Polycations as displacer in high-performance bioseparation

Non-aqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the determination of nicotine. The measurements were performed using an acetonitrile-based buffer. Nicotine was shown to yield well defined voltammetric signals suitable for oxidative detection. The precision of NACE-ED regarding migration time and peak height for samples containing 8 micrograms/ml nicotine is expressed by relative standard deviations of 0.1% and 1.6%, respectively (n = 8). The limit of detection for nicotine was 13 ng/ml (286 fg). For nicotine determination in tobacco samples various solutions were studied regarding the extraction efficiency in an ultrasonic bath. The highest extraction efficiency was obtained using a solvent mixture consisting of acetonitrile-acetic acid-water (20:5:75, v/v). The results for nicotine determination in tobacco were evaluated using tobacco reference material with certified nicotine content. Analytical aspects such as accuracy, reproducibility and selectivity were addressed in this work. The measurements were based on the use of a newly developed electrochemical detector cell which was found to enable user-friendly operation of NACE-ED measurements.     

Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van 't Hoff plots (In Kcec versus 1/T) were observed for these peptides between 15 and 60 degrees C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the Kcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative Kcec values can, in principle, thus arise with these charged analytes. However, the Kcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water-50 mM NH4OAc/AcOH, pH 5.2-acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.     

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.     

CE hydrogen deuterium exchange-MS in peptide analysis

CE and hydrogen-deuterium (H/D) exchange MS are useful tools in the analysis and characterisation of peptides. This study reports the facile coupling of these tools in the H/D exchange CE-MS analysis of model and pharmaceutically important peptides, using a sheath flow interface. The peptides varied in mass from 556 (leucine enkephalin) to 1620 Da (bombesin), and in charge state from 0.33 (leucine enkephalin) to 3.0 (substance P). The application of a BGE composed of ammonium formate buffer (25 mM, pD 3.5 in D(2)O (>98% D atom)), a sheath liquid composed of formic acid (0.25% v/v in D(2)O) and ACN (30:70 v/v), and dissolving the samples in a mixture of ACN/D(2)O (50:50 v/v) facilitates complete H/D exchange. Because of complete H/D exchange the ESI mass spectra produced are easy to interpret and comparable to those obtained from LC-MS analysis. The CE-H/D-MS approach has the advantage of requiring lower volumes of deuterated solvents. The b- and y-series fragments produced by using in-source decomposition correspond to those predicted. With the peptides studied, the complete exchange H/D exchange observed with both the molecular and fragment ions helps to confirm both amino acid composition and sequence.     

Cation effects in the separation of calix[4]pyrroles by nonaqueous capillary electrophoresis with tetraalkylammonium chloride salts as background electrolytes

The migration behavior of three calix[4]pyrroles (C4Ps) in acetonitrile (ACN) and in the mixtures of ACN/ethanol (EtOH) (95:5; v/v)with tetraalkylammonium chloride as background electrolyte (BGE) was studied. Electroosmotic flow (EOF) mobilities and absolute mobilities in ACN and mixture of ACN/EtOH (95:5; v/v) were investigated. A decrease of EOF mobilities in EtOH/ACN was observed compared with that of in pure ACN. Variation of the C4Ps effective mobilities as a function of the square root of concentration of tetraakylammonium chloride salts such as tetramethylammonium chloride (Me4NCl), tetraethylammonium chloride (Et4NCl) and tetrabutylammonium chloride (Bu4NCl) in both solvents was plotted, and straight lines were exhibited when Bu(4)NCl was used as BGE. Absolute mobilities of C4Ps were obtained by extrapolation from the mentioned plots. It was found that the absolute mobilities of C4Ps is independent of the nature of BGE and the values are: meso-octamethylcalix[4]pyrrole (OMCP)>meso-tetracyclopentylcalix[4]pyrrole (TPCP)>meso-tetracyclohexylcalix[4]pyrrole (THCP). The ion-pairing constants follow the order under given solvent: Me4N+>Et4N+>Bu4N+, and ion-pairing constant of C4P follows the order: THCP>TPCP>OMCP. As though, ion-pairing association is intensified by steric hindrance.     

