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The mechanisms controlling the generation of PbH 4 by reaction of inorganic Pb(II) with aqueous NaBH 4 were investigated both in the presence and in the absence of the additive K 3 Fe(CN) 6 . For the first time PbH 4 has been identified in analytical chemical vapor generation (CVG) by using gas chromatographic mass spectrometry (GC-MS), which allows the use of deuterium labelled experiments. In the absence of the additive, under reaction conditions typically employed for trace lead determination by CVG, Pb(II) is converted to solid species and no volatile lead species can be detected by either atomic or mass spectrometry for Pb(II) concentration up to 100 mg L -1 . In alkaline conditions Pb(II) substrates are unreactive towards NaBH 4 . In the presence of K 3 Fe(CN) 6 , deuterium labelled experiments clearly indicated that the generated PbH 4 is formed by the direct transfer of hydride from borane to lead atoms. Kinetic experiments were carried out to evaluate the rate of reduction of K 3 Fe(CN) 6 by NaBH 4 , the rate of hydrolysis of NaBH 4 both in the presence and in the absence of K 3 Fe(CN) 6 , and the rate of dihydrogen evolution following NaBH 4 hydrolysis. The effect of delayed addition of Pb(II) to NaBH 4 -HCl- K 3 Fe(CN) 6 , and K 3 Fe(CN) 6 to NaBH 4 -HCl-Pb(II) reaction mixtures on the efficiency of plumbane generation was investigated by continuous flow CVG coupled with atomic fluorescence spectrometry. The collected evidences, complemented with thermodynamic considerations and literature data, have made it possible to clarify long-standing controversial aspects related to the mechanism of plumbane generation and the role of K 3 Fe(CN) 6 additive., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mitochondria are organelles of bacterial origin historically identified as the cell power plant. In addition to energy, mitochondria produce reactive oxygen species and they have been found to have a key role in cell defense regulation, cell stress and damage. All the investigations regarding the nature of the molecules mediating these processes include compounds from mammalian cell metabolism. We hypothesize that the bacterial origin of mitochondria brings them to produce small fermentation products when cell is subjected to stress. In this work we studied the effect of hyperglycemia on the metabolome of hippocampal HN9.10e neurons, an in vitro model of one of the most vulnerable regions of central nervous system. Targeted metabolites were analyzed in the cell culture medium by liquid chromatography - diode array detection and headspace - gas chromatography - mass spectrometry. Twenty-two low molecular weight metabolites were identified and quantified in the growth medium of the cells, treated with 25, 50 or 75 mM glucose, sampled along 8 days to mimic a prolonged hyperglycemia. The results of statistical analysis showed the clear impairment of neuronal metabolism already after 48 h, represented by a significant reduction of the metabolic activity, together with the production of typical fermentative compounds., (Copyright © 2022 Elsevier Inc. All rights reserved.)

