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.     

On-line concentration sample stacking coupled with water-in-oil microemulsion electrokinetic chromatography

This study describes for the first time, the ability of a normal stacking mode (NSM) on-line concentration step coupled with water-in-oil (W/O) microemulsion electrokinetic chromatography (MEEKC), using six common penicillin antibiotics (oxacillin, penicillin V, penicillin G, nafcillin, ampicillin, and amoxicillin) as test analytes. Optimization of penicillin separation in the conventional W/O MEEKC system demonstrated that change in the type and concentration of the oil phase (1-butanol) and column temperature had a pronounced effect on the separation. With the subsequent development of the NSM coupled with W/O MEEKC, improved separation and detection sensitivities were observed when an organic solvent plug (1-propanol; 1.04 cm) was placed between the W/O microemulsion and the sample solutions. This could be attributed to the solution viscosity difference between the aqueous sample zone and the organic solvent plug causing the penicillin to be stacked in this 1-propanol plug. The optimal NSM W/O MEEKC provided about 12-fold increase in detection sensitivity compared with conventional sample injection (50 mbar, 3 s). Finally, this proposed method was successfully applied in the analyses of several food samples (porcine organs) spiked with penicillin.     

Separation and on-column preconcentration of some nonsteroidal anti-inflammatory drugs by microemulsion electrokinetic capillary chromatography using high-speed separations

Various strategies have been investigated for separating a group of nonsteroidal anti-inflammatory drugs (NSAIDs) by microemulsion electrokinetic capillary chromatography (MEEKC) using high-speed separations. The parameters that of affect the separation, such as the nature of the oil droplet and the buffer, and the surfactant concentration have been studied. In addition, several organic solvents were used to decrease the retention of the analytes in the oil droplet phase and to improve the resolution of the NSAIDs. The optimum microemulsion background electrolyte (BGE) solution made of 0.8% w/w ethyl acetate, 6.6% w/w butan-1-ol, 6.0% w/w acetonitrile, 1.0% w/w sodium dodecyl sulfate (SDS), and 85.6% w/w of 10 mM sodium tetraborate at pH 9.2 resolved the drugs within 8 min. The short-end injection procedure is an alternative for reducing the analysis time. When this procedure was used, the microemulsion BGE solution consisted of 0.8% w/w ethyl acetate, 6.6% w/w butan-1-ol, 17.0% w/w methanol, 1.0% w/w SDS, and 74.6% w/w of 10 mM sodium tetraborate, pH 9.2, and the NSAIDs were separated within 3 min. The reversed electrode polarity stacking mode (REPSM) technique was applied to the on-line concentration of the NSAIDs. In this technique, the sample matrix was pumped out of the capillary using a polarity-switching step. When this technique was applied, the sensitivity was enhanced up to 40-fold and the limits of detection (LODs) were in the low microg.L(-1) levels.     

On-line preconcentration of weak electrolytes by electrokinetic accumulation in CE: experiment and simulation

A new on-line preconcentration technique was developed that makes possible to determine nanomolar concentrations of weak acidic analytes in CE. The method consists of long-running electrokinetic sample injection and stacking (electrokinetic immobilization) of the analytes at a boundary of two electrolytes with different pH values (pH 9.5 and 2.5) and consequent mobilization of the stacked uncharged analytes in a micellar system (containing SDS micelles). Several factors including buffer concentration, pH, applied voltage, time of preconcentration, and SDS concentration were tested to optimize the analysis method. An about 4600-fold increase of the sample concentration (in comparison with the standard CZE) can be achieved during the preconcentration step. Two preservatives applied in food industry -- benzoic acid and sorbic acid were used as model samples. The applicability of the proposed method in food analysis was demonstrated by determination of nanomolar concentrations of benzoic acid in sunflower oil. An extended version of the computer program Simul was used for modeling both the preconcentration and mobilization processes taking place in the capillary.     

Separation and determination of two sesquiterpene lactones in Radix inulae and Liuwei Anxian San by microemulsion electrokinetic chromatography

A novel microemulsion electrokinetic chromatography (MEEKC) method for separating and determining two sesquoterpene lactones, alantolactone (AL) and isoalantolactone (IAL), in Radix inulae and Liuwei Anxian San has been developed. The effects of several important factors such as internal organic phases, concentration of microemulsion, concentration of acetonitrile, injection time and running voltage were systematically investigated to determine the optimum conditions. The optimum microemulsion system was composed of n-hexane (0.32% w/w), SDS (1.24% w/w), 1-butanol (2.64% w/w), acetonitrile (10% w/w) and 10 mm sodium tetraborate buffer (85.80% w/w, pH 9.2). The applied voltage was 20 kV. The analytes were detected at 214 nm. Regression equations revealed linear relationships (correlation coefficients 0.9950 for AL and 0.9946 for IAL) between the peak area of each analyte and the concentration. The limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 0.45 microg/mL for AL and 0.56 microg/mL for IAL. The levels of the analytes were successfully determined with recoveries ranging from 98.2 to 104.3%. Furthermore, a simple and effective extraction method, with methanol in an ultrasonic water bath for 60 min, was used for sample preparing. Also, MEEKC was compared with micellar electrokinetic chromatography (MEKC) and shown better separation results.     