Determination of cannabinoids in hair using high-pH* non-aqueous electrolytes and electrochemical detection. Some aspects of sensitivity and selectivity

Non-aqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the determination of cannabinoids in hair. The effect of different electrolyte compositions on the selectivity of the separation of tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and tetrahydrocannabinol carboxylic acid (THCA) was studied. Complete electrophoretic resolution was obtained using a strongly basic background electrolyte consisting of 5 mM sodium hydroxide dissolved in acetonitrile-methanol (1:1). Electrochemical detection yielded well defined signals in the oxidation mode. In order to obtain low limits of detection experimental parameters, which determine the sensitivity and the noise level, were optimized. A crucial parameter for sensitive measurements using a wall-tube flow cell as end-column detector is the distance between the capillary outlet and the working electrode. The highest signal-to-noise ratio using a 50 microm I.D. capillary was obtained at a distance of 25 microm. When the capillary outlet was moved away from the working electrode, thus reducing the strength of the separation field present at the working electrode, a large low frequency noise developed. This rise was attributed to disturbances of the hydrodynamic pattern in the flow cell. Analytical aspects such as sensitivity, reproducibility and selectivity were addressed in this work. The precision of NACE-ED regarding migration time and peak height for a sample containing 1 microg/ml THC was 0.4% and 1.1% (RSD), respectively (n=5). The calibration curve was linear for concentrations ranging between 0.1 and 10 microg/ml (r=0.998). The limit of detection for THC was 37 ng/ml, which is almost two orders of magnitude lower when compared with on-column UV detection. The method was evaluated using hair samples containing cannabinoids as sample material.     

On-line coupling of SPE and CE-MS for peptide analysis

An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.     

葫［7］环联脲作为毛细管电泳分离介质的研究

以葫［7］环联脲(CB［7］)作为毛细管电泳添加剂,成功地分离了6种硝基苯类化合物。考察了pH值对电渗流的影响,初步评价了其基本的电泳性能,结果表明在所考察的pH值范围内(pH 2~6.5),葫［7］环联脲是质子化的,且吸附到毛细管内壁上,这使得毛细管内壁带上正电荷,电渗流反向;初步考察了葫［7］环联脲的浓度对分离的影响,证明了用葫［7］环联脲作为添加剂可完全分离对硝基乙苯、对硝基甲苯、对氯硝基苯、间二硝基苯、2,4-二硝基氯苯和硝基苯这6种物质,最佳的缓冲液为10 mmol/L Na2HPO4（用盐     

Simultaneous analysis of methionine- and leucine-enkephalin from rat brain: quantification by liquid chromatography-electrochemistry

This study focuses on the application of liquid chromatography with electrochemical detection (LC-ED) for the analysis of methionine-enkephalin (ME) and leucine-enkephalin (LE) extracted from rat brain regions. The high applied potentials necessary for enkephalin detection required the development of an efficient sample processing protocol. Brain extracts were processed using chromatographic mode sequencing (CMS). The decrease in electroactive interfering substances by CMS improved the chromatographic resolution of ME and LE and the electrode performance. Other qualitative and analytical methods were used to evaluate the enkephalin data obtained by LC-ED for rat brain regions. This study demonstrates that LC-ED provides both the sensitivity and specificity necessary for the analysis of enkephalins from rat brain regions.     

Discriminative affinity labelling of opioid receptors by enkephalin and morphiceptin analogues containing 3-nitro-2-pyridinesulphenyl-activated thiol residues.

The thiol groups of leucinthiol, cysteamine and cysteine incorporated into opioid peptides enkephalin and morphiceptin were activated by the 3-nitro-2-pyridinesulphenyl (Npys) group to form mixed disulphides highly reactive to a free thiol. Enkephalin analogues containing Npys-leucinthiol or -cysteine at positions 4, 5 and 6 exhibited high affinities for both mu and delta receptors, while morphiceptin analogues containing Npys-cysteine at positions 4 and 5 showed relatively weak affinity only for mu receptors. When these S-activated opioid peptides were incubated with rat brain membrane preparations, it was found, by binding assay using radiolabelled and non-labelled [D-Ala2,MePhe4,Gly-ol5]enkephalin, that they label mu opioid receptors in a dose-dependent manner. The concentrations required to label half of the receptors were 0.2-2 microM for enkephalins and 10-30 microM for morphiceptins. These results suggested that the thiol group labelled by S-activated enkephalins and morphiceptins is present in the ligand binding site of receptor protein, but not in GTPase-binding protein.     