When working with humid gaseous samples, the amount of water vapour collected in a needle trap along with volatile analytes may vary from sample to sample and decrease during the storage. This has a major impact on desorption efficiency and recovery. We propose the addition of a labelled internal standards to nullify the effect of variable humidity on the analytical performance of needle trap micro-extraction combined with gas chromatography mass spectrometry. Triple-bed (Divinylbenzene/Carbopack X/Carboxen 1000) and single-bed (Tenax GR) needles were tested with standard gaseous mixtures prepared at different relative humidity levels (85%, 50% and 10%). The standard mixtures contained twenty-five analytes representative of breath and ambient air constituents, including hydrocarbons, ketones, aldehydes, aromatics, and sulphurs, in the concentration range 0.1-700 ppbv. The two needles showed different behaviours, as recovery was independent of humidity for single-beds, whereas a low recovery (10-20%) was observed when triple-beds trapped very volatile compounds at low humidity (e.g. pentane and ethanol, 10% relative humidity. Triple-beds showed an almost quantitative recovery (>90%) of all the analytes at 50% and 85% relative humidity. This big difference was probably due to the reduced action of water vapour pressure during the desorption step. The addition of 6 D-acetone and 8 D-toluene to the sorbent material before gas sampling and the normalization of raw data nullified this effect, thereby lowering the variations of analyte recovery at different humidity levels down to 20%. Internal standards were also exploited to limit within 10-20% alterations in peak areas of very volatile compounds during needle storage at room temperature. This variation may results from a loss of water vapour either retained from the sorbent material and/or condensed on triple-bed needle walls. After normalization, the inter- and intra-day precision were halved to 5% and 10% in the case of single-beds, respectively, and to 15% and 20% with three-beds. The addition of an internal standard to the sorbent helps to keep the overall analytical procedure under control and improves the reliability of needle trap micro-extraction for the analysis of volatile organic compounds at ultra-trace levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heavy metals (HMs) and polychlorobiphenyls (PCBs) enter the Arctic environment through a variety of anthropogenic sources with deleterious effects towards biota and public health. Bacteria first transfer toxic compounds to higher trophic levels and, due to the tight link existing between prokaryotic community functions and the type and concentration of contaminants, they may be useful indicator of pollution events and potential toxicity to other forms of life. The occurrence and abundance of HM-tolerant and PCB-oxidizing bacteria in the sub-Arctic Pasvik river area, heavily impacted by anthropogenic modifications, was related to HM and PCB contamination. This latter more likely derived from local inputs rather than a global contamination with higher PCB and HM amounts (and higher bacterial viable counts) that were determined in inner and middle sections of the River. Finally, a panel of bacteria with potential applications in the bioremediation of cold environments were selected and phylogenetically identified., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, we propose a novel approach for the determination of total dissolved nitrogen (TDN) in seawater combining high-precision isotope dilution GC-MS with persulfate digestion. A 2 mL sample aliquot was digested with an alkaline solution of persulfate to convert nitrogen containing compounds to nitrate. Digested samples were spiked with 15 NO 3 - internal standard and treated with aqueous triethyloxonium to convert the analyte into volatile EtONO 2 . This derivative was readily separated from the matrix under gaseous form and could be sampled from the headspace before GC-MS analysis. The resulting chromatograms showed a stable flat baseline with EtONO 2 as the only eluting peak (retention time 2.75 min on a DB 5.625 column). Such an approach provides specificity and obviates the shortcomings of current detection methods employed to analyze seawater samples after digestion with persulfate. In negative chemical ionization mode, the method reached a detection limit of 0.5 μmol/kg TDN (7 ng/g N) and could be applied to quantify seawater samples with 1-25 μmol/kg TDN. On the upper end of the range, quantitation could be repeated within 1%, whereas on a 6 μmol/kg TDN sample repeatability was 2.3% on eight measurements. The method was employed in two proficiency testing exercises providing results in agreement with consensus values. We investigated the impact of reagent blank and we implemented a blank-matching optimal design to account for such contribution. Finally, we performed a study on the yield of persulfate oxidation for organic and inorganic nitrogen compounds typically present in seawater. Whilst nitrite and ammonium are fully converted to nitrate, more complex organic molecules showed recoveries varying from 70% to 100%., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

