Aqueous Buffer System Research Articles

Determination of quinolizidine alkaloids inSophora flavescens and its preparation using capillary electrophoresis

A simple and accurate capillary electrophoresis method was developed for the determination of four quinolizidine alkaloids in Sophora flavescens and Kuhuang injection. Optimum separation of the analytes was obtained on a 65 cm x 75 microm i.d. uncoated fused-silica capillary using a aqueous buffer system of 60 mmol L(-1) sodium borate at pH 8.5, with applied voltage and capillary temperature of 12 kV and 25 degrees C, respectively. Detection wavelength was set at 204 nm and jatrorrhizine was used as the internal standard. Good linear relationships between peak-area ratios and concentrations of the analytes were observed over the concentration range 0.044-0.792 mg mL(-1) for matrine, 0.142-1.926 mg mL(-1) for oxymatrine, 0.0377-0.3393 mg mL(-1) for sophocarpine and 0.0664-1.062 mg mL(-1) for sophoridine. The recoveries of four alkaloids ranged between 93.08 and 101.4% with relative standard deviations from 0.7 to 9.2% (n = 6) as determined by standard addition. The limits of detection for four alkaloids were determined to be over the range 8.8-48.0 microg mL(-1). Contents of four alkaloids in Sophora flavescens and three alkaloids in Kuhuang injection were successfully determined under the optimum 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.

Capillary electrophoresis‐laser induced fluorescence‐electrospray ionization‐mass spectrometry: A case study

The simultaneous hyphenation of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and electrospray ionization-mass spectrometry (ESI-MS) as a novel combined detection system for CE is presented. beta-Carbolines were chosen as model analytes with a forensic background. Nonaqueous CE as well as conventional CE with an aqueous buffer system are compared concerning efficiency and obtainable detection limits. The distance between the optical detection window and the sprayer tip was minimized by placing the optical cell directly in front of the electrospray interface. Similar separation efficiencies for both detection modes could thus be obtained. No significant peak-broadening induced by the MS interface was observed. The high fluorescence quantum yield and the high proton affinity of the model analytes investigated resulted in limits of detection in the fg (nmol/L) range for both detection methods. The analysis of confiscated ayahuasca samples and ethanolic plant extracts revealed complementary selectivities for LIF and MS detection. Thus, it is possible to improve peak identification of the solutes investigated by the use of these two detection principles.

Analysis of positional isomers of hydroxylated aromatic cytokinins by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method was developed for the separation of six positional isomers of hydroxylated aromatic cytokinins (topolin and topolin riboside), including ortho-topolin, meta-topolin, para-topolin, ortho-topolin riboside, meta-topolin riboside, and para-topolin riboside. Optimum resolution and analysis time (ca. 20 min) for the six aromatic cytokinin standards were achieved with a running buffer at pH 8.0 consisting of 20 mM boric acid, 50 mM sodium dodecyl sulfate (SDS), and 20% v/v methanol. The method has good reproducibility and has been successfully applied to detect the presence of a putative ortho-topolin in coconut water extract sample purified using C(18) and mixed-mode solid-phase extraction (SPE) columns. Other advantages of this MEKC method are short analysis time, low solvent consumption, and separation of positional isomers which could be achieved by a simple aqueous buffer system without the use of expensive chromatographic columns. In addition, a high-performance liquid chromatography (HPLC) method with baseline separation of the six topolin and topolin riboside standards was developed for the confirmation of the endogenous ortho-topolin in coconut water sample. Finally, the presence of ortho-topolin was further confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on its characteristic fragmentation pattern.

A molecularly imprinted polymer-based synthetic transaminase.

