PKW would like to thank the Voss Environmental Fellowship at Brown University for funding and support. This research was funded by a National Science Foundation CAREER Award (AGS-1351932) to MGH. The authors thank Ruby Ho for her support of this project. The authors would also like to thank Rebecca Ryals; Jim Tang and his research group at the Marine Biological Laboratory, specifically Elizabeth de la Reguera, for access to the LMA-3d and Li-COR 7000 analyzers; Barbara Morin at the Rhode Island Department of Environmental Management; Roy Heaton and Paul Theroux at the Rhode Island Department of Health; Adam McGovern at the Warren Alpert Medical School; and Charlie Vickers and Tom Keifer for collection system machining. We also thank the editorial staff and two anonymous reviewers for their excellent suggested edits that improved the manuscript.

<ol>
	<li>Galloway, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nitrogen+Cascade.">The Nitrogen Cascade.</a> BioScience. 53 (4), 341-356 (2003).</li><li>Levy, H., Moxim, W. J., Klonecki, A. A., Kasibhatla, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+tropospheric+NOx:+Its+evaluation,+global+distribution+and+individual+source+contributions.">Simulated tropospheric NOx: Its evaluation, global distribution and individual source contributions.</a> J. Geophysical Research. 104 (D21), 26279-26306 (1999).</li><li>Hastings, M. G., Casciotti, K. L., Elliott, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Isotopes+as+Tracers+of+Anthropogenic+Nitrogen+Sources,+Deposition,+and+Impacts.">Stable Isotopes as Tracers of Anthropogenic Nitrogen Sources, Deposition, and Impacts.</a> Elements. 9 (5), 339-344 (2013).</li><li>Fibiger, D. L., Hastings, M. G., Lew, A. F., Peltier, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+NO+and+NO2+for+Isotopic+Analysis+of+NOx+Emissions.">Collection of NO and NO2 for Isotopic Analysis of NOx Emissions.</a> Anal. Chem. 86 (24), 12115-12121 (2014).</li><li>Ammann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+uptake+of+traffic-derived+NO2+from+15N+abundance+in+Norway+spruce+needles.">Estimating the uptake of traffic-derived NO2 from 15N abundance in Norway spruce needles.</a> Oecologia. 118 (2), 124-131 (1999).</li><li>Felix, J. D., Elliott, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+composition+of+passively+collected+nitrogen+dioxide+emissions:+Vehicle,+soil+and+livestock+source+signatures.">Isotopic composition of passively collected nitrogen dioxide emissions: Vehicle, soil and livestock source signatures.</a> Atmospheric Environment. 92, 359-366 (2014).</li><li>Heaton, T. H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=15N/14N+ratios+of+NOx+from+vehicle+engines+and+coal-fired+power+stations.">15N/14N ratios of NOx from vehicle engines and coal-fired power stations.</a> Tellus. 42 (3), 304-307 (1990).</li><li>Walters, W. W., Goodwin, S. R., Michalski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+Stable+Isotope+Composition+(&#948;15N)+of+Vehicle-Emitted+NOx.">Nitrogen Stable Isotope Composition (&#948;15N) of Vehicle-Emitted NOx.</a> Environ. Sci. Technol. 49 (4), 2278-2285 (2015).</li><li>Zaveri, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ozone+production+efficiency+and+NOx+depletion+in+an+urban+plume:+Interpretation+of+field+observations+and+implications+for+evaluating+O3-NOxVOC+sensitivity.">Ozone production efficiency and NOx depletion in an urban plume: Interpretation of field observations and implications for evaluating O3-NOxVOC sensitivity.</a> J. Geophys. Res. 108 (D14), 4436 (2003).</li><li>Margeson, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+method+for+determining+nitrogen+oxide+(NOx)+emissions+at+nitric+acid+plants.">An integrated method for determining nitrogen oxide (NOx) emissions at nitric acid plants.</a> Anal. Chem. 56 (13), 2607-2610 (1984).</li><li>Drummond, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> New Technologies for Use in Acid Deposition Networks, ASTM STD. 1052. , 133-149 (1990).</li><li>Sigman, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bacterial+Method+for+the+Nitrogen+Isotopic+Analysis+of+Nitrate+in+Seawater+and+Freshwater.">A Bacterial Method for the Nitrogen Isotopic Analysis of Nitrate in Seawater and Freshwater.</a> Anal. Chem. 73 (17), 4145-4153 (2001).</li><li>Casciotti, K. L., Sigman, D. M., Hastings, M. G., B&#246;hlke, J. K., Hilkert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+oxygen+isotopic+composition+of+nitrate+in+seawater+and+freshwater+using+the+denitrifier+method.">Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method.</a> Anal Chem. 74 (19), 4905-4912 (2002).</li><li>B&#246;hkle, J. K., Mroczkowski, S. J., Coplen, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+isotopes+in+nitrate:+new+reference+materials+for+18O:17O:16O+measurements+and+observations+on+nitrate-water+equilibrium.">Oxygen isotopes in nitrate: new reference materials for 18O:17O:16O measurements and observations on nitrate-water equilibrium.</a> Rapid Commun. Mass Spectrom. 17 (16), 1835-1846 (2003).</li><li>Kaiser, J., Hastings, M. G., Houlton, B. Z., R&#246;ckmann, T., Sigman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tripe+oxygen+isotope+analysis+of+nitrate+using+the+denitrifier+method+and+thermal+decomposition+of+N2O.">Tripe oxygen isotope analysis of nitrate using the denitrifier method and thermal decomposition of N2O.</a> Anal Chem. 79 (2), 599-607 (2007).</li><li>Sun, K., Tao, L., Miller, D. J., Khan, M. A., Zondlo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-Road+Ammonia+Emissions+Characterized+by+Mobile,+Open-Path+Measurements.">On-Road Ammonia Emissions Characterized by Mobile, Open-Path Measurements.</a> Environ. Sci. Technol. 48 (7), 3943-3950 (2014).</li><li>Dahal, B., Hastings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+considerations+for+the+use+of+passive+samplers+to+quantify+the+isotopic+composition+of+NOx+and+NO2+using+the+denitrifier+method.">Technical considerations for the use of passive samplers to quantify the isotopic composition of NOx and NO2 using the denitrifier method.</a> Atmospheric Environment. , (2016).</li></ol>

