Micellar Electrokinetic Capillary Chromatography Separation Research Articles

Simultaneous determination of gaseous and particulate carbonyls in air by coupling micellar electrokinetic capillary chromatography with molecular imprinting solid-phase extraction

A novel method coupling molecular imprinting solid-phase extraction (MISPE) and micellar electrokinetic capillary chromatography (MEKC) was developed to enable the hourly determination of low level of ambient carbonyls, and study their partition between gaseous phase and particulate phase. With 2,4-dinitroaniline (DNAN) as dummy imprinting template, the unreacted 2,4-Dinitrophenylhydrazine (DNPH) in sampling solution could be removed effectively using MISPE, and an average recovery of 97±5.3% (n=5) for the carbonyl-DNPH derivatives was achieved. Owing to the high enrichment due to sample clean-up, and the improvement of MEKC separation efficiency, many low abundant carbonyls could be detected by hourly in the field study.

Field-amplified sample injection for the determination of isonicotinamide and nicotinamide in whitening cosmetics and supplemented foodstuffs by MEKC

A new micellar electrokinetic capillary chromatography (MEKC) method combined with field-amplified sample injection (FASI) has been developed for the analysis of isonicotinamide and nicotinamide in whitening cosmetics and supplemented foodstuffs. Parameters affecting FASI preconcentration and MEKC separation were optimized systematically. Compared with direct MEKC, the sensitivity of FASI-MEKC was improved by 12.8- and 9.8-fold for isonicotinamide and nicotinamide, respectively. The detection limits (LODs) of isonicotinamide and nicotinamide were 0.051 μg mL−1 and 0.069 μg mL−1, respectively, with RSD (intraday) < 4.9% (n = 7, c = 0.5 μg mL−1) and RSD (interday) < 6.4% (n = 5, c = 0.5 μg mL−1). The proposed method was successfully applied in determination of isonicotinamide and nicotinamide in whitening cosmetics and supplemented foodstuffs with acceptable recoveries of 84.7–108.4%.

Separation of 1,4-benzodiazepine Derivates by Micellar Electrokinetic Capillary Chromatography Using Cyclodextrines as Buffer Modifiers

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the separation of benzodiazepine (BZD) derivates with different structural characteristics has been studied. Efforts were first focused on the optimization of the analytical conditions, on the effects of buffer concentration, surfactant concentration and buffer pH on the separation. To manipulate selectivity of the separation βcyclodextrin (CD) was added to the buffer solution, and urea was used to improve the solubility of the CD in water. The CD-modified MEKC separation of the eight BZDs was achieved within 15 minutes using 25 mmol dm natrium tetraborate-50 mmol dm sodium dodecyl sulphate-15 mmol dm β-CD-2 mol dm urea (pH 9.3) as the running buffer. The proposed separation method was evaluated on the basis of precision, linearity and limit of detection. (doi: 10.5562/cca1763)

The multi-concentration and two-dimensional capillary electrophoresis method for the analysis of drugs in urine samples

A novel method has been developed by integration of multi-concentration and two-dimensional (2D) capillary electrophoresis (CE) for simultaneous enhancement of detection sensitivity and separation power in complex samples. Capillary zone electrophoresis (CZE) was used as the first dimension separation according to mobilities, from which the effluent fractions were further analyzed by micellar electrokinetic capillary chromatography (MEKC) acting as the second dimension. Cation-selective exhaustive injection (CSEI) preconcentration method was used to introduce more analytes into the capillary. Furthermore, pH junction and sweeping dual concentration strategies were employed to avoid sample zone diffusion at the interface. The resulting electrophoregram was quite different from that of either CZE or MEKC separation. Up to (0.5–1.2) × 104 fold improvements in sensitivity were obtained relative to the conventional electrokinetic injection method. The proposed method was successfully applied to the determination of drugs in human urine.

