Micellar electrokinetic chromatography - From theoretical concepts to real samples (Review)

Micellar electrokinetic chromatography (MEKC) is an alternative to liquid chromatographic separations. It is a highly efficient separation technique that is performed with the same experimental set-up as is used for capillary electrophoresis (CE), thus extending the applicability of CE to neutral solutes. MEKC can be regarded as a separation technique with a similar scope to that of reversed-phase high-performance liquid chromatography (RP-HPLC), having advantages over HPLC with regard to the efficiency of the separation system, separation speed, cost, and tolerance to matrix constituents. This paper discusses the applicability of MEKC to real samples and also addresses developments widening the scope of this emerging technique: on-line concentration by stacking or sweeping and sensitive detection schemes.     

Micellar electrokinetic chromatography: A practical overview of current methodological and instrumental advances

This review highlights recent methodological and instrumental advances in MEKC focused on the improvement of this CE technique in routine analyses. As in the previous reviews, the present one deals with the last 2 years' most relevant methodological contributions for improving sensitivity and resolution in MEKC as well as instrumental research related to MS and LIF detection. The most widespread approaches are discussed in detail, including enhancements in sensitivity arising from the use of on-line sample concentration (stacking, sweeping, and a combination of both of these protocols) and improvements in resolution, obtained by changing the composition of the BGE (e.g. with additives to the aqueous phase or employing alternative micellar phases to classical SDS) and using 2-D separation systems. The advantages and restrictions in MS and LIF detection as applied to MEKC analysis are also examined, especially in the direct coupling of MEKC and MS detection. Finally, some thoughts on potential future directions are also considered.     

Solid-phase extraction and sample stacking-micellar electrokinetic capillary chromatography for the determination of multiresidues of herbicides and metabolites

Micellar electrokinetic capillary chromatography (MEKC) with diode array detection was used for the separation of 13 compounds (eight herbicides widely used in agriculture: metribuzin, lenacil, ethofumesate, atrazine, terbutryn, isoproturon, chlorotoluron and linuron, and five of their principal degradation products; namely, deethylatrazine, 2-hydroxyatrazine, deethyl-2-hydroxyatrazine, deisopropylatrazine and 3-chloro-4-methylphenylurea). Peak separation for the 13 analytes was not successful when a single surfactant system was employed, neither sodium dodecyl sulfate (SDS) nor dioctyl sulfosuccinate (DOSS) sodium salt. However, a mixture of these herbicides was successfully separated using a mixed micellar system involving SDS–DOSS in less than 14 min. An application study of an on-line concentration technique for MEKC was carried out to enhance sensitivity. The optimized on-line stacking procedure consisted simply of the addition of 50 mM of sodium chloride to the injection sample, the stacking effect being more intensive as analyte polarity increased. When this stacking procedure was combined with an off-line sample preconcentration step, based on solid-phase extraction, analytes could be detected in the ppb range. The whole method was applied to ultra-high-quality and natural waters. Linear relationships between the analytical signal and the initial analyte concentration were found to be independent of the type of water, except for the more polar analytes for which small differences were observed.     

Analysis of ecdysteroids by micellar electrokinetic chromatography with on-line preconcentration

In order to enhance the UV detection sensitivity, an application study of an on-line preconcentration technique for micellar electrokinetic chromatographic (MEKC) was carried out. The simultaneous determination of four test ecdysteroids, 20-hydroxyecdysone, ajugasterone C, polypodine B and ponasterone A has been investigated by using the normal stacking mode in MEKC with UV detection. The effects of anionic surfactant composition and concentration, the applied voltage, the pH buffer, the kind and the amount of organic solvent and the injection time on the analyte resolution were evaluated. The optimised conditions for the separation involved the use of a 50 mM borate as the running buffer containing 50 mM of a mixture of sodium dodecyl sulphate (SDS) and sodium cholate (SC) in the ratio of 1:1 together with a concentration of 10% (v/v) of 2-PrOH at pH 9.0. Hydrodynamic injection of 12 s at 50 mbar and separation voltage of 20 kV at temperature of 20 degrees C were employed. These conditions allowed a repeatability separation within 21 min. Concentration detection limit for the neutral analytes studied improve about an order of magnitude. The method was also applied to the determination of ecdysteroids in a real sample.     