The authors declare that they have no competing financial interests.

The protocol above details the steps involved, from the field collection of air samples in solution to the laboratory processing of these solutions, to yield concentration and isotopic results. The critical steps in this protocol include comparing NOx analyzer measurements, minimizing the time before the reduction of solutions. and maintaining stable flow rates. If directly comparing solutions with in situ measurements of NOx concentrations, it is very important that an NOx analyzer is calibrated for ranges relevant for the chosen environment and that short-term variability in NOx concentrations be understood in the context of the longer time collections for the solutions. The accurate determination of solution NO3- concentrations is also important, both for calculation of the airborne NOx concentrations and for determining accurate injection volumes for the isotopic denitrifier method. The time period of solution stability before sample reduction is important to ensure consistent isotope ratios. As a result of the oxidizing potential of the solution, it is possible to oxidize in solution other reactive nitrogen species, most notably NH3, as it can be in high enough concentrations in certain areas to potentially affect the concentration of NO3- in solution. The oxidation of NH4+ to NO3- is expected to take longer than the oxidation of NOx to NO3-, so it had been recommended to reduce the samples (and thus stop the reaction) within 1 day of sample collection. Given that field conditions may result in the requirement of longer solution storage times, the stability of solutions was tested by examining solutions with and without added ammonium. With and without the addition of ammonium chloride, concentration and isotope values were stable within the 1&#963; uncertainty range (1.5&#8240;) for up to one week (Figure 3). At two weeks after sampling, solutions with or without added NH4+ were not stable, in that NO3- concentration decreases were observed in some cases and blank corrections were no longer robust. Although it was expected that NO3- might increase over time due to NH4+ oxidation, decreases in concentration were actually observed in some cases, suggesting that even after two weeks, NH4+ interference is not causing the instability. As such, solutions should be reduced within one week, particularly if sampling is done in an environment with high NH3 concentrations (e.g., &#62;200 ppbv). Finally, it is also critical to record the flow rate during field collections. The flow rate measured at the inlet was found to vary considerably and is difficult to control, even with a critical orifice in the system, since it can be influenced by the clogging of the hydrophobic filters and/or the frit. It is recommended to record the flow rate periodically (e.g., at 5 min intervals) throughout collections periods, such that the volume of air collected over time for each sample can be accurately determined (see step 5).
There are several alternatives or possible modifications of the protocols presented. For example, an important advantage of the denitrifier method is the low sample size requirement12,13. However, other isotopic methods may be used. Similarly, we use colorimetric determination of concentration, but other methods can yield accurate NO3- concentration results.
Collection efficiency in the field, as detailed in Figure 2, is 92 &#177; 10%. This is critical to ensure that there is no fractionation during the collection process. With collection efficiency less than 100%, fractionation in the collection process can occur, biasing the resulting isotopic ratios measured. The efficacy of this new collection method across a range of conditions in urban-influenced air has been shown. Table 1 outlines the multiple tests that were done under ambient-air, near-roadside, and smog-chamber sampling conditions to determine the reproducibility of the method. All isotope ratio differences between systems are &#60;1.57&#8240;. This demonstrates the reproducibility of this method over a range of different sampling conditions. The field-based method has a precision and reproducibility significantly better than the ~12&#8240; isotopic ratio variations observed in the environment (Figure 4).
The most significant limitation of the method is the NO3- blank or background associated with the KMnO4 solution. A variety of KMnO4 types have been tested (e.g., crystals, powders, and stock solutions)4, and all contained NO3- before being exposed to NOx in the air. As a result, it is necessary to collect enough NOx as NO3- in solution to achieve a concentration above the blank. Further studies are currently underway to quantify the level at which the sample should exceed the blank concentration for the most accurate results. Under very low ambient NOx concentrations, it may be necessary to modify the collection conditions to maximize the sample concentration. For instance, the flow rate could be increased to collect more air in a shorter timeframe or the solution volume could be reduced to increase the air-to-solution volume and to concentrate the air collection. In any case, the solution must remain above the frit in the collection vessel to maintain the bubbling of air through the solution.
This method of NOx collection for isotopic analysis is unique among existing methods (e.g., passive samplers6,17 and sulfuric acid and hydrogen peroxide solution8) in that it has been laboratory- and field-verified with respect to field applicability, reproducibility, sample solution stability, and efficiency of collection under a range of field conditions. This novel method is unique in its capabilities to actively collect NOx in field environments for isotopic analysis at ambient concentrations at a 30-120 min time resolution. It collects NOx at near 100% efficiency and has been demonstrated repeatedly to be reproducible within the range of uncertainty of the method. Sample solutions collected in the field remain stable for up to 1 week before needing to be reduced. The method can collect samples over a range of concentrations and isotope ratios, and it is shown to be reproducible from collection to collection. This technique can be used for sampling under a variety of different conditions, including on-road, using the mobile laboratory approach outlined in the protocol. The interpretation of the spatiotemporal variability in vehicular emissions of NOx is the subject of a separate manuscript, in preparation (Miller, D.J., et al. 2016.&#160; J. Geophys. Atmos. Submitted).
Future sampling includes the application of this method to other types of NOx emissions (e.g., microbially produced emissions in soils and biomass fires). Isotopes are a potential way to track NOx sources, but only if different source signatures can be quantified and understood. Our new method makes it possible to quantify the isotopic composition of NOx from a variety of NOx emission sources and to directly test whether the impacts of emissions in the environment can be directly and quantitatively tracked.

