Chemical conversion of nitrate and nitrite to nitrous oxide for nitrogen and oxygen isotopic analysis in freshwater and seawater

We present a novel method for nitrogen and oxygen natural isotopic abundance analysis of nitrate and nitrite of seawater and freshwater at environmental concentrations. The method involves the reduction of nitrate to nitrite using spongy cadmium with further reduction to nitrous oxide using sodium azide in an acetic acid buffer. For separate nitrite analysis, the cadmium reduction step is simply bypassed. Nitrous oxide is purged from the water sample and trapped cryogenically using an automated system with subsequent release into a gas chromatography column. The isolated nitrous oxide is then analyzed on a continuous flow isotope ratio mass spectrometer via an open split. This paper describes the basic protocol and reaction conditions required to obtain reproducible natural abundance level nitrogen and oxygen isotopic ratios from nitrate, nitrite, or both, and the results obtained to support these conclusions. A standard deviation less than 0.2 per thousand for nitrogen and 0.5 per thousand for oxygen was found for nitrate samples ranging in concentration from 40 to 0.5 microM (better for nitrite), with a blank of 2 nmol for 50-mL samples. Nitrogen and oxygen isotopic fractionation and oxygen atom exchange were consistent within each batch of analysis. There was no interference from any seawater matrixes. Only one other method published to date can measure the nitrate oxygen isotopic abundance in seawater and none that do so for nitrite alone in the presence of nitrate. This method may prove to be simpler, faster, and obtain isotopic information for lower concentrations of nitrate and nitrite than other methods.     

Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method

We report a novel method for measurement of the oxygen isotopic composition (18O/16O) of nitrate (NO3-) from both seawater and freshwater. The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (15N/14N) analysis of nitrate. (1) Here, we address the additional issues associated with 18O/16O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N20 analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N2O. Experiments with 18O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N20 product for Pseudomonas aureofaciens. In addition, both oxygen isotope fractionation and oxygen atom exchange are consistent within a given batch of analyses. The analysis of appropriate isotopic reference materials can thus be used to correct the measured 18O/16O ratios of samples for both effects. This is the first method tested for 18O/16O analysis of nitrate in seawater. Benefits of this method, relative to published freshwater methods, include higher sensitivity (tested down to 10 nmol and 1 microM NO3-), lack of interference by other solutes, and ease of sample preparation.     

Technical updates to the bacterial method for nitrate isotopic analyses

The bacterial conversion of aqueous nitrate (NO(3)(-)) to nitrous oxide (N(2)O) for isotopic analysis has found widespread use since its introduction (Sigman, D. M.; Casciotti, K. L.; Andreani, M.; Galanter, M.; Bo?hlke, J. K. Anal. Chem.2001, 73, 4145-4153; Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Bo?hlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). The bacterial strain Pseudomonas aureofaciens (ATTC no. 13985) was shown to convert NO(3)(-) to N(2)O while retaining both N and O isotopic signatures, and automation of the isotopic analysis of N(2)O greatly increased the throughput of the method (Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Bo?hlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). Continued development of the denitrifier method has led to increased precision and throughput of NO(3)(-) isotopic analysis. Presented here are several recent procedural modifications and the demonstration of their effectiveness.     

A new method for concentration analysis of bacterial endotoxins in perfluorocarbon

This communication demonstrates the feasibility of the gel-clot method for the analysis of bacterial endotoxins in water extracts of perfluorocarbon which is a water insoluble liquid medical device. Perfluorocarbon （10 mL） was shaken with 10 mL water for 15 min at 2000 r/min and the endotoxin present was extracted to the aqueous phase without interference inhibition/enhancement of the product and the recovery of endotoxin added to perfluorocarbon was determined, A validation study confirmed that endotoxins presented in perfluorocarbon pass over into the aqueous phase at concentrations of 20, 10 and 5EU/mL with recoveries from 86.8% to 96.8%. Therefore, the gel-clot test is suitable for detecting bacterial endotoxins in perfluorocarbon which is a water insoluble medical device.     

