ANALYSIS METHODS MANUAL
FOR HUMIC-CONTAINING ESTUARINE WATERS
Joan E. Sheldon and
Dr. William J. Wiebe
Revision Date: June 7, 1997
Initially we tried the standard Koroleff (1976, 1983) method which
uses no magnesium reagent and instead uses citrate to complex any
natural magnesium and calcium and thus avoid a precipitate.
Humics remained in solution and interfered with recovery of
ammonium. A color blank using nitroprusside solution without
phenol (Flebbe, 1982) could not completely correct for the
interference: "corrected" recoveries of ammonium added to humic-
colored water were low.
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
Dissolve 45 g NaCl and 20 g MgSO4.7H2O in
about 200 mL water. Add 1 N NaOH dropwise until a slight
precipitate forms. Add a few boiling chips and boil (to
drive off ammonia) until < 200 mL. Cool and dilute to 200
mL. A slight precipitate will remain. Use an automatic
Dissolve 0.2 g Na2Fe(CN)6NO.2H2O and 19 g
C6H5OH in 500 mL water. Use an automatic
dispenser. Store in a dark bottle in refrigerator.
Dilute 750 mg available Cl- as Clorox® bleach (see
protocol below) to 500 mL with 0.5 N NaOH. Use an automatic
dispenser. Store in a dark bottle in refrigerator.
Dissolve 7.9 g Na2S2O3
or 12.4 g Na2S2O3.5H2O
in 500 mL water. Store in refrigerator.
Dissolve 0.5 g KI in 50 mL 1 N
H2SO4. Make fresh each time.
10 mM Primary:
Dry (NH4)2SO4 overnight at
50 oC. Dissolve 0.6607 g and make up to 1 L with water.
Store in refrigerator.
1 mM Secondary:
1 mL Primary Standard + 9 mL water, or similar
convenient amount. Make fresh each day.
Make fresh each day.
5 uM: 4.975 mL water + 25 uL Secondary Std.
10 uM: 4.950 mL water + 50 uL Secondary Std.
20 uM: 4.900 mL water + 100 uL Secondary
Pyrex®, test tubes, 20 x 150 mm, with
Teflon®-lined screw caps, kept
only for this procedure. Before first use, wash and dry
tubes and caps and ash tubes as usual, then run reagent
blanks in all tubes to "burn out" any remaining ammonia.
After each use, leave any remaining sample in tube, cap, and
keep until next use.
Basically the Koroleff (1983) method but using slightly more than half the
suggested phenol and hypochlorite (Koroleff, 1976; D. Bronk,
pers. comm.) to reduce phenol vapors in the lab. The Mg
hydroxide precipitation procedure is used to clear humic
color from the water. The sum of NH4++
NH3 is determined.
Make up potassium iodide solution and add 1 mL
Clorox®. Titrate with thiosulfate solution until color is
gone. 1 mL thiosulfate reacts with 3.54 mg available Cl-,
so (mL Clorox® needed to give 750 mg available Cl-) =
(750 ÷ 3.54) ÷ (mL thiosulfate used). Discard Clorox® if < 12 mL
thiosulfate is used (Parsons, et al. 1984).
Discard old sample in test tubes (collect as hazardous
waste), rinse tubes 4 times with 18 megohm-cm water, and
shake out as much water as possible. Small drops will
remain, but these are fairly consistent among all tubes and
thus will not affect the analysis.
Make up triplicate blanks and working standards in tubes, 5
mL each. Pipette 5 mL of each sample into 3 tubes, noting
the salinity of each sample. Add 100 uL of magnesium
reagent to each blank, standard, and sample with salinity <=
5 ppt and vortex. To each tube add 250 uL
nitroprusside/phenol, vortex, immediately add 250 uL
hypochlorite, vortex, and cap. Keep tubes in a dark place
until read at least 6 hours but not more than 30 hours
Handle carefully to avoid resuspending the precipitate.
Read the absorbance in a 1 cm cell at 630 nm (against the
absorbance of water), using a sample sipper or Pasteur
pipette to draw off the supernatant. Settings for the
sample sipper for the Shimadzu Model UV160U
spectrophotometer are pump speed medium, sip time 4 sec,
dwell time 1 sec, purge time 4 sec, 1 rinse. If using a
conventional cell and Pasteur pipette, rinse once with
Corrections for salinity are obtained by running standards in
waters of various salinities. For example, salt factors
were obtained for 0, 5, 10, ... 34 ppt water made by mixing
aged Gulf Stream water with 18 megohm-cm water, and factors
for other salinities were interpolated. Salt factor = 1 +
(salinity in ppt) × 0.0062
Correct standards for dilution by magnesium reagent because not
all samples will receive it (e.g. 20 uM standard is really
19.61 uM after addition of magnesium reagent).
Plot the corrected standard concentration vs. absorbance. The
slope of this line is the calibration factor F. F should be
approximately 55-60 for a 1 cm cell.
Correct sample calculations for dilution by magnesium reagent
where necessary: Dilution factor = 1.02 if sample salinity
was <= 5 ppt (i.e. magnesium reagent was added) or Dilution
factor = 1 if sample salinity was > 5 ppt (i.e. no magnesium
Sample concentration (uM) = F × Salt factor × (sample abs -
average blank abs) × Dilution factor
Limit of Detection:
2 std. dev. above blank approx. = 0.1 uM
approx. 40 uM directly or up to 150 uM if diluted after color
development (Koroleff, 1983).
95% confidence limits for prediction of a concentration
near the mean for a typical standard curve (8.75 uM) approx. =
± 0.48 uM for a triplicate determination. (Confidence limits
for prediction near the ends of a standard curve are, of course,
somewhat larger.) For some trials with ammonium-spiked river
water, recoveries were still 10-20% lower than expected but these
results showed no pattern with humic fluorescence or salinity, so
perhaps other factors such as heavy metals interfere with the
analysis in some rivers (Koroleff, 1983).
Flebbe, P. 1982. Biogeochemistry of carbon, nitrogen, and
phosphorus in the
aquatic subsystem of selected Okefenokee
Swamp sites. Okefenokee Ecosystem Investigations no.
Koroleff, F. 1976. Determination of ammonia. Pages 126-133 in
editor. Methods of seawater analysis.
Verlag Chemie, Weinheim.
Koroleff, F. 1983. Determination of ammonia. Pages 150-157 in
Grasshoff, K., M.
Ehrhardt, and K. Kremling, editors.
Methods of seawater analysis: second, revised and extended
edition. Verlag Chemie, Weinheim.
Parsons, T.R., Y. Maita, and C.M. Lalli. 1984. A manual of
chemical and biological
methods for seawater analysis.
Pergamon Press, New York.