LOW LEVEL NITRATE AND NITRATE BY CHEMILUMINESCENCE
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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.
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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
Niskin bottles and rosette/CTD unit
acid-washed 125 ml polyethylene bottles
nitrogen oxide analyzer (Antek model #720, operated in vacuum mode)
reaction apparatus including cold finger and drying tube
ultra-high purity O2 and Ar, with drierite/ascarite scrubbers
volumetric flasks and pipettes, syringe, septa, and glass beakers
8. Reagents
glass distilled deionized water (DDW)
dilute HCl (10%): add 500 ml concentrated reagent grade HCl to 4500
ml DDW.
glacial acetic acid
sodium iodide (3% wt/vol): dissolve 3 g reagent grade NaI in 100 ml DDW.
Prepare fresh daily.
concentrated H2SO4 (36 N)
ferrous ammonium sulfate (4% wt/vol): dissolve 4 g reagent grade
ferrous ammonium sulfate in 100 ml DDW. Prepare fresh daily.
ammonium molybdate (2% wt/vol): dissolve 2 g reagent grade ammonium
molybdate in 100 ml DDW. 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 sodium hydroxide
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-.
9. References
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.
Garside, C. 1982. A chemiluminescent technique for the determination
of nanomolar concentrations of nitrate and nitrite in seawater. Marine
Chemistry, 11, 159-167.
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