An erratum was issued for: <a href="https://www.jove.com/video/54962/automated-high-resolution-mobile-collection-system-for-nitrogen">Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx</a>. An author name was updated.
One of the authors&#39; names was corrected from:
Mary O&#39;Conner
to:
Mary O&#39;Connor

An erratum was issued for: <a href="https://www.jove.com/video/54962/automated-high-resolution-mobile-collection-system-for-nitrogen">Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx</a>. An author name was updated.

Erratum: Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Atmospheric nitrogen oxides (NOx = NO + NO2) are important species in the global reactive nitrogen cycle1,2. NOx in the atmosphere is highly reactive and directly contributes to the oxidizing capacity of the atmosphere through its interactions with ozone (O3) and hydroxyl radical (OH). NOx is removed from the atmosphere on the scale of hours to days in the lower troposphere via oxidation to nitric acid (HNO3) or nitrate (NO3-), both of which are highly soluble and can be dry deposited on surfaces in gaseous and particulate aerosol forms or wet deposited by precipitation (e.g., acid rain)2. NOx is emitted from a variety of sources, including fossil fuel combustion, biomass burning, microbial processes in soils, and lightning. Source apportionment is crucial for understanding the impacts of individual sources, but the variety of sources, their variability in space and time, and the relatively short lifetimes of NOx and HNO3 make concentration analyses alone an inadequate metric. Stable isotopes may be useful as a way to better track the spatial patterns and temporal trends of sources and the chemistry of NOx and NO3- in the environment and to add new constraints on atmospheric models3. To date, the isotopic signatures associated with different NOx sources remain highly uncertain, particularly because of large uncertainties associated with previous methods4.
Previous studies represent a number of different active and passive collection methods and yield large ranges in reported isotopic values, even for the same emission source. Fibiger et al. found that previously used methods often varied greatly in terms of their efficiency in capturing NOx, with changes in conditions greatly influencing field collection (e.g., temperature, humidity, flow rates, age of solution)4. The inefficient uptake of previous NO and NO2 capture methods could lead to fractionations. For example, higher rates of oxidation for 14N relative to 15N could yield low biases in &#948;15N-NOx that are not representative of atmospheric values. In addition to methodological issues4,17, a variety of different types of air sampling may also contribute to differences in the reported ranges for isotope values associated with the same source. For example, isotopic signatures associated with vehicle emissions of NOx have been suggested based on collections at near-road sites5, in traffic tunnels6, and directly from the tailpipes of vehicles7,8. Furthermore, previous methods have time resolutions of 24 h at best, and significant changes in ambient NOx concentrations are observed on hourly (or shorter) timescales9, potentially limiting the application of isotopic detection for different sources. Many of the NOx collection methods require very strong oxidizing solutions capable of oxidizing NOx, but also other collected reactive nitrogen species (e.g., ammonium), to nitrate over time, potentially contributing an isotopic measurement interference. Some previous methods are also limited to collecting NO2 in solution, which provides only a limited understanding of NOx isotopes, as it does not collect NO (the primary emission). Thus, there is a need to capture NOx from different emissions sources using a consistent, validated method to better constrain whether the variability in isotopes of NOx (and NO3-) in the environment can be used to directly track sources and chemistry.
This paper reports on a field-based NOx collection technique for isotopic analysis with the requisite time resolution, collection efficiency (100%), and reproducibility (&#8804;1.5&#8240;) for application in multiple field environments. The method, originally described by Fibiger et al.4, is further validated through the demonstration of its collection efficiency under changing NOx and meteorological conditions in the field, the test of solution stability and ammonia interferences, and the substantiation of its reproducibility in urban environments. Spatial and temporal differences in isotopic values are investigated using a single laboratory- and field-verified method that can capture NOx in solution at high efficiency. This paper demonstrates the application of the method for near-road, on-road, and ambient urban air collections at time resolutions of 30 to 120 min.
In brief, NOx (NO and NO2) is collected from the atmosphere in a highly oxidizing solution as NO3-. At the same time, ambient NOx, NO2, and CO2 concentrations and other relevant data, such as GPS location and time of collection, are recorded. After a sample is collected, the solution is processed in the laboratory, which involves reducing the solution to stop the reaction, then neutralizing the solution pH for subsequent NO3- concentration and isotopic analyses. The NO3- concentration is determined here by an automated spectrophotometric (i.e., colorimetric) process. The nitrogen isotopic composition is determined using the denitrifier method, which quantitatively converts the NO3- in solution to gaseous N2O that is subsequently measured on an isotope ratio mass spectrometer. Laboratory and field blanks are also collected and measured as part of the collections to ensure the sample integrity. Below is a detailed step-by-step protocol.

