SUMMARY: Seawater is collected from known depths using CTD-rosette sampling procedures. Subsamples are drawn and stored frozen (-20°C) in acid-washed, HDPE bottles. Nitrate/nitrite is converted to nitric oxide by wet chemical reduction in an acidic solution. The nitric oxide produced is measured using a commercial chemiluminescent detector.
Surface water samples (<100 m) from oligotrophic open ocean ecosystems typically have nitrate (NO3-) plus nitrite (NO2-) concentrations below the 0.03 µM detection limit of standard colorimetric measurement procedures. Except for a narrow band of elevated NO2- around 100 m (the primary NO2- maximum), NO2- concentrations are also below standard detection limits for the entirely of the water column. To achieve high-precision and high-accuracy measurements at low concentrations, we employ the chemiluminescent method of Cox (1980), which is described for seawater by Garside (1982). Our current protocol and instrumentation are detailed in Foreman et al. (2016). In this method, NO3- and NO2- are both chemically reduced to gaseous NO by an acidic solution of concentrated H2SO4 and TiCl3. To determine NO2- concentrations only, NO2- is reduced to NO using the less aggressive mixture of glacial acetic acid and sodium iodide. The NO is carried by an inert carrier gas (Ar) through a cold finger filled with NaOH to remove acid, then through a column filled with anhydrous NaCO3 and quartz chips (4:1 ratio) to further scrub protons and a subsequent nafion membrane drier to remove water vapors. Counter-current drying air for the nafion membrane tube comes from house compressed air, which is scrubbed of moisture using a drierite canister. The gas stream is then routed into the chemiluminescent analyzer, where the NO is combined with ozone (O3) to produce an excited state NO2*. The NO2* emits a photon as it returns to ground state, and the emitted light is detected by a photomultiplier. The integrated electrical signal produced by the photomultiplier is linearly proportional to the content of NO2- or [NO3- +NO2-] originally present in the sample. The high-precision and high-sensitivity of chemiluminescence detection provide a convenient analytical detection system that is capable of NO3- and NO2- detection at nM concentrations.
As with automated analysis of these nutrients, contamination is the primary concern. All sample bottles and laboratory glassware must be meticulously cleaned with dilute HCl (1 M) and DIW before use, and both the argon carrier gas and the O2 supplied to the O3 generator must be of the highest purity available. An additional concern with the chemiluminescent technique is the quenching effect that water vapor has on the sample signal as the result of collisional degradation of NO2* with H2O instead of the desired photon release. Thus, all drying and scrubbing parts must be well maintained to ensure that no water vapor enters the detector.
Concerns about the potential "freezing effect" on these samples are heightened due to the low levels of the nutrients and the high precision of the measurements. However, results from experiments conducted using Sta. ALOHA seawaters indicate no evidence for any discrepancy between replicate samples analyzed immediately on ship and those frozen and analyzed later in the laboratory (Dore et al., 1996). Samples are therefore collected and stored exactly as described for autoanalysis of these nutrients.
NO3- and NO2- analyses are performed using an Antek model 7090 nitrogen oxide analyzer. The reaction apparatus is a slight variation of that described by Garside (1982).
[NO2-] determinations: One ml NaI solution (3% wt/vol) and 3 ml glacial acetic acid are dispensed into the reaction chamber with flow-through carrier gas. The reagent mix is allowed to react into order remove any background NO2- in the reagents. When this step is complete, 10 ml of sample or standard are introduced into the reaction chamber. The signal produced is integrated and peak area is recorded using a PeakSimple data system from SRI Instruments. The waste is then discarded, and the procedure repeated. Generally, each sample or standard analysis takes about 5 min.
[NO3- + NO2-] determinations: Seven ml concentrated H2SO4 (36 N) and 2 ml TiCl3 solution (4% wt/vol) are dispensed into the reaction tube. Unlike the nitrite-only analysis, the heat generated by the dilution of the acid is important for the rapid stripping of the NO produced from the solution, therefore the reaction must remain warm for proper reaction kinetics. The reagents are degassed, as above, and the sample added immediately after degassing is complete to maintain an elevated reaction temperature. If the reaction temperature gets too low, the sample may not degas rapidly enough for accurate quantitative results. The signal is integrated and recorded as for the NO2- only analysis.
[NO3-] determinations: Our preferred method for low level NO3- determination is to analyze the samples for both [NO3- + NO2-] and [NO2-] and calculate [NO3-] by difference. An alternative method is to first bind the NO2- in the sample with sulfanilamide to form a diazonium salt, then analyze the sample for [NO3- + NO2-]. Two hundred µl of a sulfanilamide solution (1% wt/vol in dilute HCl) is added to the sample and allowed to react with the NO2- for at least 2 min. Then the treated sample is analyzed for [NO3- + NO2-], as described above.