Multi-concentration and two-dimensional capillary electrophoresis method for the analysis of drugs in urine samples

A novel method has been developed by integration of multi-concentration and two-dimensional (2D) capillary electrophoresis (CE) for simultaneous enhancement of detection sensitivity and separation power in complex samples. Capillary zone electrophoresis (CZE) was used as the first dimension separation according to mobilities, from which the effluent fractions were further analyzed by micellar electrokinetic capillary chromatography (MEKC) acting as the second dimension. Cation-selective exhaustive injection (CSEI) preconcentration method was used to introduce more analytes into the capillary. Furthermore, pH junction and sweeping dual concentration strategies were employed to avoid sample zone diffusion at the interface. The resulting electrophoregram was quite different from that of either CZE or MEKC separation. Up to (0.5-1.2)×104 fold improvements in sensitivity were obtained relative to conventional electrokinetic injection method. The proposed method was successfully applied to the determination of drugs in human urine.

1-Hexadecyl-3-methylimidazolium Ionic Liquid as a New Cationic Surfactant for Separation of Phenolic Compounds by MEKC

The ionic liquid 1-hexadecyl-3-methylimidazolium bromide ([C16MIm]Br) has been used as a novel cationic surfactant for separation of phenolic compounds, including quinol, phloroglucinol, resorcinol, phenol, p-cresol, and m-nitrophenol, by micellar electrokinetic capillary chromatography (MEKC). The effects of buffer concentration and pH, concentration of [C16MIm]Br, and applied potential were studied. Use of the optimized buffer (25 mmol L−1 NaH2PO4), 10 mmol L−1 [C16MIm]Br, and an applied potential of −15 kV enables optimum separation with regard to resolution and migration time. The phenolic compounds were detected at 214 nm. The micelle of this long-alkyl-chain imidazolium ionic liquid acts as a pseudo-stationary phase in this MEKC separation.

Heart-cut capillary electrophoresis for drug analysis in mouse blood with electrochemical detection

A novel separation method, which interfaced capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC), was developed for analysis of weak basic compounds. CZE was used as the first separation, from which the effluent components were transferred and further analyzed by MEKC. As the key to successful integration of CZE and MEKC, a new microhole interface was designed. On-line concentration strategies, namely pH junction and sweeping, were employed to avoid sample zone diffusion at the interface. For evaluation of the feasibility and effectiveness of the capillary electrophoresis (CE) system, basic cardiovascular drugs were used as model compounds. The resulting electropherogram was quite different from that of either CZE or MEKC separation. The relative standard deviations (RSDs) of peak height, peak area, and migration time were in the ranges of 2.7% to 4.5%, 1.3% to 3.6%, and 0.8% to 1.4%, respectively, and detection limits (signal/noise = 3) were 0.015 to 0.052 mg/L. The proposed method was successfully applied to the determination of basic cardiovascular drugs in mouse blood.

Sodium cholate aggregation and chiral recognition of the probe molecule (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate (BNDHP) observed by 1H and 31P NMR spectroscopy.

Bile salt micelles can be employed as a pseudostationary phase in micellar electrokinetic capillary chromatography (MEKC) separations of chiral analytes. To improve MEKC separations of chiral analytes, a molecular level understanding of micelle aggregation in the presence of analyte is needed. Here, aggregation of sodium cholate has been observed by exploiting the presence of a model analyte molecule. The 31P and 1H nuclear magnetic resonance spectroscopy (NMR) chemical shifts of (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate ((R,S)-BNDHP), a model analyte in chiral MEKC separations, are demonstrated to be very sensitive to the aggregation state of the bile salt sodium cholate. In addition to probing micellar aggregation, the NMR spectral resolution of enantiomeric species is also stronglycorrelated with chiral separations in MEKC. In this work, the aggregation of sodium cholate in basic solutions (pH 12) has been observed over the concentration range 0-100 mM. The primary critical micelle concentration (cmc) was found to be 14 +/- 1 mM for basic solutions of sodium cholate. In addition, a primitive aggregate is clearly observed to form at 7 +/- 1 mM sodium cholate. The data also show pseudo-cmc behavior for secondary aggregation observed in the regime of 50-60 mM cholate. Finally, the H5-H7 edge of BNDHP is shown to be sensitive to chirally selective interactions with primary cholate micelles.