<table><tbody><tr><td>Gas Washing Bottle</td><td></td><td></td><td>Custom Design used, numerous companies sell other gas washing bottles. The bottle needs to have a frit inside it.</td></tr><tr><td>Syringe Pump</td><td>Kloehn</td><td>Kloehn Versa Pump 6, 55 Series</td><td></td></tr><tr><td>PTFE Isolation Valves</td><td>Parker</td><td>002-00170-900</td><td>Both 2 three way and double two-way normally closed, electronically actuated valves</td></tr><tr><td>Gas Handling Teflon Tubing</td><td>McMaster Carr</td><td>5033K31</td><td>Quarter inch outer diameter, eigth inch inner diameter, PTFE tubing</td></tr><tr><td>Liquid Handling Tygon Tubing</td><td>McMaster Carr</td><td>5103K32</td><td>Quarter inch outer diameter, eigth inch inner diameter, PTFE tubing</td></tr><tr><td>Compression gas fittings and ball valves (assorted)</td><td>Swagelok</td><td>Assorted</td><td>Stainless Steel</td></tr><tr><td>Flow calibrator</td><td>MesaLabs</td><td>Defender 520</td><td></td></tr><tr><td>Compression PFA fittings</td><td>Cole Parmer</td><td>Assorted</td><td>Gas and liquid handling</td></tr><tr><td>Data Acquisition Board</td><td>National Instruments</td><td>NI USB-6001</td><td>Used for valve switching</td></tr><tr><td>Solid State Relay</td><td>Crydom</td><td>DC60S5</td><td>Used for valve switching</td></tr><tr><td>Single Stage Filter Assembly</td><td>Savillex</td><td>401-21-25-50-21-2</td><td>Use 25 mm and 47 mm diameter holders</td></tr><tr><td>Nylon Membrane Filter</td><td>Pall Corporation</td><td>66509</td><td>1 &amp;mu;m filter, both 25 mm and 47 mm diameter filters</td></tr><tr><td>Hydrophobic Membrane Filter</td><td>Millipore</td><td>LCWP04700</td><td>10.0 &amp;mu;m, 25 mm and 47 mm diameter filters</td></tr><tr><td>Particle Membrane Filters</td><td>Millipore</td><td>FALP04700</td><td>1 &amp;mu;m filter, both 25 mm and 47 mm diameter filters</td></tr><tr><td>Mini Diaphragm Pump</td><td>KNF</td><td>UN 816.1.2 KTP</td><td>Used for stationary lab</td></tr><tr><td>Mini Diaphragm Pump</td><td>KNF</td><td>PJ 26078-811</td><td>Used for mobile lab</td></tr><tr><td>Aluminum</td><td>Onlinemetals.com</td><td>6061-T6</td><td>Cut to size to build system</td></tr><tr><td>Deep Cycle Power Battery</td><td>EverStart</td><td>24DC</td><td></td></tr><tr><td>MilliQ Water</td><td>Millipore</td><td>ZMQSP0DE1</td><td></td></tr><tr><td>Potassium Permanganate 1 N Solution</td><td>Fischer Scientific</td><td>SP282-1</td><td></td></tr><tr><td>Sodium Hydroxide Pellets</td><td>Fischer Scientific</td><td>S318-1</td><td></td></tr><tr><td>Ohaus Benchtop scale</td><td>Pioneer</td><td>EX224N</td><td></td></tr><tr><td>4 ounce Amber Glass Bottles</td><td>Qorpak Bottles</td><td>GLC-01926</td><td>(60 ml and WM 125 ml bottles)</td></tr><tr><td>Amber HDPE Bottles</td><td>Fischerbrand</td><td>300751</td><td>Part number given for 125 ml narrow mouth bottles, Two varieties (125 wide mouth and narrow mouth of some volume)</td></tr><tr><td>Pre-cleaned EPA Amber Wide-mouth Bottle, 500 ml</td><td>Cole Parmer</td><td>EW-99540-55</td><td></td></tr><tr><td>Hydrogen Peroxide 30%</td><td>Fischer Chemical</td><td>H325-500</td><td>Corrosive</td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td>5821020010</td><td></td></tr><tr><td>50 ml Polypropylene Conical Tube</td><td>Falcon</td><td>14-432-22</td><td></td></tr><tr><td>12 N Hydrochloric acid</td><td>Fischer Scientific</td><td>A114SI212</td><td>Corrosive</td></tr><tr><td>Colorimetric Nutrient Analyzer</td><td>Westco Scientific Instruments</td><td>SmartChem 170</td><td>In purchasing the Colorimetric Nutrient Analyzer, this comes with buffers, cleaning solutions, rinse solutions, and solutions for running the instrument, including the solutions to be able to activate the cadmium column in the instrument for nitrate analysis.</td></tr><tr><td>Automatic Titrator</td><td>Hanna Instruments</td><td>HI 901</td><td></td></tr><tr><td>20 ml Clr Headspace Vial</td><td>Microleter, a WHEATON Company</td><td>W225283</td><td>Information listed&amp;nbsp; is for 20 ml vials. 50 ml vials can also be purchased from the vendor listed.</td></tr><tr><td>Septa, 20 mm Gray Butyl Stopper</td><td>Microleter, a WHEATON Company</td><td>20-0025</td><td></td></tr><tr><td>Seal, 20 mm Standard Aluminium</td><td>Microleter, a WHEATON Company</td><td>20-0000AS</td><td></td></tr><tr><td>25 G x 1 1/2 BD PrecisionGlide Needle</td><td>BD</td><td>305127</td><td></td></tr><tr><td>26 G x 1/2 BD PrecisionGlide Needle</td><td>BD</td><td>305111</td><td></td></tr><tr><td>Helium</td><td></td><td>05078-536</td><td>Can order from many different soures</td></tr><tr><td>Crimper/Uncrimper</td><td>WHEATON</td><td>61010-1</td><td></td></tr><tr><td>Isopropanol</td><td>Fischer Chemical</td><td>A459-1</td><td></td></tr><tr><td>Syringes of varying size for mass spec injection</td><td>BD</td><td>Varies based on size</td><td></td></tr><tr><td>Antifoam B Emulsion</td><td>Sigma-Aldrich</td><td>A5757-500ML</td><td></td></tr><tr><td>IRMS</td><td>ThermoFischer Scientific</td><td>IQLAAMGAATFADEMBHW</td><td>The actual isotope ratio mass spectrometer is listed here. Our set up also includes a gas bench and an autosampler.</td></tr><tr><td>Gass Bench II</td><td>ThermoFischer Scientific</td><td>IQLAAEGAATFAETMAGD</td><td></td></tr><tr><td>TriPlus RSH&amp;trade; Autosampler</td><td>ThermoFischer Scientific</td><td>1R77010-0200</td><td>Choose product for headspace injection</td></tr><tr><td>42i NOx Concentration Analyzer</td><td>ThermoFischer Scientific</td><td>101350-00</td><td></td></tr><tr><td>NOx Box</td><td>Drummond Technologies</td><td>LMA-3D/LNC-3D</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; analyzer</td><td>Licor 7000</td><td>7000</td><td></td></tr><tr><td>GPS</td><td>Garmin</td><td>010-00321-31</td><td></td></tr><tr><td>Model 146i Dynamic Gas Calibrator</td><td>ThermoFischer Scientific</td><td>102482-00</td><td></td></tr><tr><td>Model 111 Zero Air Supply</td><td>ThermoFischer Scientific</td><td>7734</td><td></td></tr><tr><td>50.2 ppm NO in N&lt;sub&gt;2&lt;/sub&gt; Gas standard</td><td>Praxis Air</td><td>Will vary with each tank of standard air purchased</td><td></td></tr></tbody></table>

