HPLC simultaneous analysis of thiols and disulfides: on-line reduction and indirect fluorescence detection without derivatization

A liquid chromatography method with indirect fluorescence detection has been developed for simultaneous detection of cysteine, cystine, homocysteine, homocystine, glutathione and glutathione disulfide. After separation in their native forms, a post-column solution of tris(2-carboxyethyl)phosphine (TCEP) at 32 mM, pH 8 is added to reduce the disulfides on-line to the corresponding thiol. The effluent is then merged with a second post-column solution of the highly fluorescent complex Cd(HQS)(2)(2-). The cadmium is complexed by the eluting thiols, effectively quenching the fluorescence. Optimization of the separation, the on-line reduction and the indirect fluorescence detection are discussed. Detection limits from 0.3-4.3 microM (0.04 to 2.6 ppm) are achieved for the six analytes in a 20 min separation.     

Flow-injection and liquid chromatographic determination of aluminum based on its fluorimetric reaction with 8-hydroxyquinoline-5-sulphonic acid in a micellar medium

Aluminum reacts with 8-hydroxyquinoline-5-sulphonic acids (HQS) in cationic micelles of cetyltrimethylammonium bromide (CTAB) to form a strongly fluorescent compound. In the micellar media the formation of the Al—HQS complex is very fast, allowing a useful continuousdetermination of aluminum in flow systems. At pH 6.3, for 2 × 10^-4 M HQS and 2 × 10^-3 M CTAB the detection limit for a luminum by flow injection is 0.1 ng ml^-1. The calibration is linear up to 100 ng ml^-1 and the peak-height precision is 2% at the 10 ng ml^-1 level. Interfences are greatly decreased compared with the batch method. The reaction has also been applied to the post-column detection by ion chromatography of aluminium in fresh and high-salinity wates and for speciation of aluminium in blodd serum after separation of serum proteins by ion-exchange liquid chromatography.     

Metal Ion Chromatography with Fluorescence Detection

Abstract

Nonpolar, agglomerated anion exchanger, and surface-sulfonated cation exchanger stationary phases have been used in conjunction with 8-hydroxyquinoline-5-sulfonic acid (HQS) in the eluent or as a postcolumn reagent for the separation and detection of a number of metals that form fluorescent HQS complexes. Several metals, notably those classified as transition metals, form nonfluorescent HQS chelates and quenches the fluorescence of other metal-HQS metal chelates. Such transition metals have been detected by introducing the fluorescent Al-HQS chelate postcolumn. Cation exchange stationary phases are the most useful for chromatographic applications involving HQS and are able to provide a variety of useful separations by tailoring elution conditions. Although not sensitive to Ba, the approach may be particularly good for the determination of the other alkaline earth metals. Fluorescence quenching resulting from Fe and Ni leaching from stainless steel chromatographic systems present a problem for trace analysis and accentuate the need for nonmetallic hardware. Subpicomole detection limits are attainable for Cd, Mg and Zn.     

Effects of Ca2+, Zn2+ and Cd2+ on uridine diphosphate-glucuronyltransferase and beta-glucuronidase activities in rat liver microsomes

The effect of various metals on uridine diphosphate (UDP)-glucuronyltransferase and beta-glucuronidase activities in rat liver microsomes was investigated. The presence of Mn2+, Cd2+, Zn2+, V5+, Ni2+, Co2+, Cu+ or Ca2+ (20 microM) in the enzyme reaction mixture did not cause a significant alteration of UDP-glucuronyltransferase activity in hepatic microsomes. Of these metals, Zn2+ and Cd2+ (20 microM) caused a remarkable increase in hepatic microsomal beta-glucuronidase activity. Appreciable effects of Zn2+ and Cd2+ on beta-glucuronidase activity were seen at 5.0 microM, and the effects were saturated at 50 microM. Ca2+ (5.0-50 microM) and/or the Ca2(+)-binding protein regucalcin (2.0 microM) did not have an appreciable effect on UDP-glucuronyltransferase and beta-glucuronidase activities in hepatic microsomes. Thus, Zn2+ and Cd2+ uniquely increased beta-glucuronidase activity. The Zn2(+)- and Cd2(+)-induced increase in beta-glucuronidase activity was completely reversed by the presence of an SH group-protecting reagent (dithiothreitol). The response of the microsomal enzyme to Zn2+ and Cd2+ (20 microM) was no longer seen after treatment with 0.2% Triton X-100 [polyoxyethylene(10)octylphenyl ether], indicating that the stimulation by these metals is dependent on membrane association. The present study suggests that, of various metals tested, Zn2+ and Cd2+ can uniquely increase hepatic microsomal beta-glucuronidase activity and that their effect is based on binding to membranous SH groups, beside the enzyme protein.     

