Analysis of orotic acid in human urine by on-line combination of capillary isotachophoresis and zone electrophoresis.

The techniques of the on-line combination of capillary isotachophoresis with zone electrophoresis in two coupled capillaries (ITP-CZE) and a single capillary zone electrophoresis (CZE) were used for the sensitive determination of orotic acid (OA) in human urine. The simple CZE system was successfully applied for fast and reliable analyses of urine of healthy adult volunteers (the detection limit 1.7.10(-6) M OA, the total time of analysis 6 min). However, this method failed in analyses of OA in urine of ill children due to more complex matrix of the samples. Here, the ITP preconcentration and preseparation step coupled on-line with CZE proved to serve well with an electrolyte system developed and optimized for this purpose. The maximum selectivity and resolution of OA from other sample constituents in ITP-CZE was achieved by use of an electrolyte system of very low pH 2.15 both for ITP and CZE stage. The sensitivity of detection and simplicity of OA identification were enhanced by use of an external UV scanning detector. High sensitivity of ITP-CZE combination (limit of detection 3.10(-7) M OA), low sample consumption (1 microliter), good reproducibility of migration times (inter-day RSD < 1.86%) and acceptable reproducibility of the determination of OA in urine samples (average RSD = 7.27%) make this technique suitable for routine determination of trace concentration of OA especially in urine of ill children under various pathological conditions and medication.     

Determination of ammonium in wastewaters by capillary electrophoresis on a column-coupling chip with conductivity detection

Analytical potentialities of a chip-based CE in determination of ammonium in wastewaters were investigated. CZE with the electric field and/or ITP sample stacking was performed on a column-coupling (CC) chip with integrated conductivity detectors. Acetate background electrolytes (pH ～3) including 18-crown-6-ether (18-crown-6) and tartaric acid were developed to reach rapid (in 7-8 min) CZE and ITP-CZE resolutions of ammonium from other cations (sodium, potassium, calcium and magnesium) present in wastewater samples. Under preferred working conditions (suppressed hydrodynamic flow (HDF) and EOF on the column-coupling chip), both the employed methods did provide very good repeatabilities of the migration (RSD of 0.2-0.8% for the migration time) and quantitative (RSD of 0.3-4.9% for the peak area) parameters in the model and wastewater samples. Using a 900-nL sample injection volume, LOD for ammonium were obtained at 20 and 40 μg/L concentrations in CZE and ITP-CZE separations, respectively. Very good agreements of the CZE and ITP-CZE determinations of ammonium in six untreated wastewater samples (only filtration and dilution) with the results obtained by a reference spectrometric method indicate a very good accuracy of both the CE methods presented.     

Quantitative monitoring by high-performance liquid chromatography of the dissociation of human serum cholinesterase by limited proteolysis

Proteolytic action on human serum cholinesterase, a tetrameric enzyme, results in a partial disintegration which can be recorded only qualitatively by time-consuming electrophoretic techniques. In this study, a rapid high-performance liquid chromatographic method was used for the separation and determination of the active dissociation products. Separation of the cholinesterase subunits was accomplished by high-performance gel permeation chromatography on a combination of DIOL columns (Zorbax GF 450/GF 250) in 0.2 M phosphate buffer (pH 7.0). Detection and quantification of enzyme activity in the fractionated eluate were carried out using a Flexigem analyser (substrate, butyrylthiocholine). On limited tryptic digestion of partially purified human ChE, up to three peaks of enzyme activity could be identified. Their elution volumes corresponded to apparent molecular masses of 480,000, 270,000 and 120,000, indicating, in addition to the tetrameric holoenzyme, a dimeric and a monomeric form. Quantification of the relative amounts of individual enzyme activity peaks revealed that in the course of degradation, the dimer appeared first, followed by the monomer. This suggests that the first step in the sequence of dissociation is cleavage of the tetramer into a pair of dimers, then further into the monomeric subunit. During the incubation with trypsin, a significant change in the pattern of the different peaks had already occurred when the total enzyme activity was only slightly reduced.     

