Determination of salicylic acid and its metabolites in urine by derivative synchronous spectrofluorimetry

The simultaneous determination of salicylic acid in binary and/or ternary mixtures and its two main urinary metabolites is proposed. Mixtures of salicylic, salicyluric and gentisic acids are resolved by synchronous spectrofluorimetry, in combination with first-derivative measurements. The urine is extracted with diethyl ether in acid medium. Salicylic and salicyluric acids are re-extracted into glycine-sodium hydroxide buffer solution of pH 11.6 and determined at that pH, and salicylic and gentisic acids are re-extracted into boric acid-sodium hydroxide buffer solution of pH 8.5 and determined at pH 6.     

Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4 x 10(-4) cm2 V(-1) s(-1) The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte pKa values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.     

Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins.

The chromatographic behaviour of phosphoamino acids, phosphopeptides and phosphoproteins and their non-phosphorylated counterparts was studied on Fe(III)-Chelating Sepharose and Fe(III)-Chelating Superose. The phosphorylated compounds, in contrast to their non-phosphorylated or dephosphorylated counterparts, adsorb to immobilized iron(III) ions at pH 5.5 and can be desorbed by an increase in pH. Phosphoamino acids were eluted at pH 6.5-6.7, whereas monophosphopeptides and phosphoprotamine eluted in the pH range 6.9-7.5. Molecules possessing clusters(s) of carboxylic groups are weakly retained (gamma-carboxyglutamic acid, Ala-Ser-Glu5) or bound (polyglutamic acid, beta-casein) to the immobilized iron(III) ions at pH 5.5. Dephosphorylated beta-casein was desorbed at pH 7.0, whereas for elution of native (non-dephosphorylated) beta-casein, phosphate buffer of pH 7.7 was required. The homopolymer of polyglutamic acid was desorbed in the pH range 6.0-6.3, whereas copolymers of glutamic acid and tyrosine require pH 7.0-7.3 or even phosphate buffer at pH 7.7 for elution.     

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.     

Aqueous size-exclusion chromatography of humic acids on a sephadex gel column with diluted phosphate buffers as eluents

The elution behavior of humic acids on a Sephadex gel column is senstive to the composition, concentration, and pH of the eluent. the concentrations of the eluent in the literature are too high to obtain the correct molecular size distribution of humic acids. By reducing the concentration of phosphate buffer eluents to about a hundredth of conventional concentrations, the correct distribution is obtained. In the proposed method, 1 ml of 0.1% sample solution is introduced into a Sephadex G-50 column (2.2-cm diameter, 55 cm long) and humic acids are eluted with a 10^?3 M phosphate buffer solution (pH 7–9) at a flow rate of 1 ml min^?1.     

Similarity of salt influences on the pH of buffers, polyelectrolytes, and proteins

Changes in pH induced by the addition of electrolytes to buffers, polyelectrolytes (a polycarboxy polymethylene and a polyethyleneimine), and proteins (casein, whey, and lysozyme) solutions are explored systematically. The two buffer systems are triethanolamine/triethanolammonium chloride and citric acid/sodium citrate. These are chosen because of the similarity of their acid-base equilibria with those of amino acids predominant in most proteins, that is, amino acids that include carboxylate or ammonium groups in their structures. The pH of triethanolamine and of citrate buffers respectively increases and decreases when salt is added. At low electrolyte concentrations (<0.15 mol/kg), the phenomenon is well accounted for by standard electrostatic theories. pH values at higher salt concentrations are not reliable when measured with a commercial glass electrode without cross-checking by a standard hydrogen electrode. The changes of the pH values of polyelectrolyte and protein solutions with added salts turn out to be remarkably similar to the salt induced pH changes in the buffer solutions. It is even possible to qualitatively predict these changes in protein solutions simply from the primary protein structure. At least in the systems considered here, the specific ion effects on pH seem to correlate with the bulk activity coefficients of the added electrolytes, at least at moderate salt concentrations.     

Optimization of the separation of phenolic compounds by micellar electrokinetic capillary chromatography

A group of phenolic compounds including phenolic aldehydes, acids and flavonoids are separated by micellar electrokinetic chromatography (MECC). The influence of buffer (concentration and pH), concentration of sodium dodecylsulphate (SDS) and applied voltage were studied. To increase the selectivity of the separation and the resolution of the solutes organic solvents are added to the separation buffer, the best results were obtained when methanol was used at lower percentages. An optimized buffer (150 mM boric acid (pH 8.5)-50 mM SDS-5% methanol) provides the optimum separation with regard to resolution and migration time. This method was applied to the determination of these compounds in wine samples with good results.     