automated, high-resolution mobile collection system for the nitrogen isotopic analysis of nox

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

In the original method development work by Fibiger et al., the NOx collection method was rigorously tested in the laboratory under a variety of conditions4. Here, the focus is on updates to the method and field applications under a variety of environmental conditions. Results are reported on (1) field collection efficiency, (2) sample solution stability in terms of time before sample reduction and sensitivity to high concentrations of ammonium (NH4+) in solution, and (3) reproducibility in the field. The versatility of the method is demonstrated in its application for ambient air, near-road, and on-road measurements.
The average concentrations collected in solution were compared with those from 1 min NOx concentrations from the chemiluminescence NOx analyzer over a two-day diurnal study in ambient urban air in Providence, RI. Figure 2 details the collection efficiency during a period when concentrations varied over a large range, from ~2.5-18 ppbv NOx. Figure 2A displays a direct comparison of median NOx concentrations from the NOx analyzer compared with concentrations calculated from the solution and flow measurements, indicating that, on average, solution concentrations are 92% of median in situ concentrations. This falls within the expected uncertainty range of &#177;10%, but the difference likely reflects varying concentrations during the collection periods (Figure 2B). Based upon examination of the percentiles of the distribution of the 1-min NOx concentration data, the solution-based NOx concentrations are within the distribution for every collection interval (Figure 2B).
It has been recommended to complete the reduction of the samples collected in the field within 1 day after collection is complete (i.e., complete all of step 3). This target was suggested to reduce the potential for interference from the collection of other soluble nitrogen species, such as NH3, that could be converted to nitrate in the highly oxidizing KMnO4/NaOH solution over time. To test this more specifically, samples were collected in May and July 2015 in Providence, RI on the campus of Brown University, at a loading dock that is near a regularly traveled local road where diesel delivery trucks are regularly running in idling mode to unload. Samples were collected, and then aliquots of the samples were separated and reduced at different times (1 day, 4-7 days, and 13-15 days) after the sample collection (Figure 3A). Samples in Figure 3B were also collected during May and July but were prepared by adding 5 ml of 10 mM ammonium chloride to 450 ml of solution. This yielded a concentration of 111 &#181;M NH4+ in solution, corresponding to collecting 220 ppbv of NH3 in the air, if only NH3 was collected. These concentrations are the maximum expected during on-road measurements near vehicle NH3 sources16. With or without the added NH4+, samples reduced within 7 days after collection had consistent isotope ratios when compared to the first reduction (within 1 day of collection), all falling within the expected uncertainty range of &#177;1.5% (Figure 3A and 3B). Note that the &#177;1.5% uncertainty is representative of isotopic determinations of repeated collections of tank NOx4. The uncertainty associated with repeated measures of isotopic reference materials alone is typically 0.3%. After two weeks, however, samples with or without added NH4+ were not necessarily stable. While in some cases the isotope values still appear to be consistent (e.g., Figure 3A), samples exhibited small NO3- concentration increases (&#60;1 &#181;M) when compared with the first reduction and, in some cases, decreases in NO3- concentrations. With the added NH4+, it would have been expected that NO3- concentration would increase over time above the expected uncertainty range (~0.8 &#181;M) for concentration measurements, suggesting that even after two weeks, the NH4+ was not the source of interference. Further experiments are needed to better understand the source of this instability, though it is noted that blank solutions left untreated over the same time course consistently showed no change or slight increases in concentrations, and therefore, the instability must be created by the presence of other species found in the ambient urban air. Until this is resolved, it is recommended that sample solutions be reduced within 7 days from the time of collection.
