Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis

Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.     

Separation of open-cage fullerenes using nonaqueous capillary electrophoresis

In this study, nonaqueous capillary electrophoresis (NACE) was used to separate three open-cage fullerenes. Trifluoroacetic acid (TFA) was used as the nonaqueous background electrolyte to change the analytes’ mobilities. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the choice of organic solvent, and the concentrations of TFA and sodium acetate (NaOAc) in the background electrolyte. The optimized separation occurred using 200 mM TFA/20 mM NaOAc in MeOH/acetonitrile (10:90, v/v), providing highly efficient baseline separation of the open-cage fullerenes within 5 min. The migration time repeatability for the three analytes was less than 1% (relative standard deviation). Thus, NACE is a rapid, useful alternative to high-performance liquid chromatography for the separation of open-cage fullerenes.     

Separation of transition metals in nonaqueous media with capillary electrophoresis

The separation of transition metal Ni2+, Cu2+, Co2+, Zn2+, Cd2+ and Fe3+ in methanol was investigated by using different types of organic acids as complexing agents. In pure methanol, the weaker and simpler acetic, propionic, butyric and valeric acids could enhance metal ions selectivity by increasing acid concentration and metal ions could be separated with high efficiency. However, hydroxycarboxylic acids obviously made separation efficiency worse. The effect of mixed organic acids, mixture solvent (methanol-acetonitrile, methanol-water) on metal ions separation was discussed further. The advantages of using nonaqueous solvent over aqueous for metal ions separation were shown finally.     

Rapid separation of basic drugs by nonaqueous capillary electrophoresis

Summary Nonaqueous capillary electrophoresis (NACE) has been used to achieve rapid separations of basic drugs. A high electric field was obtained by using short capillaries. Baseline separations of basic drugs, including amphetamines, tropane alkaloids and local anesthetics, were achieved in 1 min by selection of the appropriate organic solvent and electrolyte composition. Thus, high-throughput analyses can be performed. Peak efficiency up to 9154 theoretical plates s⁻¹ was achieved in a separation performed at 923V cm⁻¹. No discernible loss in resolution was observed when a conventional capillary (64.5cm) was replaced by a short (32.5 cm) capillary.     

Dual-opposite injection capillary electrophoresis for the determination of anionic and cationic homologous surfactants in a single run

An electrophoretic method for the simultaneous separation and determination of cationic and anionic surfactants based on double electrokinetic injection from the two ends of the capillary is proposed here. Nonaqueous capillary electrophoresis (NACE) with methanol as solvent was used to reduce the electroosmotic flow so that under these conditions the analytes migrate toward the corresponding electrode. The optimization step was the key to solve the problems associated with surfactants analysis (namely, adsorption on the capillary wall, micelle formation, and those issues related to the separation of homologous compounds). Good results were obtained with the proposed method both for the analysis of both spiked and natural samples, thus demonstrating the applicability of the proposed method for routine analysis. Finally, a comparison between the proposed method and two methods for independent analysis of cationic and anionic surfactants was made. The results showed that the precision (between 1.90 and 4.10% for repeatability and 7.43 and 8.98% for within-laboratory reproducibility, both expressed as relative standard deviation) and sensitivity (limits of detection and quantification between 0.52 and 1.88 microg/mL and between 1.73 and 6.20 microg/mL, respectively) are not affected by the CE mode. The resolution was similar to or better than that of the comparison methods and the analysis time was considerably shortened as both types of compounds were determined in a single run in only 9 min.     

Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers

Nonaqueous capillary electrophoresis was evaluated for its potential to separate peptides in methanolic background electrolytes in comparison to aqueous-methanol (50% v/v) and water. Isomeric aspartyl dipeptides and Leu- and Met-enkephalin served as model compounds. pK(a) values were determined in the three solvent systems based on the apparent pH scale and in the case of methanol additionally based on the conventional pH scale. Changing from water to methanol led to an increase of the ionization constants describing the dissociation equilibria of the carboxyl group and the amino group, respectively. The pK(a) shift was more pronounced for the carboxylic acid function leading to a compression of the mobility-pH curve. As reported for aqueous buffers, efficient separations of the peptides were achieved in methanolic background electrolytes including the resolution of the diastereomers of the isomeric alpha- and beta-aspartyl dipeptides. In contrast to aqueous buffers, the separation of Leu- and Met-enkephalin could also be obtained in buffers in methanol at high pH.     

