ANALYSIS METHODS MANUAL
FOR HUMIC-CONTAINING ESTUARINE WATERS
Joan E. Sheldon and
Dr. William J.
Revision Date: June 7, 1997
NITRATE (+ NITRITE)
Initially we tried to use a standard autoanalyzer procedure with a
cadmium column, but humic colored water reduced column reduction
efficiency drastically after only a few samples passed through
Filter through ashed GF/F filter in glass or
plastic apparatus, ashed or used only for similar samples.
in polyethylene bottle at -20 oC or lower
Same as for nitrite analysis plus those below.
Ammonium Chloride Buffer (0.7 M):
Dissolve 37.4 g NH4Cl in approx. 800 mL water. Add
approx. 3 mL 1 M NaOH to adjust pH to 8.5. Dilute to 1 L
Cadmium Sulfate Solution, 20% w/v:
Dissolve 200 g CdSO4 and make up to 1 L with water.
6 N Hydrochloric Acid:
492 mL conc. HCl made up to 1 L with water.
5 mM Primary:
Dry KNO3 for several hours at 60 oC. Dissolve
0.5055 g and make up to 1 L with water. Store in refrigerator.
Make fresh each day:
10 uM: 9.980 mL water + 20 uL Primary Std.
20 uM: 9.960 mL water + 40 uL Primary Std.
40 uM: 9.920 mL water + 80 uL Primary Std.
Make fresh each day:
40 uM: 9.920 mL water + 80 uL Primary NO2
Same as for nitrite analysis plus that below.
Shaker, linear motion preferred, capable of > 100 excursions/min.
Plastic centrifuge tubes, 15 mL, with water-tight screw caps, suggested.
Acid-wash before and between uses.
Spongy cadmium method (Jones, 1984; D.
Bronk, pers. comm.) for
reducing nitrate to nitrite, but using twice as much cadmium
as in the standard protocol to keep acceptable reduction
efficiency in the presence of humic material. Determination
of nitrite by autoanalyzer method. The sum of nitrate +
nitrite is determined.
Cd IS TOXIC. Handle carefully and use appropriate
protective clothing, especially against accidental
inhalation or ingestion! Gloves and a particle mask
recommended. Put zinc sticks in individual test tubes or
cylinders and cover with CdSO4 solution. Let stand for
approx. 8 hours, not much longer. Remove zinc sticks (wash
with acid and water and dry before putting away). Acidify
remaining CdSO4 with a few drops 6 N HCl and then drain from
Cd. Cover Cd with 6 N HCl and stir to break up. Drain acid
and rinse Cd with water until pH goes above 5 (at least 10
rinses). Store under water. Regenerate after use by
repeating 6 N HCl wash and rinses.
Make up triplicate working standards in ordinary test tubes, 10
Put 1 mL NH4Cl buffer and 0.4 g spongy Cd in
each 15 mL screw-cap centrifuge tube. Add 5 mL of standard,
blank (water) or sample to each tube (do triplicates).
(Include NO2 standard to test for over-reduction or to be
able to distinguish reduction problems from reagent problems
in the event of a bad standard curve.) Cap tubes and lay
horizontally on shaker in the direction of shaking for
maximum mixing. Shake at 100-125 excursions/min for approx.
90 min. Transfer to polyethylene autoanalyzer cups (use
approx. 2 mL and be careful not to get any Cd particles) set
up in sampler tray in the following order (all in
triplicate): 40 uM NO2 std., 40 uM NO3 std., 20 uM
10 uM NO3 std., water + NH4Cl blank, 8 samples. (Carefully
collect Cd and remaining solution. A vortex mixer may be
useful to dislodge Cd from tubes. Drain and regenerate
before next use.)
Run nitrite analysis but set Standard Calibration setting 0.4 and
Damp setting "normal". Also re-run samples only for nitrite
Same as for nitrite except account for dilution by ammonium
chloride buffer: "Adjusted peak height" = (top of peak -
baseline) × 1.2
No correction for salinity is necessary.
Nitrate alone is calculated by subtraction of nitrite, analysed
Limit of Detection:
2 std. dev. above blank approx. = 0.3 uM
approximately 65 uM by changing Standard Calibration setting.
Nitrate + nitrite range is higher than nitrite range because of
dilution by ammonium chloride buffer.
95% confidence limits for prediction of a concentration
near the mean for a typical standard curve (17.5 uM) approx. =
± 4.04 uM for a triplicate determination. (Confidence limits
for prediction near the ends of a standard curve are, of course,
Jones, Martha N. 1984. Nitrate reduction by shaking with cadmium: alternative to
cadmium columns. Water Res. 18(5): 643-646.