Nonaqueous capillary electrophoresis with indirect electrochemical detection

Nonaqueous capillary electrophoresis (NACE) which makes use of organic solvents in place of conventional aqueous electrophoresis buffers is gaining increasing importance among modern separation techniques. Recently, it has been shown that amperometric detection in conjunction with acetonitrile-based NACE offers an extended accessible potential range and an enhanced long-term stability of the amperometric responses generated at solid electrodes. The present contribution takes advantage of the latter aspect to develop reliable systems for NACE with indirect electrochemical detection (IED). In this context, several compounds such as (ferrocenylmethyl)trimethylammonium perchlorate, tris(1,10-phenanthroline)cobalt(III) perchlorate and bis(1,4,7-triazacyclononane)nickel(II) perchlorate were studied regarding their suitability to act as electroactive buffer additives for IED in NACE. The performance characteristics for the respective buffer systems were evaluated. Tetraalkylammonium perchlorates served as model compounds for the optimization of the NACE-IED system. Target analytes choline and acetylcholine could easily be separated and determined by means of NACE-IED. In the case of a buffer system containing 10(-4) M tris(1,10-phenanthroline)cobalt(III) perchlorate the limits of detection were 2.5 x 10(-7) M and 4.6 x 10(-7) M for choline and acetylcholine, respectively. With the elaborated analytical procedure choline could be determined in pharmaceutical preparations.     

Determination of hydrazine, monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine by nonaqueous capillary electrophoresis with amperometric detection

The present study is concerned with the application of nonaqueous capillary electrophoresis (NACE) with electrochemical detection (ED) to the separation and quantitative determination of hydrazine (Hy) and its methyl derivatives. The best performance of NACE-ED was found when using 4 mM sodium acetate/10 mM acetic acid/methanol: acetonitrile = 1:2 as the running buffer, with a bare platinum working electrode set at +1.0 V in an end-column amperometric detection cell. The choice and ratio of suitable solvents for the separation and injection media played an essential role for the performance characteristics of the method. The limits of detection for Hy, methylhydrazine, symmetrical dimethylhydrazine, and unsymmetrical dimethylhydrazine were 5, 2, 12, and 1 ng/mL, respectively. This is between one and two orders of magnitude lower than that achieved by previously reported CE-ED methods in aqueous buffer systems in conjunction with various types of chemically modified electrodes. The practical utility of the new NACE-ED methodology is demonstrated in terms of the determination of traces of Hys in spiked environmental samples containing a wide range of explosives and related compounds.     

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.     

Comparative study of aqueous and non-aqueous capillary electrophoresis in the separation of halogenated phenolic and bisphenolic compounds in water samples

Capillary zone electrophoresis methods, based on either aqueous and non-aqueous solutions as running buffers and UV spectrophotometric detection, have been developed and optimized for the separation of several halogenated phenolic and bisphenolic compounds, suspected or proved to exhibit hormonal disrupting effects. Both aqueous capillary electrophoresis (CE) and non-aqueous capillary electrophoresis (NACE) methods were suitable for the analysis of compounds under study. The separation of the analytes from other 25 potentially interfering phenolic derivatives was achieved with NACE method. Large-volume sample stacking using the electroosmotic flow pump (LVSEP) was assayed as on-column preconcentration technique for sensitivity enhancement. LVSEP-CE and LVSEP-NACE improved peak heights by 5-26 and 16-330 folds, respectively. To evaluate their applicability, the capillary electrophoresis methods developed were applied to the analysis of water samples, using solid-phase extraction as sample pre-treatment process.     

Hydrogen-bond effect and ion-pair association in the separation of neutral calix[4]pyrroles by nonaqueous capillary electrophoresis

A study on the migration behavior of four neutral macrocyclic compounds calix[4]pyrroles (C4Ps) by nonaqueous capillary electrophoresis (NACE) with tetrabutylammonium halide salts as background electrolyte (BGE) is described. Systematic studies were carried out with different BGE in acetonitrile (ACN) to elucidate the involved phenomena. The effect of BGE concentration on analytes effective mobilities mu eff,i was studied. Rough values of analytes absolute mobilities mu infinity,i were obtained by extrapolation of effective mobilities to zero ionic strength of BGE. Stokes radius of C4Ps in the form of C4PF(-) and C4PCl(-) was calculated. The complex constants of C4Ps with F(-) anion and Cl(-) anion and ion-pair association constants of each C4PF(-) and C4PCl(-) with tetrabutylammonium cations was evaluated and confronted. Hydrogen bonding effect is prerequisite in the separation; meanwhile ion-pair association which is intensified by steric hindrance effect plays an important role.     