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.     

Study on the effects of electrolytes and solvents in the determination of quaternary ammonium ions by nonaqueous capillary electrophoresis with contactless conductivity detection

A study on the separation of lipophilic quaternary ammonium cations in NACE coupled with contactless conductivity detection (NACE‐C⁴D) is presented. The suitability of different salts dissolved in various organic solvents as running electrolytes in NACE‐C⁴D was investigated. A solvent mixture of methanol/acetonitrile at a ratio of 90%:10% v/v showed the best results. Deoxycholic acid sodium salt as BGE was found to provide exceptional high stability with low baseline noise that leads to highest S/N ratios for the target analytes among all BGEs tested. Under the optimum conditions, capillaries with different internal diameters were examined and an id of 50 μm was found to give best detection sensitivity. The proposed method was validated and showed good linearity in the range from 2.5 to 200 μM, low limits of detection (0.1–0.7 μM) and acceptable reproducibility of peak area (intraday RSD 0.1–0.7%, n = 3; interday RSD 5.9–9.4%, n = 3).     

Novel nonaqueous capillary electrophoresis separation and determination of bioactive flavone derivatives in Chinese herbs

Nonaqueous CE (NACE) coupled to UV detection is described for the separation and determination of bioactive flavone derivatives in Chinese herbs extraction. After optimization of electrophoresis parameters, including the electrolyte nature and the organic solvent composition, a reliable separation of the analytes in an ACN/methanol (60:40, v/v) mixture containing 80 mM Tris and 10 mM sodium cholate was performed. The detection was performed at 254 nm. Method performances, including migration time and peak area reproducibility, linearity, sensitivity, and accuracy, were evaluated. The method was applied to determine bioactive flavone derivatives in seven Chinese herbs.     

Enhanced detection of seven glucoconjugated and hydroxylated porphyrins and chlorins by nonaqueous capillary electrophoresis combined with stacking

The nonaqueous capillary electrophoresis mode which includes a preconcentration step based on a transient pseudo-isotachophoresis to the simultaneous separation of seven glucoconjugated and hydroxylated porphyrins and chlorins, exhibiting very close structures, is reported. A high methanol content, of the buffer solution, was necessary in order to prevent self-assembly of the compounds and to enhance their solubility during separation. With the addition of 66% (v/v) methanol and 1% (w/v) NaCl in the aqueous sample solution, large volumes could be injected (44% capillary volume) without a loss in resolution. Sensitivity of detection was therefore improved by a 100-fold factor with regard to the method employing normal injection (2% capillary volume). Optimum electrophoretic conditions, in terms of sensitivity and performance, were obtained by using 20 mM phosphoric acid buffer, pH 2.2 and 50% methanol. The method was validated and applied to qualitative analysis of glucoconjugates in serum samples.     

Application of nonaqueous capillary electrophoresis for quantitative analysis of quinolizidine alkaloids in Chinese herbs

An easy, rapid method for simultaneous determination of tetrandrine (TET), fangchinoline (FAN), sinomenine (SIN) and tetrahydropalmatine (TEP) in Chinese herbs was developed by nonaqueous capillary electrophoresis without pretreatment for the first time. Optimum separation was achieved with a fused-silica capillary column ( i.d.) and a running buffer containing 50 mM ammonium acetate, 1.0% acetic acid and 20% acetonitrile in methanol medium. The applied voltage was 20.0 kV. The analytes were detected by UV at 214 nm. The effects of concentration of ammonium acetate, acetic acid and organic modifier on electrophoretic behavior of the analytes were studied. Regression equations revealed linear relationships (correlation coefficients: 0.9991-0.9999) between the peak area of each analyte and the concentration. The levels of analytes in Stephania tetrandra S. Moore, Rhizoma corydalis and Sinomenium acutum Rehd. et. Wils were easily determined with recoveries ranging from 96 to 107%.     