Determination of triazine herbicide residues in water samples by on-line sweeping concentration in micellar electrokinetic chromatography

A new method for the determination of atrazine, simazine and prometryn in water samples by on-line sweeping concentration technique in micellar electrokinetic chromatography （MEKC） was developed. Various parameters affecting sample enrichment and separation efficiency were systematically studied. Compared with the conventional MEKC method, up to 60-200-fold improvement in concentration sensitivity was achieved in terms of peak height by using this sweeping injection technique. The compound strychnine was used as the internal standard for the improvement of the experimental reproducibility. The limits of detection （S/ N = 3：1） for atrazine, simazine and prometryn were 9, 10 and 0.5 ng mL-1, respectively. This method has been successfully applied to the analysis of atrazine, simazine and prometryn in lake, steam and ground water.     

Concentration and determination of cotinine in serum by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC), combined with on-line concentration techniques, cation-selective exhaustive injection (CSEI) and sweeping, was developed for the analysis of cotinine, the primary biomarker for exposure to secondhand smoke. Experimental parameters including sample matrix, surfactant concentration, injection length and concentration of high-conductivity buffer, and sample electrokinetic injection time were optimized for electrophoretic enrichment and separation processes. Under the optimal conditions, the detection sensitivity of cotinine was enhanced by about 5000-fold using CSEI-sweeping MEKC compared to normal MEKC. The limit of detection for cotinine was found to be 0.2 ng/mL using ultraviolet absorbance detection. Furthermore, the developed method was successfully applied to the detection of cotinine in mouse serum samples.     

Micellar electrokinetic chromatography on microchips

This review highlights the methodological and instrumental developments in microchip micellar EKC (MCMEKC) from 1995. The combination of higher separation efficiencies in micellar EKC (MEKC) with high-speed separation in microchip electrophoresis (MCE) should provide high-throughput and high-performance analytical systems. The chip-based separation technique has received considerable attention due to its integration ability without any connector. This advantage allows the development of a multidimensional separation system. Several types of 2-D separation microchips are described in the review. Since complicated channel configurations can easily be fabricated on planar substrates, various sample manipulations can be carried out prior to MCMEKC separations. For example, mixing for on-chip reactions, on-line sample preconcentration, on-chip assay, etc., have been integrated on MEKC microchips. The application of on-line sample preconcentration to MCMEKC can provide not only sensitivity enhancement but also the elucidation of the preconcentration mechanism due to the visualization ability of MCE. The characteristics of these sample manipulations on MEKC microchips are presented in this review. The scope of applications in MCMEKC covers mainly biogenic compounds such as amino acids, peptides, proteins, biogenic amines, DNA, and oestrogens. This review provides a comprehensive table listing the applications in MCMEKC in relation to detection methods.     

On-line concentration and determination of all-trans- and 13-cis- retinoic acids in rabbit serum by application of sweeping technique in micellar electrokinetic chromatography

A simple and rapid micellar electrokinetic chromatography (MEKC) method with UV detection was developed for the simultaneous separation and determination of all-trans- and 13-cis-retinoic acids in rabbit serum by on-line sweeping concentration technique. The serum sample was simply deproteinized and centrifuged. Various parameters affecting sample enrichment and separation were systematically investigated. Under optimal conditions, the analytes could be well separated within 17min, and the relative standard deviations (RSD) of migration times and peak areas were less than 3.4%. Compared with the conventional MEKC injection method, the 18- and 19-fold improvements in sensitivity were achieved, respectively. The proposed method has been successfully applied to the determination of all-trans- and 13-cis-retinoic acids in serum samples from rabbits and could be feasible for the further pharmacokinetics study of all-trans-retinoic acid.     

Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast.     

Twenty-five years of micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC), which can separate neutral analytes as well as charged analytes by the capillary electrophoretic technique, was developed in 1982 and the first paper was published in 1984. The authors’ group concentrated their effort into the characterization of MEKC as a separation technique until early 1990s. Most issues in MEKC separations were successfully solved and wide applicability of MEKC was verified in 1990s. In particular, sweeping, an on-line sample preconcentration technique, was very successful for the concentration of neutral analyte as well as ionic ones. In this paper, our studies on MEKC will be summarized from the personal viewpoint of the author.     