A method for the extraction of ammonium from freshwaters for nitrogen isotope analysis

The measurement of delta15N values of inorganic nitrogen species is an important analytical tool to trace nitrogen species in order to understand nitrogen cycling in aquatic systems. Nitrogen isotope analysis of freshwater ammonium has, however, been hindered by the lack of a simple and reliable technique to measure delta15N values at natural abundance levels. We present a simple and rapid method to concentrate ammonium from freshwater samples for on-line N-isotope ratio determination. Ammonium is collected by adsorption on N-free cation exchange resins. The dried N-loaded exchange resin is then directly combusted to produce N2 gas for subsequent delta15N analysis. The method was evaluated with simulated freshwater solutions containing varying amounts of standard NH4+-N (delta15N = 2.1 per thousand) and potentially interfering inorganic and organic compounds. In general, the cation exchange resin method gives accurate and reproducible delta15N values (sigma1 < 0.3 per thousand; n = 10). Because of adsorption interference, high concentrations of cations in solution may cause ammonium loss but do not result in measurable isotope fractionation. Replicate extractions of the ammonium standard added to water collected from four Swiss lakes demonstrate the good performance of this method when applied to low ionic strength natural water samples with modest concentrations of dissolved organic nitrogen.     

A novel chemosynthetic method for the production of nitrate sodalite

This paper presents a novel method for the synthesis of nitrate sodalite from an uncalcined, dry Al₂O₃-2SiO₂ gel powder at low temperature. Nitrate sodalite particles were successfully synthesized utilizing a dry-gel conversion method (DGC) and characterized by SEM, HRTEM and SAED techniques. The mechanism of formation of the nitrate sodalite and its cation exchange capacity (CEC) were studied and the experimental results indicate that the nitrate sodalite particles were composed of numerous small grains, incompletely crystallized and arranged in a spherical cage-like structure.     

A simple cleanup method for the isolation of nitrate from natural water samples for O isotope analysis.

The analysis of O-isotopic composition of nitrate has many potential applications in studies of environmental processes. O-isotope nitrate analysis requires samples free of other oxygen-containing compounds. More than 100% of non-NO3- oxygen relative to NO3- oxygen can still be found in forest soil water samples after cleanup if improper cleanup strategies, e.g., adsorption onto activated carbon, are used. Such non-NO3- oxygen compounds will bias O-isotopic data. Therefore, an efficient cleanup method was developed to isolate nitrate from natural water samples. In a multistep cleanup procedure using adsorption onto water-insoluble poly(vinylpyrrolidone), removal of almost all other oxygen-containing compounds, such as fulvic acids, and isolation of nitrate was achieved. The method supplied samples free of non-NO3- oxygen which can be directly combusted to CO2 for subsequent O-isotope analysis.     

A preconcentration/matrix reduction method for the analysis of rare earth elements in seawater and groundwaters by isotope dilution ICPMS

A simple method of simultaneous preconcentration and matrix reduction was developed for the analysis of rare earth elements (REEs) in seawater and groundwater by ID ICPMS. The method utilizes partitioning of the REEs with solid hydroxides to separate them from soluble matrix species (e.g., Ba2+, NOM, seawater salts). Acidified samples were spiked and equilibrated with an enriched isotope cocktail (142Ce, 145Nd, 161Dy, 171Yb). Aqueous NH3 was then added to the spiked samples to induce the coprecipitation of the REEs with a small fraction of the natural Mg2+ as Mg(OH)2. The samples were centrifuged and the precipitate was rinsed to remove more than 99.8% of the Ba2+ along with the matrix salts. The precipitate was dissolved in 400 microL of 10% HNO3 for ICPMS analysis. The four spiked elements, determined by isotope dilution, served as internal standards for the remaining REEs. Analysis of NASS-4 and NASS-5 seawater reference materials showed good agreement with published values. Calculated limits of detection for a 1.65-g sample ranged from 0.1 pg/g for the light REEs to 0.02 pg/g for the heavy REEs. The reagent blanks ranged from a high of 0.28 pg/sample for Ce to a low of 0.0036 pg/sample for Tb.     

Determination of the total oxygen isotopic composition of nitrate and the calibration of a delta 17O nitrate reference material

A thermal decomposition method was developed and tested for the simultaneous determination of delta 18O and delta 17O in nitrate. The thermal decomposition of AgNO3 allows for the rapid and accurate determination of 18O/ 16O and 17O/16O isotopic ratios with a precision of +/- 1.5 per thousand for delta 18O and +/- 0.11 per thousand for delta 17O (delta 17O = delta 17O - 0.52 x delta 18O). The international nitrate isotope reference material IAEA-NO3 yielded a delta 18O value of +23.6 per thousand and delta 17O of -0.2 per thousand, consistent with normal terrestrial mass-dependent isotopic ratios. In contrast, a large sample of NaNO3 from the Atacama Desert, Chile, was found to have delta 17O = 21.56 +/- 0.11 per thousand and delta 18O = 54.9 +/- 1.5 per thousand, demonstrating a substantial mass-independent isotopic composition consistent with the proposed atmospheric origin of the desert nitrate. It is suggested that this sample (designated USGS-35) can be used to generate other gases (CO2, CO, N2O, O2) with the same delta 17O to serve as measurement references for a variety of applications involving mass-independent isotopic compositions in environmental studies.     

