The dynamics of band (peak) shape development in capillary zone electrophoresis in light of the linear theory of electromigration

The continuity equations that describe the movement of ions in liquid solutions under the influence of an external stationary electric field, as it is utilized in electrophoresis, were introduced a long time ago starting with Kohlrausch in 1897. From that time on, there have been many attempts to solve the equations and to discuss the results. In electrophoresis, special attention has always been devoted to the peak shapes obtained by the detector since the shapes have a tight connection with the phenomena taking place during electromigration and influence the efficiency and selectivity of the separation. Among these phenomena, the most important is electromigration dispersion. In this commented review paper, we compare various models of electromigration, try to find points that connect them, and discuss the range of their validity in light of the linear and nonlinear theory of electromigration.     

Characterization of surfactant and phospholipid vesicles for use as pseudostationary phases in electrokinetic chromatography

The physical, electrophoretic and chromatographic properties (mean diameter, electroosmotic flow, electrophoretic mobility, elution range, efficiency, retention, and hydrophobic, shape, and chemical selectivity) of three surfactant vesicles and one phospholipid vesicle were investigated and compared to a conventional micellar pseudostationary phase comprised of sodium dodecyl sulfate (SDS). Chemical selectivity (solute-pseudostationary phase interactions) was discussed from the perspective of linear solvation energy relationship (LSER) analysis. Two of the surfactant vesicles were formulated from nonstoichiometric aqueous mixtures of oppositely charged, single-tailed surfactants, either cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) in a 3:7 mole ratio or octyltrimethylammonium bromide (OTAB) and SDS in a 7:3 mole ratio. The remaining surfactant vesicle was comprised solely of bis(2-ethylhexyl)sodium sulfosuccinate (AOT) in 10% v/v methanol, and the phospholipid vesicle consisted of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and phosphatidyl serine (PS) in 8:2 mole ratio. The mean diameters of the vesicles were 76.3 nm (AOT), 86.9 nm (CTAB/SOS), 90.1 nm (OTAB/SDS), and 108 nm (POPC/PS). Whereas the coefficient of electroosmotic flow (10(-4) cm2 V(-1) s(-1)) varied considerably (1.72 (OTAB/SDS), 3.77 (CTAB/SOS), 4.05 (AOT), 5.26 (POPC/PS), 5.31 (SDS)), the electrophoretic mobility was fairly consistent (-3.33 to -3.87 x 10(-4) cm2 V(-1) s(-1)), except for the OTAB/SDS vesicles (-1.68). This resulted in elution ranges that were slightly to significantly larger than that observed for SDS (3.12): 3.85 (POPC/PS), 8.6 (CTAB/SOS), 10.1 (AOT), 15.2 (OTAB/SDS). Significant differences were also noted in the efficiency (using propiophenone) and hydrophobic selectivity; the plate counts were lower with the OTAB/SDS and POPC/PS vesicles than the other pseudostationary phases (< or = 75,000/m vs. > 105,000/m), and the methylene selectivity was considerably higher with the CTAB/SOS and OTAB/SDS vesicles compared to the others (ca. 3.10 vs. < or = 2.6). In terms of shape selectivity, only the CTAB/SOS vesicles were able to separate all three positional isomers of nitrotoluene with near-baseline resolution. Finally, through LSER analysis, it was determined that the cohesiveness and hydrogen bond acidity of these pseudostationary phases have the greatest effect on solute retention and selectivity.     

Separation of binaphthyl enantiomers by capillary zone electrophoresis and electrokinetic chromatography

The capillary electrophoretic (CE) separation of the enantiomers of three binaphthyl compounds is investigated. Several CE modes such as cyclodextrin (CD) modified capillary zone electrophoresis (CZE) (CD-CZE), micellar electrokinetic chromatography (MEKC), cyclodextrin electrokinetic chromatography (CD-EKC), etc. are employed for the simultaneous enantiomer separation of the three solutes. The successful separation was achieved by combining two modes, in other words by using more than two chiral selectors. A development of the CE enantiomer separation is demonstrated for the binaphthyl compounds. The enantioselectivity of binaphthyl compounds is alo briefly discussed.     