Electromigration peak dispersion of isotachophoretic protein zones during capillary zone electrophoresis

The relationships between electromigration dispersion (EMD) and on-line isotachophoresis-capillary zone electrophoresis (ITP-CZE) are described for several basic model proteins and interleukin-6 (rhIL-6). During CZE separation of the highly concentrated analyte zones which were generated during the initial ITP step EMD evolves from intrinsic differences in conductivity between the focused ITP zones and the leading electrolyte. Nearly triangular peaks with a sharp front and diffuse rear side were observed. An electromigration dispersion factor (F_EMD) was introduced to measure peak asymmetry. EMD of individual peaks was shown to increase with the absolute amount of the respective analyte injected and with analyte mobility. Good linearity was observed when F_EMD was plotted against protein mobility (r > 0.95). The slope of the graphs describing this relationship increased with the amount of analyte injected. The influence of EMD on the separation efficiency of neighboring peaks appeared to be less pronounced than expected. Consecutive release from the ITP-stack during transition from ITP to CZE might be an explanation for this observation.     

Study of Electrochemical Oxidation of Xanthohumol by Ultra-Performance Liquid Chromatography Coupled to High Resolution Tandem Mass Spectrometry and Ion Mobility Mass Spectrometry

Electrochemically assisted oxidation off-line combined with UPLC/ESI–MS and ion mobility mass spectrometry enabled us to gain insight into the oxidation mechanisms of xanthohumol. Several types of monomeric oxidation products were identified, i.e., monohydroxylated and dehydrogenated derivatives and related quinones. Besides, high contents of dimers were observed. The structures of four main oxidative condensation products of two xanthohumol molecules were proposed based on combination of retention time, exact mass measurement, fragmentation pattern, data from on-line ion mobility mass spectrometric experiments and with the support of independent electrochemical experiments. To the best of our knowledge, this is the first evidence on formation of xanthohumol dimers. The effect of the pH on the generation of oxidation products was further investigated. The monomeric and dimeric oxidation products are favored at pH of 5.5 and 4.5, respectively.

     

Antioxidant activity of propofol and related monomeric and dimeric compounds

This study was carried out to investigate the antioxidant activity of propofol (2,6-diisopropylphenol) and its related compounds, butylated hydroxyanisole (BHA), 2,6-dimethylphenol, 2,6-di-t-butylphenol, and their dimeric compounds. The degree of antioxidant activity was evaluated based on the degree of peroxidation induced with Fe-ascorbic acid in egg phosphatidylcholine through the determination of thiobarbituric acid-reactive substances (TBARS) formed during peroxidation. Their antioxidant activities were in the order of dipropofol>di(2,6-di-t-butylphenol)>diBHA>di(2,6-dimethylphenol). Dipropofol, a dimeric compound of propofol, showed the highest antioxidant activities. Dimeric compounds had higher activities than monomeric compounds, and the 1,1-diphenyl-p-picryhydrazyl-trapping ability of dimeric compounds was also greater than those of monomeric compounds (4-10-fold). These results suggest that dimeric phenols may increase their antioxidant activities along with increments in the conjugation system and play a inhibitory role in the propagation of free radical chain reactions.     

Isotachophoresis and isotachophoresis--zone electrophoresis separations of inorganic anions present in water samples on a planar chip with column-coupling separation channels and conductivity detection