The behavior of NaBH 4 (THB) and the amine boranes, NH 3 BH 3 (AB), tertbutylNH 2 BH 3 (TBAB), Me 2 NHBH 3 (DMAB) was investigated in continuous flow chemical vapor generation of H 2 Se from aqueous Se IV coupled with atomic absorption spectrometry. Unexpected higher efficiency of H 2 Se generation was obtained with amine boranes compared to THB (TBAB > AB > THB) using millimolar concentration of reductant (0.001-0.1 mol L -1 ) under strongly acidic conditions (HCl, HClO 4 , H 2 SO 4 , HNO 3 , 0.5-5 mol L -1  H + ). Analytical applicability of the CVG system was tested by the determination of Se IV in natural water samples certified reference materials, using 0.01 mol L -1 TBAB in 0.5 M H 2 SO 4 . In order to explain this unexpected higher efficiency of amine boranes with respect of THB, the kinetic of hydrolysis of AB, TBAB and DMAB was investigated in acid media typically employed in chemical vapor generation for trace element determination. The kinetic was investigated by monitoring the rate the hydrogen gas evolved during hydrolysis, using a laboratory made thermostated reaction cell. Kinetics were measured for AB, TBAB and DMAB in 0.1, 0.5, 5 mol L -1 HCl or HClO 4 reaction media and in 0.1 mol L -1 cysteine +0.1 mol L -1 HCl or HClO 4 buffer, for reaction times from 0 to 30 min. Under strongly acidic conditions, the rates of hydrogen evolution produced by amine boranes hydrolysis appear to be much slower than those predicted by a pseudo-first order reaction and using the rate constants reported in the literature. This suggests that, at elevated acidities (5 mol L -1 HCl or HClO 4 ), the hydrolysis of amine boranes takes place in two steps, generating a first amount of H 2 (0.67-1.15 mol) much faster than the remaining about 2 mol. This evidence indicates a different mechanism of hydrolysis to the one accepted in the literature for amine boranes. The relatively high efficiencies of H 2 Se observed with amine borane reduction of inorganic Se IV at elevated acidities can be addressed to the action of borane intermediates, most probably amine borane cations, formed during amine borane hydrolysis in the same reaction conditions., (Copyright © 2017. Published by Elsevier B.V.)

The present work describes a non invasive lactate sensing in sweat during workout. The sensing system is based on a non-equilibrium potentiometric measure performed using disposable, chemically modified, screen printed carbon electrodes (SPCEs) that can be wetted with sweat during the exercise. The potentiometric signal, which is proportional to lactate concentration in sweat, is produced by a redox reaction activated by UV radiation, as opposed to the enzymatic reaction employed in traditional, blood-based measuring devices. The sensing system exhibits chemical selectivity toward lactate with linearity from 1 mM up to 180 mM. The dynamic linear range is suitable for measurement of lactate in sweat, which is more than 10 times concentrated than hematic lactate and reaches more than 100 mM in sweat during workout. The noninvasive measure can be repeated many times during exercise and during the recovery time in order to get personal information on the physiological and training status as well as on the physical performance. The device was successfully applied to several human subjects for the measurement of sweat lactate during prolonged cycling exercise. During the exercise sweat was simultaneously sampled on filter paper and extracted in water, and the lactate was determined by HPLC for method validation. The lactate concentration changes during the exercise reflected the intensity of physical effort. This method has perspectives in many sport disciplines as well as in health care and biomedical area., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this work we present an advantageous method for the simultaneous separation and detection of Tl(I) and Tl(III) species through ion chromatography coupled with on-line inductively coupled plasma - mass spectrometry. Chromatographic separation between Tl(III) and Tl(I) was achieved in less than two minutes. The method was validated by recovery experiments on real samples, and by comparing the sum of the concentrations of individual Tl species with total thallium values obtained from continuous flow ICP-MS. The experimental procedure offers an accurate, sensitive and interference-free method for Tl speciation at trace levels in environmental samples. This allowed us to investigate the Tl speciation in acid mine drainages (AMD), surface waters and springs in a mining catchment in Valdicastello Carducci (Tuscany, Italy), where severe Tl contamination ad been evidenced previously. This study shows for the first time that Tl(III), in addition to Tl(I), is present in considerable amounts in water samples affected by acid mining outflow, raising the question of the origin of this thermodynamically unstable species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study we present a novel isotope dilution gas chromatography method for the determination of nitrate in vegetables. The analyte was extracted in water at 70 ∘ C and mixed with 15 N isotopically enriched nitrate internal standard. The sample was centrifuged and the supernatant reacted with sulfamic acid for removal of nitrite, and with triethyloxonium tetrafluoroborate for converting nitrate into volatile EtONO 2 . This simple aqueous chemistry allowed for separation of analyte from sample matrix in the form of a gaseous derivative which could be sampled in the headspace before GC-MS analysis. This key-feature of the method made possible the collection of clean chromatograms within an elution time of only 1.8 min. Detection of EtONO 2 could be performed using electron impact ionization with a standard GC-MS setup. The method was optimized and validated for the analysis of nitrate in fresh vegetables in the 10-10,000 μg/g range with a detection limit of 2 μg/g. Due to the use of primary isotope dilution quantitation, traceable results of high-precision were attained., (Copyright © 2017. Published by Elsevier B.V.)