Determination of traces of zinc by fluorimetrically indicated complexometric titrations

Zinc forms fluorescent complexes with 8-hydroxyquinoline-5-sulphonic acid (HQS) in the pH region 7-10. The stability of these complexes is such that two different titration procedures are possible: the titration of zinc with HQS and the titration of zinc with EDTA in the presence of HQS. For both procedures the choice of the titration conditions is described. Also two separation procedures for zinc are described, especially suitable in connection with the EDTA titration. The practical results show that zinc in concentrations larger than 10(-6)M (0.05 mug/ml) can be titrated with good precision.     

Selectivity enhancements for the determination of thorium by flow-injection analysis through the formation of the Th-DTPA-HQS fluorescent ternary complex

Addition of diethylenetriaminepentaacetic acid (DTPA) to the fluorescent binary complex of thorium and 8-hydroxyquinoline-5-sulfonic acid (HQS) forms the Th-DTPA-HQS fluorescent ternary complex. The formation of this ternary complex enhances the selectivity for the determination of thorium. Excesses of DTPA and HQS are used as reagents in flow-injection analysis to detect thorium. The excess DTPA effectively masks potentially interfering ions by preventing the formation of fluorescent binary metal-HQS complexes. The presence of lanthanides and transition metals does not interfere with the thorium detection with this method (the ratio of molar intensity for metals to molar intensity for thorium is <0.3% with the exception of lutetium, for which molar intensity ratio is 1.34%). The detection limit for thorium is 12 ng ml(-1).     

Preparation and characterization of thiacalix[4]arene coated water-soluble CdSe/ZnS quantum dots as a fluorescent probe for Cu2+ ions

Highly fluorescent water-soluble CdSe/ZnS (core/shell) quantum dots (QDs) as a fluorescent Cu2+ ion probe were synthesized using thiacalix[4]arene carboxylic acid (TCC) as a surface coating agent. Hydrophobic trioctylphosphine oxide (TOPO) capped CdSe/ZnS QDs were overcoated with TCC in tetrahydrofuran at room temperature, and deprotonation of the carboxyl groups of TCC resulted in the formation of water-soluble QDs. The surface structure of the QDs was characterized by using transmission electron microscopy (TEM) and fluorescence correlation spectroscopy (FCS). TEM images showed that TCC-coated QDs were monodispersed with the particle size (core-shell moiety) of approximately 5 nm. Hydrodynamic diameter of the TCC-coated QDs was determined to be 8.9 nm by FCS, showing that the thickness of the surface organic layer of the QDs was approximately 2 nm. These results indicate that the surface layer of TCC-coated QDs forms a bilayer structure consisting of TOPO and TCC molecules. TCC-coated CdSe/ZnS QDs were highly fluorescent (quantum yield, 0.21) compared to the QDs surface-modified with mercaptoacetic acid and mercaptoundecanoic acid. Fluorescence of the TCC-coated QDs was effectively quenched by Cu2+ ions even in the presence of other transition metal ions such as Cd2+, Zn2+, Co2+, Fe2+, and Fe3+ ions in the same solution. The Stern-Volmer plot for the fluorescence quenching by Cu2+ ions showed a linear relationship up to 30 microM of Cu2+ ions. The ion selectivity of TCC-coated QDs was determined by measurements of fluorescence responses towards biologically important transition metal ions (50 microM) including Fe2+, Fe3+, Co2+>Zn2+, Cd2+. The fluorescence of TCC-coated QDs was almost insensitive to other biologically important ions such as Na+, K+, Mg2+, and Ca2+, suggesting that TCC-coated QDs can be used as a fluorescent Cu2+ ion probe for biological samples. A possible quenching mechanism by Cu2+ ions was also discussed on the basis of a Langmuir-type adsorption isotherm.     