The use of a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to electrophoretic separations of a group of inorganic anions (chloride, nitrate, sulfate, nitrite, fluoride and phosphate) that need to be monitored in various environmental matrices was studied. The electrophoretic methods employed in this study included isotachophoresis (ITP) and capillary zone electrophoresis (CZE) with on-line coupled ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the separations performed by these methods. ITP separations on the chip provided rapid resolutions of sub-nmol amounts of the complete group of the studied anions and made possible rapid separations and reproducible quantitations of macroconstituents currently present in water samples (chloride, nitrate and sulfate). However, concentration limits of detection attainable under the employed ITP separating conditions (2-3 x 10(-5) mol/l) were not sufficient for the detection of typical anionic microconstituents in water samples (nitrite, fluoride and phosphate). On the other hand, these anions could be detected at 5-7 x 10(-7) mol/l concentrations by the conductivity detector in the CZE stage of the ITP-CZE combination on the CC chip. A sample clean-up performed in the ITP stage of the combination effectively complemented such a detection sensitivity and nitrite, fluoride and phosphate could be reproducibly quantified also in samples containing the macroconstituents at 10(4) higher concentrations. ITP-CZE analyses of tap, mineral and river water samples showed that the CC chip offers means for rapid and reproducible procedures to the determination of these anions in water (4-6 min analysis times under our working conditions). Here, the ITP sample pretreatment concentrated the analytes and removed nanomol amounts of the macroconstituents from the separation compartment of the chip within 3-4 min. Both the ITP and ITP-CZE procedures required no or only minimum manipulations with water samples before their analyses on the chip. For example, tap water samples were analyzed directly while a short degassing of mineral water (to prevent bubble formation during the separation) and filtration of river water samples (to remove particulates and colloids) were the only operations needed in this respect.     

Influence of ionic strength and organic modifier concentrations on characterization of aquatic fulvic and humic acids by high-performance size-exclusion chromatography

Aquatic fulvic acid (FA) and humic acid (HA) were characterized by an aqueous high-performance size-exclusion chromatography (HPSEC) using a hydrophilic polymeric stationary phase and an aqueous eluent at neutral pH and low-ionic strength (5 mM Na2HPO4; final ionic strength, 13 mM). Employed HPSEC showed low sensitivity of FA to variations in ionic strength (13 and 100 mM) and contents of organic modifier (0.1 or 40% methanol) in aqueous eluent. Under these analytical conditions, peak maxima of relative UV signals versus molecular mass (Mr) defined as M'p and peak maxima of relative mass concentrations versus Mr defined as Mp of FA were shown to be located at 548-690 and 500, respectively. Organic modifier concentrations of 40% methanol in aqueous eluent enabled not only analysis of FA, but also analysis of some aquatic HA by HPSEC. Analysis showed M'p and Mp values of aquatic HAs around 1000 and 600, respectively. Measured molecular mass data of FA were found to be consistent with the recently published data describing low molecular masses of FA. Results recommend the use of the described HPSEC as a simple, rapid, reproducible, low-cost method giving consistent molecular sizes/masses of FA and some aquatic HAs.     

Molecular size fractionation of soil humic acids using preparative high performance size-exclusion chromatography

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12-25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.     

Monomeric and dimeric anionic surfactants: A comparative study of self-aggregation and mineralization

A study on the phase behavior and structure of the alkanolamine salts of the dimeric amphiphile 3,4-bis-dodecyloxycarbonyl-hexanedioic acid (GS-H) is presented for the first time. Data are compared to those of the corresponding monomeric surfactant (lauric acid, LA). The alkanolamine salts of GS-H show very low Krafft points (<0 °C) and form hexagonal liquid crystals at concentrations lower than its monomeric counterpart, indicating that aggregation is favored for dimeric surfactants. The minimum concentration for liquid crystal formation increases for bulky alkanolamines with a structure-disrupting effect, such as triethanolamine (TEA). However, the specific surface areas per molecule in the liquid crystals derived from small-angle X-ray scattering (SAXS) are similar for monoethanolamine (MEA) and TEA salts; the same can be said when comparing monomeric (LA) and dimeric (GS-H) salts. GS-H can also form hexagonal and lamellar liquid crystals with organic aminosilanes acting as reactive counterions, as revealed by solvent penetration experiments with polarized optical microscopy (POM). Consequently, mineralization with silica and alumina was carried out by a sol–gel method using GS-H as a possible structure-directing agent. Both silica and alumina samples possessed a lamellar structure, which disappears on calcination; however, calcined alumina has indeed a high surface area coming mainly from micropores. It was found that the surfactant/aminosilane ratio is critical for obtaining structured silica before calcination.     