The high toxicity of cyanide, along with its widespread industrial use, has fuelled interest in the development of analytical methods for its determination in complex matrices. In this study, we propose a novel approach for the measurement of total cyanide in soil samples based on single-step derivatization with pentafluorobenzyl bromide (F 5 Bn-Br) followed by quantitation with gas chromatography mass spectrometry in negative chemical ionization mode. The reaction between CN - and F 5 Bn-Br resulted in the identification of several derivatives such as F 5 Bn-CN, (F 5 Bn)(F 5 Ph)CH-CN, and (F 5 Bn) 2 (F 5 Ph)C-CN. The relative proportion between such compounds was dependent on experimental conditions. When a 100 μL aliquot of an alkaline-aqueous extract was reacted with 700 μL of 1.3% F 5 Bn-Br in acetone, the tri-alkylated derivative was the most abundant. In such conditions a detection limit of 0.5 ng/g of CN - was attained. Soil samples were initially spiked with an alkaline solution of K 13 C 15 N internal standard and suspended in 7.5% aqueous NaOH. Determination of total cyanide was achieved by digestion of the alkaline extract with H 3 PO 4 to produce HCN which was then trapped in 0.1% NaOH in a sealed double vial system, followed by reaction with F 5 Bn-Br. Isotope dilution calibration was chosen for quantitation, and the validity of the novel method was demonstrated by analysis of soil Certified Reference Materials (CRMs) and by spike recovery tests., (Copyright © 2017. Published by Elsevier B.V.)

The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called "indole-3-acetamide pathway" to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis. Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

A geological study evidenced the presence of thallium (Tl) at concentrations of concern in groundwaters near Valdicastello Carducci (Tuscany, Italy). The source of contamination has been identified in the Tl-bearing pyrite ores occurring in the abandoned mining sites of the area. The strongly acidic internal waters flowing in the mining tunnels can reach exceptional Tl concentrations, up to 9000μg/L. In September 2014 Tl contamination was also found in the tap water distributed in the same area (from 2 to 10μg/L). On October 3, 2014 the local authorities imposed a Do Not Drink order to the population. Here we report the results of the exposure study carried out from October 2014 to October 2015, and aimed at quantifying Tl levels in 150 urine and 318 hair samples from the population of Valdicastello Carducci and Pietrasanta. Thallium was quantified by inductively coupled plasma - mass spectrometry (ICP-MS). Urine and hair were chosen as model matrices indicative of different time periods of exposure (short-term and long-term, respectively). Thallium values found in biological samples were correlated with Tl concentrations found in tap water in the living area of each citizen, and with his/her habits. Thallium concentration range found in hair and urine was 1-498ng/g (values in unexposed subjects 0.1-6ng/g) and 0.046-5.44μg/L (reference value for the European population 0.006μg/L), respectively. Results show that Tl levels in biological samples were significantly associated with residency in zones containing elevated water Tl levels. The kinetics of decay of Tl concentration in urine samples was also investigated. At the best of our knowledge, this is the first study on human contamination by Tl through water involving such a high number of samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A novel method for the determination of salivary thiocyanate is presented. Thiocyanate was converted into ethyl thiocyanate by single-step aqueous derivatization based on triethyloxonium tetrafluoroborate and measured by gas chromatography-mass spectrometry (15 min runtime). The ethyl thiocyanate derivative is volatile and can be sampled from the headspace. The derivatization chemistry proposed allows for separation of the analyte from saliva matrix whose introduction in the measurement system is avoided. Quantitation of the analyte was obtained by isotope dilution, employing a (13)C-enriched thiocyanate as internal standard. Technical details and fundamental aspects of derivatization chemistry and calibration strategy are presented. The method was validated by comparison with a standard method based on ion chromatography. The two independent methodologies produced results in agreement within 3%. Also a three level spike recovery test was carried out for validation purpose and quantitative recoveries were attained. The method is fast, simple, safe, and sensitive. Measurement of a 1 mL volume 50 ng/g of thiocyanate standard produced a signal-to-noise ratio of 250 for the analytical peak. This method is therefore suitable for ultra-trace determination of thiocyanate (low part-per-billion range). For the application described the full detection potential of the method was not required and the sample preparation presented has been designed for quantitation of saliva samples containing 1-400 μg/g of thiocyanate with a combined standard uncertainty of 2% relative for saliva samples containing 25 μg/g of thiocyanate. This method was applied for the determination of thiocyanate in human saliva samples., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

