Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.     

Determination of biogenic amines in HeLa cell lysate by 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

An MEKC-LIF method using 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxy-carbonyl) fluorescein (SAMF) newly synthesized in our lab as a labeling reagent for the separation and determination of eight typical biogenic amines was proposed. After careful study of the derivatization condition such as pH value, reagent concentration, temperature, and reaction time, derivatization reaction was accomplished as quickly as 10 min with stable yield. Optimal separation of SAMF-labeled amines was achieved with a running buffer (pH 9.3) containing 30 mM boric acid, 25 mM SDS, and 20% v/v ACN. The proposed method allowed biogenic amines to be determined with LODs as low as 0.25-2.5 nmol/L and RSD values from 0.4 to 4.5%. The present method has been successfully used to monitor biogenic amines in HeLa cells and fish samples. This study exploits the potential of MEKC-LIF with SAMF labeling as a tool for monitoring biogenic amines involved in complex physiological and behavioral processes in various matrices.     

Sensitive determination of biogenic amines by capillary electrophoresis with a new fluorogenic reagent 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

An effective approach was proposed to the derivatization of seven biogenic amines using 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA) as a fluorogenic reagent. The sensitive determinations of these derivatives were achieved by micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection. The derivatization and electrophoretic conditions have been optimized. A running buffer was composed of mixtures of 25 mM pH 9.5 boric acid, 25 mM SDS, and 27% ACN. At 25 °C and 22.5 kV, the baseline separation of the derivatives was accomplished in 13 min. The detection limit (S/N = 3) was found as low as 0.4 nM. The proposed method was validated by the linearity of two orders magnitude and correlation coefficient in the range 0.9969–0.9998. Also, the procedure was successfully applied to the determination of biogenic amines in soy sauce, fish and wine samples.     

Separation of natural pyrethrum extracts using micellar electrokinetic chromatography

The separation of the six pyrethrin esters in a technical pyrethrum extract (Riedel-de-Ha?n, Cresent Chemical Co. Inc. Hauppauge, NY, USA) by micellar electrokinetic chromatography (MEKC) using both sodium dodecyl sulfate (SDS) and a polymerized surfactant as pseudo-stationary phases has been investigated and optimized. Parameters such as pH, SDS and polymerized sodium N-undecyl sulfate (poly-SUS) concentration, type and concentration of background electrolyte and organic modifier, as well as the acetonitrile/water ratio in the sample were studied to optimize the resolution, efficiency, and analysis time. An optimized separation of the six pyrethrin esters was achieved in 25 min with 25 mM Tris, buffered at pH 9, containing 30 mM SDS, 25% (v/v) acetonitrile, and an equal volume ratio of acetonitrile/water sample matrix at a voltage of 25 kV. The use of 0.5% (w/v) poly-SUS enhanced resolution of the pyrethrin esters and shortened the total analysis time from 25 to 20 min, compared to the SDS mediated separation. The optimized MEKC results are compared to the HPLC separation of these esters and show an improvement in efficiency and total analysis time.     

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AA's, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AA's using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AA's yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.     

Microchannel electrophoretic separation of biogenic amines by micellar electrokinetic chromatography

Fast, efficient separation of most common biogenic amines was successfully performed on a glass microchip capillary electrophoresis device. The amines putrescine, histamine, tyramine, cadaverine, phenethylamine, tryptamine, spermidine and spermine were derivatized prior to fluorescence detection with fluorescein isothiocyanate. Separation was carried out using a channel length of 28 mm, a cross section of 50 x 8 microm, and a field strength of 600 V/cm. After optimization of buffer electrolyte conditions (120 mM boric acid, pH 9.4, modified with 40 mM SDS), fluorescein thiocarbamyl amine derivatives were successfully resolved. Analysis time was as short as 75 s. Determination of the biogenic amines was achieved in soy sauce samples.     

Micellar electrokinetic capillary chromatography as an alternative method for the determination of ethinylestradiol and levo-norgestrel

A method for quantifying of ethinylestradiol (ETE) and levo-norgestrel (LEV) in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. The separation was carried out at 25 degrees C and 25 kV, using a 20 mM borate buffer (pH 9.2), 15 mM sodium dodecylsulfate (SDS) in 30% acetonitrile/water (v/v). Under these conditions the analysis takes about 7 min. The method has been applied for quantifying both compounds in six different commercial contraceptives and the proposed method gave good results when compared with a reference liquid chromatographic (LC) method.     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

