LOW LEVEL NITRATE AND NITRATE BY CHEMILUMINESCENCE


SUMMARY: Seawater is collected from known depths using CTD-rosette sampling protocols. Subsamples are carefully drawn and stored in acid-washed polyethylene bottles. Nitrate/nitrite is converted to nitric oxide by wet chemical reduction in a highly acidic solution. The nitric oxide produced is measured by a chemiluminescent detector.


1. Principle

Surface water samples (<100m) in oligotrophic waters usually have nitrate concentrations below the 0.03 µM detection limit of the Technicon Autoanalyzer. Except for a narrow band of elevated nitrite around 100 m (the primary nitrite maximum), nitrite concentrations are also below standard detection limits. To achieve high-precision high-accuracy measurements at these low levels, we employ the chemiluminescent method of Cox (1980) and Garside (1982). In this method nitrite and/or nitrate are chemically reduced to gaseous nitric oxide by an acidic solution of glacial acetic acid and sodium iodide (nitrite only) or concentrated sulfuric acid, ferrous ammonium sulfate and ammonium molybdate (nitrate plus nitrite). The reduced nitric oxide is carried by an inert carrier gas (argon) through a cold finger filled with 6M sodium hydroxide, a drying tube filled with anhydrous sodium carbonate, and a membrane drier in order to remove acid and water vapors. The gas stream is then routed into the chemiluminescent analyzer, where the nitric oxide is combined with ozone to produce a metastable nitrogen dioxide. The nitrogen dioxide subsequently emits a photon as it returns to ground state, and the emitted light is detected by a photomultiplier tube. The integrated electrical signal produced by the photomultiplier is proportional to the content of nitrite (or nitrate plus nitrite) in the sample.

2. Precautions

As with the autoanalysis of these nutrients, contamination is the primary concern. Because nanomolar concentrations are to be measured, all sample bottles and laboratory glassware must be meticulously cleaned with dilute HCl (10%) and distilled deionized water (DDW) before use, and both the argon carrier gas and the oxygen supplied to the ozone- 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 signal. All drying and scrubbing tubes must be well maintained to ensure that no water 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 by Constantinou (unpubl.) indicate no evidence for any discrepancy between replicate samples analyzed immediately on ship and those frozen and analyzed later in the laboratory. Samples are therefore collected and stored exactly as described for autoanalysis of these nutrients.

4. Analysis

Nitrate and nitrite analyses are performed on an Antek model 720 nitrogen oxide analyzer. The reaction apparatus is a slight variation of that described by Garside (1982).

4.1. [NO2-] determinations

One ml sodium iodide solution (3% wt/vol) and 3 ml glacial acetic acid are dispensed into the reaction tube. The reaction tube is inserted into the carrier gas line and the reagents degassed. This degassing step removes any contaminating nitrite from the reagents. The extent of the degassing is monitored by the detector signal. When this step is complete, the detector is reset and 10 ml of sample or standard are introduced into the reaction tube with a syringe through a septum fitted to the side arm of the tube. The signal produced is integrated and peak area is recorded. The waste is then discarded, the detector reset and the procedure repeated. Generally each sample or standard analysis takes about five minutes.

4.2. [NO3- + NO2-] determinations

Ten ml concentrated sulfuric acid (36 N) followed by 2 ml ferrous ammonium sulfate (4% wt/vol) and 2 ml ammonium molybdate (2% wt/vol) are dispensed into the reaction tube (the order of addition is important). Unlike in the nitrite- only analysis, the heat generated by the dilution of the acid is important for the rapid stripping of the nitric oxide produced from the solution, therefore an insulating sleeve is used around the reaction tube. The reagents are degassed as above, and the sample added immediately after degassing is complete, in order to maintain an elevated reaction temperature. If the reaction temperature gets too low, the sample may not degas rapidly enough for quantitative results. A strip chart recorder is useful for troubleshooting: if asymmetrical peak tailing is observed, the reaction temperature is probably not being maintained at a high enough level (Garside, 1982). The signal is integrated and recorded as for the nitrite-only analysis.

4.3. [NO3-] determinations

Our preferred method for low level nitrate 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 nitrite in the sample with sulfanilamide to form a diazonium salt, then analyze the sample for nitrate plus nitrite. 0.2 ml of a sulfanilamide solution (1% wt/vol in dilute HCl) is added to the sample and allowed to react with the nitrite for at least two minutes. Then the treated sample is analyzed as for [NO3- + NO2-] above. The disadvantages of using the latter method are that: (1) a small blank correction for contaminating nitrate in the sulfanilamide solution must be determined and applied to the results and (2) some precision is sacrificed in using the [NO3- + NO2-] assay twice instead of using the [NO3- + NO2-] assay once and the more precise [NO2-] assay once. The advantage is that two separate sets of analyses involving the time-consuming generation of two standard curves are not required.

5. Calibration, Data Reduction and Calculations

5.1. Calibration stocks and regression standards

The calibration of the low level nitrate/nitrite analysis is performed using standard solutions of NO3- or NO2- in DDW. Dried (60°C, 72 hours) analytical grade reagent chemicals are dissolved in DDW in 500 ml acid-washed glass volumetric flasks. A few drops of chloroform are added to inhibit microbial activity. The stock solutions are stored at 4°C and discarded about every three months. The reagent chemicals and concentrations are KNO3 (10 mM) and NaNO2 (10 mM).

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 DDW. 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. Typical correlations produce r2 values of 0.9999 or better.

In order to maintain the accuracy of the analysis, a few certified reference standards (CSK or equivalent), diluted volumetrically with DDW until within the concentration range of interest, are included in every sample run in case stock solutions become contaminated or microbially altered during storage.

5.2. Blank corrections

For the [NO3- + NO2-] analysis, a small blank is observed due to traces of nitrate in the DDW diluent. This blank is corrected for in the calculation of standard concentrations, but is not subtracted from sample results because they contain no diluent. For samples, therefore, only the slope of the regression line is used. For the [NO2-] analysis, no such diluent blank is observed. 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 nitrate plus nitrite is approximately 1-2 nM. The nitrite-only analysis produces less noisy signals; standards with as little as 0.4 nM nitrite and samples with as little as 0.1 nM nitrite have been detected. Precision and accuracy of the nitrate plus nitrite analysis are approximately 2-3 nM while for nitrite-only they are generally <2 nM.

7. Equipment/Supplies

8. Reagents

9. References