Activation of hepatic microsomal Ca2+-adenosine triphosphatase by calcium-binding protein regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+-adenosine triphosphatase (ATPase) activity in hepatic microsomes was investigated. Mg2+-ATPase activity was clearly increased by the presence of 50 microM Ca2+. Regucalcin (1.0-4.0 microM) caused a remarkable elevation (about 3-fold) of Ca2+-ATPase activity. Also, Mg2+-ATPase activity was increased (about 1.6-fold) by the presence of regucalcin (2.0 and 4.0 microM). Guanosine-5'-O-(3-thiotriphosphate) (GTPrs; 10(-5) and 10(-4) M) and nicotinamide adenine dinucleotide phosphate oxidized form (NADP+; 10(-5) to 10(-3) M) or reduced form (NADPH; 10(-4) and 10(-3) M) significantly increased Ca2+-ATPase activity. These increases were not enhanced by the presence of regucalcin (2.0 microM). Of various metal ions, a comparatively low concentration of V5+ (10(-5) M) or Cd2+ (10(-6) M) significantly increased Ca2+-ATPase activity, while Hg2+, Zn2+, Cu2+ and Mn2+ did not have such an effect. Regucalcin (2.0 microM) did not enhance the effect of V5+ and Cd2+ on Ca2+-ATPase activity. The present finding, that regucalcin activates hepatic microsomal Ca2+-ATPase, suggests a cell physiological role of regucalcin as an activator in the microsomal Ca2+-pump activity. This action of regucalcin may not be influenced by other regulators.     

A Comparative Study of 8‐Hydroxyquinoline and 8‐Hydroxyquinoline‐5‐sulfonic Acid for Antimony(III) Determination by AdSV. Substituent Effect on Sensitivity II

A sensitive and selective method for the determination of Sb³⁺ based on the formation of its complexes with 8‐hydroxyquinoline (HQ) and 8‐hydroxyquinoline‐5‐sulfonic acid (HQS) is proposed. The best analytical conditions are: pH 5.4 and 2.2 for HQ and HQS, respectively; C_HQ from 15.0 to 25.0 µmol L^?1 and C_HQS from 70.0 to 200.0 µmol L^?1. The detection limits are 100.0 and 14.0 ng L^?1 (t_acc=30 s) for Sb³⁺ with HQ and HQS, respectively. The method using HQS as ligand has a 2.2‐fold higher sensitivity than that with HQ and the former was chosen for Sb³⁺ determination.     

Use of ion chromatography for the determination of heavy and transition metals in biochemical samples.

A novel, highly sensitive method for simultaneous separation and determination of Cu2+, Ni2+, Zn2+, Cd2+, Co2+, Mn2+ and Pb2+ in biochemical samples was developed and evaluated by ion chromatography. All of these metals were well separated on a bifunctional ion-exchange column by a concentration gradient of oxalic acid and sodium chloride eluents, coupled with spectrophotometric detection after post-column derivatization with 2-[(5-bromo-2-pyridyl)azo]-5-diethylaminophenol at 560 nm. The method detection limits (signal-to-noise 3:1) were at microg l(-1) levels. The calibration graphs were linear (r2>0.999) over two-orders of magnitude. This technique was optimized and validated by analyzing five standard biochemical references.     

8-Hydroxyquinoline derivative as a fluorescent chemosensor for zinc ion.

8-Hydroxyquinoline derivative 1 as a fluorescent chemosensor for Zn2+ was synthesized. Because Cd2+ is often found with Zn2+ in the environment and can form fluorescent complexes with chelating fluorophores, a potentially important property of chemosensors for Zn2+ is their selectivity for Zn2+ over Cd2+. The Zn2+ or Cd2+ complexes of 1 gave an emission band from the 1:1 complex, but the fluorescence intensity for Cd2+ was a half of that for Zn2+. Ligand 1 is suited for use as a fluorescent chemosensor for Zn2+.     