Mobilities of metal β-diketonato complexes in micellar electrokinetic chromatography

Electrically neutral acetylacetonato complexes of chromium(III), cobalt(III), rhodium(III), and platinum(II) (or palladium(II)) have been separated successfully by capillary zone electrophoresis in a micellar solution of sodium dodecyl sulfate (SDS). The distribution coefficient of each metal complex between the SDS micelle and the aqueous solution surrounding the micelle was calculated from the capacity factor. A linear log-log relationship was found between the distribution coefficient and the partition coefficient of the complex between dodecane and water. The linear relationship was effectively used for prediction of both the distribution coefficients and the migration times of other metal complexes, such as palladium(II) acetylacetonate and chromium(III) 3-methyl acetylacetonate.     

Study of aldehyde oxidase by micellar electrokinetic chromatography separation of O6‐benzylguanine and 8‐oxo‐O6‐benzylguanine

A separation method for O⁶‐benzylguanine (O⁶‐BG) and 8‐oxo‐O⁶‐benzylguanine (8‐oxo‐O⁶‐BG) is developed by using MEKC. This study includes the optimization of separation and incubation parameters for both off‐line and on‐line procedures. The BGE consisted of 25 mM sodium phosphate buffer‐methanol (70:30, v/v), apparent pH 7.4, in which SDS and methyl‐β‐cyclodextrin were dissolved yielding final concentrations of 50 and 15 mM, respectively. Separations were performed at 15 kV using an untreated fused‐silica capillary (40 cm length, effective length is 30 cm) with the detection wavelength at 195 nm. The capillary was kept at 15°C. Good performances were demonstrated for the repeatability and linearity. The LOQ was determined to be 14 μM for 8‐oxo‐O⁶‐BG (S/N = 10). The accuracy values showed a bias of +7.9% for 50 μM and –7.0% for 100 μM. Premix and transverse diffusion of laminar flow profiles (TDLFP) methods were used for on‐line mixing and reaction of the substrate O⁶‐BG with aldehyde oxidase. Both procedures were successful in mixing as well as subsequent separation of the substrate and the metabolite, while the repeatability of TDLFP (14.7% (n = 3)) was much better than the premix technique.     

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Summary Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.     

Twenty‐one years of microemulsion electrokinetic chromatography (1991–2012): A powerful analytical tool

Microemulsion electrokinetic chromatography (MEEKC) is a CE separation technique, which utilizes buffered microemulsions as the separation media. In the past two decades, MEEKC has blossomed into a powerful separation technique for the analysis of a wide range of compounds. Pseudostationary phase composition is so critical to successful resolution in EKC, and several variables could be optimized including surfactant/co‐surfactant/oil type and concentration, buffer content, and pH value. Additionally, MEEKC coupled with online sample preconcentration approaches could significantly improve the detection sensitivity. This review comprehensively describes the development of MEEKC from the period 1991 to 2012. Areas covered include basic theory, microemulsion composition, improving resolution and enhancing sensitivity methods, detection techniques, and applications of MEEKC.     

Synthesis of poly(N,N‐dimethylacrylamide)‐block‐poly(ethylene oxide)‐block‐poly(N,N‐dimethylacrylamide) and its application for separation of proteins by capillary zone electrophoresis

A series of well‐defined triblock copolymers, poly(N, N‐dimethylacrylamide)‐block‐poly(ethylene oxide)‐block‐poly(N, N‐dimethylacrylamide) (PDMA‐b‐PEO‐b‐PDMA) synthesized by atom transfer radical polymerization, were used as physical coatings for protein separation. A comparative study of EOF showed that the triblock copolymer presented good capillary coating ability and EOF efficient suppression. The effects of the M_r of PDMA block in PDMA‐b‐PEO‐b‐PDMA triblock copolymer and buffer pH on the separation of basic protein for CE were investigated. Moreover, the influence of the copolymer structure on separation of basic protein was studied by comparing the performance of PDMA‐b‐PEO‐b‐PDMA triblock copolymer with PEO‐b‐PDMA diblock copolymer. Furthermore, the triblock copolymer coating showed higher separation efficiency and better migration time repeatability than fused‐silica capillary when used in protein mixture separation and milk powder samples separation, respectively. The results demonstrated that the triblock copolymer coatings would have a wide application in the field of protein separation.     

