Capillary electrochromatographic separation of metal ion species with on-line detection by inductively coupled plasma mass spectrometry

A bonded phase capillary column containing macrocyclic polyamine, [28]ane-N₆O₂ functional groups was used for the electrophoretic separation of arsenic, chromium and selenium species. A simple device interfacing this capillary electrochromatography (CEC) systems to an inductively coupled plasma mass spectrometer (ICPMS) is described. The dimension of the capillary column was 160 cm×100 μm i.d. To accommodate this electrophoretic separation, an auxiliary capillary was used with nitric acid (0.05 M) as makeup liquid. With the electrokinetic method at –20 kV, 20 s and a nebulizer gas flow rate of 1 l min⁻¹, the sample injected was analyzed with an applied potential of −20 kV. The background electrolyte buffer for the separation of CrO₄²⁻–Cr³⁺ was phosphate (20 mM, pH 6.5). That for HAsO₄²⁻–Ph₄As⁺ was pyromellitate (20 mM, pH 6.0) and for SeO₄²⁻–SeO₃²⁻ was acetate (20 mM, pH 6.0). The role of the buffer’s anion was also discussed. The separation efficiency of the bonded phase was compared with the bare fused silica. Concentration detection limits for these metal ions were in the low ppb range. In addition, the matrix effect of the established system with the bonded phase was found smaller than that with the bare fused silica.     

Macrocyclic polyamine as a selective modifier in a bonded-phase capillary column for the electrophoretic separation of aromatic acids.

The use of a macrocyclic polyamine, 28[ane]-N6O2, as a selective modifier in a bonded-phase capillary column for the electrophoretic separation of 14 aromatic acids is described. Parameters that affect the performance of the separations, such as the type of buffer, the pH and concentration of buffer, the applied potential and the injection mode were studied. By changing the buffer pH (4.0-5.0), buffer concentration (10-50 mM) and applied potential (-10 approximately -20 kV), optimum conditions were obtained at -20 kV, using an acetate buffer (20 mM, pH 4.5), hydrodynamic injection with a vacuum at the buffer reservoir on the detector side and detection at 220 nm. The results showed that the separation was effective under these conditions. The plate number was greater than 4 x 10(4) m-l. Due to the wide variation in the mobilities of the test compounds, injection studies suggested that a vacuum at the buffer reservoir on the detector side would produce a result that is more representative of the initial sample composition. Benzoic acid in soy sauce, salicylic acid in Salic ointment and Aspirin were sampled and analyzed using the established conditions.     

卤代乙酸及其结构相近化合物的高效毛细管电泳分离

氟、氯、溴等卤代乙酸是结构非常相近的离子型化合物,对它们的分离测定比较困难。用高效毛细管电泳法在碱性或酸性缓冲液条件下可将它们分离。在酸性缓冲液条件下,可提高有机酸分离的选择性。较低的操作电压有利于提高阴离子的分离度,而改变温度对分离度的影响不大。     

Preparation and evaluation of an imidazole-coated capillary column for the electrophoretic separation of aromatic acids

An imidazole-coated capillary column for electrophoresis has been prepared by means of organosilanization. With mesityl oxide as neutral marker, the results indicated that the electroosmotic flow of the bonded phase displays a dramatic difference in pH dependence in comparison with that of the bare fused-silica column. The presence of positive charges on the coating surface and the anionic exchange property, due to the cationic property of the imidazole group at pH values below 6, allows the separation of geometric isomers that are very similar in ionic mobility. Separation parameters including buffer composition and concentration, pH, applied voltage, and the influence of other additives were investigated. By using acetate buffer (100 mM, pH 5.2) and an applied voltage of -15 kV with UV detection at 212 nm, the separation of 11 aromatic acids including mono-, di-, tri- and tetra-carboxylic acids could be achieved in less than 14 min. The average plate number was 3 x 10(5)/m. With acetate buffer (25 mM, pH 5.5) and an applied voltage of -25 kV, the addition of silver nitrate or beta-cyclodextrin significantly improved the resolution of some more highly charged carboxylic acids.     