Comparative study of multi walled carbon nanotubes-based electrodes in micellar media and their application to micellar electrokinetic capillary chromatography

This work reports on a comparative study of the electrochemical performance of carbon nanotubes-based electrodes in micellar media and their application for amperometric detection in micellar electrokinetic capillary chromatography (MEKC) separations. These electrodes were prepared in two different ways: immobilization of a layer of carbon nanotubes dispersed in polyethylenimine (PEI), ethanol or Nafion onto glassy carbon electrodes or preparation of paste electrodes using mineral oil as binder. Scanning electron microscopy (SEM) was employed for surface morphology characterization while cyclic voltammetry of background electrolyte was used for capacitance estimation. The amperometric responses to hydrogen peroxide, amitrol, diuron and 2,3-diclorophenol (2,3CP) in the presence and in the absence of sodium dodecylsulphate (SDS) were studied by flow injection analysis (FIA), demonstrating that the electrocatalytic activity, background current and electroanalytical performance were strongly dependent on the electrodes preparation procedure. Glassy carbon electrodes modified with carbon nanotubes dispersed in PEI (GC/(CNT/PEI)) displayed the most adequate performance in micellar media, maintaining good electrocatalytic properties combined with acceptable background currents and resistance to passivation. The advantages of using GC/(CNT/PEI) as detectors in capillary electrophoresis were illustrated for the MEKC separations of phenolic pollutants (phenol, 3-chlorophenol, 2,3-dichlorophenol and 4-nitrophenol) and herbicides (amitrol, asulam, diuron, fenuron, monuron and chlortoluron).

Simultaneous Determination of Various Classes of Pesticides Using Micellar Electrokinetic Capillary Chromatographyin Combination with Solid-Phase Extraction

The potential of micellar electrokinetic capillary chromatography (MEKC) with UV-detection for the simultaneous determination of various selected pesticides in water samples was investigated. The developed method using solid-phase extraction showed high efficiency and good resolution with detection limits in the 0.2 to 0.5 ppm range. Comparison of several SPE cartridges demonstrates their suitability for the extraction of pesticides with different hydrophobicity achieving 5000-fold enrichment. The described method involving SPE procedure and MEKC separation enables the successful determination of a wide spectrum of pesticides in water in the range of maximum residue limits (MRL’s).

Determination of impurities in the drug 5-aminosalicylic acid by micellar electrokinetic capillary chromatography using an electrolyte pH that approaches the isoelectric point of the parent compound

Process impurities found in the drug substance 5-aminosalicylic acid were determined by micellar electrokinetic capillary chromatography (MECC). In order to enhance the detection of trace impurities, the parent drug was dissolved to an unusually high concentration of 5 mg/mL. To reduce the dispersive processes of electromigration dispersion and anti-stacking, both of which occur at high solute concentration, the electrolyte pH was adjusted to be close to the apparent isoelectric point (p I) of the zwitterionic drug. In this fashion, the net charge on the solute should approach zero thereby minimizing the aforementioned sources of band-broadening. Two additional developments are reported. Short sodium hydroxide washes were used to optimize the MECC reproducibility. The elimination of anti-stacking permitted the use of peak heights to quantitate low level impurities with improved precision. The method was compared to the high-performance liquid chromatographic (HPLC) method found in the United States Pharmacopoeia (USP). Both the MECC and HPLC methods were found to be similar with regard to migration-retention time reproducibility, peak area-height reproducibility, linearity and limit of quantitation. The MECC separation is 2× faster than HPLC. Both methods meet the system suitability requirements described in the USP.