Trace analysis of glyphosate in water by capillary electrophoresis on a chip with high sample volume loadability

A new method for the determination of trace glyphosate (GLYP), non-selective pesticide, by CZE with online ITP pre-treatment of drinking waters on a column-coupling (CC) chip has been developed. CC chip was equipped with two injection channels of 0.9 and 9.9 μL volumes, two separation channels of 9.3 μL total volume and a pair of conductivity detectors. A very effective ITP sample clean-up performed in the first channel at low pH (3.2) was introduced for quick CZE resolution and detection of GLYP carried out at higher pH (6.1) in the second channel on the CC chip. The LOD for GLYP was estimated at 2.5 μg/L (15 nmol/L) using a 9.9 |mL volume of the injection channel. ITP-CZE analyses of model and real samples have provided very favorable intra-day (0.1-1.2% RSD) and inter-day (2.9% RSD) repeatabilities of the migration time for GLYP while 0.2-6.9% RSD values were typical for the peak area data. Recoveries of GLYP in spiked drinking water varied in the range of 99-109%. A minimum pre-treatment of drinking water (degassing and dilution) and a short analysis time (ca. 10 min) were distinctive features of ITP-CZE determinations of GLYP on the CC chip with high sample volume loaded, as well.     

Binding of monomeric and dimeric Concanavalin A to mannose-functionalized dendrimers

Because of the central role of Concanavalin A (Con A) in the study of protein-carbohydrate interactions, a thorough understanding of the multivalent functions of Con A is imperative. Here, the association of monomeric and dimeric derivatives of Con A with mannose-functionalized generation two through six PAMAM dendrimers is reported. Hemagglutination assay results indicate relatively low activity of the dendrimers for monomeric Con A, with small increases as the dendrimer generation increases. Isothermal titration microcalorimetry experiments indicate monovalent binding by the dendrimers with monomeric Con A and divalent binding by the dendrimers with dimeric Con A. Continuous (and comparable) but narrowing increases in enthalpy and entropy and the slight increase in association constants with monomeric Con A as the dendrimer generation increases suggest favorable proximity effects on binding. Both the hemagglutination assay and the calorimetry experiments suggest that statistical binding enhancements can be observed with monomeric Con A. The results described here should allow for a more quantitative evaluation of the enhancements that are often observed in protein-carbohydrate interactions for glycosylated frameworks binding to Con A.     

Copper uptake by silica and iron oxide under high surface coverage conditions: surface charge and sorption equilibrium modeling

A sorption modeling approach based on surface complexation concepts was applied to predict copper uptake and its effects on the surface electrostatic potential of ferric oxide and silica colloids. Equilibrium modeling of copper uptake by ferric oxide using the traditional surface complexation model (SCM) was reasonably successful with some discrepancies especially in the acidic pH ranges and high colloid concentration cases. Good predictions of the ferric oxide charge reversals during uptake were obtained from the modeling. Based on the SCM predictions, copper removal from solution is due to the outer-sphere complexation of the first hydrolysis product, resulting in the surface-metal complex SO(-)CuOH(+). The SCM was found to be insufficient to describe copper uptake by silica particles. To address discrepancies between experimental data and SCM predictions, the SCM was modified to include attributes of the surface polymer model (SPM), which incorporates sorption of the dimeric copper species Cu(2)(OH)(2)(2+). The continuum model (CM) was also studied as a second modification to the SCM to include formation of surface precipitates. Both the SPM and the CM were successful in modeling copper uptake and zeta potential variations as a function of pH at various solution conditions and colloid concentrations. From the SPM and CM predictions, it was concluded that for systems with high surface loadings, copper removal from solution occurs due to the formation of both monomeric and dimeric surface complexes, as well as through precipitation mechanisms.     

Application of solid-phase extraction on anion-exchange cartridges to quantify 5'-nucleotidase activity.