The design, synthesis, and evaluation of a molecularly imprinted polymer transaminase mimic is described. Methacrylic acid-ethylene glycol dimethacrylate copolymers were synthesized using, as a template, a transition state analogue (TSA) for the reaction of phenylpyruvic acid and pyridoxamine to yield phenylalanine and pyridoxal. Polymer suitability was established on the basis of (1)H NMR studies of template-functional monomer interactions. Polymer recognition characteristics were examined in a series of HPLC studies using the polymers as chromatographic stationary phases. Selectivity for the TSA, relative to substrates and products, was observed in both aqueous and nonpolar media. In the latter case (chloroform/AcOH, 96:4), an enantioseparation factor (alpha) of 2.1 was obtained, and frontal chromatographic studies revealed the presence of 11.9 +/- 0.2 micromol g(-1) (dry weight) of enantioselective sites. Polymers imprinted with the l-form of the oxazine-based TSA induced a 15-fold enhancement of the apparent reaction rate (app. V(max) 2.5 x 10(-7) mol s(-1); app. K(m) 8.2 x 10(-3) M) and enantioselective production of phenylalanine (32 +/- 4% ee) for reactions conducted in an aqueous buffer system. Substrate selectivity was evident, and a turnover number (k(cat)) of 0.1 s(-)(1) was determined. This is the first example of the catalysis of sigmatropic shifts in aqueous media by molecularly imprinted polymers.

Dynamics of human serum albumin studied by acoustic relaxation spectroscopy.

Sonic absorption spectra of solutions of human serum albumin (SA) in water and in aqueous phosphate buffer systems have been measured between 0.2 and 2000 MHz at different temperatures (15-35 degrees C), pH values (1.8-12.3), and protein concentrations (1-40 g/L). Several spectra, indicating relaxation processes in the whole frequency range, have been found. The spectra at neutral pH could be fitted well with an analytical function consisting of the asymptotic high frequency absorption and two relaxation contributions, a Debye-type relaxation term with discrete relaxation time and a term with asymmetric continuous distribution of relaxation times. Both relaxation contributions were observed in water and in buffer solutions and increased with protein concentration. The contribution represented by a Debye-type term is practically independent of temperature and was attributed to cooperative conformational changes of the polypeptide chain featuring a relaxation time of about 400 ns. The distribution of the relaxation times corresponding to the second relaxation contribution was characterized by a short time cutoff, between about 0.02 and 0.4 ns depending on temperature, and a long time tail extending to microseconds. Such relaxation behavior was interpreted in terms of solute-solvent interactions reflecting various hydration layers of HSA molecules. At acid and alkaline pH, an additional Debye-type contribution with relaxation time in the range of 30-100 ns exists. It seems to be due to proton transfer reactions of protein side-chain groups. The kinetic and thermodynamic parameters of these processes have been estimated from these first measurements to indicate the potential of acoustic spectra for the investigation of the elementary kinetics of albumin processes.

Enzymatic Synthesis of N‐Acetyllactosamine in Aqueous‐Organic Reaction Media.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Thermodynamics of partitioning of some sulfonamides in 1‐octanol–buffer and liposome systems

AbstractThe thermodynamics of partitioning of some structurally related sulfonamides have been determined in 1‐octanol–aqueous buffer and liposome systems. The partition coefficients were approximately tenfold higher in liposomes compared with 1‐octanol. The enthalpies of transfer from aqueous media to organic systems were positive in liposomes but negative or positive in 1‐octanol, whereas the entropies of transfer were positive in almost all cases. Copyright © 2002 John Wiley & Sons, Ltd.

Broadband acoustic spectra of aqueous solutionsof human serum albumin

Between 0.2 and 2000 MHz, sonic absorption spectra of solutionsof albumin in water and in aqueous phosphate buffer systems havebeen measured at different temperatures (15-35 °C), pHvalues (1.8-12.3), and protein concentrations (0.2-4% w/w).Broad absorption spectra, indicating the presence of variousrelaxation processes, have been found. Depending on the albuminconcentration, the spectra can be analytically represented by asum of the asymptotic high-frequency contribution and up to fourDebye-type relaxation terms. Other relaxation models, includingone based on a continuous distribution of relaxation times,also appropriately describe the measured spectra. Low-frequencyrelaxation processes with relaxation times between about 30 and300 ns appear to be due to cooperative conformational changes ofthe polypeptide chain. High-frequency processes with relaxationtimes between 0.3 and 3 ns seem to reflect elementary reactionsof the albumin molecule that are associated with changes in thehydration properties.