Sample stacking and sweeping in microemulsion electrokinetic chromatography under pH-suppressed electroosmotic flow

Two on-line sample concentration techniques, sample stacking and sweeping under pH-suppressed electroosmotic flow, were evaluated in microemulsion electrokinetic chromatography. The concept of stacking with anion selective electrokinetic injection and a water plug in a reverse-migrating microemulsion (SASIW-RMME) was brought forward in this article. Six flavonoids were concentrated using a microemulsion consisting of 80 mM sodium dodecyl sulfate, 1.2% (v/v) ethyl acetate, 0.6% (v/v) 1-butanol, 10% acetonitrile (v/v) and 50 mM phosphoric acid (pH* 1.8). Significant detector response improvements were achieved. The limits of detection were in the low ng/ml level. Finally, the sample of Fructus aurantii Immaturus was analyzed using sweeping technique.     

On-line concentration of neutral analytes by complexation and acetonitrile sweeping in nonionic microemulsion electrokinetic chromatography with direct ultraviolet detection

To separate and detect neutral solutes in nonionic microemulsion electrokinetic chromatography (MEEKC), a novel method was developed, combining complex formation and acetonitrile (ACN) sweeping. In this report, dynamic borate complexation and on-line sweeping occurred simultaneously during a run. The operating parameters which affected the performance of analyte sweeping in nonionic MEEKC were examined in terms of borate complexation, ACN content, Brij-35 concentration and sample plug length. In addition, the validation of the method included tests of the limit of detection, reproducibility and sensitivity enhancement. 60–110-Fold of magnitude improvement in detection sensitivity for model compounds (ginsenoside Rf, ginsenoside Rb2, ginsenoside Re) using Brij-35 microemulsion was demonstrated. Furthermore, the method was applied to the determination of glucosides in the plant extract.     

Simultaneous electrokinetic and hydrodynamic injection with on-line sample concentration via micelle to solvent stacking in micellar electrokinetic chromatography

Simultaneous electrokinetic and hydrodynamic injection (SEHI) of organic cations (tricyclic antidepressant and beta blocker drugs) with on-line sample concentration using micelle to solvent stacking (MSS) was studied in micellar electrokinetic chromatography. Compared to conventional injection, >300-fold improvements in signals were obtained by hydrodynamic injection. However, with SEHI the amount of sample ions introduced into the capillary was increased which afforded a higher gain of up to 4000-fold without compromise to separation efficiency. The electrokinetic injection at negative polarity (anode at the detector end) introduced the micelle bound analytes. The hydrodynamic injection also maintained the MSS boundary inside the capillary. The stability of the MSS boundary affected SEHI where mild conditions that were low voltage as well as pressure injection were desired. The limits of detection were in the range from 0.6–4.2 ng mL⁻¹. A strategy for optimization was described and the method was applied to the ng mL⁻¹ analysis of spiked wastewater after simple dilution and centrifugation.     

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.     

Determination of food colorants by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method was developed to analyze and detect eight food colorants (tartrazine, fast green FCF, brilliant blue FCF, allura red AC, indigo carmine, sunset yellow FCF, new coccine, and carminic acid), which are commonly used as food additives in various food products. The effects of sodium dodecyl sulfate (SDS) surfactant, organic modifier, cosurfactant, and oil were examined in order to optimize the separation. The amount of organic modifier (acetonitrile) and SDS surfactant were determined as apparent influences on the separation resolution while the type of oil and cosurfactant rarely affected the separation selectivity of the eight colorants. A highly efficient MEEKC separation method, where the eight colorants were separated with baseline resolution within 14 min, was achieved by using a microemulsion solution of pH 2.0 containing 3.31% SDS, 0.81% octane, 6.61% 1-butanol, and 10% acetonitrile. This optimal MEEKC method has a higher separation efficiency and similar detection limit when compared to conventional capillary electrophoresis (CE) method. Furthermore, a sample pretreatment is rarely needed when this MEEKC technique is used to analyze colorants in food products, whereas a suitable sample pretreatment (for example solid-phase extraction) has to be employed prior to CE separation in order to eliminate matrix interferences resulting from the constituents of the food sample.     