Analysis of illicit drugs by nonaqueous capillary electrophoresis and electrochemical detection

Nonaqueous capillary electrophoresis (NACE) was applied to the determination of illicit drugs. The complete separation of amphetamine, methamphetamine, 3,4-methylene dioxy amphetamine (MDA), 3,4-methylene dioxy methamphetamine (MDMA), mescaline, cocaine and benzoylecgonine was obtained using an acetonitrile based buffer solution containing 10 mM sodium acetate and 1 M acetic acid. Electrochemical detection using a Pt microdisk electrode set to a potential of +1.8 V was found to be selective for MDA, MDMA and mescaline. The detection limits for these compounds were in the low ng/mL range which is between 2 and 3 orders of magnitude lower compared to UV-detection.     

Analysis of illicit drugs by nonaqueous capillary electrophoresis and electrochemical detection

Nonaqueous capillary electrophoresis (NACE) was applied to the determination of illicit drugs. The complete separation of amphetamine, methamphetamine, 3,4-methylene dioxy amphetamine (MDA), 3,4-methylene dioxy methamphetamine (MDMA), mescaline, cocaine and benzoylecgonine was obtained using an acetonitrile based buffer solution containing 10 mM sodium acetate and 1 M acetic acid. Electrochemical detection using a Pt microdisk electrode set to a potential of +1.8 V was found to be selective for MDA, MDMA and mescaline. The detection limits for these compounds were in the low ng/mL range which is between 2 and 3 orders of magnitude lower compared to UV-detection.     

Micelle to solvent stacking of two alkaloids in nonaqueous capillary electrophoresis

This paper for the first time describes the development of micelle to solvent stacking (MSS) to nonaqueous capillary electrophoresis (NACE). In this proposed MSS-NACE, sodium dodecyl sulfate (SDS) micelles transport, release, and focus analytes from the sample solution to the running buffer using methanol as their solvent. After the focusing step, the focused analytes were separated via NACE. The focusing mechanism and influencing factors were discussed using berberine (BBR) and jatrorrhizine (JTZ) as model compounds. And the optimum condition was obtained as following: 50 mM ammonium acetate, 6% (v/v) acetic acid and 10 mM SDS in redistilled water as sample matrix, 50 mM ammonium acetate and 6% (v/v) acetic acid in pure methanol as the running buffer, -20 kV focusing voltage with 30 min focusing time. Under these conditions, this method afforded limits of detection (S/N=3) of 0.002 μg/mL and 0.003 μg/mL for BBR and JTZ, respectively. In contrast to conventional NACE, the concentration sensitivity was improved 128-153-fold.     

CZE separation of amitrol and triazine herbicides in environmental water samples with acid-assisted on-column preconcentration

A simple analytical scheme for the detection and quantification of amitrol and triazine herbicides (atrazine, ametryn and atraton) and degradation product (2‐hydroxyatrazine) in environmental water samples by CZE is reported. On‐column preconcentration of analytes from untreated water samples (mineral, spring, tap and river water) is accomplished by introducing an acid plug (200 mM citrate of pH 2.0) after the sample and then proceeding with the CZE separation, using 100 mM formiate buffer of pH 3.5 as running buffer and 25.0 KV as separation voltage. UV detection at 200 nm provides LODs from 50 to 300 nM in untreated samples and they were lowered tenfold by sample preconcentration by evaporation. Calculated recoveries were typically higher than 90%. Minimal detectable concentration of the electroactive amitrol could be decreased about 20‐fold when electrochemical detection was employed by monitoring the amperometric signal at +800 mV using a carbon paste electrode (LOD of 9.6 nM, 0.81 μg/L, versus 170 nM, 14.3 μg/L, using amperometric and UV detection, respectively) in untreated water samples.     