We developed a novel and straightforward derivatization method for the determination of lactate by reversed phase high-performance liquid chromatography (RP-HPLC) with fluorescence and UV detection in biological matrices as urine and saliva. The derivatization of lactate was achieved employing 9-chloromethyl anthracene (9-CMA) as fluorescence reagent, which has never been previously used to obtain a lactate derivative. Lactate reacts with 9-CMA with high selectivity in a very short time, without requiring extraction procedures from the aqueous solution, and the reaction reaches 70% completion in 30 min. The ester derivative obtained can be easily determined by RP-HPLC with fluorescence detection at 410 nm (λ ex=365 nm) and UV detection at 365 nm. The method was also optimized in order to allow for the simultaneous determination of lactate and creatinine for the application to urine samples. The lactate calibration curve was linear in the investigated range 2 × 10(-4)-3 × 10(-2)mM and the limit of detection, calculated as three times the standard deviation of the blank divided by the calibration curve slope, was 50 nM for both fluorescence and UV detection. Intra-day and inter-day repeatability were lower than 5% and 6%, respectively. The method proposed was successfully applied to the analysis of urine and saliva samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein-pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC-CVG-AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75-100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced SH groups before mercury labelling. We obtained a detection limit of 100 nmol L(-1) based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L(-1), depending on the number of cysteines in the protein sequence., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We developed a flow injection (FI) method for the determination of thiomersal (sodium ethylmercurithiosalicylate, C9H9HgNaO2S) based on the UV/microwave (MW) photochemical, online oxidation of organic mercury, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. Thiomersal was quantitatively converted in the MW/UV process to Hg(II), with a yield of 97±3%. This reaction was followed by the reduction of Hg(II) to Hg(0) performed in a knotted reaction coil with NaBH4 solution, and AFS detection in an Ar/H2 miniaturized flame. The method was linear in the 0.01-2 μg mL(-1) range, with a LOD of 0.003 μg mL(-1). This method has been applied to the determination of thiomersal in ophthalmic solutions, with recoveries ranging between 97% and 101%. We found a mercury concentration in commercial ophthalmic solutions ranging between 7.5 and 59.0 μg mL(-1)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two analytical procedures are presented for the determination of the total content and unbound fraction of both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried out in isocratic conditions at 25°C on a C-18 reversed-phase column with a mobile phase consisting of a 70% buffer phosphate 25mM at pH=7, 25% methanol and 5% acetonitrile at a flow rate of 1.2mL/min. Fluorescence detection was performed at 390nm (excitation wavelength 310nm). Neither method showed any detectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000ng/mL was 89±3% and 73±3%, respectively, whereas for their unbound fraction (at a concentration level of 10ng/mL) was 66±8% and 90±7%, respectively. The intra- and inter-day precision (assessed as relative standard deviation) was <10% for both methods. The limits of detection were 0.4 and 0.2ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfully applied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A dynamic surface tension detector (DSTD) has been equipped with an additional pressure sensor for simultaneous viscosity measurements, as a detector for flow injection analysis. The viscosity measurement is based on a single capillary viscometer (SCV) placed in parallel configuration with the DSTD. The viscometer in the optimized conditions consists of a PEEK capillary (i.d.=0.25 mm, L=75 cm) kept at constant temperature using a thermostatic bath, which leads on the two sides to the two arms of a differential piezoelectric pressure transducer with a range of 0-35 psi. The DSTD, described previously, measures the changing pressure across the liquid/air interface of 2 μL drops repeatedly forming at the end of a capillary. SCV performance has been evaluated by measuring dynamic viscosity of water/glycerol mixtures analysed in flow injection and comparing the results with the values reported in the literature. The detection limits of SCV and DSTD, calculated as 3σ of the blank, were 0.012 cP and 0.6 dyn cm(-1), respectively. The FI-SCV-DSTD system has been applied to the study of temperature-induced denaturation/aggregation process in bovine serum albumin (BSA). The results have been supported and discussed with respect to BSA conformational analysis performed using Fourier Transform infrared spectroscopy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The simultaneous application of microwave (MW) power and UV light leads to improved results in photochemical processes. This study investigates the oxidative decomposition of atrazine in water using an innovative MW and UV photochemical reactor, which activates a chemical reaction with MW and UV radiation using an immersed source without the need for a MW oven. We investigated the influence of reaction parameters such as initial H(2)O(2) concentrations, reaction temperatures and applied MW power and identified the optimal conditions for the oxidative decomposition of atrazine. Atrazine was completely degraded by MW/UV/H(2)O(2) in a very short time (i.e. t(1/2) = 1.1 min for 20.8 mg/L in optimal conditions). From the kinetic study, the disappearance rate of atrazine can be expressed as dX/dt = k(PH)[M](0)(b-X)(1-X), where b ≡ [H(2)O(2)](0)/[M](0)+k(OH)[·OH]/k(PH)[M](0), and X is the atrazine conversion, which correlates well with the experimental data. The kinetic analysis also showed that an indirect reaction of atrazine with an OH radical is dominant at low concentrations of H(2)O(2) and a direct reaction of atrazine with H(2)O(2) is dominant when the concentration of H(2)O(2) is more than 200 mg/L., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Multidimensional analysis of instant coffee and barley beverage samples using size exclusion chromatography (SEC) combined with a dynamic surface tension detector (DSTD) and a UV-vis absorbance detector (UV) is reported. A unique finding of this study was the action of the tetrabutylammonium (TBA) cation as a modifying agent (with bromide as the counter anion) that substantially increased the surface pressure signal and sensitivity of many of the proteins in the chromatographically separated samples. The tetrabutylammonium bromide (TBAB) enhancement of the surface pressure signal was further investigated by studying the response of 12 commercial standard proteins (alpha-lactalbumin, beta-lactoglobulin, human serum albumin (HSA), albumin from chicken egg white (OVA), bovine serum albumin (BSA), hemoglobin, alpha-chymotrypsinogen A, cytochrome C, myoglobin, RNase A, carbonic anhydrase, and lysozyme) in buffer performed using flow injection analysis (FIA) coupled with the DSTD with and without various concentrations of TBAB. The FIA-DSTD data show that 1mM TBAB enhances sensitivity of HSA detection, by lowering the limit of detection (LOD) from 2mg/mL to 0.1mg/mL. Similarly, the LOD for BSA was reduced from 1mg/mL to 0.2mg/mL. These FIA-DSTD experiments allowed the detection conditions to be optimized for further SEC-UV/DSTD experiments. Thus, the SEC-UV/DSTD system has been optimized and successfully applied to the selective analysis of surface-active protein fractions in a commercial instant coffee sample and in a soluble barley sample. The complementary selectivity of using the DSTD relative to an absorbance detector is also demonstrated.