5.1. | Calibration stocks and regression standards
The calibration of the low-level NO3-/NO2- analysis is performed using standard solutions of NO3- (from KNO3) or NO2- (from NaNO2) in DIW. Dried (60°C, 72 hr) analytical grade reagent chemicals are dissolved in DIW in 1000 ml acid-washed glass volumetric flasks to a final analyte concentration of 10,000 µM. One ml of chloroform is added to inhibit microbial activity. The stock solutions are stored at room temperature for up to a year. Working standards are prepared fresh by volumetric dilutions of the stock using acid-washed glass pipettes and flasks. Since there is no salt effect in the chemiluminescent analysis, working standards are all prepared using DIW. These working standards are used to generate a standard curve and are analyzed at intervals throughout the sample run in order to detect any drift in the detector response. To maintain the accuracy of the analysis, a few certified reference standards (CSK or equivalent), diluted volumetrically with DIW into the concentration range of interest, are included in every sample run in case stock solutions become contaminated during storage. |
5.2. | Blank corrections
For the [NO3- + NO2-] analysis and [NO2-] only analysis, there is no blank detected in the DIW diluent. If the sulfanilamide method is used for [NO3- ] measurements, the blank due to the sulfanilamide solution should be determined and subtracted from results. |
The detection limit for [NO3- + NO2-] is approximately 1-2 nM. The NO2- only analysis produces less noisy signals; standards and samples with as little as 0.4 nM NO2- have been detected. Precision and accuracy of the [NO3- + NO2-] analysis are approximately 2-3 nM, while for NO2- only they are generally <2 nM.
Acid-washed, 125 ml high-density polyethylene bottles
Nitrogen oxide analyzer (Antek model 7090, operated with vacuum)
Reaction chamber and cold finger with NaOH (acid trap)
Sodium carbonate drying column and quartz chips
Nafion drying column with counter-current dry air
Ultra-high purity O2 and Ar, with drierite/ascarite scrubbers
Volumetric flasks, pipettes, and glass beakers
Sample and Reagent injection manifold
DIW
HCl (10%): add 500 ml concentrated reagent grade HCl to 4500 ml DIW.
glacial acetic acid
sodium iodide (3% wt/vol): dissolve 3 g reagent grade NaI in 100 ml DIW. Prepare fresh daily.
concentrated H2SO4 (36 N)
titanium trichloride (4% wt/vol solution): dilute concentrated solution to 4% TiCl3 using DIW. Prepare fresh daily.
sulfanilamide (1% wt/vol in 10% HCl): dissolve 1 g reagent grade sulfanilamide in 100 ml 10% HCl.
sodium hydroxide (6 M): dissolve 240 g reagent grade NaOH and make up to 1 L with DDW.
nitrite stock solution (10 mM): dissolve 0.345 g dried NaNO2 in 500 ml DDW. Add 0.5 ml chloroform and store in the dark at 4°C.
nitrate stock solution (10 mM): dissolve 0.506 g dried KNO3 in 500 ml DDW. Add 0.5 ml chloroform and store in the dark at 4°C.
CSK (or equivalent) certified reference standards for seawater: NO2- and NO3-.
Cox, R. D. 1980. Determination of nitrate at the parts per billion level by chemiluminescence. Analytical Chemistry, 52, 332-335.
CSK Certified Seawater Reference Standards. Sagami Chemical Research Center, Sagimahara, Japan.
Dore, J. E., T. Houlihan, D. V. Hebel, G. Tien, L. M. Tupas and D. M. Karl. 1996. Freezing as a method of seawater preservation for the analysis of dissolved inorganic nutrients in seawater. Marine Chemistry, 53, 173-185.
Foreman, R. K., M. Segura-Noguera, and D. M. Karl. 2016. Validation of Ti(III) as a reducing agent in the chemiluminescent determination of nitrate and nitrite in seawater. Marine Chemistry, 186, 83-89.
Garside, C. 1982. A chemiluminescent technique for the determination of nanomolar concentrations of nitrate and nitrite in seawater. Marine Chemistry, 11, 159-167.
analyte-compound class: nitrate [NO3- ]/nitrite [NO2-] (high sensitivity)
method: chemiluminescence
precision: - laboratory: 15% at 2 nmol kg-1 - field: 30% at 1.5 nmol kg-1
accuracy: 2-3 nmol kg-1 ; 1% for HOT 41-47 data set using CSK reference standard
reference standard: - primary: KNO3 - secondary: CSK-NO3
analysis history for HOT program:
analysis of NO3-/NO2- by the high sensitivity method began on HOT-1 (Oct 1988) and has continued without substantive change. From HOT-287 to present the Foreman et al., (2016) protocol has been implemented.
Notes, comments or additions:
detection limit is 1-2 nM NO3- and 0.8 nM NO2-
this method could also be used to measure NH4+, after oxidation to NO2- with hypochlorite in alkali using excess potassium bromide as a catalyst; this method is currently under development in the HOT program and may later be added as a core measurement.
Nitrile gloves will contaminate the sample both at sampling and during analysis.