Simultaneous determination of inorganic and organic anions,alkali, alkaline earth and transition metal cations by capillary electrophoresis with contactless conductometric detection

Simultaneous separation of up to 22 inorganic and organic anions, alkali, alkaline earth and transition metal cations was achieved in less than 3 min in the capillary electrophoresis system with contactless conductometric detector. The sample was injected from both capillary ends (dual opposite end injection) and anionic and cationic species were detected in the center of the separation capillary. The parameters of the separation electrolyte, such as pH, concentration of the electrolyte, concentration of complexing agents and concentration of 18-crown-6 were studied. Best results were achieved with electrolytes consisting of 8 mM L-histidine, 2.8 mM 2-hydroxyisobutyric acid, 0.32 mM 18-crown-6 at pH 4.25 or 9 mM L-histidine, 4.6 mM lactic acid, 0.38 mM 18-crown-6 at pH 4.25. Other electrolytes containing complexing agents such as malic or tartaric acid at various concentrations could also be used. The detection limits achieved for most cations and anions were 7.5 - 62 micro gL(-1) except for Ba2+ (90 micro gL(-1)), Cd 2+, Cr 3+ and F- (125 micro gL(-1)), and fumarate (250 micro gL(-1)). The repeatability of migration times and peak areas was better than 0.4% and 5.9%, respectively. The developed method was applied for analysis of real samples, such as tap, rain, drainage and surface water samples, plant exudates, plant extracts and ore leachates.     

Fluorescence anisotropy in studies of solute interactions with covalently modified colloidal silica nanoparticles

Time-resolved anisotropy decays of a fluorescent cationic solute, rhodamine 6G (R6G), in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles after modification of the surface with neutral or cationic silane coupling agents. The anisotropy decays provided direct evidence for distribution of the dye between the aqueous solution (picosecond decay component) and silica particles (nanosecond decay component and residual anisotropy component, which were attributed to the wobbling motion of dye on the silica surface and to the ionically bound probe, respectively). The dye was strongly adsorbed to unmodified silica nanoparticles, to the extent that less than 1% of the dye was present in the surrounding aqueous solution. Significant decreases in the degree of probe adsorption were obtained upon covalent modification of the silica with neutral or cationic silanes, with up to 80% of the probe being present in the aqueous solution in cases where the surface was coated with (3-aminopropyl)triethoxysilane. The addition of such agents also altered the fractional distribution between the nanosecond decay component and the residual anisotropy component in favor of the nanosecond component, indicative of weaker interactions between the dye and the modified surface (i.e., more wobbling motion). The data clearly show the power of time-resolved fluorescence anisotropy decay measurements for probing the modification of silica surfaces and should prove useful in characterization of new chromatographic stationary phases.     

Ion-exchange chromatography with an oxalic acid-α-hydroxyisobutyric acid eluent for the separation and quantitation of rare-earth elements in monazite and xenotime

Summary An oxalic acid-α-hydroxyisobutyric acid eluent has been used for the separation and determination of rare-earth elements by high-performance ion-exchange chromatography. Fifteen rare-earth elements were separated within less than 25 min on a 150×4.6 mm i.d. column packed with 5-μm sulfonic acid-bonded silica particles by elution with a combined gradient of 0.60–9.0 mM oxalic acid and 19.0–5.0 mM α-hydroxyisobutyric acid at pH 4.6. Detection and quantitation of the separated rare-earth elements was accomplished by visible-absorbance measurements at 600 nm after postcolumn reaction with arsenazo I. The gradient of the two complexing agents was optimized to enable the separation of yttrium(III) without interference from other elements, especially dysprosium(III) and terbium(III). Mass detection limits of the elements were in the range of 2–4 ng. Finally, the chromatographic system was applied to the quantitative analysis of rare-earth elements in monazite and xenotime.     