Simultaneous determination of phosphorus-containing amino acid-herbicides by nonionic surfactant micellar electrokinetic chromatography with laser-induced fluorescence detection

The potential of micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the separation and determination of phosphorus-containing amino acid-herbicides (glufosinate and glyphosate), and aminomethylphosphonic acid (the major metabolite of glyphosate), involving derivatization with fluorescein isothiocyanate (FITC) isomer I, was investigated. Different variables that affect derivatization (pH, FITC concentration, time and temperature) and separation (pH and concentration of the buffer, kind and concentration of surfactants and applied voltage) were studied. The analysis was conducted within about 8 min and the use of the nonionic surfactant Triton X-100 improved the selectivity, thus indirectly enhancing sensitivity by shifting of the interfering peaks of the FITC excess. Dynamic ranges of 2.0-3,000 microg/L, limits of detection at microgram or submicrogram-per-liter level, and relative standard deviations from 4.7 to 6.4% were obtained. The ensuing method--nonionic surfactant MEKC-- is a useful choice for the determination of these herbicides as it provides limits of detection similar or lower than those reported by existing chromatographic alternatives without the use of an additional preconcentration technique such as solid-phase extraction. The separation of a mixture of nine FITC-derivatized amino acids, selected as target compounds, was also carried out to assess the discrimination power of the nonionic surfactant MEKC method for the analysis of closely related anionic analytes.     

Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.     

Application of sweeping to micellar electrokinetic chromatography-atmospheric pressure chemical ionization-mass spectrometric analysis of environmental pollutants.

An application study of sweeping, an on-line sample concentration technique, to micellar electrokinetic chromatography (MEKC) directly combined with mass spectrometry (MS) using an atmospheric pressure chemical ionization (APCI) interface, namely MEKC-APCI-MS, was investigated to enhance the concentration sensitivity for the analysis of environmental pollutants. Under a neutral condition, around 100-fold increase in the concentration sensitivity was achieved for several aromatic amines and alkyl phthalates as test samples by sweeping-MEKC-APCI-MS compared to conventional MEKC-APCI-MS, whereas under an acidic condition, 100 to 600-fold sensitivity enhancement was gained for similar solutes. Linearity of the corrected peak area obtained in the mass chromatogram against the sample concentration was examined for 3,4-dichloroaniline and diisopropyl phthalate (DIPP). The estimated limits of detection for 3,4-dichloroaniline and DIPP were 0.6 and 0.4 ppm, respectively, in terms of the injected sample concentration.     

On-line concentration by field-enhanced sample injection with reverse migrating micelles in micellar electrokinetic capillary chromatography for the analysis of flavonoids in Epimedium brevicornum Maxim

A simple and sensitive micellar electrokinetic capillary chromatography (MEKC) method was developed for the separation and determination of six flavonoids in Epimedium brevicornum Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM) was used for on-line concentration of the flavonoids. An electrolyte containing 20 mM H3PO4, 100 mM SDS, 20% acetonitrile and 2% 2-propanol (pH 2.0) was chosen as the electrophoretic buffer. By optimizing the stacking conditions, about 40-360-fold improvement in the detection sensitivity was obtained for the flavonoids.     

Anion-selective exhaustive injection-sweeping microemulsion electrokinetic chromatography

In this study, anion-selective exhaustive injection-sweeping (ASEI-sweeping) technique, which is a selective on-line sample concentration technique, was first proposed in microemulsion electrokinetic chromatography (MEEKC) for analyses of eight acidic phenolic compounds. In contrast to a capillary that is typically filled with nonmicellar background solution in conventional ASEI-sweeping MEKC method, in the proposed ASEI-sweeping MEEKC method, a capillary is filled with a low pH microemulsion solution (pH 2.0), and then with a short acid plug (pH 2.0, 1.9 cm) before field-amplified sample injection. This proposed design has two functions. First, the microemulsion solution that is present at the front of capillary column is able to avoid phase separation of microemulsion solution during MEEKC separation. Second, the presence of the short acid plug would effectively limit the partition behavior of acid analytes with the oil droplets in the microemulsion during field-amplified sample injection; otherwise, the stacking effect of acid analytes would be markedly reduced. This optimal ASEI-sweeping MEEKC method afforded about 96,000-fold to 238,000-fold increases in detection sensitivity in terms of peak areas without any separation efficiency loss when compared to normal MEEKC separation. Furthermore, trace levels (about 3 ng/g) of gallic acid and catechin in foods were also detected successfully by the proposed ASEI-sweeping MEEKC technique.     