We report on the application of a commercially available mercury analyzer, which is based on vapour generation of Hg(0) by NaBH(4) reduction and atomic absorption detection, to the quantification and characterization of -SH groups and its application to wine samples. The behaviour of Hg(II) and thiol-Hg(II) (RS-Hg) complexes at nanomolar level (RS=l-cysteine, dl-penicillamine, N-acetyl penicillamine, glutathione, cysteinylglycine, homocysteine) has been studied following their reduction with alkaline NaBH(4) to give Hg(0). In the absence of thiol-Hg(II) is quantitatively converted to Hg(0) by stoichiometric amount of NaBH(4) (reaction ratio 1/4mole NaBH(4)/mole Hg), while the complete reduction of Hg(II)-thiol complexes to Hg(0) requires molar excess of NaBH(4) up to six orders of magnitude, depending on the type of complex and on the pK(a) of the thiolic group. Under an appropriate excess of reductant, Hg(II) and its thiol complexes are not distinguishable giving the same response. These properties allow the discrimination of Hg(II) from Hg(II)-thiol complexes without any preliminary separation and the quantification of thiol groups. Instrumental detection limits are as low as 2.5pg, permitting sample dilution, therefore, minimizing the risk of possible interferences occurring with complex real matrices. The method has been applied to quantification of thiol groups in wine samples. Comparison with results obtained by HPLC coupled to atomic fluorescence detection confirmed the promising potentialities of the method.