Separation of chlorophenoxyacetic acids and chlorophenols by using capillary zone electrophoresis

In this study, the choice of electrolyte systems for the separation and detection of a range of chlorophenoxyacetic acids and chlorophenols by means of capillary zone electrophoresis (CZE) is discussed. A series of acetate buffers over the buffering capacity pH range 4.03-5.5 were initially chosen for the separation. It was found that chlorophenoxyacetic acids could be separated at pH 4.03 and 4.5 but the most satisfactory separation of chlorophenols was obtained at pH 5.5. The factors affecting separation selectivity, including the addition of organic modifiers, was also studied. The use of 25% 2-butanol, 5% ethylene glycol and 10% acetonitrile as organic solvents resulted in the total separation of both classes of these compounds but poor peak shape of chlorophenols resulted and a number of chlorophenoxyacetic acids were not well separated. A borate-phosphate buffer gave improved peak shape of chlorophenols. Further improved separation of the components of the mixture was obtained by the addition of 2 mM fully methylated-beta-cyclodextrin to the 35 mM borate- 60 mM phosphate buffer at pH 6.5, maintaining good peak shape. In this case, separation of the two compound classes, chlorophenoxyacetic acids and chlorophenols, is achieved, with complete resolution of individual compounds in less than 5 min with high efficiency (of the order of 150,000 plates for the ca. 40 cm column). The method is applied to a commercial 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide mixture.     

Capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs with a histidine bonded phase

An open tubular wall-coated capillary column containing histidine functional groups was prepared and employed for the capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs. The anion exchange along with the hydrogen bonding and hydrophobic properties of the surface coating allowed the separation of analytes with very similar ionic mobility. Selectivity and resolution were studied by changing the pH over the range from 3.5 to 5.0 and the concentration of the buffer from 10 to 25 mM, as well as variation of the organic modifier, such as methanol, ethanol and 1-propanol over the range 7.5 to 20%. The optimum experimental conditions for the separation of a drug mixture, which consisted of indoprofen, ketoprofen, suprofen, naproxen, flurbiprofen, fenoprofen and ibuprofen were using a mixture of acetate buffer (20 mM, pH 5.0)-ethanol (1:5, v/v) as background electrolyte and an applied voltage of -20 kV with UV detection at 220 nm. The separation of these drugs could be achieved with an average plate number of 1.0 x 10(5) m(-1).     

Development and validation of a capillary electrophoresis method for direct measurement of isocitric, citric, tartaric and malic acids as adulteration markers in orange juice

Fruit juices each have very distinct organic acids profiles that can be used as fingerprints for establishing authenticity. A method has been developed, optimised and validated for measuring by capillary electrophoresis citric, isocitric, malic and tartaric acids as authenticity markers in orange juices, without any sample treatment other than dilution and filtration. Final conditions were phosphate buffer 200 mM, pH 7.50, -14 kV as applied potential, and 57 cm length neutral capillary. Detection was direct UV at 200 nm. Different kinds and marks of orange juice, chosen from the great variety existent in the market, were analysed and clear differences could be found between them and just pressed orange juice.     

Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization

With 3-trimethoxysilylpropyl chloride as the spacer, a proline-coated capillary column was prepared for the capillary electrochromatographic (CEC) separation of amino acids by in-column derivatization. Nine standard mixtures, including aspartic acid, glutamic acid, valine, phenylalanine, alanine, isoleucine, leucine, tyrosine, and tryptophan, were injected. o-Phthalaldehyde (OPA), OPA/2-mercaptoethanol (2-ME) and OPA/N-acetylcysteine (NAC) in borate buffer were tested as the derivatizing agent. Among them, OPA (50 mM) in borate buffer (pH 9.5, 50 mM) gave the best performance. The formation of isoindole could be detected by UV detection. The sandwich-type injection was carried out in hydrostatic mode (10 cm) with the program R(10 s)S(10 s) R(10 s)W(10 min) with R, S, and W being the reagent, sample, and waiting times. Mesityl oxide, benzyl alcohol, and acetone showed some interaction with the column. A current monitoring method was used instead of the determination of the electroosmotic flow (EOF). The direction of EOF was from anode to cathode even under acidic condition lower than the pI value (6.31) of the bonded group due to some unreacted silanol groups. Some parameters including pH, nature, and concentration of the mobile phase and the effect of organic modifier with regard to the CEC separation were investigated. With the proline-coated column (75 (50) cm x 75 microm ID) the best separation was performed in phosphate buffer (pH 4.00, 100 mM) with an applied voltage of -15 kV. The established method was also compared with those precolumn derivatized prior to the separation with proline-coated column as well as with in-capillary derivatization and separation with a bare fused-silica column.     