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

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

Fast CE for combinatorial catalysis

Two solvent-modified MEKC methods were developed for the quantitative analysis of heterocyclic amines synthesised using intramolecular ring closure via catalysed hydroamination. The first method was capable of resolving six of the amines (precursors and products) with a sample-to-sample injection time of 2 min employing a 20 mM borate buffer, pH 9.2 with 20 mM SDS and 5% v/v n-butanol (n-BuOH). A second low-pH method using 20 mM phosphate buffer, 100 mm SDS, 5% v/v n-BuOH and 20% v/v iso-propanol (i-PrOH) was able to resolve an additional pair of compounds with a sample-to-sample time of 3.5 min. Application of the first method to the analysis of a sample containing catalyst as well as amines placed directly in a 96-well plate showed excellent performance, with migration time and peak height and area reproducibility being less than 0.9 and 9.6%, respectively. The quantity of conversion by catalyst was calculated to be 68 +/- 7%, which was in excellent agreement with the 67 +/- 5% obtained by more conventional (1)H NMR experiments, with the added advantage that this method is also cheaper, quicker and more amendable to high-throughput screening of combinatorial libraries.     

Use of SDS micelles for improving sensitivity, resolution, and speed in the analysis of beta-lactam antibiotics in environmental waters by SPE and CE

This study dealt with the potential of MEKC with LIF detection involving derivatization with sulfoindocyanine succinimidyl ester (Cy5) for the separation and determination of beta-lactam antibiotics (ampicillin, amoxicillin, cephradine, and cephalexin) in environmental water samples. Water samples of 50 mL were enriched by SPE by passage through a weak base-cation Amberlite(R) IRA-93 exchange column. SDS micelles play important roles in the whole analytical process by improving the yield (sensitivity) and the kinetics of the labeling reaction, the elution of the retained antibiotics from the SPE preconcentration system and the electrophoretic resolution of their Cy5-derivatives. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 10 min and direct injection for MEKC analysis, which is conducted within about 15 min using 15 mM SDS in the running buffer (35 mM sodium borate at pH 9.3). LODs from 30 to 45 ng/L and RSDs (within-day precision) from 3.5 to 5.9% were obtained for the antibiotics in water samples with average recoveries ranging from 96.4 to 99.4%. These results indicate that the method proposed is a straightforward and sensitive tool for the determination of these antibiotics in environmental water samples providing similar quantitative results to those using more expensive equipment like LC-electrospray MS/MS.     

Separation of sympathomimetic amines of abuse and related compounds by micellar electrokinetic chromatography.

Separation of twelve sympathomimetic amines and related compounds by micellar electrokinetic chromatography (MEKC) with UV absorbance detection is described. These amines were well separated within 25 min using 50 mM sodium tetraborate solution containing 15 mM sodium dodecylsulfate (SDS) of pH 9.3 as a running solution and detected at 210 nm. MEKC was performed with an applied voltage of 13 kV at 25 degrees C using a fused-silica capillary (50 cm x 75 mm i.d.) with effective length of 37.5 cm. The detection limits of these compounds were in the range from 4 to 97 fmol/injection at a signal-to-noise ratio (S/N) of 3. The reproducibility of the method expressed as relative standard deviation (RSD) for within-day (n=6) and between-day (n=5) assays was less than 4.8 and 8.8%, respectively. The proposed method could be applied to the determination of an anorectic drug, phentermine, in Chinese tea with a detection limit of 99 microg/g (105 fmol/injection, S/N=3).     

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.     

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25 degrees C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed-phase high-performance liquid chromatography (RP-HPLC).     

Enantioseparation of esbiothrin by cyclodextrin‐modified microemulsion and micellar electrokinetic chromatography

A comparison between chiral cyclodextrin‐modified microemulsion electrokinetic chromatography (CD‐MEEKC) and cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) for the enantiomeric separation of esbiothrin was carried out. For both methods, the separation conditions were optimized by varying CD types and concentration, running buffer pH and compositions, organic modifiers, and temperature. The optimal CD‐MEEKC conditions were 0.8% n‐heptane, 2.3% SDS, 6.6% n‐butanol, 90.3% 10 mM sodium tetraborate containing 3% (w/v, the ratio of CD mass to microemulsion volume) methyl‐β‐cyclodextrin, pH 10, 25°C. The optimized CD‐MEKC conditions were 3.3% SDS, 96.7% 10 mM sodium tetraborate containing 5% (w/v) β‐CD, pH 10, 25°C. The difference in physicochemical properties of the buffer and CDs resulted in different optimal CD type. The competitive distribution between the microemulsion (or micelle) and chiral CD contributed to the chiral separation. Both methods provided excellent separation (R_s ～? 3) with similar migration time (ca. 15 min). CD‐MEEKC provided higher separation efficiencies (>300000) than CD‐MEKC (>200000). The LODs for CD‐MEEKC and CD‐MEKC were 4.7 μg/mL and 3.2 μg/mL, respectively. The RSDs of migration time and peak area for CD‐MEEKC were slightly higher than for CD‐MEKC. Both the demonstrated CD‐MEEKC and CD‐MEKC methods provided high efficiencies, low LODs, and reproducible enantioseparations of esbiothrin.     