A method for testing the mechanical strength and kinetic features of fracture of lacquer coatings in seawater

A method for testing lacquer coatings in seawater is developed. This method enables one to predict the cohesion and adhesion strength of these coatings under conditions close to operating. The mechanical strength of the coatings is estimated with the help of a dimensionless parameter. Within the framework of the proposed testing method, one can also investigate the kinetic properties and other characteristics of fracture of lacquer coatings by using an optical technique and photography.Karpenko Physicomechanical Institute, Ukrainian Academy of Sciences, L'viv. Translated from Fiziko-Khimicheskaya Mekhanika Materialov, Vol. 29, No. 6, pp. 108–110, November–December, 1993.     

普通克里金法在海水温度剖面插值中的应用

海水温度剖面在水声学领域扮演着重要的角色,而目前大面积实时温度剖面测量能力尚存不足,因此对于海水温度剖面的网格插值研究就有一定的意义。基于普通克里金法对某海域获得的剖面样本进行插值,通过逐点逐面的拟合最终得到该海域三维温度场。通过拟合温度剖面和部分样本剖面的对比,证实普通克里金法对于海水温度剖面拟合具有很好的效果,具有应用的可行性。     

Oxidation-reduction reactions: A novel method in the synthesis of nitrate cancrinite-type zeolites

A novel method using redox reactions among nitrite and permanganate anions in NaOH solutions i.e. 8 and 16 mol/L was used for the synthesis of nitrate cancrinites. Reactions were carried out at 80 °C under autogenous pressure for 40 h. Zeolite X was used as silicon and aluminum source. Obtained polycrystals were characterized by using powder X-ray diffraction (XRD) and FT-IR spectroscopy. Results showed that only the nitrated cancrinite was obtained in a wide range of basicities and NO₂⁻/MnO₄⁻ compositions.     

On-line technique to determine the isotopic composition of total dissolved nitrogen

A new on-line analytical setup for 15N measurements of total dissolved nitrogen (TDN) has been developed through the coupling of a high-temperature catalytic (Ce(IV)O2) oxidation furnace, a Cu reduction furnace, and an isotope ratio mass spectrometer. The detection limit for accurate delta15N measurements is 20 mg of N L-1. For N-containing compounds dissolved in water, a standard deviation on N concentration measurements of 0.2 mg of N L-1, independent of N concentration, has been found. Reproducibility on delta15N increased with increasing N concentration, with standard deviations varying from 0.8 to 0.1 per thousand in the range of 20-100 mg of N L-1. Salt matrixes, in which the N species might be dissolved, could influence the analysis capacity and continuity, mainly at concentrations above 0.1 M. To our knowledge, this system is the first successful on-line setup capable of performing routine delta15N and N concentration measurements of the TDN pool. Potential applications are large and are believed to result in an increased insight in N cycling and dissolved organic nitrogen behavior in terrestrial and aquatic ecosystems.     

Isotopic analysis of N and o in nitrite and nitrate by sequential selective bacterial reduction to N2O

Nitrite is an important intermediate species in the biogeochemical cycling of nitrogen, but its role in natural aquatic systems is poorly understood. Isotopic data can be used to study the sources and transformations of NO2- in the environment, but methods for independent isotopic analyses of NO2- in the presence of other N species are still new and evolving. This study demonstrates that isotopic analyses of N and O in NO2- can be done by treating whole freshwater or saltwater samples with the denitrifying bacterium Stenotrophomonas nitritireducens, which selectively reduces NO2- to N2O for isotope ratio mass spectrometry. When calibrated with solutions containing NO2- with known isotopic compositions determined independently, reproducible delta15N and delta18O values were obtained at both natural-abundance levels (+/-0.2-0.5 per thousand for delta15N and +/-0.4-1.0 per thousand for delta18O) and moderately enriched 15N tracer levels (+/-20-50 per thousand for delta15N near 5000 per thousand) for 5-20 nmol of NO2- (1-20 micromol/L in 1-5 mL aliquots). This method is highly selective for NO2- and was used for mixed samples containing both NO2- and NO3- with little or no measurable cross-contamination. In addition, mixed samples that were analyzed with S. nitritireducens were treated subsequently with Pseudomonas aureofaciens to reduce the NO3- in the absence of NO2-, providing isotopic analyses of NO2- and NO3- separately in the same aliquot. Sequential bacterial reduction methods like this one should be useful for a variety of isotopic studies aimed at understanding nitrogen cycling in aquatic environments. A test of these methods in an agricultural watershed in Indiana provides isotopic evidence for both nitrification and denitrification as sources of NO2- in a small stream.     