Sample stacking for determination of aromatic acid impurities by microemulsion electrokinetic chromatography

In this study, a sample stacking step coupled with microemulsion electrokinetic chromatography (MEEKC) was used to detect and analyze nine aromatic acids (benzoic acid (BA), isophthalic acid (IPA), terephthalic acid (TPA), p-toluic acid (p-TA), 4-carboxylbenzaldehyde (4-CBA), trimesic acid (TSA), trimellitic acid (TMA), o-phthalic acid (OPA), and hemimellitic acid (HMA)) which are common impurities produced during aromatic acid synthesis. First, the presence of both acid and water plugs at the front of the capillary improved the reproducibility in retention time and peak intensity of the tested analytes in the stacking method. Second, the pH and the electrolyte type of acidic plug and sample matrix were found to be the predominant influences on the aromatic acid stacking. The detection limits of these aromatic acids were reduced to the range of 0.00007-0.00032 μg mL⁻¹ by this optimal sample stacking step. This proposed on-line concentration MEEKC method was able to detect trace levels of aromatic acid impurities in commercial aromatic acid products that were not previously possible by the normal MEEKC method. Furthermore, these results in comparison with our previous studies on sample stacking MEEKC method indicated that all acidic species were concentrated by this simple stacking procedure. The sensitivity enhancement, however, was highly dependent on the types of functional groups present in the structures of analytes, and the enhancement was in the order of first the compounds carrying both carboxy and hydroxy groups (e.g. phenolic acid), followed by carboxylic acid compounds (e.g. aromatic acid), and then phenol compounds (e.g. polyphenol).     

Microemulsion electrokinetic chromatography of drugs varying in charge and hydrophobicity: I. Impact of parameters on separation performance evaluated by multiple linear regression models

The separation of anionic, cationic and neutral drugs in microemulsion electrokinetic chromatography (MEEKC) was studied with a statistical experimental design. The concentration of sodium dodecyl sulfate (SDS, surfactant), 1-butanol (co-surfactant) and borate buffer and the factors Brij 35 (surfactant), 2-propanol (organic solvent) and cassette temperature were varied simultaneously, while the parameters pH (9.2), the concentration of octane (oil, 0.8% w/w), the voltage (10 kV) and the dimension of the fused-silica capillary, were kept constant. Eight different model substances were chosen with different hydrophobicities. Two of the analytes were positively charged, two were negatively charged, and the remaining four were neutral or close to neutral at the pH explored. The importance of each parameter on the separation window, the plate height and the retention factor for each of the analytes was studied by means of multiple linear regression (MLR) models. A new response was evaluated for anions, the quotient between the effective mobility in the microemulsion and the effective mobility in the corresponding buffer. Factors affecting selectivity changes were also explored, and it was found that SDS and 2-propanol had the largest effect on selectivity.     

Separation and sweeping of flavonoids by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants

In this report, a novel means for the separation and sweeping of flavonoids (quercetin, rutin, calycosin, ononin and calycosin-7-O-β-d-glucoside) by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants as modified pseudostationary phase was presented. The optimized background electrolyte consisted of 0.5% (w/v) ethyl acetate, 2.0% (w/v) SDS, 9 mM DTAC, 4.0% (w/v) 1-butanol and 10 mM sodium borate or 25 mM phosphoric acid. We systematically investigated the separation and preconcentration conditions, including the concentrations of surfactant, types of sweeping, sample matrix, the effect of high salt or acetonitrile, and sample injection volume. It was found that the use of mixed surfactants significantly enhanced the separation efficiency through the change of the efficient electrophoretic mobility of analytes. Compared with normal sample injection, 185-508-fold sensitivity enhancement in terms of limit of detection was achieved through effective sweeping of large sample volume at 50 mbar pressure (up to 45% capillary length). At last, the proposed method was suitable for the determination of Radix Astragali sample.     

Microemulsion electrokinetic chromatography of proteins.

Microemulsion electrokinetic chromatography was used to separate a test mixture of proteins effectively. The separation was carried out in a 42.5 cm (to the detector) x 50 microns I.D. fused-silica capillary using a microemulsion system consisting of 80 mM heptane, 120 mM SDS, 900 mM butanol in 2.5 mM borate buffer, pH 8.5-9.5. Optimum separation conditions were investigated with respect to the running voltage, temperature, pH and the composition of microemulsion. Results were compared with those obtained in micellar electrokinetic chromatography and capillary zone electrophoresis. The examined method is practical and successfully applied to the assay of genetically engineering pharmaceuticals, recombinant human granulocyte macrophage colony stimulating factor injection and recombinant human granulocyte colony stimulating factor injection.     

High-speed microemulsion electrokinetic chromatography.