Analysis of Calix[4]arenes Using Nonaqueous Capillary Electrophoresis

In nonaqueous capillary electrophoresis (NACE), an organic solvent is used in place of an aqueous medium as the background solution to improve the solubility and selectivity for hydrophobic analytes. In this study, we employed NACE with UV detection for the analysis of eight calix[4]arenes. We examined the influence of several parameters—the buffer composition, the nonaqueous solvent‘s composition and proportion, and the concentration of the electrolyte of the nonaqueous buffer—on the efficiency of the electrophoretic separation. The separation was achieved through the analyte's different effective mobility via different degrees of deprotonation on the phenolic OH groups of the calix[4]arene. This deprotonation can further affect the analyte's ability to form a complex with the metal ion. The optimized background electrolyte (BGE), comprising a mixture of N‐methylformamide/acetonitrile (30:70, v/v) and 100 mM AcOH/20 mM NH₄OAc, provided rapid (<11 min) separation of the calix[4]arenes with good resolution. The relative standard deviations of the migration times for the eight analytes were all less than 1%. Within the calibration concentration range, the coefficients of determination (R²) were all greater than 0.9914. Thus, the present study demonstrated NACE can provide adequate separation for the analysis of calix[4]arenes.     

Comparison of CZE, open-tubular CEC and non-aqueous CE coupled to electrospray MS for impurity profiling of drugs

Open-tubular CEC and non-aqueous CE (NACE) methods were developed for the analysis of six pharmaceutical compounds and their respective process-related impurities, comprising 22 analytes in total with a range of functional groups and lipophilicities. These methods were assessed for orthogonality of analyte separation with respect to existing CZE-ESI-MS and HPLC-ESI-MS methods, in order to complement a generic analytical strategy for impurity profiling of pharmaceutical compounds. Open-tubular CEC, using etched and chemically modified capillaries, induced weak reversed-phase-type interactions between some of the analytes and the bonded phases (0.811相似文献   

A sensitive non‐aqueous capillary electrophoresis‐mass spectrometric method for multiresidue analyses of β‐agonists in pork

Non‐aqueous capillary electrophoresis–mass spectrometry (NACE‐MS) was developed for trace analyses of β‐agonists (i.e. clenbuterol, salbutamol and terbutaline) in pork. The NACE was in 18 mM ammonium acetate in methanol–acetonitrile–glacial acetic acid (66 : 33 : 1, v/v/v) using a voltage of 28 kV. The hyphenation of CE with a time‐of‐flight MS was performed by electrospray ionization interface employing 5 mM ammonium acetate in methanol–water (80 : 20, v/v) as the sheath liquid at a flow rate of 2 μL/min. Method sensitivity was enhanced by a co‐injection technique (combination of hydrodynamic and electrokinetic injection) using a pressure of 50 mbar and a voltage of 10 kV for 12 s. The method was validated in comparison with HPLC–MS‐MS. The NACE‐MS procedure provided excellent detection limits of 0.3 ppb for all analytes. Method linearity was good (r² > 0.999, in a range of 0.8–1000 ppb for all analytes). Precision showed %RSDs of <17.7%. Sample pre‐treatment was carried out by solid‐phase extraction using mixed mode reversed phase/cation exchange cartridges yielding recoveries between 69 and 80%. The NACE‐MS could be successfully used for the analysis of β‐agonists in pork samples and results showed no statistical differences from the values reported by the Ministry of Public Health, Thailand using HPLC‐MS‐MS method. Copyright © 2009 John Wiley & Sons, Ltd.     

Nonaqueous capillary electrophoresis-mass spectrometry for separation of venlafaxine and its phase I metabolites

Aqueous and nonaqueous capillary electrophoresis (NACE) were investigated for separation of venlafaxine, a new second-generation antidepressant, and its three phase I metabolites. Working at basic pH, around the venlafaxine pKa value, was effective in resolving the investigated drugs, but created considerable peak tailing. To overcome electrostatic interactions between analytes and silanol groups, investigations were also carried out at acidic pH. However, despite the addition of up to 50% v/v of organic solvents (e.g., methanol or acetonitrile), complete separation of the studied compounds was not possible. NACE was found to be an appropriate alternative to resolve venlafaxine and its metabolites simultaneously. Using a conventional capillary (fused-silica, 64.5 cm length, 50 microm inner diameter), and a methanol-acetonitrile mixture (20/80 v/v) containing 25 mM ammonium formate and 1 M formic acid, complete resolution of these closely related compounds was performed in less than 3.5 min. Selectivity, efficiency and separation time were greatly affected by the organic solvent composition. As the electric current generated in nonaqueous medium was very low, the electric field was further increased by reducing the capillary length. This allowed a baseline resolution of venlafaxine and its three metabolities in 0.7 min. Selectivity was compared in aqueous and nonaqueous media in relation to the acid-base properties of the analytes as well as to the solvation degree. Finally, the method successfully coupled on-line to mass spectrometry with electrospray ionization interface allowed significant sensitivity enhancement.     