Retention mechanisms and selectivity in internal-surface reversed-phase liquid chromatography. Studies with cyanobacterial peptide toxins

Summary Microcystins-LA,-LR,-RR,-YR and nodularin, cyanobacterial peptide toxins, were separated by internal-surface reversed-phase (ISRP), high-performance liquid chromatography. The capacity factors of the toxins were measured in the range pH 2–8 using acetonitrile, isopropanol or tetrahydrofuran in potassium dihydrogenphosphate mobile phase. The main retention mechanism of the ISRP column was reversed-phase interaction but cation-exchange offered additional selectivity at neutral and slightly acidic pH. At neutral pH (10% modifier, 0.1 M buffer) the elution order was microcystin-LA (two nonpolar residues leucine and alanine as the variable amino acids), nodularin, microcystin-LR,-YR and-RR (two basic arginines as the variable amino acids). The retention times of all toxins except microcystin-RR were substantially longer at acidic pH. At pH 2 (10% modifier, 0.1 M buffer) where the cation-exchange mechanism was inoperative the elution order was changed to microcystin-RR, nodularin, microcystin-LR,-YR and-LA. The best separation was achieved at pH 2 where even two desmethylated microcystin-RR analogs could be separated from microcystin-RR.     

Enantioseparation of α-amino acids on an 18-crown-6-tetracarboxylic acid-bonded silica by capillary electrochromatography

(−)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid-bonded silica was used as the chiral stationary phase in capillary electrochromatography (CEC) for enantioseparation of some α-amino acids. Separation data in CEC were measured in mobile phases of varying pH, and composition of methanol and buffer, and compared with those in capillary liquid chromatography (CLC). In CEC better enantioseparation was generally obtained in the eluent of lower pH, higher buffer concentration and intermediate MeOH content, usually at the expense of analysis time. CEC showed generally better enantioselectivity and resolutions than CLC for the amino acids investigated.     

Synthesis and application of single-isomer 6-mono(alkylimidazolium)-beta-cyclodextrins as chiral selectors in chiral capillary electrophoresis

Novel single isomers of positively charged beta-CDs were prepared via nucleophilic substitution of 6-monotosyl-beta-CD with alkylimidazoles to afford 6-mono(alkylimidazolium)-beta-CD tosylates and then 6-mono(alkylimidazolium)-beta-CD chlorides by anion exchange. The chiral resolution abilities of these cationic CDs were studied by CE using dansyl (Dns)-amino acids as model analytes. From the experimental results, it was found that both resolution and selectivity of these cationic CDs were dependent on the following parameters: concentration of chiral selectors, pH of the running buffer, counteranions of the CDs, side chain length of the n-alkyl-imidazolium cation, temperature of the capillary column, and organic modifier used. The concentration of chiral selectors required for enantioseparation varied from 3 to 30 mM. The BGE pH played an important role in the resolution of Dns-amino acids. For acidic BGEs, chiral resolution increased with pH (4.0-6.0) and reached a local maximum at pH 6.0. However, better resolutions were obtained with basic phosphate buffer at pH 9.6. Methanol was found to be an effective organic modifier for the resolution of Dns-amino acids by CE.     

毛细管区带电泳法测定油田水中的脂肪酸

采用二硝基本甲酸一十六烷基三甲基溴化脓缓冲体系对油田水中短链脂肪酸进行了分析。应用间接紫外法,使脂肪酸在数分钟之内得以分离检测。研究表明,当缓冲溶液pH值为9.0、电解质浓度为5.0mmol／L、表面活性剂浓度为0.5mmol／L和甲醇含量为5％时可达到最佳的分高效果。     

Electro chromatography on paper

The term 'electrochromatography' is used to denote the specific cases where chromatography and ionophoresis operate simultaneously. The electric field may be applied either in the direction of flow of the solvent or at right angles to it. The filter paper is previously treated with the relevant buffer solution and the developing solvent too is saturated with it. Colloidal graphite painted on the two sides of the paper serves as electrodes.A detailed study has been made of a number of amino acids at different pH's with phenol-buffer as the developing solvent. On ordinary paper acidic amino acids move towards the anode, basic ones towards the cathode, neutral amino acids do not move at all. Using buffer-treated papers, however, the movement depends upon the pH of the buffer and the isoelectric points of the amino acids. By choice of suitable pH and application of electric field in proper direction amino acids having very close RF values (except isomers) can be separated from one another.     