Study on separation of aristolochic acid I and II by micellar electrokinetic capillary chromatography and competition mechanism between SDS and beta-cyclodextrin

In this study, a rapid MEKC method using 40 mM sodium borate buffer containing 50 mM SDS as surfactant was developed for the analysis of aristolochic acid (AA) in Aristolochia plants. Baseline separation of AA-I and AA-II was achieved within 3 min with high separation efficiency, satisfactory sensitivity, repeatability, and recovery. Resolution between AA-I and AA-II is above 5 and great performance with higher than 200,000 theoretical plate numbers was obtained. The detection limits (based on 3 S/N) were both 1.0 microg/mL. Two kinds of AA in 35 herbal samples of Aristolochia plants were successfully determined. The competition mechanism between beta-CD and SDS was also investigated by changing the content ratio of beta-CD and SDS.     

Highly efficient separation of isomeric epoxy fatty acids by micellar electrokinetic chromatography

A capillary electrophoresis (CE) method has been developed for simple and direct separation of cis- and trans-12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid isomers. Separation was performed in micellar electrokinetic capillary chromatography (MEKC) using a buffer consisting of 25 mM borate (pH 9.20), 10 mM sodium dodecyl sulfate (SDS) and 10% v/v acetonitrile. The key variables, concentrations of SDS and organic modifier, were optimized by the application of a factorial experimental design. The use of a low micellar concentration, just above critical micelle concentration (CMC), in a background electrolyte containing an organic modifier not only made it possible to dissolve and separate highly hydrophobic fatty acid isomers, but also resulted in improved separation efficiency and selectivity. Separation efficiency up to 4 x 10(5) theoretical plates/m was achieved under an optimized condition. Also investigated were the influence of temperature on separation and the effect of organic modifier concentration on the dynamic exchange of the analytes between micelles and the bulk of the buffer solution. Direct UV was applied for detection of the fatty acids.     

Micellar electrokinetic chromatography estimation of size and composition of procyanidins after thiolysis with cysteine

This paper describes the characterization of procyanidin mixtures by acid depolymerization in the presence of cysteine (thiolysis with cysteine) and micellar electrokinetic chromatography (MEKC). Reversed-phase liquid chromatography (RP-HPLC) and MEKC were investigated for the separation of the major components of the depolymerized mixtures (catechins and their cysteinyl derivatives). The solutes could only be effectively separated using MEKC. Two background electrolytes (BGEs) are recommended: (i) 50 mM phosphate at pH 7, containing 40 mM sodium cholate (SC) and 10 mM sodium dodecyl sulfate (SDS); (ii) a BGE with the same composition but containing only 50 mM SDS. The MEKC procedures here reported, are cheap, reliable and fast, and their potential in the determination of the size and composition in procyanidin mixtures has been shown. The proposed MEKC methods were validated by comparison with our intralaboratory reference RP-HPLC method using cysteamine as thiol donor.     

Profiling of cocaine by micellar electrokinetic chromatography

The potential of micellar electrokinetic chromatography (MEKC) for the profiling of cocaine samples is described. An MEKC system containing sodium dodecyl sulfate (SDS) and methanol was optimized using a test mixture of cocaine, its common impurities (benzoylecgonine, norcocaine, tropacocaine, and trans-cinnamoylcocaine), and several degradation products. The effect of pH, percentage modifier, and concentration surfactant on the separation has been investigated. The optimal separation buffer for cocaine samples consisted of 75 mM SDS, 17.5% methanol, and 25 mM borate (pH 8.3) and was well suited to separate components of diverse polarity in one run. Various cocaine seizures have been analyzed with the MEKC system and their signatures were compared. The electrokinetic chromatograms obtained were characteristic, and differences and similarities among the samples could easily be observed. Several impurities were identified in the samples by means of migration times and comparison of recorded and library UV spectra. The composition of the samples was determined semiquantitatively using relative corrected peak areas.     

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.