Sensitive indirect photometric detection of inorganic and small organic anions by capillary electrophoresis using Orange G as a probe ion

This study addresses the two major problems in the use of dyes as highly absorbing probes for indirect photometric detection in capillary electrophoresis (CE). First, effective electroosmotic flow (EOF) modification or suppression to allow separation and detection of a wide mobility range of analytes is not straightforward when electrolytes containing increased dye concentrations are used. The suppression of EOF to less than + 5x10(-9) m(2)V(-1)s(-1) was achieved with a combination of a poly(ethylenimine) (PEI)-coated capillary and the addition of the neutral polymer hydroxypropylmethylcellulose (HPMC) to the background electrolyte. Second, the deterioration of baselines due to adsorption of the dye probe to the capillary wall is generally a problem. In this work, baseline quality at higher probe concentrations was significantly improved by a rather unusual but highly effective combination of a simultaneous application of a slight overpressure (25 mbar) at the injection end during the separation, and the use of a relatively narrow capillary of 50 microm inner diameter. Both measures would appear to be counterproductive. Optimisation of the probe concentration with regard to signal-to-noise ratio resulted in an electrolyte of 4 mM Orange G, 0.05% HPMC buffered at pH 7.7 by the addition of 10.0 mM histidine isoelectric buffer. Very high separation efficiencies of 128 000-297 000 plates were made possible by the relatively high probe concentration. Combined with excellent detection sensitivity, even with the introduction of hydrodynamic flow and a reduced optical path length, these measures resulted in limits of detection ranging from 0.216 to 0.912 microM with a deuterium lamp light source (248 nm) and from 0.147 to 0.834 microM with a 476 nm blue light-emitting diode (LED) light source. Reproducibility over 30 consecutive runs without changing the electrolyte was excellent, with relative standard deviation (RSD) values of 0.14-0.80% for migration time, 1.27-3.36% for peak area and 0.88-5.12% for peak heights. The optimised electrolyte was used for the analysis of inorganic anions in air filter samples, providing good agreement with results obtained by ion chromatography.     

β-cyclodextrin as the vehicle for forming ratiometric mercury ion sensor usable in aqueous media, biological fluids, and live cells

The selective and sensitive detection methods for toxic transition-metal ions, which are rapid, facile, and applicable to the environmental and biological milieus, are of great importance. In this study, we designed a β-CD-based ratiometric sensor for detecting mercury ions in aqueous media, some biological fluids, and live cells. In this sensing platform, the thiocarbamido-containing probe dye was covalently linked onto the hydrophilic β-CD rim, which is conducive to complexing with metal ion, while the donor dye was anchored inside hydrophobic β-CD cavity via the adamantyl moiety, which is good for avoiding self-aggregation and enhancing the quantum yield of the donor dye. Upon associating with mercury ion, the probe dye undergoes ring-opening process and serves as the energy acceptor and constitutes the FRET system with the donor dye; by this way ratiometric detection of mercury ion in water can be realized with the detection limit of 10 nM. The cyclodextrin plays a crucial role for the sensing system; it not only accommodates both the donor dye and the probe dye which can form FRET system upon addition of Hg(2+) but also makes the sensor water-soluble and cell membrane permeable. This nontoxic sensing platform can be used for mercury ion detection in aqueous medium, biological fluids, and live cells (L929 and Hela). We also found that, upon being taken up by L929 cells, the sensor exhibited no cytotoxicity, and the cell proliferation was not affected.     

Development of a highly sensitive and specific immunoassay for determining chrysoidine, a banned dye, in soybean milk film

A highly specific and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA）was developed for the first time for the detection of chrysoidine, a dye banned in soybean milk film. Two haptens with different spacer arms were synthesized to produce antibodies. Both homologous and heterologous immunoassay formats were compared to enhance the icELISA sensitivity. The heterologous icELISA exhibited better performance, with an IC(50) (50% inhibitory concentration) of 0.33 ng/mL, a limit of detection (LOD, 10% inhibitory concentration) of 0.04 ng/mL, and a limit of quantitation (LOQ, 20%-80% inhibitory concentration) from 0.09 to 4.9 ng/mL. The developed icELISA was high sensitive and specific, and was applied to determine chrysoidine in fortified soybean milk film samples. The results were in good agreement with that obtained by high-performance liquid chromatography (HPLC) analyses.     