Methods for the stable isotopic analysis of chlorine in chlorate and perchlorate compounds.

Chlorate and perchlorate compounds, used as herbicides, solid fuel propellants, and explosives, are increasingly recognized as pollutants in groundwater. Stable isotope characterization would permit both environmental monitoring of extent of remediation and forensic characterization. Stoichiometric reduction to chloride (greater than 98% yield), by Fe(II) for chlorate and alkaline fusion-decomposition for perchlorate, allows analysis by standard methods to give highly reproducible and accurate delta37Cl results (0.05/1000, 2 x standard error). Analysis of various compounds from different suppliers yielded delta37Cl values for chlorate samples near to +0.2/1000 (SMOC), but one has within-sample heterogeneity of 0.5/1000, possibly due to crystallization processes during manufacture. Results for perchlorate samples also are generally near +0.2/1000, but one is +2.3/1000 (SMOC). The initial results suggest that both forensic and environmental applications might be feasible.     

An extraction chromatography method for Hf separation prior to isotopic analysis using multiple collection ICP-mass spectrometry

A novel method for the single-step separation of Zr + Hf from all matrix elements of geological samples has been developed for Hf isotopic measurements using multiple collector-ICP-mass spectrometry. The method combines an effective sample decomposition by LiBO2 fusion with a selective separation of Hf + Zr by a solid-phase extraction material based on dipentyl pentyl phosphonate, commercially available as U-TEVA.Spec. Using this simple and rapid procedure, Hf and Zr can be isolated in a single separation step with good recoveries (>90%) and satisfactory blank levels (approximately 55 pg of Hf), so that a subsequent isotopic measurement with ICPMS is possible. An excellent separation from rock-forming constituents is achieved, including those elements (Al, P, Ti, Cr, Fe, Mo, etc.) known to interfere in conventional separation methods based on ion-exchange techniques. The potential of this new method for Hf isotopic analysis is demonstrated by replicate MC-ICPMS measurements of 176Hf/177Hf ratios in seven international reference materials of silicate rocks, spanning a range of Hf contents and bulk compositions.     

Capillary electrophoretic method for the detection of bacterial contamination

There has been growing interest in separations-based techniques for the identification and characterization of microorganisms because of the versatility, selectivity, sensitivity, and short analysis times of these methods. A related area of analysis that is scientifically and commercially important is the determination of the presence or complete absence of microbes (in essence, a test for sample sterility). In such a test, it is not of immediate importance to identify a particular microorganism, but rather, to know with a high degree of certainty whether any organism(s) is (are) present. Current regulations require culture-based tests that can take up to 2 weeks to complete. As a rapid alternative, capillary electrophoresis-based methods are examined. Experimental formats are developed that promote the consolidation of all cell types into a single zone (peak) which is separated from the electroosmotic flow front and any other interfering molecular constituents. This process can be accomplished using a segment of dilute cetyltrimethylammonium bromide, which serves to temporarily reverse the migration direction of the cells, and another segment of solution containing a "blocking agent", which serves to stop the cell migration and focus them into a narrow zone. Relatively wide-bore capillaries can be used to increase sample size. This approach appears to be effective for a broad spectrum of bacteria, and analyses times are less than 10 min.     

Triple oxygen isotope analysis of nitrate using the denitrifier method and thermal decomposition of N2O

Interaction with ozone transfers its anomalous (non-mass-dependent) 17O enrichment to atmospheric nitrogen oxides and nitrate. The 17O anomaly (Delta17O) in nitrate can be used to identify atmospheric nitrate inputs into terrestrial and aquatic environments as well as to study the role of ozone in the atmosphere's reactive nitrogen cycle. We report here on an online method for analysis of the 17O anomaly, using a strain of denitrifiers to convert nitrate to N2O, which decomposes quantitatively to N2 and O2 in a gold furnace at 800 degrees C, followed by gas chromatographic separation and isotope analysis of O2. This method requires approximately 50 nmol of nitrate, 2-3 orders of magnitude less than previous offline thermal decomposition methods to achieve a similar analytical precision of 0.5 per thousand for Delta17O. There is no significant memory effect, but calibration via nitrate or N2O reference materials is required for scale normalization. The N2O decomposition method is shown to be well-suited for nitrate analysis in freshwater and seawater samples from various environments.