Separation of arsines and trinitrotoluene by reversed phase high performance liquid chromatography and micellar electrokinetic capillary chromatography

Reversed phase high performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MEKC) successfully separated mixtures of arsine compounds and explosives. HPLC separation was accomplished with a methanol/water gradient and detection with an UV-DAD detector. MEKC separation was accomplished with a borate (5 mM) and sodium dodecyl sulfate (50 mM) buffer at pH 9.2. Both methods permitted qualitative and quantitative determination but the HPLC technique was found to yield better reproducibility and thus lower detection limits. HPLC limits of detection (using the 3σ-method) for phenyldichloroarsine, diphenylchloroarsine and triphenylarsine were to be found at 1.87, 1.94 and 0.12 ng, respectively. The detection limit for TNT was found at 0.39 ng. These low detection limits show the possibility of the selected methods to quantify high concentrations found in samples of gas shells, as well as very low concentrations to be detected after destruction experiments of the arsenicals. Determination of a typical World War I 105 mm Blaukreuz-munition-type yield following results: TNT (51.6±0.4%) and Clark I (41.0±0.5%).

Applications of capillary electrophoresis with laser-induced fluorescence for analysis of dGMP-BPDE adduct.

DNA adducts are thought to be crucial to the initiation of mutational and carcinogenic processes. Polycyclic aromatic hydrocarbons (PAHs) have been identified as one major source of carcinogenic risk since they can bind to DNA thus forming an adduct. Quantification of this adduct is important because it may correlate to the risk for cancer development. In this study, the adduct formed between 2'-deoxyguanosine 5'-monophosphate and benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was analyzed by capillary electrophoresis. Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) modes with laser-induced fluorescence detection were used for the separation and analysis of DNA adducts. The exploration of capillary electrophoresis in several modes provided different separation mechanisms in which the stereochemical forms of the adduct could be separated. The best result obtained was using a coated fused-silica capillary in Tris-TAPS buffer, which provided high sensitivity with a detection limit of 2.5x10(-9) mol L(-1). MECC separation of the BPDE adduct, although less sensitive, provided an efficient enantioselective separation option.

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A 2 on the Caliper 250 HTS system: throughput of 80 min per 384-well plate can be achieved with unattended running time of 5.4 h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

Mixed micellar electrokinetic capillary chromatography separation of depolymerized grape procyanidins

Oligomeric procyanidins are potent antioxidant polyphenols of potential interest as disease-preventing agents. Their efficiency depends on the size and composition of their oligomeric structures. The mean degree of polymerization of these compounds is usually estimated by thiolysis with thiol-alpha-toluene followed by analysis using high-performance liquid chromatography (HPLC). We show the development of a mixed micellar electrokinetic chromatography (MEKC) method for the separation of the major components obtained after thiolysis with cysteamine (catechins and their cysteamine conjugates). MEKC studies using sodium dodecyl sulfate (SDS as pseudostationary phase led to long migration times, e.g., with 100 mM SDS, at pH 7, the solutes were separated in about 40 min), while the use of sodium cholate (SC) produced an elution window relatively short. Using a mixed micellar SC-SDS system (50 mM phosphate at pH 7 containing 40 mM SC and 10 mM SDS), it is possible to separate these compounds in less than 15 min. The proposed method is useful to separate the major components of the thiolysate in effluents from food processing (e.g., skins and seeds from grape and apple) considered as potential procyanidin sources.

Predicting and evaluating separation quality of micellar electrokinetic capillary chromatography by artificial neural networks.

Computer-aided optimization of micellar electrokinetic capillary chromatography (MEKC) separations was demonstrated by artificial neural networks (ANNs) using a Levenberg-Marquardt algorithm and an orthogonal experimental design. A novel criterion, named Q, for evaluating the separation quality of MEKC was firstly presented, which considered both separation selectivity and analysis time. MEKC separation conditions of seven plant hormones were then simulated and optimized using ANNs based on this novel criterion. The result was further compared to that obtained using ANNs based on a traditionally used criterion of overall normalization resolution (named r). Finally, the separation under optimum conditions predicted by ANNs using the criterion Q was compared to, and proved to be better than that obtained by empirical step-by-step optimization procedures. This method may also be adapted to other separation methods due to its generality.