Determination of organic acids by capillary electrophoresis with simultaneous addition of Ca and Mg as complexing agents

Summary A capillary electrophoretic method, with divalent cations as complexing agents in the electrolyte, has been developed for separation and determination of the low molecular weight organic acids most commonly found in wine, viz. formic, fumaric, succinic, oxalic, malic, tartaric, acetic, lactic, and citric acids. The separation conditions optimized were electrolyte concentration, organic flow modifier concentration, type and concentration of complexing agents in the electrolyte, and injection time. The best resolution of some of the acids studied was achieved by use of an electrolyte containing tetraborate buffer (10mm) at pH 9.3, an organic flow modifier (tetradecyltrimethylammonium hydroxide), and Ca²⁺ (10 ppm) and Mg²⁺ (10 ppm) as complexing agents. Other conditions used in the method were hydrostatic injection (10 cm height for 30 s), detection at 185 nm, and temperature 20°C. For all the acids studied detector response was linear for the concentration ranges considered. The repeatability of each point on the calibration plot for standards (n=4) was generally better than 1% the method was applied to samples of must, wine, brandy, and vinegar from the Jerez region.     

Monitoring solute interactions with poly(ethylene oxide)-modified colloidal silica nanoparticles via fluorescence anisotropy decay

The fluorescence-based nanosize metrology approach, proposed recently by Geddes and Birch (Geddes, C. D.; Birch, D. J. S. J. Non-Cryst. Solids 2000, 270, 191), was used to characterize the extent of binding of a fluorescent cationic solute, rhodamine 6G (R6G), to the surface of silica particles after modification of the surface with the hydrophilic polymer poly(ethylene oxide) (PEO) of various molecular weights. The measurement of the rotational dynamics of R6G in PEO solutions showed the absence of strong interactions between R6G and PEO chains in water and the ability of the dye to sense the presence of polymer clusters in 30 wt % solutions. Time-resolved anisotropy decays of polymer-modified Ludox provided direct evidence for distribution of the dye between bound and free states, with the bound dye showing two decay components: a nanosecond decay component that is consistent with local motions of bound probes and a residual anisotropy component due to slow rotation of large silica particles. The data showed that the dye was strongly adsorbed to unmodified silica nanoparticles, to the extent that less than 1% of the dye was present in the surrounding aqueous solution. Addition of PEO blocked the adsorption of the dye to a significant degree, with up to 50% of the probe being present in the aqueous solution for Ludox samples containing 30 wt % of low molecular weight PEO. The addition of such agents also decreased the value and increased the fractional contribution of the nanosecond rotational correlation time, suggesting that polymer adsorption altered the degree of local motion of the bound probe. Atomic force microscopy imaging studies provided no evidence for a change in the particle size upon surface modification but did suggest interparticle aggregation after polymer adsorption. Thus, this redistribution of the probe is interpreted as being due to coverage of particles with the polymer, resulting in lower adsorption of R6G to the silica. The data clearly show the power of time-resolved fluorescence anisotropy decay measurements for probing the modification of silica surfaces and suggest that this method should prove useful in characterization of new chromatographic stationary phases and nanocomposite materials.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.     

In-capillary non-covalent labeling of insulin and one gastrointestinal peptide for their analyses by capillary electrophoresis with laser-induced fluorescence detection

The potential of the commercially available dye sypro orange for in-capillary derivatization was evaluated for the detection of insulin and one gastrointestinal peptide (Arg-Arg-gastrin) by capillary electrophoresis with laser induced fluorescence (CE-LIF). The fluorescent emission intensity (lambda(ex) = 488 nm, lambda(em) = 610 nm) of this probe is very low in aqueous medium, and increases strongly in less polar solvent, e.g. methanol. The hydrophobic character of the two analyzed peptides is too low to induce sufficient interaction with the fluorescent probe for good sensitivity when the latter is alone in the background electrolyte. Thus, the potential of several neutral, zwitterionic, cationic and anionic surfactants to favor probe/peptide interactions has been evaluated. It was demonstrated that a borate buffer (pH 8.5) containing tetradecyltrimethylammonium bromide (TTAB) in sub-micellar conditions can be considered as the most suitable buffer for insulin CE-LIF analysis. In addition, the method showed a good linearity between insulin concentration and the peak area of the labeled insulin, allowing quantitative measurements. The sensitivity achieved so far is comparable with that achieved with UV absorption detection, but even at this level it is interesting for microchip analysis, in which fluorescence detection is much more commonly available than UV absorption detection.     