Improved scheme of chelation ion chromatography with a mixed eluent for the simultaneous analysis of transition metals at microg l(-1) levels

An improved scheme of chelation ion chromatography (CIC) system and a mixed eluent for the simultaneous determination of transition metals are described. A method based on the improved CIC system and the mixed eluent (PDCA/Na2C2O4/LiOH/NaCl) for the analysis of seven metals (Pb2+, Cu2+, Ni2+, Zn2+, Co2+, Cd2+ and Mn2+) at microg l(-1) levels in a single isocratic elution is developed. The optimize conditions which are different from references for analyte concentration and chromatographic separation are studied in detail. D418 chelation resin is used to further reduce values of the reagent blank. The above seven metals are measured at 565 nm using 2-[(5-Bromo-2-Pyridyl)-Azo]-5-Diethyl-AminoPhenol(5-Br-PADAP) as the post-column derivatizing reagent. Detection limits range from 0.3 to 12 microg l(-1) when 4 ml of sample is pre-concentrated. The results of real sample analysis are satisfactory.     

Effects of Ca2+ and V5+ on glucose-6-phosphatase activity in rat liver microsomes: the Ca2+ effect is reversed by regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on glucose-6-phosphatase in the microsomes of rat liver was investigated. Addition of Ca2+ up to 2.5 microM to the enzyme reaction mixture caused a significant increase of glucose-6-phosphatase activity in hepatic microsomes, while Ni2+, Zn2+, Cd2+, Cu2+, Mn2+ and Co2+ (20 microM) did not have an appreciable effect. Vanadate (V5+) markedly inhibited the enzyme activity; a significant inhibitory effect was seen at 10 microM V5+. The Ca2+-induced increase of glucose-6-phosphatase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 microM of the protein. Regucalcium had no effect on the basal activity of the enzyme. Meanwhile, the inhibitory effect of V5+ (10-100 microM) on glucose-6-phosphatase was not appreciably blocked by the presence of regucalcin (up to 2.0 microM). The present data suggest that hepatic microsomal glucose-6-phosphatase is uniquely regulated by Ca2+ and V5+, of various metals, and that the Ca2+ effect is reversed by regucalcin. The present study supports the view that regucalcin plays an important role as a regulatory protein in liver cell function related to Ca2+.     

Divalent Cd and Pb uptake on calcite {1014} cleavage faces: an XPS and AFM study

The interaction of divalent Cd and Pb with the {101 4} cleavage faces of calcite has been investigated with X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Analysis of the {101 4} cleavage planes of calcite was carried out with X-ray photoelectron spectroscopy (XPS) after exposure to divalent metal-bearing solutions in the 0.1-100 microM concentration range for times ranging from 1 to 24 h. The uptake of Cd2+ by calcite was determined to be greater than that of Pb2+ under similar experimental conditions (1 microM, pH 8.2, 24 h exposure time). In both cases, the majority of the divalent metal was postulated to exist in a surface precipitate. AFM results showed that the exposure of calcite to a 1 microM Pb2+ solution resulted in ellipsoidal surface growths that were attributed to the nucleation of a PbCO3 bulk phase. In the Cd circumstance, AFM showed comparatively flat growth features forming on the calcite surface even at concentrations down to 0.1 microM, where the solution would be expected to be undersaturated with respect to Cd bulk phases. These features were attributed to a (Ca,Cd)CO3 solid solution. The individual exposure of these Cd/CaCO3 and Pb/CaCO3 samples to water pre-equilibrated with calcite (metal free) for 1 h led to the removal of no more than 20% of the divalent metal, suggesting that if there was an adsorbed Pb or Cd complex initially on the calcite surface, it was an minority species compared to the precipitate phase. Exposure of calcite to 100 microM Cd and Pb resulted in the accumulation of precipitate on the calcite surface presumably due to the divalent metal initial solution concentrations exceeding the solubility products of CdCO3 and PbCO3, respectively.     

Mechanistic study of enzyme catalyzed polymerization of 8-hydroxyquinoline-5-sulfonate using nuclear magnetic resonance spectroscopy

Horseradish peroxidase catalyzed polymerization of 8-hydroxyquinoline-5-sulfonate (HQS) has been studied by in-situ NMR spectroscopy. The 2, 4 and 7 positions of the HQS are involved in oxidative free radical coupling with other HQS molecules with the order of reactivity being 7 ≥ 2 > 4. A mechanism for the oxidative free radical coupling and structure of the resulting polymer is proposed, which is supported by ¹³C NMR spectroscopy.     