Figure 4 details the collection of samples with the mobile setup over various field campaigns in urban, near-road, and on-road settings. The NOx concentration range spans three orders of magnitude, and the isotope ratios range from -1 to -13&#8240;. This sampling set includes 51 on-road samples taken over 52 hr, covering over 4,000 km, and in a myriad of driving conditions (e.g., heavy stop-and-go traffic to very light traffic at high speeds on the highway). The sampling took place on roads in and between 6 major cities, including Providence, RI, Philadelphia and Pittsburgh, PA, and Cleveland, Columbus, and Cincinnati, OH. Average vehicle speeds ranged from 12.4 km/hr to 119.7 km/hr. The near-roadside samples (N = 27) were obtained at a monitoring site at I-95 in Providence, RI. The ambient urban air samples (N = 44 samples taken over 117.5 hr) were taken from two rooftop locations in Providence, RI, one near the I-95, I-195 interchange and one 775 meters away from the interchange site. This represents the first steps towards building new capabilities to resolve the ranges of isotopic signatures from NOx sources, in this case, vehicle emissions and ambient urban sources. The variations in daytime on-road and road-side &#948;15N-NOx (Figure 4) were not correlated with variations in driving conditions and occurred across relatively constant vehicle fuel-class traffic counts. A more detailed discussion of the variations of isotopic signatures due to vehicle fuel types is the subject of another manuscript (Miller, D.J., et al. 2016. J. Geophys. Atmos. Submitted).
Finally, Table 1 details field and laboratory collections where two collection systems were deployed at the same time to test reproducibility. The comparisons show excellent agreement for the isotopic data, quantified here as the absolute deviation between the two data points for each collection period. The data are displayed from urban air collections at a rooftop location in Providence, RI; near-roadside collections in Providence, RI; and from collections in a laboratory-based smog chamber at the University of Massachusetts, Amherst.
<img alt="Figure 1" src="/files/ftp_upload/54962/54962fig1.jpg" /> 
Figure 1: Collection Schematic and Image. (A) Diagram of the Automated NOx Collection System. Gray is airflow, blue is water/solution flow, green is electronics connections, yellow is the frit, and purple is the permanganate solution. The syringe pump is used to add and remove rinsing solution (ultrapure water) and to add new solution for the start of sample collection (the syringe pump is a commercially available stepper motor syringe pump with a 50 ml syringe, a 5-port distribution valve, and driver/control boards equipped with an RS-232 serial interface). The sample is removed manually via the black valve at the bottom of the gas washing bottle. (B) Picture of the NOx collection system and NOx box in the mobile laboratory. <a href="http://cloudflare.jove.com/files/ftp_upload/54962/54962fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54962/54962fig2.jpg" /> 
Figure 2: Collection Efficiency of the Automated Collection System. (A) The NOx concentrations calculated from the NO3- concentrations measured in solution and the flow data compared against the median concentration measured by a chemiluminescent NOx concentration analyzer at a rooftop site in Providence, RI. The error bars are the standard deviation (&#177;1&#963;) of the solution-based NOx mixing ratio estimates derived from the propagated errors of the pooled standard deviations of the quality controls (0.4 &#181;M) across colorimetric concentration measurement runs and the flow rate uncertainty (&#177;1%). The NOx analyzer concentration uncertainties are &#177;5%. (B) The time series of NOx concentration distributions during diurnal measurements at a rooftop site in Providence, RI. The boxes represent the 25th, 50th, and 75th percentiles. The whiskers represent the extremes without outliers. <a href="http://cloudflare.jove.com/files/ftp_upload/54962/54962fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54962/54962fig3.jpg" /> 
Figure 3: Comparison of the reduction times for NOx samples collected at Brown University in May and July 2015. (A) The results are recorded as deviations from the first reduction, performed within 1 day of sampling. May samples are displayed as triangles and July samples as circles, with colors denoting different collection periods. Samples in (B) were pre-treated with ammonium chloride prior to air collection to test the interference of NH4+ in solution over time. The dashed lines represent the expected overall precision of the isotopic collection method, expressed as a standard deviation of &#177;1.5%. <a href="http://cloudflare.jove.com/files/ftp_upload/54962/54962fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54962/54962fig4.jpg" /> 
Figure 4: The &#948;15N-NOx (&#8240;) and NOx concentration of samples collected in ambient urban air, on-road, and near-road sites. The types of samples are delineated by different colors, and represent a range of conditions (see the text) and NOx concentrations. <a href="http://cloudflare.jove.com/files/ftp_upload/54962/54962fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
 