Separation and determination of aconitine alkaloids in traditional Chinese herbs by nonaqueous capillary electrophoresis

An easy, rapid, and simple nonaqueous capillary electrophoresis (NACE) method was developed for the identification and determination of three aconitine alkaloids, hypaconitine (HN), aconitine (AN), and mesaconitine (MN) within 6 min. The most suitable running buffer was composed of 60 mM ammonium acetate, 0.5% acetic acid, and 15% acetonitrile (ACN) in methanol with a fused-silica capillary column (50 cm x 75 microm ID). In the concentration range 12.5-1000 mg/L the calibration curves reveal linear relationships between the peak area for each analyte and its concentration (correlation coefficients: 0.9997 for HN, 0.9999 for AN, and 0.9995 for MN). The relative standard deviations of the migration time and peak area of the three alkaloids were 0.13, 0.57, 0.33 and 2.87, 1.06, 3.49%, respectively. The method was successfully applied to determine the three alkaloids in two commonly used traditional Chinese herbal medicines, the recoveries of the three constituents ranging between 94.7-101.9% for HN, 98.3-102.3% for AN, and 98.1-104.6% for MN.     

Simultaneous separation of organomercury species by nonaqueous capillary electrophoresis using methanol containing acetic acid and imidazole

A simple and rapid nonaqueous capillary electrophoresis method for simultaneous separation of four kinds of mercury species, namely inorganic mercury, methylmercury, ethylmercury, and phenylmercury, is reported. The effective mobilities of organomercury in aqueous and nonaqueous electrolytes were compared. Imidazole was confirmed not only as a co-ion for the separation but also as an online complexing reagent for mercury species. The optimum conditions for separation were achieved by using methanol solvent containing 0.15 M acetic acid and 15 mM imidazole as electrolyte. The sensitive detection of mercury species was accomplished at 191 nm.     

Development and validation method for determination of Paroxetine and its metabolites by nonaqueous capillary electrophoresis in human urine. Experimental design for evaluating the ruggedness of the method

A simple, rapid, and sensitive procedure using nonaqueous capillary electrophoresis (NACE) to measure Paroxetine (one of the mostly used antidepressants for mental diseases treatment) and three metabolites has been developed and validated. Optimum separation of paroxetine and metabolites was obtained on a 57 cm x 75 microm capillary using a nonaqueous buffer system of 9:1 methanol-acetonitrile containing 25 mM ammonium acetate and 1% acetic acid, with temperature and voltage of 25 degrees C and 15 kV, respectively, and hydrodynamic injection. Fluoxetine was used as an internal standard. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy, and precision. Detection limits between 9.3 and 23.1 microg.L(-1) were obtained for paroxetine and its metabolites. A ruggedness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. This method has been used to determine paroxetine and its main metabolite B at clinically relevant levels in human urine. Prior to NACE determination, the samples were purified and enriched by means of an extraction-preconcentration step with a preconditioned C18 cartridge and eluting the compounds with methanol.     

Micro-porous membrane liquid-liquid extraction as an enrichment step prior to nonaqueous capillary electrophoresis determination of sulfonylurea herbicides

A method based on micro-porous membrane liquid-liquid extraction (MMLLE) enrichment and nonaqueous capillary electrophoresis (CE) separation, was established for the analysis of sulfonylurea herbicides in water samples. After MMLLE, the analyte trapped in the chloroform was treated mildly with nitrogen flow to dryness and then dissolved in 200 μl of 4 mM Tris methanol solution for CE analysis. Five sulfonylurea herbicides were separated by nonaqueous CE with Tris/acetate of methanol solution as the run buffer. MMLLE related parameters such as organic solvent used as acceptor, sample flow rate, sample pH, enrichment time, and salt effect were investigated with tribenuron methyl (TBM) as a model compound. Results showed that with a sample flow rate of 3.0 ml min⁻¹ and an enrichment time of 20 min, the proposed method has good linear relationship over the scope of 1-15 ng ml⁻¹ with related coefficient of R²=0.9911, and a detection limit of 0.4 ng ml⁻¹. This method was applied to determine TBM in realworld water samples with recoveries over the range of 89-97%.     

