, kappa and lambda-carrageenans by capillary electrophoresis

The present study reports a novel method for the separation of the high-molecular-weight anionic polysaccharides, iota, kappa, and lambda carrageenans, in capillary electrophoresis (CE). Carrageenan samples are first derivatised with 9-aminopyrene-1,4,6-trisulfonic acid (APTS), separated in an ammonium acetate background electrolyte (BGE) and detected with laser-induced fluorescence (LIF). The effects of changes of instrumental parameters (temperature, injection mode, field strength) and the composition of the BGE (concentration and pH) are reported, and are explained in terms of the physical chemistry of the BGE and the biopolymers. Optimal separation conditions for kappa, iota, and lambda carrageenans, including an APTS internal standard, were found in a polyvinyl alcohol coated capillary with an ammonium acetate BGE of low concentration (25 mM) and moderate pH (8.0). This BGE gave the best reproducibility in tests on iota/kappa mixtures, with relative standard deviations (RSDs) in migration times and normalised peak areas (relative to the APTS internal standard) of less than 0.1% and 1%, respectively. Using this BGE at 50 degrees C and a voltage of 30 kV, all three carrageenan subtypes were separated in a run time of 3 min.     

Influence of pH on formation and stability of phosphatidylcholine/phosphatidylserine coatings in fused-silica capillaries

The effect of pH on the formation and stability of phospholipid coatings in fused-silica capillaries in electrophoresis was investigated. A liposome solution consisting of 3 mM of 80:20 mol% phosphatidylcholine/phosphatidylserine (PC/PS) in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer was used as coating material. The coating was prepared by a method described earlier and five steroids were used as neutral model analytes. First, the effect of pH of the coating solution on the formation and stability of phospholipid coatings was studied at pH 6.5-8.5. The pH of the background electrolyte (BGE) solution (HEPES) was either kept constant at pH 7.4 or made similar to the pH of the liposome coating solution. Results showed that attachment of the coating on the fused-silica wall mostly depends on the protonation of amines of the phospholipids and HEPES. The ability of the phospholipid coating to withstand changes in pH was then investigated by coating at pH 7.5 and separating steroids with acetic acid, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, or glycine BGE, adjusted to pH between 4.5 and 10.8. The results showed that with use of BGE solution at pH 10.8, the separation of steroids was not successful and the electroosmotic flow was high because of leakage of the phospholipid coating during preconditioning of the capillary with BGE solution. There was no phospholipid leakage with a BGE solution of pH 4.5, indicating that the protonated form of the functional groups of PS and HEPES participating in the attachment of the phospholipid coating to the capillary play an essential role in the success of the coating.     

Nonaqueous capillary electrophoretic behavior of 2-aryl propionic acids in the presence of an achiral ionic liquid. A chemometric approach

Ionic liquids (ILs) appear really attractive as electrolyte additives in nonaqueous capillary electrophoresis (NACE). These salts may offer new possibilities of interactions to modulate analyte effective mobilities. The presence of 1-n-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (BMIM NTf2) in acetonitrile/alcohol background electrolytes (BGEs) was investigated in this work. The aim of this study was to elucidate the influence of the IL concentration on the electrophoretic behavior of four arylpropionic acids and to identify the interactions between the analytes and the IL cation. The influence on mobility of the IL concentration, the nature and the proportion of the organic solvents, and the concentration of the ionic components of the BGE was first studied by a univariate approach. A four-factor D-optimal experimental design was then applied to provide a deeper insight into analyte interaction with IL cation present both free in BGE and adsorbed onto the capillary wall.     

The role of organic solvents in the separation of nonionic compounds by capillary electrophoresis

Although nonionic compounds can be separated by micellar electrokinetic chromatography (MEKC), application of this technique is restricted by a somewhat limited elution range. Incorporation of organic solvents in the background electrolyte (BGE) greatly extends the scope of MEKC and provides a major variable in optimizing the separation of neutral analytes. This paper provides a systematic review of the principles and scope of the separation of neutral analytes by capillary electrophoresis (CE) in organic-aqueous solution. The methods surveyed include those that use tetraalkylammonium salts, dioctyl sulfosuccinate, lauryl poly(oxyethylene) sulfate. Polyaromatic hydrocarbon (PAH) compounds can be separated using sodium hexadecyl sulfate in 70% methanol (30% aqueous) to 100% methanol.     

Impact of substituent position in monosubstituted α-cyclodextrins on enantioselectivity in capillary electrophoresis

In this study, our three recently synthesized regiospecifically monosubstituted carboxymethyl-α-cyclodextrins (CMACDs) were successfully applied for the enantiomeric separation of several biologically important low-molecular weight compounds by capillary electrophoresis. The enantioselectivity of the individual monosubstituted CMACDs added into the background electrolyte (BGE) was studied and compared with the mixture of three monosubstituted CMACDs and with native α-cyclodextrin at pH of the BGE ranging from 2.5 to 11. Our experiments revealed a significant influence of the position of the carboxymethyl group on the α-cyclodextrin skeleton on the enantioselectivity for all the studied analytes. Interestingly, the least common 3(I)-O regioisomer was revealed as the most effective chiral selector.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.