Enzymatic Synthesis of Aliphatic β-Lactosides as Mimic Units of Glycosphingolipids by Use of Trichoderma reesei Cellulase

Aliphatic β-lactosides were directly synthesized by β-lactosyl transfer reaction from p-nitrophenyl β-lactoside (Lacβ-pNP) to various 1-alkanols (n = 2–12), utilizing commercially available cellulase preparation of Trichoderma reesei C1. With ethanol acceptor, the enzyme induced ethyl β-lactoside (1) in 18% yield based on the donor added in aqueous buffer system. When 1-octanol and dodecanol were acceptors, octyl β-lactoside (2) and dodecyl β-lactoside (3) were also obtained as transfer products, respectively. In both cases, the addition of sodium cholate as detergent to the reaction system ensured a sufficient solubility of these acceptors and resulted in a remarkable increase of the desired compounds(5–13% yields based on the donor added). Furthermore, the enzyme catalyzed the N-acetyllactosaminyl transfer reaction from p-nitrophenyl β-N-acetyllactosaminide(LacNAcβ-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides, Galβ1-4GlcNAcβ1-4Man (4) and Galβ1-4GlcNAcβ1-4Glc (5), respectively. The enzyme activities transferring lactosyl and N-acetyllactosaminyl groups were not separated by chromatographies using DEAE–Sepharose Fast Flow and Sephadex 75 pg columns, indicating that the two reactions were catalyzed by a single enzyme. It was specified that a single enzyme works both transglycosylations, based on the substrate competition assay on hydrolysis.

Separation and determination of the macrolide antibiotics (erythromycin, spiramycin and oleandomycin) by capillary electrophoresis coupled with fast reductive voltammetric detection.

Separation and determination of erythromycin, spiramycin and oleandomycin by capillary zone electrophoresis coupled with fast reductive voltammetric detection using an Hg-film electrode was investigated in a simple aqueous phosphate buffer system. The influence of pH, concentration of phosphate, applied voltage, capillary length and dimension on the separation was examined and optimized. The entire separation of erythromycin, spiramycin, and oleandomycin was achieved in a 0.2 mol/L phosphate buffer system without organic modifiers. The electrochemical detection parameters, such as electrode material, applied waveform, scan rate, preconcentration potentials and preconcentration times, were investigated and discussed. This approach provides high separation efficiency and high sensitivity for all compounds, with detection limits (3 x peak-to-peak baseline noise) of 7.5 x 10(-8) mol/L for spiramycin, and 3 x 10(-7) mol/ L for erythromycin and oleandomycin. The calibration plot of peak areas for each separated peak vs. concentration of analyte was found to be linear over three orders of magnitude.

Determination of the p Ka of 12- S-hydroxyheptadecatrienoic acid by studies of its vial surface adsorption using high-performance liquid chromatography and liquid scintillation technique

When dissolving 12- S-hydroxyheptadecatrienoic acid (HHT) in aqueous buffer systems, a partition equilibrium is achieved between the liquid buffer phase and the solid vial phase. This adsorption equilibrium is dependent on the vial material, the ratio of solvent volume to moistened vial surface area ( V/ A), the pH of the aqueous solution, the buffer additives and the contact time. Partition ratios ( E) were established following determination of the recoveries ( ϖ) of HHT in the liquid phases. The p K a of HHT could be calculated to be 4.4±0.2. The HHT concentration in the liquid phase rose considerably when increasing the pH value (17% at pH 1.9, up to 97% of applied HHT at pH 8.0) and increasing the V/ A-ratio, as well as when using glass instead of polypropylene (PP) or adding acetonitrile as an organic solvent and HHT-affine substances like albumins or globulin. The HHT concentration was found to decrease during contact to PP surfaces as a result of HHT adsorption and diffusion into the hydrophobic vial material. This effect could be minimized by adding a larger percentage of acetonitrile (50–60%).

Separation of micrometer-size oxide particles by capillary zone electrophoresis

High quality separations of three-component mixtures of suspended micrometer-size metal oxide particles have been achieved by capillary zone electrophoresis in two different high-pH aqueous buffer systems. Mixtures of Al 2O 3, Fe 3O 4 and TiO 2 and of Al 2O 3, Fe 3O 4 and Fe 2O 3 were separated in 5–10 min in either carbonate buffer, pH 10.6 or pyrophosphate buffer, pH 10.2. The effects of electric field strength, buffer concentration and buffer type on separation quality were determined. Under optimized conditions, the separations were nearly baseline resolved. Complete separation of a mixture of Al 2O 3 and UO 2 particles was also accomplished. Different sized particles/aggregates of a given oxide were found to co-migrate so that, for the systems studied, particle size effects were negligible and the separations were electrophoretic in nature. Reproducibilities of migration times and peak widths, heights and shapes were found to be excellent, with relative standard deviations of migration times consistently being <1%.