A new procedure is proposed for the sampling and storage of hydrogen sulphide (H2S) and volatile thiols (methanethiol or methyl mercaptan, ethanethiol and propanethiol) for their determination by liquid chromatography. The sampling procedure is based on the trapping/pre-concentration of the analytes in alkaline aqueous solution containing an organic mercurial probe p-hydroxymercurybenzoate, HO-Hg-C6H4-COO- (PHMB), where they are derivatized to stable PHMB complexes based on mercury-sulfur covalent bonds. PHMB complexes are separated on a C18 reverse phase column, allowing their determination by liquid chromatography coupled with sequential non-selective UV-vis (DAD) and mercury specific (chemical vapor generation atomic fluorescence spectrometry, CVGAFS) on-line detectors. PHMB complexes, S(PHMB)2CH3S-PHMB, C2H5S-PHMB and C3H7S-PHMB, are stable alt least for 12 h at room temperature and for 3 months if stored frozen (-20 degrees C). The best analytical figures of merits in the optimized conditions were obtained by CVGAFS detection, with detection limits (LODc) of 9.7 microg L(-1) for H2S, 13.7 microg L(-1) for CH(3)SH, 17.7 microg L(-1) for C2H5SH and 21.7 microg L(-1) for C3H7SH in the trapping solution in form of RS-PHMB complexes, the relative standard deviation (R.S.D.) ranging between 1.0 and 1.5%, and a linear dynamic range (LDR) between 10 and 9700 microg L(-1). Conventional UV absorbance detectors tuned at 254 nm can be employed as well with comparable R.S.D. and LDR, but with LODc one order of magnitude higher than AFS detector and lower specificity. The sampling procedure followed by LC-DAD-CVGAFS analysis has been validated, as example, for H2S determination by a certified gas permeation tube as a source of 3.071+/-0.154 microg min(-1) of H2S, giving a recovery of 99.8+/-7% and it has been applied to the determination of sulfur compounds in real gas samples (biogas and the air of a plant for fractional distillation of crude oil).

In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-mul drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, alpha-lactalbumin (alpha-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, alpha-chymotrypsinogen A, beta-lactoglobulin (beta-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, alpha-Lac and beta-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.

Reverse phase chromatography (RPC) coupled on-line with UV-vis diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) is proposed for the speciation and determination of inorganic and organic mercury (methylmercury, ethylmercury and phenylmercury) in the form of cysteine, penicillamine and glutathione complexes. The mercury-thiol complexes are separated on a C(18) Reverse Phase column and oxidized on-line with bromine, generated in situ by KBr/KBrO(3) in HCl medium, in order to fast convert organic mercury species to inorganic mercury in less than 2.5s, at room temperature, in a 30cm knitted coil. Hg(II) is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg(0). Under optimized conditions, on-line bromine treatment gives recoveries of thiol-complexed methylmercury, ethylmercury and phenylmercury with respect to inorganic mercury ranging between 79 and 85%, 80 and 85%, 63 and 76%, respectively, depending on the complexing thiol employed. Optimized elution conditions were provided in the three complexing agents. The detection limits (LODc) for inorganic mercury, methylmercury, ethylmercury and phenylmercury, in the optimized conditions complexed with thiols were about 16, 18, 18 and 20pg (as mercury), respectively, a relative standard deviation (R.S.D) ranging between 1.5 and 2.0%, and a linear dynamic range between 0.1 and 100ng injected. LC-DAD-CVGAFS method has been validated by analysing two certificate reference material, DORM-2 and NIES CRM 13, obtaining 98+/-6 and 97+/-5% of methylmercury recovered, respectively.