Determination of rofecoxib,in the presence of its photodegradation product,in pharmaceutical preparations by micellar electrokinetic capillary chromatography

A simple and rapid micellar electrokinetic capillary chromatographic (MEKC) method for analysis of rofecoxib (ROF) and its photodegradation product (PDP) in pharmaceutical preparations has been developed and validated. Analyses were conducted in a fused silica capillary (72 cm effective length, 50 m i.d.) with a background electrolyte consisting of 25 mmol L^–1 borate buffer at pH 7.0 containing 15 mmol L^–1 sodium dodecyl sulfate (SDS) and 10% acetonitrile (ACN). The separation was performed by voltage-controlled system, applying 30 kV at 30 °C, detecting at 225 nm; injection was hydrodynamic at 50 mbar for 2 s. Nifedipine was used as internal standard (IS). Under the optimum conditions ROF, PDP, and IS were well separated with in 10 min. The method was validated with regard to linearity, limit of detection and quantitation, precision, accuracy, specificity, and robustness. The detection limit of the method was low, 0.8 g mL^–1, and the linearity range was wide, 2.5 to 125 g mL^–1. The method was highly efficient—5×10⁵ plates m^–1 for ROF. The method was applied to the tablet form of ROF-containing pharmaceutical preparations. The data were compared with those from the voltammetric method described in literature. No statistically significant difference was found.     

Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement,quality assurance,and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused‐silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium, and the applied voltage were as reported previously [16]. The change resulted in a significant lower current, higher resolution, and increased detection time intervals. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. Quality assurance data of a 2‐year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a 3‐year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis.     

Microemulsion electrokinetic chromatography: an application for the simultaneous determination of suspected fragrance allergens in rinse-off products

The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4-24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.     

A first approach using micellar electrokinetic capillary chromatography for the determination of fipronil and fipronil-sulfone in eggs

Fipronil is an insecticide that is not approved in the European Union in food. In 2017, fipronil was involved in a European health alert due to its presence in fresh hen eggs because of an illicit use in poultry farms, so reliable methods are needed to determine fipronil and its main metabolites in these matrixes. In this work, we report the first approach to the study of fipronil and two metabolites, fipronil-sulfone and fipronil-sulfide by CE. MEKC mode was employed using a solution of 50 mM ammonium perfluorooctanoate pH 9.0 with 10% (v/v) methanol as background electrolyte. The proposed method was combined with a simple sample treatment based on salting-out assisted LLE (SALLE) using acetonitrile as extraction solvent and ammonium sulfate as salt. The SALLE–MEKC–UV method allowed the simultaneous quantification of fipronil and fipronil-sulfone. Validation parameters yielded satisfactory results, with precision, expressed as relative SD, below 14% and recoveries higher than 83%. Limits of detection were 90 µg/kg for fipronil and 150 µg/kg for fipronil-sulfone, so in terms of sensitivity further studies of sample treatments allowing extra preconcentration or the use of more sensitive detection, such as MS, would be needed.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for the analysis of methadone and its metabolites in serum of heroin addicts

Methadone (MET) metabolism has been largely demonstrated with a high inter-individual variability and, therefore, quantification of MET is very important for therapeutic drug monitoring. A cation-selective exhaustive injection and sweeping MEKC (CSEI-Sweeping) was first developed to analyze MET and its two metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), in human serum. After pretreatment, the samples were electrokinetically injected into capillary (10 kV, 500s) and swept by the separation phosphate buffer (100 mM, pH 4.0) containing 20% tetrahydrofuran and 100 mM SDS at -15 kV. The LODs were 200 pg/mL for MET and EMDP, and 400 pg/mL for EDDP. Ten volunteers were administered MET (5.0-120.0 mg/day) orally for 84 days and serum samples were taken after the daily dose of MET (days 1, 2, 7, 14, 28, 56 and 84) individually. This method was used for monitoring MET and its metabolites in heroin addicts and for pharmacokinetic investigations.     