&#160;
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample Name</td>
			<td>System Number</td>
			<td>Collection Date</td>
			<td>Hours of Collection</td>
			<td>Temperature (&#186;C)</td>
			<td>[NO3-] (&#181;M)</td>
			<td>Blank/total N</td>
			<td>&#948; 15N (&#8240;)</td>
			<td>Deviation&#160;&#948; 15N (%)</td>
		</tr>
		<tr>
			<td rowspan="2">Urban Air PVD 1</td>
			<td>1</td>
			<td rowspan="2">10/8/2013 - 10/9/2013</td>
			<td rowspan="2">6.75</td>
			<td rowspan="2">15.8</td>
			<td>14.43</td>
			<td>0.3</td>
			<td>-0.6</td>
			<td rowspan="2">0.7</td>
		</tr>
		<tr>
			<td>2</td>
			<td>16.78</td>
			<td>0.26</td>
			<td>-1.3</td>
		</tr>
		<tr>
			<td rowspan="2">Urban Air PVD 2</td>
			<td>1</td>
			<td rowspan="2">11/6/2013 - 11/7/2013</td>
			<td>2.5*</td>
			<td rowspan="2">17.1</td>
			<td>30.86</td>
			<td>0.2</td>
			<td>-7.7</td>
			<td rowspan="2">1</td>
		</tr>
		<tr>
			<td>2</td>
			<td>5.25</td>
			<td>37.05</td>
			<td>0.17</td>
			<td>-6.7</td>
		</tr>
		<tr>
			<td rowspan="2">Urban Air PVD 3</td>
			<td>1</td>
			<td rowspan="2">11/20/2013 - 11/21/2013</td>
			<td rowspan="2">8.9</td>
			<td rowspan="2">3.28</td>
			<td>44.29</td>
			<td>0.14</td>
			<td>-7.1</td>
			<td rowspan="2">0.4</td>
		</tr>
		<tr>
			<td>2</td>
			<td>29.66</td>
			<td>0.21</td>
			<td>-6.7</td>
		</tr>
		<tr>
			<td rowspan="2">Near Roadside 1</td>
			<td>1</td>
			<td rowspan="2">8/14/2014 - 8/15/2014</td>
			<td rowspan="2">29</td>
			<td rowspan="2">19.2</td>
			<td>13.3</td>
			<td>0.37</td>
			<td>-9.47</td>
			<td rowspan="2">0.69</td>
		</tr>
		<tr>
			<td>2</td>
			<td>16.4</td>
			<td>0.3</td>
			<td>-10.16</td>
		</tr>
		<tr>
			<td rowspan="2">Near Roadside 2</td>
			<td>1</td>
			<td rowspan="2">8/17/2014 - 8/18/2014</td>
			<td rowspan="2">30</td>
			<td rowspan="2">21.85</td>
			<td>9.4</td>
			<td>0.68</td>
			<td>-8.95</td>
			<td rowspan="2">1.56</td>
		</tr>
		<tr>
			<td>2</td>
			<td>11.6</td>
			<td>0.55</td>
			<td>-7.39</td>
		</tr>
		<tr>
			<td rowspan="2">Near Roadside 3</td>
			<td>1</td>
			<td rowspan="2">5/25/2015</td>
			<td rowspan="2">3.5</td>
			<td rowspan="2">20</td>
			<td>6.86</td>
			<td>0.51</td>
			<td>-7.67</td>
			<td rowspan="2">0.86</td>
		</tr>
		<tr>
			<td>2</td>
			<td>9.49</td>
			<td>0.42</td>
			<td>-8.53</td>
		</tr>
		<tr>
			<td rowspan="2">Near Roadside 4</td>
			<td>1</td>
			<td rowspan="2">5/26/2015</td>
			<td rowspan="2">2.75</td>
			<td rowspan="2">25.56</td>
			<td>6.07</td>
			<td>0.656</td>
			<td>-8.7</td>
			<td rowspan="2">1.57</td>
		</tr>
		<tr>
			<td>2</td>
			<td>6.49</td>
			<td>0.61</td>
			<td>-7.13</td>
		</tr>
		<tr>
			<td rowspan="2">Smog Chamber 1</td>
			<td>1</td>
			<td rowspan="2">8/26/2014 - 8/27/2014</td>
			<td rowspan="2">24.4</td>
			<td rowspan="2">21</td>
			<td>24.392</td>
			<td>0.27</td>
			<td>-12.28</td>
			<td rowspan="2">0.33</td>
		</tr>
		<tr>
			<td>2</td>
			<td>33.2</td>
			<td>0.2</td>
			<td>-12.61</td>
		</tr>
		<tr>
			<td rowspan="2">Smog Chamber 2</td>
			<td>1</td>
			<td rowspan="2">8/27/2014 - 8/28/2014</td>
			<td rowspan="2">19.8</td>
			<td rowspan="2">21</td>
			<td>10.96</td>
			<td>0.54</td>
			<td>-10.22</td>
			<td rowspan="2">1.25</td>
		</tr>
		<tr>
			<td>1</td>
			<td>14.245</td>
			<td>0.41</td>
			<td>-11.47</td>
		</tr>
		<tr>
			<td>Smog Chamber 3</td>
			<td>1</td>
			<td>8/28/2014 - 8/29/2014</td>
			<td>24.2</td>
			<td>21</td>
			<td>7.476</td>
			<td>0.8</td>
			<td>-5.86</td>
			<td>1.27</td>
		</tr>
	</tbody>
</table>
Table 1: Reproducibility of samples collected at the same time using two identical collection systems. *The collection had to be stopped due to a clogged filter. Urban Air PVD (PVD = Providence, RI) 1-3 were previously published4. Near Roadside represents roadside collections in Providence, RI; Smog chamber represents samples collected from air inside a smog chamber at the University of Massachusetts, Amherst4.