In previous reports of microemulsion electrokinetic chromatography (MEEKC), analysis times were typically in the order of 10 min as high-ionic strength buffers were used. These buffers produced high currents which limit the voltages which can be applied, therefore, analysis times could not be reduced. The primary cause of the high-ionic strength is the relatively high concentrations of surfactants required to form the microemulsion. The surfactant concentration can be lower when using an oil with a smaller surface tension. This preliminary study showed that migration times in MEEKC can be reduced to below 1 min by using a combination of an optimum microemulsion composition, high voltage, high temperature, short capillaries by injecting via the "short end", or by simultaneously applying pressure and voltage. Long injection sequences and quantitation were found to be possible with minimum buffer depletion effects.     

High-sensitive capillary zone electrophoresis analysis by electrokinetic injection with transient isotachophoretic preconcentration: electrokinetic supercharging

The principle of an on-line preconcentration method for capillary zone electrophoresis (CZE) named electrokinetic supercharging (EKS), is described and based on computer simulation the preconcentration behavior of the method is discussed. EKS is an electrokinetic injection method with transient isotachophoretic process, is a powerful preconcentration technique for the analysis of dilute samples. After filling the separation capillary with supporting electrolyte, an appropriate amount of a leading electrolyte was filled and the electrokinetic injection was started. After a while, terminating electrolyte was filled subsequently and migration current was applied. This procedure enabled the introduction of a large amount of sample components from a dilute sample without deteriorating separation. Computer simulation of the electrokinetic injection revealed that EKS was effective for the preconcentration of analytes with wide mobility ranges by proper choice of transient isotachophoresis (ITP) system and electroosmotic flow (EOF) should be suppressed to increase injectable amount of analytes under constant voltage mode. A test mixture of rare-earth chlorides was used to demonstrate the uses of EKS-CZE. When a 100 microL sample was used, the low limit of detectable concentration was 0.3 microg/L (1.8 nM for Er), which was comparable or even better than that of ion chromatography and inductively coupled plasma-atomic emission spectrometry (ICP-AES).     

Sample stacking for the analysis of eight penicillin antibiotics by micellar electrokinetic capillary chromatography

We studied the use of micellar electrokinetic capillary chromatography for separating eight penicillins. The method consists of (i) an electrophoretic separation based on micellar electrokinetic capillary chromatography, which uses sodium dodecyl sulfate (SDS) as surfactant; (ii) a sample stacking technique called reverse electrode polarity stacking mode (REPSM); and (iii) direct UV detection. The background electrolyte that gave complete separation contained 20 mM sodium borate buffer and 60 mM SDS. The sensitivity of the method was improved by an enrichment step that used on-column stacking. The limits of detection were at the microg.L(-1) level for the penicillins and did not detract from the peak resolution.     

Analyses of tobacco alkaloids by cation-selective exhaustive injection sweeping microemulsion electrokinetic chromatography

In this study, an on-line concentration method which coupled cation-selective exhaustive injection (CSEI) sweeping technology with microemulsion electrokinetic chromatography (MEEKC) was used to detect and analyze several tobacco alkaloids (nornicotine, anabasine, anatabine, nicotine, myosmine and cotinine) that are commonly found in various tobacco products. First, the effects of microemulsion compositions (oil, cosurfactant and solution pH) were examined in order to optimize the alkaloid separations in conventional MEEKC. The pH value and the injection length of basic plug were found to be the predominant influences on the alkaloid stacking. This optimal CSEI sweeping MEEKC method provided approximately 180- to 540-fold increase in detection sensitivity in terms of peak height without any loss in separation efficiency when compared to normal MEEKC separation. Furthermore, this proposed CSEI sweeping MEEKC method was applied successfully for the detection of the minor alkaloids nornicotine, anabasine and anatabine in tobacco products.     

Analysis of catechins in extracts of Cistus species by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.     

On-line concentration by field-enhanced sample injection with reverse migrating micelles in micellar electrokinetic capillary chromatography for the analysis of triterpenoids from traditional chinese medicine

In this work, a simple, reproducible and sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of five triterpenoids, lupeol (1), 1β-hydroxy-lupeol (2), lup-3β,1α-diol (3), lup-1β,3β,11α-triol (4) and 30-norlupan-3β,11α-diol-20-one (5) in traditional Chinese medicine of Salvia roborowskii Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM) was used for on-line concentration of triterpenoids. The optimum buffer contained 50 mM H₃PO₄, 160 mM SDS, 20% acetonitrile and 15% 2-propanol and pH of buffer was 2.0. The sample solution was diluted with 10 mM H₃PO₄ (pH 2.5, containing 10 mM SDS) and injected for 15 s with −8 kV after injection of 4 s water plug. The effects of concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection time of water plug, the injection voltage and injection time of sample on the separation and stacking efficiency were investigated. Under the optimum conditions, the analytes were well separated and by optimizing the stacking conditions, about 28–96-fold improvement in the detection sensitivity was obtained for triterpenoids. The contents of five triterpenoids in Salvia roborowskii Maxim were successfully determined with satisfactory repeatability and recovery.