Novel modes of capillary electrophoresis for the determination of endocrine disrupting chemicals

The synthesis and usage of a wide range of organic chemicals has increased dramatically over the last five decades. These compounds sometimes termed endocrine disrupting chemicals include agricultural pesticides, industrial solvents, dyes, plasticisers, detergents and heat exchangers. Concerns have been raised about the potential adverse effects of these compounds on humans and wildlife species. Our objectives are to develop a method to identify, using novel capillary electrophoretic techniques, the endocrine disrupting compounds that are reported to be present in environmental samples. The CE modes, capillary zone electrophoresis, micellar electrokinetic chromatography (MEKC), cyclodextrin-modified MEKC (CD-MEKC) and electroosmotic flow-suppressed CD-MEKC were investigated for the determination of a range of endocrine disrupting chemical compounds. This paper shows some initial results obtained.     

On-line preconcentration and determination of ketamine and norketamine by micellar electrokinetic chromatography. Complementary method to gas chromatography/mass spectrometry

We have investigated a rapid, simple, and highly efficient on-line preconcentration method using in micellar electrokinetic chromatography (MEKC) for the analysis of abused drugs. Ketamine is an anesthetic that has been abused as a hallucinogen. We applied the sample sweeping technique first to ketamine and its major metabolite, norketamine, and separated the analytes with MEKC. Several of the sweeping MEKC parameters to effect successful separations, such as the concentration of sodium dodecyl sulfate (SDS), the injection time, and the applied voltage were optimized. The improvements in the number of theoretical plates under the different separation conditions are presented clearly in a three-dimensional representation. The limits of detection were 2.8, 3.4, and 3.3 ng/mL for ketamine, norketamine, and ketamine-D(4), respectively. The enrichment factor for each compound was within the range of 540-800. Experimental results are in agreement with those of analysis conducted by gas chromatography/mass spectroscopy (GC/MS). Therefore, we believe that sweeping, combined with MEKC, represents a suitable complementary method to GC/MS for use in clinical and forensic analyses of ketamine and norketamine.     

A comparative study of micellar and microemulsion EKC for the analysis of benzoylurea insecticides and their analogs

An investigation of the basic factors which govern the microemulsion EKC (MEEKC) and MEKC for the separation of four benzoylurea (BU) insecticides and their four analogs was carried out. In MEEKC, the separation of eight BU compounds was optimized by changing the microemulsion composition, such as concentration of SDS, octane, n-butanol, and isopropanol percentages, as well as capillary temperature. Separation optimization was also carried out for MEKC, showing that ACN and a high level of another additive gamma-CD were needed to achieve effective separation of these analytes. Although separation with baseline resolution was achieved by either MEEKC or MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC. In addition, analytical time in MEEKC was longer than that in MEKC, but in view of theoretical plate numbers, detection limits, and reproducibility, both methods were effective for the analysis of BU insecticides and their analogs.     

Micellar electrokinetic chromatography: current developments and future

This review highlights recent methodological and instrumental advances in micellar electrokinetic chromatography (MEKC). Enhancements in sensitivity and selectivity of the technique through the use of on-line preconcentration approaches (stacking and sweeping) and nonconventional pseudostationary phases, namely nonionic and zwitterionic surfactants, mixed micelles and polymers, are discussed in detail. Laser-induced fluorescence and mass spectrometry, as alternatives to UV-absorption detection, have been covered to evaluate their advantages and limitations when applied to analysis in an MEKC format. Some thoughts on future directions in this area such as in-capillary reactions, coated capillaries and MEKC on microchips are also presented.     

Pressurized capillary electrochromatographic analysis of water-soluble vitamins by combining with on-line concentration technique

A pressurized capillary electrochromatography (pCEC) system was developed for the separation of water-soluble vitamins, in which UV absorbance was used as the detection method and a monolithic silica-ODS column as the separation column. The parameters (type and content of organic solvent in the mobile phase, type and concentration of electrolyte, pH of the electrolyte buffer, applied voltage and flow rate) affecting the separation resolution were evaluated. The combination of two on-line concentration techniques, namely, solvent gradient zone sharpening effect and field-enhanced sample stacking, was utilized to improve detection sensitivity, which proved to be beneficial to enhance the detection sensitivity by enabling the injection of large volumes of samples. Coupling electrokinetic injection with the on-line concentration techniques was much more beneficial for the concentration of positively charged vitamins. Comparing with the conventional injection mode, the enhancement in the detection sensitivities of water-soluble vitamins using the on-line concentration technique is in the range of 3 to 35-fold. The developed pCEC method was applied to evaluate water-soluble vitamins in corns.