Metal ion capillary zone electrophoresis with direct UV detection: determination of transition metals using an 8-hydroxyquinoline- 5-sulphonic acid chelating system

The application of capillary zone electrophoresis to the separation and determination of metal ions after the precolumn formation of negatively charged chelates is described. Multi-component mixtures of transition metal complexes with 8-hydroxyquinoline-5-sulphonic acid (HQS) were separated in about 10 min in a fused-silica capillary column with a borate buffer of pH 9.2 at an applied voltage of 15 kV followed by direct UV detection. The capillary pretreatment with an electroosmotic flow modifier, namely a tetraalkylammonium salt, is necessary to achieve resonable migration times of these metal complexes. Incorporating the chelating reagent in the electrophoretic buffer markedly improves the detectability of relatively unstable chelates, such as those of Co(II), Zn(II) and Cd(II), and allows the separation of metal ions that form unstable HQS chelates, such as Mn(II) and alkaline earth metals. The effects of electrophoretic buffer parameters affecting the complexation reaction and migration behaviour are discussed. Linearity of calibration graphs is observed for about three orders of magnitude with sub-ppm detection limits. The applicability of the method to the analysis of real samples is demonstrated.     

Determination of metal complexes of ethylenediaminetetraacetate in the presence of organic matter by high-performance liquid chromatography

A high-performance liquid chromatography method is described here for the determination of the Cd(II), Co(II), Cu(II), Pb(II), and Zn(II) complexes of ethylenediaminetetraacetate (EDTA) in municipal wastewaters and surface waters. The method involves separation by ion-exchange chromatography on a reversed-phase C18 column coated with ion-pair reagent, followed by post-column conversion to FeEDTA and subsequent detection by UV absorbance. Although Co(II) and Cu(II) coelute, they can be quantified by analyzing absorbance by CuEDTA2- prior to post-column conversion. The method detection limit of 6-8 x 10(-8) M (5-7 ng) is an order of magnitude improvement over previous UV absorbance post-column reaction methods. The technique can be used in the presence of organic matter encountered in matrices such as untreated wastewater without pre-concentration or sample cleanup.     

Ratiometric and highly selective fluorescent sensor for cadmium under physiological pH range: a new strategy to discriminate cadmium from zinc

In a neutral aqueous environment, a new ratiometric Cd2+ fluorescent sensor 1a can successfully discriminate Cd2+ from Zn2+ by undergoing two different internal charge transfer (ICT) processes, and the high selectivity of sensor 1a to Cd2+ over some other metals was also observed. Moreover, through structure derivation and a series of NMR studies, the unique role of the 2-picolyl group (the part in red in the abstract graphic) in the sensor 1a-Cd2+ complexation was disclosed.     

On-column complexation of metal ions using 2,6-pyridinedicarboxylic acid and separation of their anionic complexes by capillary electrophoresis with direct UV detection

On-column complexation of metal ions with 2,6-pyridinedicarboxylate (2,6-PDC) to form anionic complexes enabled their separation by capillary zone electrophoresis with direct UV detection at 214 nm. Nine metal ions, Cu2+, Zn2+, Ni2+, Cd2+ Mn2+, Pb2+, Fe3+, Al3+ and Ca2+, were determined in less than 7 min using 10 mM 2.6-PDC solution containing 0.75 mM tetradecyltrimethylammonium bromide at pH 4.0. Satisfactory working ranges (20-300 microM), detection limits (3-10 microM) and good repeatability of the peak areas (RSD 2.1-4.2%, n=5) were obtained using hydrodynamic injection (30 s). The proposed method was used successfully for the determination of Mn2+, Fe3+, Al3+ and Ca2+ in groundwaters.     

Retention behaviour of metal complexes with 5-sulphoquinoline-8-ol in reversed-phase ion-pair liquid chromatography

Summary The reversed-phase ion-pair liquid chromatographic behaviour of several metal complexes with 5-sulphoquinoline-8-ol (HQS) on an octadecyl-modified silica column was investigated by using a fluorescence detector. The aluminum complex gave two peaks on the chromatograms when eluents containing phosphate buffer were used. The effects of the pH of the eluent, HQS concentration, the nature of anions in the eluent, and the column temperature on the retention behaviour of the aluminum complex are discussed on the basis of the equilibria and the kinetics of the complexation of the aluminum ion with HQS and anions in the eluent. The equilibrium constant for the interconversion reaction between the two Al(III)-HQS species which correspond to the two peaks observed and the second stepwise formation constant for the Zn(II)-HQS complex were evaluated from the HPLC data obtained.