Determination of sildenafil citrate and its main metabolite by sample stacking with polarity switching using micellar electrokinetic chromatography

Micellar electrokinetic capillary chromatography (MEKC) coupled with sample stacking and polarity switching was investigated for the determination of Viagra (sildenafil citrate, SC) and its metabolite (UK-103,320, UK) in human serum in the concentration range of clinical interest. Human serum samples spiked with SC and UK were eluted with methanol from a C 18 cartridge, the extract was evaporated and regenerated in a solution that contained 1 m M phosphate buffer (pH 12.3) and 20% methanol. The MEKC separation was performed using an injection time of 275 s, a polarity switching time of 93 s, a phosphate buffer, (pH 12.3, 15 m M) containing 25 m M sodium dodecyl sulfate as separation electrolyte and a fused-silica capillary. The analysis takes about 6 min and gives satisfactory inter-day precision with respect to migration times and linear responses over the 80–900 ng/ml concentration range investigated for SC and UK. Intra-day RSDs ( n=4 graphs) for the slopes of the calibration graphs were 4.86% for SC and 3.50% for UK. Inter-day RSDs for the slopes were 4.37% for SC and 5.39% for UK. Detection limits ( S/ N=3) were about 17 ng/ml for both compounds in human serum. A 1-ml volume of blood serum was necessary to do this determination.

Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen l-amino acid standards were labelled with NBD-F at 60°C for 1 min, and separated in a buffer system containing 20 m M borate, 25 m M sodium cholate, 10 m M Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.

Micellular Electrokinetic Capillary Chromatography in the Undergraduate Curriculum: Separation and Identification of the Amino Acid Residues in an Unknown Dipeptide Using FMOC Derivatization

This manuscript describes our efforts to introduce biochemistry students to micellar electrokinetic capillary chromatography (MEKC), a mode of capillary electrophoresis that employs micelles in the operating buffer. Unlike free solution capillary electrophoresis, MEKC is capable of resolving both charged and uncharged analytes because the micellar pseudo stationary phase allows for the separation of uncharged species. The experiment described herein includes a comparison of MEKC, employing sodium dodecyl sulfate (SDS) as the micelle-forming species, with reverse-phase HPLC. Both methods are used to determine the amino acid residues in an unknown dipeptide. Advanced undergraduate chemistry, biochemistry, and biology majors perform this experiment in the Biochemical Methods course at Bucknell University. The students cleave the peptide bond, derivatize the resultant amino acids with 9-fluorenylmethyl chloroformate (FMOC), and separate the FMOC–amino acid derivatives using HPLC and MEKC. This manuscript details the analytical procedures for the MEKC separation and presents typical student data obtained using this relatively new method.

Evaluation of a Separation-Based Fiber-Optic Sensor in a Micellar Electrokinetic Capillary Chromatography Mode of Operation

A fiber-optic sensor that integrates the separation-based selectivity of capillary electrophoresis with the sensitivity afforded by laser-induced fluorescence detection is evaluated. The evaluation involves measurements of neutral analytes that are separated via the micellar electrokinetic capillary chromatography (MECC) variation of capillary electrophoresis. This separation-based fiber-optic sensor (SBFOS) employs a single-reservoir, linear configuration that presents the challenges of overcoming gravity-driven hydrostatic flow and offsetting the effects of trapped gases. Hydrostatic flow resulting from nonlevel positioning of the separation capillary is reduced through the use of a restrictor capillary. MECC separations are performed in a frontal mode of operation using relatively short separation capillaries. Studies involve measuring the magnitude and reproducibility of retention time, efficiency, and signal over applied voltages spanning 2.0−4.0 kV for test mixtures composed of three fluorescently d...