Characterization of pharmaceutical drugs by a modified nonaqueous capillary electrophoresis--mass spectrometry method

A simple method for the separation and characterization of a group of nine basic compounds, comprising seven tricyclic antidepressant and two bronchodilator drugs, by nonaqueous capillary electrophoresis (NACE) employing ultraviolet and mass spectrometry detection is described. After optimization of the electrophoresis separation conditions, including the compositions of the electrolyte and the organic solvent, a reliable separation of all nine basic analytes was achieved in 80 mM ammonium formate dissolved in a methanol-acetonitrite (80:20 v/v) mixture, having an apparent pH of 8.7. The volatile nonaqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by mass spectrometry. When results were compared with reversed-phase gradient and isocratic high-performance liquid chromatography (HPLC) methods, the NACE method provided greater efficiency, achieving baseline resolution for all nine basic compounds in less than 30 min. The NACE method is suitable for use as a routine procedure for the rapid separation and characterization of basic compounds and is a viable alternative to HPLC for the separation of a wide range of pharmaceutical drugs.     

Indirect detection of organic acids in non-aqueous capillary electrophoresis.

Non-aqueous capillary electrophoresis (NACE) with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2-C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (pKa) values of C8-C18 FAs obtained from the slope of the linear plot -log [(mu 0/mu)-1] vs. pH, using 20% isopropanol and 40% acetonitrile as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 (MC540) as fluorophores, has been performed to the analysis of AA and its stereoisomer, isoascorbic acid (IAA), and the limits of detection of AA and IAA are 0.30 microM and 0.17 microM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with IAA as the internal standard in less than 3 min and its concentration is 76.7 +/- 0.4 mM.     

Determination of dopamine,serotonin, and their metabolites in pediatric cerebrospinal fluid by isocratic high performance liquid chromatography coupled with electrochemical detection

A method to rapidly measure dopamine (DA), dihydroxyindolphenylacetic acid, homovanillic acid, serotonin (5‐HT) and 5‐hydroxyindoleacetic acid concentrations in cerebrospinal fluid (CSF) has not yet been reported. A rapid, sensitive, and specific HPLC method was therefore developed using electrochemical detection. CSF was mixed with an antioxidant solution prior to freezing to prevent neurotransmitter degradation. Separation of the five analytes was obtained on an ESA MD‐150 × 3.2 mm column with a flow rate of 0.37 mL/min and an acetonitrile–aqueous (5 : 95, v/v) mobile phase with 75 mM monobasic sodium phosphate buffer, 0.5 mM EDTA, 0.81 mM sodium octylsulfonate and 5% tetrahydrofuran. The optimal electrical potential settings were: guard cell +325 mV, E1 ?100 mV and E2 +300 mV. Within‐day and between‐day precisions were <10% for all analytes and accuracies ranged from 91.0 to 106.7%. DA, 5‐HT, and their metabolites were stable in CSF with antioxidant solution at 4°C for 8 h in the autoinjector. This method was used to measure neurotransmitters in CSF obtained from children enrolled on an institutional medulloblastoma treatment protocol. Copyright © 2009 John Wiley & Sons, Ltd.     

Separation of acidic solutes by nonaqueous capillary electrophoresis in acetonitrile-based media. Combined effects of deprotonation and heteroconjugation

Nonaqueous capillary electrophoresis (NACE) is a chemical separation technique that has grown in popularity over the past few years. In this report, we focus on the combination of heteroconjugation and deprotonation in the NACE separation of phenols using acetonitrile (ACN) as the buffer solvent. By preparing various dilute buffers consisting of carboxylic acids and tetrabutylammonium hydroxide in ACN, selectivity may be manipulated based on a solute's dissociation constant as well as its ability to form heterogeneous ions with the buffer components. ACN's low viscosity, coupled with its ability to allow for heteroconjugation, often leads to rapid and efficient separations that are not possible in aqueous media. In this report, equations are derived showing the dependence of mobility on various factors, including the pKa of the analyte, the pH and concentration of the buffer, and the analyte-buffer heteroconjugation constant (Kf). The validity of these equations is tested as several nitrophenols are separated at different pH values and concentrations. Using nonlinear regression, the Kf values for the heteroconjugate formation between the nitrophenols and several carboxylate anions are calculated. Also presented in this report are the NACE separations of the 19 chlorophenol congeners and the 11 priority pollutant phenols (used in US Environmental Protection Agency methods 604, 625/1625 and 8270B).     

