HIGH-SENSITIVITY NITRATE PLUS NITRITE DETERMINATIONS BY CHEMILUMINESCENCE


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.


1. Rationale and Assay Principle

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.

2. Precautions

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.

3. Sample Collection and Storage

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.

4. Analysis

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).

5. Calibration, Data Reduction and Calculations

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.

6. Precision and Accuracy

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.

7. Equipment/Supplies

8. Reagents

9. References

10. HOT Program Analytical Summary