The enzyme 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes a critical reaction in intermediary metabolism, the phosphohydrolysis of nucleoside 5'-monophosphates to their corresponding nucleosides. We have evaluated solid-phase extraction on pre-packed anion-exchange cartridges as a chromatographic technique with which 5'-nucleotidase activity may be detected and quantified. Chromatographic conditions were established whereby substrate nucleotide was rapidly and completely separated from its corresponding nucleoside by solid-phase extraction. Both analytes were recovered quantitatively, without loss or degradation. This chromatographic system was integrated into a discontinuous radiochemical assay for 5'-nucleotidase which enabled both substrate utilization and product formation to be assessed simultaneously. Enzyme reaction samples could be analyzed directly for 5'-nucleotidase activity without any pre-chromatography preparation. The high capacity of the solid-phase cartridges and the inability of 5'-nucleotidase to enter the packing bed during analyte elution facilitated termination of the enzyme reaction by applying the entire reaction mixture to the cartridge. Loaded cartridges could then be stored at 4 degrees C prior to chromatography and subsequently batch-eluted. The excellent resolution between substrate and product in solid-phase extraction and the sensitivity of radioisotopic counting enabled detection/quantification of low tissue levels of 5'-nucleotidase in conjunction with ancillary assays for secondary enzyme reactions with the potential to elicit the artifactual loss of 5'-nucleotidase substrate/product when crude biological preparations are examined for 5'-nucleotidase activity. Our results demonstrate that solid-phase extraction on anion-exchange cartridges with elution solvents of appropriate pH offers several unique advantages for 5'-nucleotidase determination.     

Separation of basic proteins and peptides by capillary electrophoresis using a cationic surfactant

Incorporation of a low concentration of cetyltrimethylammonium bromide (CTAB) in the running electrolyte is shown to dynamically coat the silica capillary and to reverse the direction of electroosmotic flow. The CTAB coating prevented interaction of proteins with the capillary surface and enabled sharp peaks to be obtained in the electropherograms. A systematic study of experimental parameters demonstrated the importance of selecting a suitable buffer electrolyte and an appropriate pH. Excellent separations were obtained for five proteins, three enkephalins, and six dipeptides with an efficiency of approximately 500,000 theoretical plates per meter. The method developed is very simple to perform and was found to give excellent reproducibility.     

Formation and stability of mononuclear and dinuclear Eu(III) complexes and their catalytic reactivity toward cleavage of an RNA analog

The complex between Eu(III) and 1,7-diaza-4,10,13-trioxacyclopentadecane-N,N'-diacetic acid (L4) was characterized by pH potentiometric titration and 1H NMR spectroscopy. The conversion of the monomer to a dimeric complex is observed as the pH is increased from 7 to 10 in a reaction that releases one mol/HO- per dimer formed. The dimeric complex undergoes a further ionization with a pKa of 10.7. Kinetic parameters are reported for the cleavage of the simple phosphodiester 2-hydroxypropyl-4-nitrophenyl phosphate catalyzed by both the monomeric and the dimeric Eu(III) complexes. These data show that the monomer and dimer stabilize their bound reaction transition states with similar free energies of 7.1 and 7.6 kcal/mol, respectively. Clearly, a bridging hydroxide is not an optimal linker to promote cooperative catalysis between Eu(III) centers in macrocycles with multiple polyaminocarboxylate pendent groups.     

A comparative high frequency EPR study of monomeric and dimeric Mn4 single-molecule magnets

We compare high frequency single crystal electron paramagnetic resonance (EPR) data for monomeric and dimeric Mn₄ complexes where, in the case of the latter, intra-dimer exchange interactions are known to significantly alter the low temperature quantum behavior. The monomeric system exhibits typical single-molecule S=9/2 EPR spectra; analysis of these data have enabled us to characterize the effective spin Hamiltonian parameters for this material. In the dimeric system, the ground state transition shows a splitting over a narrow range of fields and frequencies. The transfer of spectral weight between these split peaks is consistent with the exchange bias picture which has been developed to explain the absence of a zero-field tunneling resonance in low temperature hysteresis experiments. Indeed, these measurements provide the first independent confirmation for this exchange bias model. The absence of a significant intensity in excited state EPR transitions for the dimer contrasts the results for the monomer. This likely suggests important differences concerning the coherence of excited states of the dimer versus the monomer. No clear indications for intermolecular exchange interactions were observed in the EPR for the monomer.     