Analysis of metacycline by capillary electrophoresis

The development and validation of an optimized capillary electrophoresis method for the determination of metacycline in the presence of its related substances by capillary electrophoresis is shown. The influence of methanol as organic modifier, buffer pH, buffer concentration, capillary length, column temperature, Triton X-100 and methyl-beta-cyclodextrin was investigated. A central composite design was performed in order to optimize the method. The optimal separation conditions were: uncoated fused-silica capillary (39 cm total length, 31 cm effective length, 50 microm ID); as background electrolyte a solution of 160 mM sodium carbonate and 1 mM EDTA (pH 10.35)/methanol (89:13 v/v); temperature, 15 degrees C; voltage, 12 kV. The method showed good selectivity, repeatability, linearity, and sensitivity. The limits of detection and quantitation are 0.024% and 0.06%, respectively, relative to a 2.5 mg/mL solution. Six commercial samples were analyzed quantitatively.     

Nucleoside monophosphates recognition using macrocyclic polyamine bonded phase in capillary electrochromatography

An open-tubular wall-coated macrocyclic polyamine capillary column (70 cm x 75 microm ID) with 50 cm effective length for the separation of nucleoside monophosphates is described. Some parameters with respect to concentration, pH, composition of the buffer, and voltage in order to optimize the separation were studied. The coated capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. Baseline separations were achieved for the eight nucleotides in less than 26 min using a background electrolyte consisting of H(3)PO(4)-NaH(2)PO(4) (30 mM, pH 3.10), an applied voltage of -15 kV, and detection at 254 nm. The macrocyclic polyamine on the capillary wall introduced anion coordination for the interaction with the analytes, the strength of which could be moderated by the type and concentration of the competing ion used in the background electrolyte (BGE). With a low concentration of the competing ion (phosphate ion), the migration behavior followed that obtained in the electrophoretic system. Increasing the concentration of the competing ion resulted in a faster migration and more complete elution of the analyte. The method established was also employed for the analysis of nucleotides in mushrooms. Aqueous extracts of mushrooms from different species and various extraction methods were injected directly for the analysis. Uridine 5'-monophosphate, guanosine 5'-monophosphate, adenosine 5'-monophosphate, and cytidine 5'-monophosphate, were found in the sample tested.     

Separation of amino acids in plant tissue extracts by capillary zone electrophoresis with indirect UV detection using aromatic carboxylates as background electrolytes

Summary Amino acids in extracts of plant tissue were separated and detected by capillary zone electrophoresis (CZE) with indirect UV detection. Various aromatic carboxylates such as salicylate, benzoate, phthalate and trimellitate were investigated as background electrolytes (BGEs). A BGE of benzoate gave the best resolution and detector response. Amino acids were separated at a highly alkaline pH to charge amino acids negatively. Separation was achieved by the co-electroosmotic flow (Co-EOF) by the addition of the cationic surfactant myristyltrimethyl-ammonium bromide (MTAB) to the electrolyte. The condtions affecting the separation of amino acids, including electrolyte pH, concentrations of both benzoate and MTAB, were investigated and optimised. Separation of a mixture of 17 amino acids at pH 11.20 with indirect UV detection at 225 nm was achieved with a BGE of 10 mM benzoate containing 1.0 mM MTAB at pH of 11.20. Detection limits ranged between 10 and 50 μM. The proposed method was demonstrated by the determination of amino acids in extracts of Eucalypt leaves with direct injection of samples.     