A new analytical hyphenated technique is proposed for determination and characterization of thiolic proteins, based on reverse phase chromatography (RPC) coupled on-line with cold vapour generation atomic fluorescence spectrometry (CVGAFS). Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol l(-1) urea and p-hydroxymercurybenzoate (PHMB). The derivatized proteins are separated on a C4 Vydac Reverse Phase column. Post-column on-line reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of both the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury, also in the presence of methanol in the RPC eluent phase. Hg(II) is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg degrees. Under optimized conditions, on-line bromine treatment gives a 98+/-2% recovery of both free and protein-complexed PHMB. The effect of methanol on the sensitivity of Hg(II) detection was studied and controlled. RPC-CVGAFS system has been applied to the analysis of metallothioneins from rabbit liver (MT(RL)) standard solutions, and their commercial isoforms MT-1 and MT-2. The analysis of denatured, PHMB-complexed MTs allowed the determination of the number of thiolic groups complexed by PHMB. It was found that MTs from rabbit liver have 10.0+/-0.3 (MT-1) and 6.7+/-0.3 (MT-2 and MT(RL)) -SH groups complexed by PHMB. The detection limit (LODc) for PHMB in 95% methanol in the optimized conditions was about 9.3 x 10(-9) mol l(-1) and for the denatured MTs LODc was about 8.6 x 10(-10) mol l(-1), taking into account an approximate complexating ratio PHMB:MTs of 7:1.

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0moldm(-3) urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO(3) in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg(2+), present in solution as Hg(2+)-urea complex, is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100+/-2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and beta-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10(-10)-10(-12)moldm(-3), depending on the considered protein, with linear calibration curves spanning over four decades of concentration.

Multidimensional analysis of denatured milk proteins is reported using high-performance liquid chromatography (HPLC) combined with dynamic surface tension detection (DSTD). A hydrophobic interaction chromatography (HIC) column (a TSK-Gel Phenyl-5PW column, TosoBiosep), in the presence of 3.0 M guanidine hydrochloride (GdmHCl) as denaturing agent is employed as the mobile phase. Dynamic surface tension is measured through the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 4 s) is achieved, for each eluting drop of 4 s length, providing insight into both the kinetic and thermodynamic behavior of molecular orientation processes at the liquid-air interface. An automated calibration procedure and data analysis method is applied with the DSTD system, which allows two unique solvents to be used, the HIC mobile phase for the sample and a second solvent (water for example) for the standard, permitting real-time dynamic surface tension data to be obtained. Three-dimensional data is obtained, with surface tension as a function of drop time first converted to surface pressure, which is plotted as a function of the chromatographic elution time axis. Experiments were initially performed using flow injection analysis (FIA) with the DSTD system for investigating commercial single standard milk proteins (alpha-lactalbumin, beta-lactoglobulin, alpha-, beta-, kappa-casein and a casein mixture) denatured by GdmHCl. These FIA-DSTD experiments allowed the separation and detection conditions to be optimized for the HIC-DSTD experiments. Thus, the HIC-DSTD system has been optimized and successfully applied to the selective analysis of surface-active casein fractions (alpha s1- and beta-casein) in a commercial casein mixture, raw milk samples (cow's, ewe's and goat's milk) and other diary products (yogurt, stracchino, mozzarella, parmesan cheese and chocolate cream). The different samples were readily distinguished based upon the selectivity provided by the HIC-DSTD method. The selectivity advantage of using DSTD relative to absorbance detection is also demonstrated.

Caseins alpha(s1)-, alpha(s2)-, beta- and kappa- from raw cows', ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alpha-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analyzed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (alpha(s1)/kappa, alpha(s2)/beta, beta/kappa and alpha(s2)/alpha(s1)) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.