Method for on-line derivatization and separation of aspartic acid enantiomer in pharmaceuticals application by the coupling of flow injection with micellar electrokinetic chromatography

A novel, easy and accurate capillary electrophoresis (CE) coupled with flow injection (FI) method for the separation and determination of aspartic acid (Asp) enantiomers by on-line derivatization had been developed, and it had been applied to the real sample for the first time. The derivatization reagents were o-phthalaldehyde (OPA) and mercaptoethanol (ME), which were obtained easily, the chiral selector was beta-cyclodextrin (beta-CD), the micellar chemical was sodium dodecyl sulfate (SDS), and the modifier was methanol. By on-line derivatization, aspartic acid enantiomers were automatically and reproducibly converted to the ultraviolet (UV)-absorbing diastereoisomer derivates, which were separated by micellar electrokinetic chromatography (MEKC). According to the factors affecting the separation and sensitivity of aspartic acid enantiomer and other amino acids in the real sample, the pH value and concentration of the buffer, the concentration of beta-CD and SDS, the volume percentage of the methanol (v/v) in the buffer, the applied voltage and the conversion time were selected as the investigating variates. Under the investigated separation conditions, D-aspartic acid (D-Asp), L-aspartic acid (L-Asp) and other four amino acids achieved the baseline separation in not only the standard mixture of amino acids but also the real sample (Compound Amino Acid Injection (6AA)). The repeatability (defined as relative standard deviation (RSD), n = 5) was 4.0% and 4.0% with peak area evaluation, and 4.2% and 3.7% with peak height evaluation for D-Asp and L-Asp in the real sample. Recovery at added standard levels of 1.0, 3.0 and 6.0 mM was 92%, 104% and 109%, respectively.     

Sequential injection chromatography against HPLC and CE: Application to separation and quantification of amoxicillin and clavulanic acid

Sequential injection chromatography (SIC) has been recently proposed as an alternative separation technique. SIC has the advantages of inexpensiveness, rapid operation procedure, small dimension, short stabilization time, friendly maintenance process and less reagent consumption. In contrast, SIC has had suffered from some limitations. In the current study, a higher pressure resistant selection valve with additional ports than that available in commercially SIC systems was installed. This development allows achieving solution propulsion without showing leakage and linking more standard/sample solutions. In addition, a new method for the separation and quantification of amoxicillin and clavulanic acid in pharmaceutical formulations has been optimized and validated. A comparative study on the efficiency of the SIC method with previous HPLC and CE methods was conducted as well. Besides the benefits of the instrumentation of SIC, the proposed method has shown some advantages over previous HPLC and CE methods with respect to reagent consumption and sample frequency. Other such analytical characters of the SIC method as resolution, peak symmetry, number of theoretical plates, linearity range, accuracy, precision and limits of detection and quantification, recorded comparable results with those obtained from previous HPLC and CE methods.     

Electrokinetic sample injection for high‐sensitivity CZE (part 2): Improving the quantitative repeatability and application of electrokinetic supercharging‐CZE to the detection of atmospheric electrolytes