Fast analysis of phenolic acids by electrokinetic supercharging‐nonaqueous capillary electrophoresis

A method was developed to analyze phenolic acids by nonaqueous CE after online concentration with electrokinetic supercharging. The EOF was reversed using a polyelectrolyte multilayer approach based on the successive adsorption of poly(diallyldimethylamonium chloride) and poly(styrenesulfonate) to reduce the analysis time. The results showed that the coatings were stable during 40 consecutive injections. Four phenolic acids were separated within 8 min using 30 mM ammonium acetate (pH^* 8.0). The electrokinetic injection time and terminator length of the electrokinetic supercharging method were optimized to improve the detection sensitivity. Under the optimized conditions (electrokinetic injection of 100 s, ?10 kV; terminator of 20 mM 2‐(cyclohexylamino) ethanesulfonic acid, 22 s, 0.5 psi), the sensitivity was enhanced from 300‐ to 440‐fold. The detection limits, based on three times noise, were in the range 1.0–2.5 ng/mL.     

Electroosmotic pump-assisted capillary electrophoresis of proteins

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.     

Ion-pair association and acid-base equilibria in nonaqueous capillary electrophoresis of weakly basic compounds

CE in nonaqueous media was used to study the migrating behavior of two weakly basic s-triazine pesticides and one of their metabolites. The target pesticides were selected to be representative for each of the two main groups: propazine and deethylatrazine for the chloro-s-triazines group and ametryn for the methylthio-s-triazines group. To elucidate the phenomena involved, systematic studies were carried out in the different organic media studied. Absolute mobilities were determined in 50% v/v methanol (MeOH)/ACN by extrapolation of the effective mobilities to zero ionic strength in the presence of different concentrations of perchloric acid. Conductivity measurements performed in MeOH and 50 and 20% v/v methanol/ACN permitted the evaluation of the associations of the components of the BGE. The effects of ionic strength on the actual mobilities of the compounds were determined in the presence of perchloric acid and SDS in different organic media. Two different ion-pair equilibria were considered: one due to the presence of perchlorate anions present in the BGE and second that from the added dodecyl sulfate anions. Bearing in mind that these weakly basic compounds can exhibit ion-pair and acid-base equilibria, the acid-base and ion-pair parasite reaction coefficients were determined. Finally, the effects of ionic strength, ion-pair interactions and acid-base properties on the effective electrophoretic mobilities of the analytes are discussed.     

Separation and determination of the anthraquinones in Xanthophytum attopvensis pierre by nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) method with direct on-column UV detection has been developed for the separation of the pharmaceutically important anthraquinones from the total grass of Xanthophytum attopvensis pierre extract. The separation of three main anthraquinones (1-hydroxy-2-methoxy-3-hydroxymethyl-9, 10-anthraquinone-1-O-β-d-glucoside (1), rubiadin- 1-methylether (2) and 1-methoxy-2-formyl-3-hydroxy-9, 10-anthraquinone (3)) was optimized with respect to concentration of sodium cholate (SC) and acetic acid, addition of acetonitrile (ACN), and applied voltage. Baseline separation was obtained for the three analytes within 5 min using a running buffer containing 50 mM sodium cholate (SC), 1.0% acetic acid and 40% ACN in methanol. The method of NACE for the separation and determination of bioactive ingredient in traditional Chinese medicines was discussed.     

Piperazine quaternary diammonium salts as additives to background electrolytes in capillary zone electrophoresis

The differential behavior of five different quaternary mono- and diammonium salts, among the 18 investigated, in modulating the electroendoosmotic flow (EOF) and analyte separations in capillary zone electrophoresis is evaluated. It is found that quaternary diammonium salts with positive charges separated by more than four carbon atoms, while exhibiting a very strong affinity for chromatographic silica beads, to the point of exhibiting Rf values close to zero, display, on the contrary, a very poor affinity for the silica wall of capillaries. Compounds separated only by a C2 unit (i.e., 1,4-dialkyl-1,4-diazoniabicyclo[2,2,2,]octane, salts 17 and 18) show high Rf values due to strong ion pair association. The unique behavior of quaternary monoammonium salts possessing an iodinated alkyl (butyl or octyl) tail (i.e., 1, 6, and 7) is attributed to their ability to be covalently affixed to the silica wall via alkylation of ionized silanols at alkaline pH values. They thus strongly modulate and typically invert the EOF, even when not present in the background electrolyte. On the contrary, all diammonium salts, devoid of such alkyl tails, are unable to modulate the EOF and to prevent analyte binding to the silica wall, since they are rapidly removed from the wall by the voltage gradient. However, if added in small amount to the background electrolyte, they offer excellent separations of mixtures of very similar organic acids and prevent any interaction with the capillary wall.