The use of ethylenediaminetetraacetic acid or nitrilo-triacetic acid as masking agents in the determination of palladium with dimethylglyoxime or quinaldic acid

The use of complexing agents to mask interference in the gravimetric determination of palladium has been studied. Ethylenediaminetetraacctic acid was used to complex interfering ions in the determination of palladium with dimethylglyoxime, and nitrilotriacetic acid has been employed as a masking agent in the determination of palladium with quinaldic acid. From 15 to 150 mg of palladium were determined. All the metals tested do not interfere with the exception of gold which s reduced by the complexing agents and has to be removed prior to the determination. The procedures are rapid, simple and accurate.     

Investigation of the effects of wall adsorption of the visualization agent on baseline noise characteristics for indirect UV detection in capillary electrophoresis

New insights into the effects of the wall adsorption of the visualization agent on baseline noise characteristics in indirect UV detection in capillary electrophoresis were provided. When compared to relatively small, hydrophilic cationic (1-(4-pyridyl)pyridinium chloride hydrochloride) or anionic chromophores (bromide and benzoate), the use of a UV-absorbing long-chain cationic surfactant (benzyldimethylhexadecylammonium chloride) as a probe (visualization agent) for studying baseline noise characteristics as a function of various experimental conditions, such as applied voltage, pH or buffer composition, was found to provide noise data that were very different under certain conditions. For cationic visualization agents that have a strong tendency of adsorbing onto the capillary surfaces due to electrostatic and/or hydrophobic interactions, it appeared that the interplay of both thermal as well as adsorption/desorption events occurring within the capillary is likely responsible for a fluctuation of the visualization agent concentration at the detector.     

Detection and Separation of Free Amino Acid Enantiomers by Capillary Electrophoresis with a Chiral Crown Ether and Indirect Photometric Detection

18-Crown-6 tetracarboxylic acid (18C6H₄) has been successfully used as a chiral selector for capillary electrophoretic (CE), high-performance liquid chromatographic (HPLC), and gas chromatographic (GC) separation of the enantiomers of DL-amino compounds. We have previously used X-ray crystallographic analysis and HPLC with an immobilized 18C6H₄ chiral stationary phase to study chiral recognition by 18C6H₄ of several DL amino acids (DL-AA). In this study CE was used for chiral recognition of several DL-AA in electrolyte solution containing 18C6H₄, in which the analyte (D or L amino acid) interacts freely. Among 14 DL-AA investigated, the enantiomers of nine (Glu, Ile, Met, PheG, Phe, Ser, Tyr, Val, and Thr) were successfully recognized in 4-15 mM 18C6H₄. Indirect photometric detection with a cationic dye, chrysoidine, was used to monitor non-chromophoric DL-AA. Among nine successfully recognized DL-AA, the D forms of Ser, Thr and Met migrated faster than the corresponding L forms. The strengths of interactions predicted from the order of migration of each enantiomer in CE were different from those in HPLC analysis. The different enantiomer recognition probably can be ascribed to the difference between CE in which the selector is not immobilized and HPLC in which the selector is immobilized by means of a spacer.     

Colorimetric and ratiometric fluorescent detection of bisulfite by a new HBT-hemicyanine hybrid

A novel HBT-hemicyanine hybrid was prepared. This hybrid not only displays a large red-shifted (Δλ = 125 nm) emission compared to the well known ESIPT dye HBT, but also can be used as a new probe for rapid, colorimetric and ratiometric fluorescent detection of bisulfite with high selectivity and sensitivity in aqueous solution. The detection limit of this probe for HSO₃⁻ was calculated to be about 56 nM with a linear range of 0–25 μM. The potential application of this probe was exampled by detection of bisulfite in real food samples and living cells. Overall, this work not only provided a new ratiometric sensing platform, but also provided a new promising colorimetric and ratiometric fluorescent probe for bisulfite.     

