NUTRIENT
ANALYSIS METHODS MANUAL
FOR HUMIC-CONTAINING ESTUARINE WATERS
AUTHORS/CONTACTS:
Joan E. Sheldon and
Dr. William J.
Wiebe
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
the column.
SAMPLES
Sample Volume:
15 mL
Sample Collection:
Filter through ashed GF/F filter in glass or
plastic apparatus, ashed or used only for similar samples.
Sample Storage:
in polyethylene bottle at -20 oC or lower
REAGENTS
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
with water.
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.
Zinc Sticks
STANDARDS
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.
Working:
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.
NO2 Working:
Make fresh each day:
40 uM: 9.920 mL water + 80 uL Primary NO2
Std.
SPECIAL EQUIPMENT
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.
PROTOCOL
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.
Spongy Cd:
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
mL each.
Nitrate Reduction:
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
NO3 std.,
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
color blank.
CALCULATIONS
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
separately.
Limit of Detection:
2 std. dev. above blank approx. = 0.3 uM
Range:
approximately 65 uM by changing Standard Calibration setting.
Nitrate + nitrite range is higher than nitrite range because of
dilution by ammonium chloride buffer.
Accuracy:
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,
somewhat larger.)
REFERENCES
Jones, Martha N. 1984. Nitrate reduction by shaking with cadmium: alternative to
cadmium columns. Water Res. 18(5): 643-646.
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 the column.
SAMPLES
- Sample Volume:
- 15 mL
- Sample Collection:
- Filter through ashed GF/F filter in glass or
plastic apparatus, ashed or used only for similar samples.
- Sample Storage:
- in polyethylene bottle at -20 oC or lower
REAGENTS
- 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
with water.
- 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.
- Zinc Sticks
STANDARDS
- 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.
- Working:
- 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.
- NO2 Working:
- Make fresh each day:
- 40 uM: 9.920 mL water + 80 uL Primary NO2
Std.
SPECIAL EQUIPMENT
- 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.
PROTOCOL
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.
- Spongy Cd:
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 mL each.
- Nitrate Reduction:
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 NO3 std., 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 color blank.
CALCULATIONS
- 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
separately.
- Limit of Detection:
- 2 std. dev. above blank approx. = 0.3 uM
- Range:
- approximately 65 uM by changing Standard Calibration setting.
Nitrate + nitrite range is higher than nitrite range because of
dilution by ammonium chloride buffer.
- Accuracy:
- 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,
somewhat larger.)
REFERENCES
- Jones, Martha N. 1984. Nitrate reduction by shaking with cadmium: alternative to
- cadmium columns. Water Res. 18(5): 643-646.