Mutagenicity of potassium alkanediazotates in Chinese hamster V79 cells and their alkylating activity.

Alkanediazohydroxides are the key intermediates of carcinogenic N-nitroso compounds, and exist as geometrical isomers. In this paper, the mutagenicity and cytotoxicity of (E)- and (Z)-potassium alkanediazotates, precursors of alkanediazohydroxides, in Chinese hamster V79 cells were investigated. Mutagenic and cytotoxic activities of (E)-diazotates were dose-dependent, and activity decreased with an increase in the alkyl chain length; methyl>ethyl>propyl, butyl. On the other hand, (Z)-diazotates were less mutagenic and cytotoxic than (E)-diazotates, however (Z)-potassium methanediazotate did show weak mutagenicity. To compare chemical reactivity with biological activity, alkylating activity towards nicotinamide in an aqueous phosphate buffer system was evaluated as an index of the chemical reactivity of diazotates. Using a fluorometric HPLC method, alkylated nicotinamides were detected with high sensitivity in the reaction with all diazotates tested. The alkylating activity of (Z)-methanediazotate was higher than that of the corresponding (E)-diazotate, but the other isomers with ethyl, propyl and butyl groups had similar reactivity under the conditions used. The activity decreased by increasing the alkyl chain-length, which correlated well with the mutagenicity in V79 cells and also with that in Salmonella typhimurium, which we reported earlier. The results for (E)-diazotates were similar to the corresponding N-nitroso-N-(hydroxymethyl)alkylamines, further supporting the notion that alpha-hydroxy nitrosamines decompose through alkanediazohydroxide and alkylate DNA, and suggests that geometrical isomerism influences the carcinogenicity of -nitroso compounds in mammals.

Quantitation of counter ion of a water-insoluble drug by nonaqueous capillary electrophoresis with indirect UV detection

This is a report on a capillary electrophoretic approach for the quantitation of the counter ions of drugs that are insoluble or slightly soluble in water. Using a nonaqueous run buffer, the sample solvents were not restricted, which is not the case with conventional aqueous run buffer systems. The separation selectivity could be manipulated using mixed solvents such as methanol and dimethylformamide as run buffers. Detection was carried out by indirect UV detection with phthalate. The linearity and the repeatability of the peak area of chloride was satisfactory. This approach was applied to the drug substance, azelastine hydrochloride, which is slightly soluble in water, and provided a value of chloride content consistent with the theoretical value.

Capillary electrochromatographic separation of basic compounds with bare silica as stationary phase

The feasibility of capillary electrochromatography (CEC) was demonstrated using capillary columns packed with 3-μm silica particles and a mobile phase of organic–aqueous buffer system. The retention mechanisms of basic compounds were explored. Preliminary results indicated that the separation of the basic compounds in the CEC system involved multiple retention mechanisms including reversed-phase, cation-exchange as well as normal-phase mechanisms. In addition, electrophoretic mobility of the solutes also contributed to retention. The effects of experimental parameters such as the volume fraction of the organic modifier, pH value and ionic strength of the buffer on the retention behaviours of the solutes were investigated. A comparison of differences between CEC and CE of separations was also discussed.