Matrix isolation studies of carbonyl selenide,OCSe: Evidence of the formation of dimeric species, (OCSe)2

OCSe isolated in solid Ar or N₂ at 10 K was investigated by FTIR spectroscopy. The IR spectra of OCSe diluted in a 1:1000 proportion with the matrix gases were interpreted in terms of monomeric carbonyl selenide in a single matrix site. The IR spectra of more concentrated matrices revealed several new IR absorptions, which were tentatively assigned to different dimeric structures, aided by the prediction of quantum chemical calculations. The different matrices were exposed to UV–visible broad-band radiation, finding that monomeric OCSe decomposes into CO and Se, as evidenced by the IR absorption of CO perturbed by the presence of a selenium atom in the same matrix cage, while dimeric forms of OCSe decompose giving mainly (CO)₂.     

Dimeric molecular association of dimethyl sulfoxide in solutions of nonpolar liquids

Although many vibrational spectroscopic studies using infrared (IR) absorption and Raman scattering (RS) techniques revealed that dimethyl sulfoxide (DMSO) forms intermolecular dimeric associations in the pure liquid state and in solutions, the results of a number of dielectric relaxation studies did not clearly show the presence of such dimers. Recently, we found the presence of dimeric DMSO associations in not only the pure liquid but also in solutions of nonpolar solvents, such as tetrachloromethane (CCl(4)) and benzene (Bz), using dielectric relaxation (DR) techniques, which ranged from 50 MHz to 50 GHz at 25 °C. The dimeric DMSO associations cause a slow dielectric relaxation process with a relaxation time of ca. 23 ps for solutions in CCl(4) (ca. 17 ps in Bz) due to the dissociation into monomeric DMSO molecules, while the other fast relaxation is caused by monomeric DMSO molecules with a relaxation time of ca. 5.0 ps (ca. 5.5 ps in Bz) at 25 °C. A comparison of DR and vibrational spectroscopic data for DMSO solutions demonstrated that the concentration dependence of the relative magnitude of the slow and fast DR strength corresponds well to the two IR and RS bands assigned to the vibrational stretching modes of the sulfoxide groups (S═O) of the dimeric associations and the monomeric DMSO molecules, respectively. Moreover, the concentrations of the dimeric associations ([DIM]) and monomeric DMSO molecules ([MON]) were governed by a chemical equilibrium and an equilibrium constant (K(d) = [DIM](2)[MON](-1)) that was markedly dependent on the concentration of DMSO and the solvent species (K(d) = 2.5 ± 0.5 M(-1) and 0.7 ± 0.1 M(-1) in dilute CCl(4) and Bz solutions, respectively, and dramatically increased to 20-40 M(-1) in pure DMSO at 25 °C).     

酸性媒染紫—示波计时电位法测定天然水不同形态铝

本文报道酸性媒染紫（SVRS）一示波计时电位法测定天然水中不同形态铝。对24个实际水样分别在酸性pH5.2测定了无机单核铝Ali和碱性pH8.8底液中测定总单核铝Ala,有机单核铝Al0-Ala-Alio同时还应用该法测定了酸化水样中总铝AlT,酸溶态铝AlT=AlT-Ala,从而实现了水样中五种形态铝的电化学测定,测定值与Driscoll方法进行了比较对照,结果基本一致。本法特点为：简便快捷,灵敏准确,可以直接测定与铝毒性密切相关的无机单核铝Ali,无需分离步骤,水样用量小,适用于大批量天然水样中Al形态的快速分析。