Detection of rare earth elements by post-column reaction with xylenol orange and cetylpyridinium bromide

Rare earth elements have been separated by HPLC on octadecyl-bonded silica gel, dynamically modified with ammonium dodecylsulfate, using aqueous solutions of α-hydroxyisobutyric acid as mobile phase. The requirements and characteristics of post-column reaction with xylenol orange and cetylpyridinium bromide were investigated and optimized, and the reaction found to be highly sensitive and sufficiently rapid. The absorbance of the complex at 598 nm was not affected by the presence of α-hydroxyisobutyric acid at concentrations of the latter up to 150 mmol/l; at higher concentrations of the acid, the absorbance decreased slightly. For most of the rare earth elements the response was linear up to 0.2 mmol/l; the relative standard deviation of peak area was, typically, ～1 %. The sensitivity of the method was usually better than 1 μg/ml. The results obtained from the determination of lanthanum, cerium, praseodymium, and neodymium in samples of apatite and bastnesite were in a good agreement with those obtained by ICP-AES.     

The graphite probe constant temperature furnace

A graphite probe, on which the sample is deposited, is thrust into a heated graphite furnace tube, providing a constant temperature furnace environment. By comparison with wall sampling, the graphite probe system provides equal or improved sensitivity and detection limit and considerably less memory effect. Experiments with metals that range from volatile to refractory show that the peak width is the same at the same atomization temperature. A tube was designed that provided a curtain of argon to limit the absorbance path to about 15 mm.     

Separation and direct UV detection of complexed lanthanides,thorium and uranyl ions with 2-thenoyltrifluoroacetone by using capillary zone electrophoresis

Separation and detection of lanthanides, thorium and uranyl ions by capillary zone electrophoresis in the presence of 2-thenoyltrifluoroacetone (HTTA) as UV-absorbing complexing agent were investigated. The separation of positively charged complexes is partially improved by using a competing ligand in buffer with HTTA for metal ions. When 2-hydroxyisobutyric acid (HIBA) is used as competing ligand, complete separation of thorium, uranyl and lanthanides ions were observed. Some separation parameters such as pH value, the concentration of carrier electrolyte, applied voltage, the concentration of ligand in buffer and the temperature were also optimized. Under the selected conditions, the complete separation of thorium and uranyl from each other and from lanthanides was accomplished in only 12 min using 1 mmol/L HTTA, 50 mmol/L HIBA, 5 mmol/L NaNO₃, 5 % methanol with a pH 5.2 at a capillary temperature of 25 °C. Direct photometric detection at 210 nm using a voltage of 25 kV and an electrokinetic injection (100 mm for 6 s) were used.     

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.     

Selective extraction and elution of weak bases by in-line solid-phase extraction capillary electrophoresis using a pH step gradient and a weak cation-exchange monolith

A polymer monolith bearing weak cation-exchange functionality was prepared for the purpose of demonstrating pH-selective extraction and elution in in-line solid-phase extraction-capillary electrophoresis (SPE-CE) utilising a model set of cationic analytes, namely imidazole, lutidine and 3-phenylpropanamine. Optimization of the electrolyte conditions for efficient elution of the adsorbed analytes using a moving pH boundary required that the capillary and monolith be filled with 44 mM sodium acetate at high pH (pH 6) and a low pH electrolyte of 3 mM sodium acetate pH 3 was placed in the electrolyte vials. This combination allowed the adsorbed analytes to be simultaneously eluted and focused into narrow bands, with peak widths of the eluted analytes having a baseline width of 1.2 s immediately after the monolith. Using these optimum elution conditions, the versatility of the SPE-CE approach was demonstrated by removing unwanted adsorbed components after extraction with a wash at a different pH and also by selecting a pH at which only some of the model weak bases were ionised. The analytical performance of the approach was evaluated and the relative standard deviation for peak heights, peak area and migration times were in the ranges of 1.4-5.3, 1.2-3.3 and 0.4-1.2% respectively. Analytes exhibited linear calibrations with r(2) values ranging from 0.996 to 0.999 over two orders of magnitude. Analyte pre-concentration provided excellent sensitivity, and limits of detection for the analyte used in this study were in the range 8.0-30 ng ml(-1), which was an enhancement of 63 when compared to normal hydrodynamic injection occupying 1.3% of the capillary of these bases in water.     

一种近红外花菁染料的合成及其应用于生物大分子的测定

光谱性质;蛋白质;核酸测定;一种近红外花菁染料的合成及其应用于生物大分子的测定