ANALYSIS METHODS MANUAL
FOR HUMIC-CONTAINING ESTUARINE WATERS
Joan E. Sheldon and
Dr. William J.
Revision Date: June 7, 1997
Initially we tried this standard method with no dilution (or lesser
dilution for >100 uM Si samples). Humics interfered with
recovery of silicate. A color blank using appropriately diluted
sulfuric acid solution without molybdate (APHA Standard
methods..., 1981) could not completely correct for the
interference: "corrected" recoveries of Na2SiF6 added to humic-
colored water were low.
Filter through 0.6 um polycarbonate filter (Poretics) in
plastic apparatus, used only for similar samples.
in polyethylene bottle at 4 oC
4.5 M H2SO4:
Carefully (slowly and with stirring) add 250 mL
concentrated (sp. gr. = 1.84 g cm-³
H2SO4 to 750 mL water. Cool and dilute
to 1 L with water (use plastic graduated cylinder rather
than volumetric glassware).
Dissolve 38 g
in 300 mL water. Add this to 300 mL 4.5 M H2SO4.
DO NOT ADD ACID TO MOLYBDATE. Use an all-plastic automatic dispenser.
Store in dark.
Saturated Oxalic Acid Solution:
Dissolve 80 g (COOH)2.2H2O in
800 mL water. Use an all-plastic automatic dispenser.
Dissolve 2.8 g C6H8O6 in 100 mL
water. Use an automatic dispenser (may be brown glass). Store in a
dark bottle in refrigerator.
10 mM Primary:
Dry Na2SiF6 overnight at 105 oC. Dissolve 0.9403 g
in about 100 mL water in a plastic beaker, warming if
necessary, and quickly make up to 500 mL with water in a
glass volumetric flask. Store in plastic bottle.
1 mM Secondary:
0.5 mL Primary Standard + 4.5 mL water, or similar convenient
amount. Make fresh each day.
Make fresh each day:
10 uM: 4.950 mL water + 50 uL Secondary Std.
20 uM: 4.900 mL water + 100 uL Secondary Std.
40 uM: 4.800 mL water + 200 uL Secondary Std.
Plastic test tubes (15 mL centrifuge tubes are fine), beakers,
graduated cylinders, and bottles for making and storing
reagents and performing analysis.
Koroleff (1983) method but dilute samples considerably before
analysis, for several reasons. Silicate in Georgia rivers
is very high, often above the range in which Beer's Law
applies for this analysis (which is very sensitive).
Salinity requires a correction, but this can be avoided by
dilution with fresh water. Humic material interferes with
the analysis, but this can also be avoided by dilution.
Dissolved inorganic silicate is determined.
Make up triplicate blanks and working standards in tubes, 5 mL
each. Pipette 0.5 mL (if salinity is < 25 ppt) or 1 mL (if
salinity is >= 25 ppt) of each sample into 3 tubes. Make up
to 5 mL with water. Add 200 uL of acid molybdate to each
tube and vortex. Let stand for 20 min, then add 200 uL
oxalic acid, vortex, immediately add 100 uL ascorbic acid,
and vortex. (Try to be very consistent in timing the
reagent additions to tubes in racks so that standing time
between acid molybdate and oxalic acid is the same for EACH
TUBE.) Keep tubes in a dark place until read at least 3
hours but not more than 6 hours later.
Read the absorbance in a 1 cm cell at 810 nm (against the
absorbance of water), using a sample sipper or cuvette.
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
cuvette, rinse once with sample.
Tubes may be simply rinsed 4 times with 18 megohm-cm water and
allowed to dry between uses.
Plot the standard concentration vs. absorbance. The slope of
this line is the calibration factor F. F should be
approximately 47 for a 1 cm cell and is very dependent on
timing between reagent additions.
Correct sample calculations for dilution where necessary.
Dilution factor = 10 if sample salinity was < 25 ppt (i.e.
1:10 dilution used) or Dilution factor = 5 if sample
salinity was >= 25 ppt (i.e. 1:5 dilution used).
Sample concentration (uM) = F × (sample abs - average blank abs)
× Dilution factor
With dilution of seawater samples according to this protocol,
errors due to salinity are reduced to perhaps 2% at most
(Koroleff, 1983) and therefore can be ignored. Color blanks
for diluted samples (APHA Standard methods..., 1981), even
those of originally high humic content, are zero and need
not be run.
Limit of Detection:
With 18 megohm-cm water, blanks are all 0.000 so
std. dev. = 0. With a minimum absorbance reading of 0.001, limit
of detection = 0.05 uM if sample is not diluted, or 0.5 uM if
sample is diluted 1:10.
100 uM if sample is not diluted (absorbance is near 1.9).
Dilute appropriately above this range.
95% confidence limits for prediction of a concentration
near the mean for a typical standard curve (17.5 uM) approx. =
± 2.57 uM for a triplicate determination if sample is not
diluted, or ± 25.7 uM for a sample near 175 uM which is diluted
1:10. (Confidence limits for prediction near the ends of a
standard curve are, of course, somewhat larger.)
American Public Health Association Standard methods for the
water and wastewater: 15th edition. 1981.
American Public Health Association, Washington, D.C.
Koroleff, F. 1983. Determination of silicon. Pages 174-183 in
Grasshoff, K., M.
Ehrhardt, and K. Kremling, editors.
Methods of seawater analysis: second, revised and extended
edition. Verlag Chemie, Weinheim.