Watch this Scientific Journal Video about Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx at JoVE.com

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

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11.6K Views.  Brown University. Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Video: Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Chemistry

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

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Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

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Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is crucial. There are significant challenges to overcome when measuring trace compounds in a complex matrix such as fuels. Background interferences and matrix effects can create limitations to routine analytical instrumentation, such as GC-MS. In order to facilitate specific and quantitative measurements of trace nitrogen compounds in fuels, a nitrogen-specific detector is ideal. In this method, a nitrogen chemiluminescence detector (NCD) is used to detect nitrogen compounds in fuels. NCD utilizes a nitrogen-specific reaction that does not involve the hydrocarbon background. Two-dimensional (GCxGC) gas chromatography is a powerful characterization technique as it provides superior separation capabilities to one-dimensional gas chromatography methods. When GCxGC is paired with a NCD, the problematic nitrogen compounds found in fuels can be extensively characterized without background interference. The method presented in this manuscript details the process for measuring different nitrogen-containing compound classes in fuels with little sample preparation. Overall, this GCxGC-NCD method has been shown to be a valuable tool to enhance the understanding of the chemical composition of nitrogen-containing compounds in fuels and their impact on fuel stability. The % RSD for this method is &#60;5% for intraday and &#60;10% for interday analyses; the LOD is 1.7 ppm and the LOQ is 5.5 ppm.

Here, we present a method utilizing two-dimensional gas chromatography and nitrogen chemiluminescence detection (GCxGC-NCD) to extensively characterize the different classes of nitrogen-containing compounds in diesel and jet fuels.

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NO_x

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