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization

An MEKC method was developed for the separation and characterization of phenyl-isothiocyanate (PITC)-labeled amino acids derived from Gonometa rufobrunnae silkworm after microdialysis sample cleanup. The influence of the buffer and SDS concentration on the resolution of the amino acids was investigated. A buffer system consisting of 25 mM phosphate, 10 mM borate buffer at pH 9.00, and 70 mM SDS showed the best results, with 13 PITC-amino acid derivatives being resolved out of 15 possible amino acids that were under study. Microdialysis sampling demonstrated its efficiency as a sample cleanup technique. Sericin protein from G. rufobrunnae was found to be characterized by at least 11 positively identified amino acids. These included His, Tyr, Ser, Ala, Phe, Lys, Gly, Arg, Cys, Glu, and Asp. Leu/Met and Val/Thr were coeluting pairs and hence could not be positively confirmed.     

Ionic liquids as additives for separation of benzoic acid and chlorophenoxy acid herbicides by capillary electrophoresis

Ionic liquids (ILs) were tested as additives to phosphate-acetate buffer for the separation of chlorophenoxy and benzoic herbicide acids. The effects of buffer concentration, buffer pH, IL concentration, and concentration of organic solvent were investigated. It was found that in the presence of 40 mM phosphate-acetate containing 10% acetonitrile at pH 4.5, addition of 10 mM 1-butyl-3-methylimidazoium could reverse EOF. The shoulder-merged peaks of two herbicide acids, 2,4-dichlorobenzoic acid and 3,5-dichlorobenzoic acid, were successfully resolved by the addition of IL cation. Apart from these, results showed different IL cations had different influences on the migration behavior of some of the analytes, while IL anions did not lead to obvious difference on the separation.     

Coulometric Generation of Gradient Elution Programs: Separation of Amino Acids Without Buffer Mixing

Abstract

A coulometric cell and buffer system is reported which allows control of the pH of the mobile phase by control of the electrolysis current passed through the cell. The electrolysis cell is capable of varying the pH over the range of 2.5 – 12 without gas evolution at flow rates up to 1 ml/min. The pH is flow rate dependent in the configuration reported, but appropriate pH monitoring and electronic feedback can eliminate that problem. The use of coulometric pH control for the separation of amino acids without any buffer mixing is illustrated.     

Ultrasensitive Capillary Electrophoresis of Oligoguluronates with Laser-Induced Fluorescence Detection

An ultrasensitive method, capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection, has been established for analysis of oligoguluronates. The oligoguluronates were derivatized with 9-aminopyrene-1,4,6-trisulfonic acid (APTS) by reductive amination at 60°C for 3.5 h. The effect on the separation of buffer pH and buffer concentration were examined. The APTS-oligoguluronates were successfully separated within 20 min by use of acidic (pH 2.5) phosphate buffer as electrolyte and a potential of 20 kV. The method was used to monitor the oligoguluronates produced by acidic hydrolysis of homopolymeric blocks of guluronic acids.     

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.     

Capillary electrophoresis of FITC labeled amino acids with laser-induced fluorescence detection

FITC labeled amino acids have been separated using a home-huilt capillary electrophoresis with a laserinduced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.     

Capillary electrochromatographic analysis of aliphatic mono- and polycarboxylic acids

The parameters influencing the electrochromatographic separation of aliphatic organic acids in a capillary column with a wall-coated macrocyclic polyamine have been studied. Indirect detection using chromate, pyromellitate, trimellitate, o-phthalate, benzoate and acetate as background electrolytes has been tested. A complete separation of polyprotic acids could be achieved with pyromellitate buffer (7.5 mM, pH 6.5), and satisfactory results for the simultaneous separation of monoprotic acids and polyprotic acids were found using a capillary column of 70 cm (50 cm effective length)x75 microm inner diameter, electrokinetic injection (-10 kV, 10 s), benzoate buffer (6 mM, pH 4.6), separation voltage of -10 kV, and detection at 220 nm. For the separation of the geometric isomers fumarate and maleate, acetate buffer was found the best choice among the background electrolytes tested. The method so established has been applied to the determination of organic acids in soy sauce, brandy, lemon juice, spinach juice and cigarette. From the retention behavior, it was found that the separation mechanism on the bonded phase was influenced by the macrocyclic effect, electrostatic attraction, hydrogen bonding, van der Waals forces, and anion exchange, in addition to the differences in electrophoretic mobility.