Electrokinetic supercharging (EKS) is defined as a technique that combines electrokinetic sample injection with transient ITP. Quantitative repeatability of EKS‐CZE and the other CE methods using electrokinetic sample injection process is usually inferior in comparison with the CE methods using hydrodynamic or hydrostatic injection. This is due to some effects, such as the temperature change and the convection of the sample solution in the reservoir, as well as the change of the distance between an electrode and a capillary end (D_ec). In particular, we have found that the D_ec change might most seriously affect the repeatability, especially when the electrode is a thin Pt wire that could be unintentionally bent during sampling. By using a Teflon spacer to fix D_ec to 1.1 mm, the RSD of peak area (n=5) was decreased from 20 to 3.4% in EKS‐CZE for several metal cations. This D_ec dependence of the sample amount injected was supported by computer simulation using CFD‐ACE+ software. The improved repeatability (down to 5.1% at n=5, averaged RSD for Co²⁺, Li⁺, Ni²⁺, Zn²⁺ and Pb²⁺) was also experimentally attained by increasing the D_ec to ca. 20 mm, which was also effective to obtain high sensitivity. Since the temperature and the convection effects on the repeatability are comparatively small in a proper laboratory environment, these effects were estimated from the EKS‐CZE experiments using conditions such as warming and agitating the sample solution during EKS process. Finally, EKS‐CZE was applied to the detection of ions from atmospheric electrolytes in high‐purity water exposed to ambient air for 2 h. The microgram per liter levels of anions (chloride, sulfate, nitrate, formate, acetate and lactate) and cations (ammonium, calcium, sodium and magnesium) could be detected using conventional UV detector.     

Quality by Design approach in the development of a solvent-modified micellar electrokinetic chromatography method: Finding the design space for the determination of amitriptyline and its impurities

A solvent-modified micellar electrokinetic chromatography method was set up for the simultaneous determination of the tricyclic antidepressant amitriptyline (AMI) and its main impurities. The method was developed following Quality by Design (QbD) principles according to ICH Guideline Q8(R2). QbD approach made it possible to find the design space (DS), where quality was assured. After a scouting phase, aimed at selecting a suitable capillary electrophoresis pseudostationary phase, risk assessment tools were employed to define the critical process parameters (CPPs) to be considered in a screening phase (applied voltage, concentration and pH of the background electrolyte, concentration of the surfactant sodium dodecyl sulphate, of the cosurfactant n-butanol and of the organic modifiers acetonitrile and urea). The effects of the seven selected CPPs on critical quality attributes (CQAs), namely resolution values between critical peak pairs and analysis time, were investigated throughout the knowledge space by means of a symmetric screening matrix. Response surface study was then carried out on four selected CPPs by applying a Doehlert Design. Monte-Carlo simulations were performed in order to estimate the probability of meeting the desired specifications on CQAs, and thus to define the DS by means of a risk of failure map. Additional points at the edges of the DS were tested in order to verify the requirements for CQAs to be fulfilled. A control strategy was implemented by defining system suitability tests. The developed method was validated following ICH Guideline Q2(R1), including robustness assessment by Plackett–Burman design, and was applied to the analysis of real samples of amitriptyline coated tablets.     

Effect of hydrophobic chain length of the chiral surfactant on enantiomeric separations by electrokinetic chromatography: Comparison between micellar and vesicular pseudo-stationary phases

Two novel amino acid based surfactants sodium N-(4-n-decyloxybenzoyl)-l-valinate (SDeBV) and sodium N-(4-n-octyloxybenzoyl)-l-valinate (SOBV) have been synthesized and used as chiral selectors for enantiomeric separations by micellar electrokinetic chromatography (MEKC). The aggregation behavior of the surfactants was studied in buffered aqueous solution using surface tension and fluorescence probe techniques. The microenvironment of the aggregates was studied using pyrene, and 1,6-diphenyl-1,3,5-hexatriene (DPH) as probe molecules. Results of these studies indicate that these two surfactants form micelles in buffered aqueous solution. Successful enentioseparation has been achieved for 1,1′-bi-2-naphthol (BOH), 1,1′-binaphthyl-2,2′-diylhydrogenphosphate (BNP), 2,8-dimethyl-6H-5,11-methanodibenzo[b,f] [1,5]diazocine (Tröger's base, TB), and benzoin (BZN) using the two chiral selectors SDeBV and SOBV. The separations were optimized with respect to surfactant concentration, pH, and buffer concentration. The results are discussed in light of the aggregation behavior of the surfactants. A comparison of the results of this study has been made with the data from literature to investigate the effect of self-assembly morphology on enantiomeric separations.