Biocatalytic removal of nickel and vanadium from petroporphyrins and asphaltenes

Asphaltenes from a crude oil rich in heavy metals (Castilla crude oil) were fractionated and partially characterized. Biocatalytic modifications of these fractionated asphaltenes by three different hemoproteins: chloro-peroxidase (CPO), cytochrome C peroxidase (Cit-C), and lignin peroxidase (LPO) were evaluated in both aqueous buffer and organic solvents. The reactions were carried out in aqueous buffers, ternary systems of toluene: isopropanol: water, and aqueous-miscible organic solvent solutions with petroporphyrins as substrate. The petroporphyrins were more soluble in the ternary systems and aqueous miscible-organic solvent systems than in the aqueous buffer systems. However, only the CPO-mediated reactions were effective in eliminating the Soret peak in both aqueous and organic solvent systems. The effects of CPO-mediated reactions on the release of the metals complexed with the porphyrins and asphaltenes were also determined. Chloroperoxidase was able to alter components in the heavy fractions of petroleum and remove 53 and 27% of total heavy metals (Ni and V, respectively) from petroporphyrin-rich fractions and asphaltenes.

A novel cell-free system for peptide synthesis driven by pyridine.

Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without any protein factors. Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes. Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueous buffer systems even in the presence of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins located at the protuberances of the large subunit, e. g. L7/L12 and L11 forming the elongation factor-binding domain, were released in the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the ribosomal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine system, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation. Another inhibitor of the ribosomal translocation, thiostrepton, had no effect concerning the two activities, which is consistent with the fact that this antibiotic requires L11 for its binding to the ribosome. These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.

Direct Fluorometry of Phase-Extracted Tryptamine-Based Fast Quantitative Assay ofl-Tryptophan Decarboxylase fromCatharanthus roseusLeaf

An assay for the enzymel-tryptophan decarboxylase (TDC; EC 4.1.1.28) is described. It is based on direct fluorometry of the enzymatic reaction product (tryptamine) selectively recovered in ethyl acetate from the reaction mixture. Catalytically formed tryptamine from tryptophan in the incubation mixture is selectively (free from tryptophan) physically separated as ethyl acetate solution under basic (pH ≥ 11) conditions and subjected to direct fluorescence measurement in the organic solvent using a spectrofluorometer with excitation and emission wavelengths of 280 and 350 nm, respectively. Tryptamine production rate was quantitated from the luminescence response curve of tryptamine drawn under similar extraction and measurement conditions. Luminescence calibration curves were drawn for tryptamine in aqueous (water or buffer system) as well as in organic solvent as recovered from the varied aqueous solution conditions including those similar to the enzyme incubation mixture. The luminescence calibration graphs were linear for at least 0.5 to 10 μM tryptamine. The examination of interassay variations and the comparative magnitude of fluorescence response allowed to infer that a satisfactory and sufficient sample luminescence response was retained under the varied conditions including those akin to the enzymatic assay mixture, allowing adaptation of the fluorometry for the TDC activity quantitation. The assay was found to follow the proportionality principle of product formation with respect to catalytic reaction time as well as protein concentration in the assay mixture usingCatharanthus roseusleaf crude homogenate as well as the enzyme preparation at different states of purity. The rate of tryptamine formation under the catalytic conditions was linear for at least 1 h at 30°C. Though the assay has been demonstrated to use theC. roseusleaf as the enzyme source, it should be equally applicable to other plant and nonplant sources. The merits and precautions of the protocol have been discussed.

Fraccionamiento y desmetalización biocatalítica de asfaltenos de crudo castilla

Asphalte nes obtained from a crude oil rich in heavy metals (Castilla crude oil) were fractioned by adsorption chromatography. These asphaltenes were partially characterized in orderto determine Ni and Vcontent by High pressure liquid chromatography (HPLC), UV-visible spectroscopy and atomic absorption . Petroporphyrin contents and molecular weigth in the fractions were also determined. The results indicated that Ni and V were not uniformely distributed in asphaltene fractions. Biocatalytic modifications by chloroperoxidase (CPO) of these petroporphyrins were evaluated in both aqueous buffer and organic solvents. The reactions were carried out in both aqueous buffers and ternary systems of toluene: isopropanol: water with petroporphyrins as substrate. The petroporphyrins were more soluble in the ternary system than in the aqueous buffer systems. CPO-mediated reactions were effective in eliminating the Soret peak in both aqueous and organic solvent systems. The effect of CPO-mediated reaction on the release of the metals complexed with the porphyrins and asphaltenes was determined. Chloroperoxidase was able to modify components in the heavy fractions of petroleum and remove 57 % of total heavy metals (Ni and V) and 23 % of asphaltenes from petroporphyrin-rich fractions .