An insight into the phenomena involved in a multiple-function stationary phase for the capillary electrochromatographic separation of 2'-, 3'-, and 5'-monophosphorylated nucleoside isomers

The electrochromatographic separations of 2'-, 3'- and 5'-monophosphates of adenosine, guanosine, cytidine, and uridine were carried out with an open-tubular capillary column which was wall-coated with a highly selective reagent, 28-membered macrocyclic polyamine, 4, 8, 12, 18, 22, 26-hexaaza-1,15-dioxacyclooctaeicosane ([28]ane-N6O2). The effects of pH, composition and concentration of background electrolyte (BGE), applied voltage, column length, and the additive of the BGE, such as metal ions, borate, beta-cyclodextrin and organic solvent on the separation of these monophosphorylated nucleotide isomers were investigated. The results suggested that the interactions between analytes and the bonded groups on the wall predominantly comprise anion coordination and anion exchange in addition to the electrophoresis. A well-resolved electrochromatogram was obtained with the capillary column of 100 cm (75 cm effective length) x 75 microm inside diameter (ID), citrate buffer (20 mM, pH 3.99), applied voltage of -22 kV and detection at 254 nm. Column efficiency was found with the average theoretical plate numbers of 119,500/m and a low detection limit of 0.01 microM level could be achieved for the separation of these isomers.     

Simultaneous separation of 14 antiarrhythmic drugs and determination of mexiletine and flecainide by capillary zone electrophoresis

A method using capillary zone electrophoresis was developed for the simultaneous separation of 14 antiarrhythmic drugs belonging to various classes. The drugs are separated on a fused-silica capillary, 90 cm x 75 microm (72 cm effective length), with phosphate and acetate buffers as background electrolytes and UV detection at 217 nm. The effects of buffer pH, temperature, and applied voltage on the migration of the drugs were studied. The pH was found to be the most significant factor determining effective separation. The antiarrhythmic compounds are completely separated within a relatively short time (< 7 min) by using 70 mM phosphate buffer at pH 7.91, an applied voltage of 28 kV, and a temperature of 32 degrees C. Mexiletine (MEX) and flecainide (FLE) were quantified under conditions of the optimum separation. The calibration graphs were constructed over the concentration range of 4.0-14.0 microg/mL for both drugs with good correlation (r > or = 0.9999). Detection and quantitation limits were found to be 0.5 and 1.5 microg/mL for FLE and 0.7 and 2.1 microg/mL for MEX, respectively. The proposed method was used for the determination of both drugs in their commercial forms with satisfactory precision (relative standard deviations of 0.36-1.21% for FLE and 0.78-1.66% for MEX) and accuracy (relative standard errors of 0.13-1.17% for FLE and 0.35-1.18% for MEX).     

Capillary electrophoresis for the analysis of short-chain organic acids in coffee

A simple and rapid capillary electrophoresis method for low-molecular mass carboxylic acids measurement in coffee has been optimised and validated. Regarding separation conditions, phosphate concentration in the background electrolyte, surfactant type [cetyltrimethylammonium bromide (CTAB), tetradecyltri methylammonium bromide (TTAB) and hexadimethrine bromide (HDB)], percentages of organic modifier and pH were assayed. The best conditions were: 500 mM phosphate buffer at pH 6.25 with CTAB 0.5 mM. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm i.d.) which was operated at -10 kV potential. Detection was performed at 200 nm. In such conditions 17 short-chain organic acids: oxalic, formic, fumaric, mesaconic, succinic, maleic, malic, isocitric, citric, acetic, citraconic, glycolic, propionic, lactic, furanoic, pyroglutamic, quinic acids plus nitrate were separated, identified and measured. Validation parameters of the method allow us to consider it lineal, accurate and precise and, therefore, reliable for its employment in food composition studies or for quality control. Results in coffees with different industrial treatment allow the detection of important differences in the organic acid profile.     

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.     

Highly reproducible capillary zone electrophoresis of humic acids in cyclodextrin- or oligosaccharide-modified background electrolytes

Capillary zone electrophoresis has been used for the characterization and separation of humic acids. It was found that addition of saccharides like alpha-, beta-, gamma-cyclodextrins, maltose, hydroxyethylcellulose or dextran sulfate in the background electrolyte (50 mM Na2 B4 O7, pH 9.6) yields better separation patterns and highly reproducible electropherograms. Electropherograms with higher numbers of peaks and high reproducibility were obtained with alpha- and beta-cyclodextrins or with a mixture of alpha- + gamma-cyclodextrin-modified background electrolytes. Separation was carried out with the cathode at the detector end of the column. Adsorption of humic acids to the capillary wall was diminished using an epoxy-coated capillary tube.     