Separation and determination of enkephalin-related peptides by capillary electrophoresis with amperometric detection

A simple, reliable, and reproducible method for separation and determination of five enkephalin-related peptides based on CE with amperometric detection (AD) is described in this paper. A potential of 1.0 V was applied to the carbon disk electrode, which was used as a working electrode in this system. At 15 kV of applied voltage, the five compounds were separated within 18 min in a 20 mmol/L phosphate buffer solution (PBS, pH 7.6) including 2.5% methanol v/v. LOD for five enkephalins were ranged from 6.31 to 54.3 nmol/L. The method was applied successfully to determine the five compounds added in the human cerebrospinal fluid (CSF) and the recoveries were in the range of 95.8-98.2%.     

Application of bare gold nanoparticles in open-tubular CEC separations of polyaromatic hydrocarbons and peptides

In this study, bare gold nanoparticles (GNPs) immobilized in the sol-gel-pretreated fused-silica (FS) capillary as a stationary phase for open-tubular capillary electrochromatography (OT-CEC) are for the first time shown to be able to separate both hydrophobic polyaromatic hydrocarbons (PAHs) as well as hydrophilic cationic antimicrobial peptides. Model mixture of four PAHs, naphthalene, fluorene, phenanthrene, and anthracene, was resolved by OT-CEC in the GNP-modified FS capillaries using the hydro-organic background electrolyte (BGE) composed of 20 mmol/L sodium phosphate buffer, pH 7, modified with ACN at 8:2 v/v ratio. On the other hand, three synthetic analogues of an antimicrobial peptide mastoparan PDD-B, basic tetradecapeptides INWKKLGKKILGAL-NH(2), INSLKLGKKILGAL-NH(2) and NWLRLGRRILGAL-NH(2), were separated in aqueous acidic BGEs, pH 2.1-3.1, composed of weak acids (formic and acetic) or amphoteric amino or imino acids (aspartic or iminodiacetic), utilizing the advantage of a slow reversed (anodic) EOF and slightly positive charge of the GNP-modified FS capillary suppressing the adsorption of cationic peptides on the inner capillary wall and improving their resolution.     

Sensitive method for measuring hydrolysis of enkephalins in plasma, using high-performance liquid chromatography with electrochemical detection

This paper describes a simple and sensitive method for detection of [Leu]- and [Met]enkephalin and their N-terminal tyrosine-containing metabolic fragments (Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe), using high-performance liquid chromatography with electrochemical detection. The method employs a carbon graphite working electrode with increased working electrode surface area (40 mm2). The procedures were applied to assay of the activities of enkephalin-degrading enzymes in whole plasma collected from rats, mice, and chicks.     

Analysis of phenolic xenoestrogens by pressurized CEC with amperometric detection

A new method, pressurized CEC with end‐column amperometric detection using carbon paste electrode, has been developed for the separation and determination of five phenolic xenoestrogens in chicken eggs and milk powder samples. Efficient separation of five analytes was performed by pressurized CEC using a mobile phase consisting of 60% v/v ACN and 40% v/v Tris buffer (5 mmol/L, pH 8.0), +6 kV of applied voltage and 7.0 MPa of supplementary pressure. Detection limits of 50, 5, 2, 10 and 20 ng/mL for pentachlorophenol, bisphenol‐A, 2,4‐dichlorophenol, 4‐tert‐octylphenol and 4‐nonylphenol, respectively, were achieved using carbon paste electrode as working electrode and +0.8 V as detection potential. Matrix solid phase dispersion extraction method had been employed during sample preparation procedure, and mean recoveries ranged from 79.2 to 102.6% at different concentrations of phenolic xenoestrogens for spiked egg and milk powder samples were obtained.     

Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.     

A glass capillary ultramicroelectrode with an electrokinetic sampling ability.

A glass capillary ultramicroelectrode (tip diameter approximately 1.2 microm) having an electrokinetic sampling ability is described. It is composed of a pulled glass capillary filled with an inner solution and three internal electrodes (Pt working and counter electrodes and an Ag/AgCl reference electrode). The voltammetric response of the capillary electrode is based on electrokinetic transport of analyte ions from the sample solution into the inner solution across the conical tip. It was found that the electrophoretic migration of analytes at the conical tip is faster than electroosmotic flow, enabling electrokinetic transport of analyte ions into the inner solution of the electrode. By using [Fe(CN)6]4- and (ferrocenylmethyl)trimethylammonium (FcTMA+) ions as model analytes, differential pulse voltammetric responses of the capillary electrode were investigated in terms of tip diameter of the capillary, sampling voltage, sampling time, detection limit and selectivity. The magnitude of the response depends on the size and charge of analyte ions. With a capillary electrode having a approximately 1.2-microm tip diameter, which minimizes non-selective diffusional entry of analytes, the response after 1 h sampling at +1.7 V is linearly related to [Fe(CN)6]4- concentration in the range of 0.50-5.0 mM with the detection limit of 30 microM. Application of a potential of the same sign as that of the analyte ion forces the analyte to move out from the electrode to the solution, enabling reuse of the same capillary electrode. The charge-selective detection of analytes with the capillary electrode is demonstrated for [Fe(CN)6]4- in the presence of FcTMA+.     