A three-dimensionally adjustable amperometric detector for microchip electrophoretic measurement of nitroaromatic pollutants

A microchip capillary electrophoresis (CE)–amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an amperometric detection cell containing a one-dimensionally adjustable disc detection electrode in a Plexiglas holder. It facilitates the precise three-dimensional alignment between the channel outlet and the detection electrode without a complicated three-dimensional manipulator. The performance of this unique system was demonstrated by separating four nitroaromatic pollutants (nitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and p-nitrobenzene). Factors influencing their separation and detection processes were examined and optimised. The four analytes have been well-separated within 120 s in a 75 cm long separation channel at a separation voltage of +2000 V using an electrophoretic separation medium containing 15 mM borax and 15 mM sodium dodecyl sulfate (pH 9.2). Highly linear response is obtained for the four analytes over the range of 0–5 ppm with the detection limits ranging from 12 to 52 ppb. The present system demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 9). The new approach for the microchannel–electrode alignment should find a wide range of applications in other microfluidic analysis systems.     

A high-performance polycarbonate electrophoresis microchip with integrated three-electrode system for end-channel amperometric detection

A fully integrated polycarbonate (PC) microchip for CE with end-channel electrochemical detection operated in an amperometric mode (CE-ED) has been developed. The on-chip integrated three-electrode system consisted of a gold working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, which was fabricated by photo-directed electroless plating combined with electroplating. The working electrode was positioned against the separation channel exit to reduce post-channel band broadening. The electrophoresis high-voltage (HV) interference with the amperometric detection was assessed with respect to detection noise and potential shifts at various working-to-reference electrode spacing. It was observed that the electrophoresis HV interference caused by positioning the working electrode against the channel exit could be diminished by using an on-chip integrated reference electrode that was positioned in close proximity (100 microm) to the working electrode. The CE-ED microchip was demonstrated for the separation of model analytes, including dopamine (DA) and catechol (CA). Detection limits of 132 and 164 nM were achieved for DA and CA, respectively, and a theoretical plate number of 2.5x10(4)/m was obtained for DA. Relative standard deviations in peak heights observed for five runs of a standard solution containing the two analytes (0.1 mM for each) were 1.2 and 3.1% for DA and CA, respectively. The chip could be continuously used for more than 8 h without significant deterioration in analytical performance.     

Flow injection analysis of sulfite ion with a potentiometric titanium phosphate-epoxy based membrane sensor

A novel pH sensor suitable for use in both aqueous and non-aqueous mediums is reported. The sensor is derived from polymer modified electrode obtained from electrochemical polymerisation of aniline in dry acetonitrile containing 0.5 M tetraphenyl borate at 2.0 V versus Ag/AgCl. The light yellow colour polymer modified electrode obtained under the present experimental condition has been characterised by scanning electron microscopy (SEM). The pH sensing of polymer modified electrode in both aqueous and non-aqueous mediums is examined and reported. As the typical examples, we used weak acid (acetic acid) and weak base (ammonium hydroxide) as analytes. The acetic acid is analysed in both aqueous and dry acetonitrile whereas ammonium hydroxide is analysed only in aqueous medium. The analysis in aqueous medium is conducted in 1 mM Tris-HCl buffer pH 7.0 and also in 0.1 M KCl. The slope of pH sensing is calculated from the data recorded in typical buffers and found to be approximately 86 mV per pH. The application of polymer modified electrode for the construction of urea biosensor is described based on immobilised urease within poly vinyl alcohol (PVA) matrix and also within organically modified sol-gel glass on the surface of polymer-modified electrode. The new urea sensor has shown maximum response of 160 mV at 25 degrees C with a lowest detection limit of 20 muM. The performance of new pH sensor and urea sensor has been studied and reported in this communication.