Analysis of polymyxin B sulfate by capillary zone electrophoresis with cyclodextrin as additive. Method development and validation

A capillary zone electrophoresis method for analysis of polymyxin B sulfate is described. In this method, triethanolamine (TEA)-phosphate buffer at pH 2.5 was employed to reduce the adsorption of analyte onto the capillary wall. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found to be necessary for selectivity enhancement. In order to optimize the method and to control its robustness, a central composite design was performed with four parameters, i.e. concentration of M-beta-CD, TEA, IPA and buffer pH. The optimal separation conditions were as follows: capillary, 55 cm (50 microm I.D., 47 cm effective length); 130 mM TEA-phosphate buffer (pH 2.5) containing 5 mM M-beta-CD and 5% IPA; 24 kV (51 microA) applied voltage; column temperature, 20 degrees C. Further, linearity and limits of detection quantification were examined. Three commercial samples were analyzed quantitatively.     

Novel approach for the rapid determination of water-soluble organic acids in wine by co-electroosmotic flow capillary zone electrophoresis

An innovative protocol for the fast analysis of some organic acids in red wine by co-electroosmotic capillary zone electrophoresis and indirect UV detection using hexadimethrine bromide (HDB) as coating agent was proposed. The adsorption of HDB onto the capillary wall provided a stable electroosmotic flow and separation of small anions was carried out using background electrolytes containing no polymer additive. Low RSD% values (<3.6%) in terms of migration times and effective mobilities were obtained from the analysis of a mixture of nitrate and nitrite and of a mixture of organic acids. An experimental design approach was used to investigate the effects of temperature, separation voltage, and percentage of methanol added to the running buffer solution on the separation of the analytes. A faster method allowing the separation of the organic acids involved in the malolactic fermentation of wine was developed. Using a running electrolyte consisting of 35% (v/v) methanol in a solution of 22 mM benzoic acid at pH 6.10 adjusted with 1.0 M TRIS-base buffer, the separation of tartaric, malic, succinic, acetic, and lactic acids was feasible in less than 210 s. Application of the method to the quantification of the above-mentioned organic acids in Italian red wine samples is reported.     

The design and synthesis of alanine-based indolicidin derivatives with identical physicochemical properties and their separation using capillary electrophoresis

Four novel alanine-based indolicidin peptide derivatives were designed containing one WPW motif and two alanine residues, resulting in peptides of similar sequence. The separation of these peptides with identical physicochemical properties including molar mass, charge, and secondary structure as characterized by circular dichroism spectroscopy is very difficult; and the separation of peptides with differing physicochemical properties has only previously been reported. Capillary electrophoresis parameters such as separation buffer concentration, separation buffer pH, capillary length, and separation voltage were investigated to optimize the analysis. Using optimized conditions of a background electrolyte containing 5 mM formic acid of pH 2.0, total capillary length of 51 cm and a voltage of 10 kV enabled a baseline separation of the four peptides. The relative standard deviation of the peak areas and migration times for method repeatability (n = 3) were found to be lower than 8% and 3%, respectively. In addition, reasoning for the separation of these peptides is proposed based on the acidity of the formic acid buffer and the hydrophobic grouping of the tryptophan residues in the peptide primary sequence.     

Separation and determination of flavone and xanthone glycosides in Tibetan folk medicinal species Swertia mussotii and S. franchetiana by capillary electrophoresis

The contents of five pharmacologically active flavone and xanthone glycosides, namely, swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode-array detection. The separation of five components has been optimized with a capillary column with a total length of 48.5 cm and effective length of 40 cm (50 μm i.d). The influence of the running buffer, the sodium dodecyl sulfonate (SDS) concentration, organic modifier, etc. on the resolution was evaluated. The background electrolyte contained 30 mM borate buffer, 28 mM SDS, 1.0% (v/v) acetonitrile, and was adjusted to pH 9.0 with 0.1 M NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with the working voltage of 24 kV and a column temperature of 25°C. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and franchetiana plant samples. The text was submitted by the authors in English.