Simultaneous determination of polycarboxylic acids by capillary electrophoresis with a copper electrode

The simultaneous determination of polycarboxylic acids including oxalic acid, citric acid, malonic acid, malic acid, tartaric acid, aspartic acid and glutamic acid was achieved by capillary electrophoresis with a copper disk electrode (d = 200 microm). In the system. 0.2 mmol/l cetylpridinium bromide (CPB) was used as an electroosmotic flow (EOF) modifier to reverse the direction of EOF. The effects of the solution pH and CPB concentration on separation were evaluated to achieve the optimum separation conditions. At the working potential of +0.14 V (vs. saturated calomel electrode), the calibration curves for all polycarboxylic acids studied were linear with 2 approximately 3-orders of magnitude and all the detection limits (S/N = 3) were below 15 fmol except malonic acid. Furthermore, the oxalic and citric acids in urine were successfully separated and determined with high sensitivity.     

Characterization of the SDS-induced electroosmotic flow in micellar electrokinetic chromatography with cationic polyelectrolyte-coated capillaries

Manipulation of the EOF is essential for achieving optimal separations by MEKC. In this paper, we present an extensive investigation of the effect of common experimental conditions on the EOF observed in a capillary coated with poly(diallyldimethylammonium chloride) (PDADMA) polyelectrolyte under MEKC conditions. It was found that highly reproducible cathodal EOF is achieved approximately at or just below the conditional CMC value of SDS in the electrolytes used. At concentrations of SDS greater than the CMC the EOF is independent of pH. The impact of common organic modifiers (ACN, methanol, urea, beta-CD and nonionic surfactant) on the EOF behavior in both a PDADMA-coated capillary and a bare silica capillary is compared. The suppressing effect of organic modifiers on the EOF is much stronger for coated capillary indicating that these compounds additionally reduce the negative charge density on the capillary surface due to alteration of the surfactant coating.     

Capillary liquid chromatography with MS3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely-moving rats

In vivo microdialysis sampling was coupled to capillary liquid chromatography (LC)/electrospray ionization quadrupole ion trap mass spectrometry (MS) to monitor [Met]enkephalin and [Leu]enkephalin in the striatum of anesthetized and freely-moving rats. The LC system utilized a high-pressure pump to load 2.5 microl samples and desalt the 25 microm i.d. by 2 cm long column in 12 min. Samples were eluted with a separate pump at approximately 100 nl min(-1). A rapid gradient effectively separated the endogenous neuropeptides in 4 min. A comparison was made for operating the mass spectrometer in the MS2 and MS3 modes for detection of the peptides. In standard solutions, the detection limits were similar at 1-2 pM (2-4 amol injected); however, the reproducibility was improved with MS3 as the relative standard deviation was <5% compared with 20% for MS2 for 60 pM samples. For dialysate solutions, reconstructed ion chromatograms and tandem mass spectra had much higher signal-to-noise ratios in the MS3 mode, resulting in more confident detection at in vivo concentrations. The method was successfully used to monitor the peptides under basal conditions and with stimulation of peptide secretion by infusion of elevated K+ concentration.     

EuCl_3在氯化1-丁基-3-甲基咪唑中的电化学性质

应用循环伏安法研究了Eu3+在亲水性离子液体—氯化1-丁基-3-甲基咪唑([BMIM]Cl)中的电极过程.实验表明,工作电极为玻碳电极时,[BMIM]Cl的电化学窗口为-1.7～0.80V(vs.Ag/AgCl).Eu3+在[BMIM]Cl中被还原为Eu2+,此电极反应受电荷迁移和物质扩散共同控制.当体系温度从55℃升高到75℃时,Eu3+在[BMIM]Cl中的扩散系数D从3.75×10-9cm2/s变化到1.32×10-8cm2/s,该反应活化能Ea为62.6kJ/mol.