Method 366.0
Determination of Dissolved Silicate in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
Jia-Zhong Zhang, Cooperative Institute for Marine and Atmospheric Studies,
Rosenstiel School of Marine and Atmospheric Science, Atlantic Oceanographic and
Meteorological Laboratory, National Oceanic and Atmospheric Administration,
University of Miami, Miami, FL 33149
George A. Berberian, National Oceanic and Atmospheric Administration, Atlantic
Oceanographic and Meteorological Laboratory, Ocean Chemistry Division, Miami, FL
33149
Project Officer
Elizabeth J. Arar
Version 1.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Method 366.0
Determination of Dissolved Silicate in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
1.0	Scope and Application
1.1	This method provides a procedure for the
determination of dissolved silicate concentration in
estuarine and coastal waters. The dissolved silicate is
mainly in the form of silicic acid, H4Si04, in estuarine and
coastal waters. All soluble silicate, including colloidal
silicic acid, can be determined by this method. Long chain
polymers containing three or more silicic acid units do not
react at any appreciable rate1, but no significant amount
of these large polymers exists in estuarine and coastal
waters.23 This method is based upon the method of
Koroleff,4 adapted to automated gas segmented
continuous flow analysis.5"7
Chemical Abstracts Service
Analyte	Registry Numbers (CASRN)
Silicate	12627-13-3
1.2	A statistically determined method detection limit
(MDL) of 0.0012 mg Si/L has been determined by one
laboratory in seawaters of three different salinities.8 The
method is linear to 6.0 mg Si/L using a Flow Solution
System (Perstorp Analytical Inc., Silver Spring, MD).
1.3	Approximately 60 samples per hour can be
analyzed.
1.4	This method should be used by analysts
experienced in the use of automated gas segmented
continuous flow colorimetric analyses, and familiar with
matrix interferences and procedures for their correction.
A minimum of 6-months experience under supervision is
recommended.
2.0	Summary of Method
2.1	An automated gas segmented continuous flow
colorimetric method for the analysis of dissolved silicate
concentration is described. In the method, P-
molybdosilicic acid is formed by reaction of the silicate
contained in the sample with molybdate in acidic solution.
The P-molybdosilicic acid is then reduced by ascorbic
acid to form molybdenum blue. The absorbance of the
molybdenum blue, measured at 660 nm, is linearly
proportional to the concentration of silicate in the sample.
A small positive error caused by differences in the
refractive index of seawater and reagent water, and
negative error caused by the effect of salt on the color
formation, are corrected prior to data reporting.
3.0	Definitions
3.1	Calibration Standard (CAL) — A solution
prepared from the primary dilution standard solution or
stock standard solution containing analytes. The CAL
solutions are used to calibrate the instrument response
with respect to analyte concentration.
3.2	Dissolved Analyte (DA) — The concentration of
analyte in an aqueous sample that will pass through a
0.45 /im membrane filter assembly prior to sample
acidification or other processing.
3.3	Laboratory Fortified Blank (LFB) — An aliquot
of reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine
whether method performance is within acceptable control
limits, and whether the laboratory is capable of making
accurate and precise measurements.
This is basically a standard prepared in reagent water that
is analyzed as a sample.
3.4	Laboratory Fortified Sample Matrix (LFM) — An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
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3.5	Laboratory Reagent Blank (LRB) - An aliquot
of reagent water that is treated exactly as a sample
including exposure to all labware, equipment, and
reagents that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
the reagents, or apparatus.
3.6	Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.7	Method Detection Limit (MDL) — The minimum
concentration of an analyte that can be identified,
measured and reported with 99% confidence that the
analyte concentration is greater than zero.8
3.8	Reagent Water (RW) — Type 1 reagent grade
water equal to or exceeding standards established by
American Society for Testing and Materials (ASTM).
Reverse osmosis systems or distilling units followed by
Super-Q Plus Water System that produce water with 18
megohm resistance are examples of acceptable water
sources.
3.9	Refractive Index (Ri) — The ratio of velocity of
light in a vacuum to that in a given medium. The relative
refractive index is the ratio of the velocity of light in two
different media, such as estuarine or sea water versus
reagent water. The correction for this difference is
referred to as refractive index correction in this method.
3.10	Stock Standard Solution (SSS) — A
concentrated solution of method analyte prepared in the
laboratory using assayed reference compounds or
purchased from a reputable commercial source.
3.11	Quality Control Sample (QCS) - A solution of
method analyte of known concentrations which is used to
fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.12	SYNC Peak Solution - A colored solution used
to produce a synchronization peak in the refractive index
measurement. A synchronization peak is required by
most data acquisition programs to initialize the peak
finding parameters. The first cup in every run must always
be identified as a SYNC sample. The SYNC sample is
usually a high standard, but can be any sample that
generates a peak at least 25% of full scale.
4.0	Interferences
4.1	Interferences caused by hydrogen sulfide, such
as occur in samples taken from deep anoxic basins can
be eliminated by oxidation with bromine or stripping with
nitrogen gas after acidification. Interferences of
phosphate at concentrations larger than 0.15 mg P/L is
eliminated by the use of oxalic acid in the color
development step of this method. Interferences of fluoride
at concentrations greater than 50 mg F/L can be reduced
by complexing the fluoride with boric acid.4
4.2	Glassware made of borosilicate glass should be
avoided for use in silicate analysis. Plastic labware such
as polyethylene volumetric flasks and plastic sample
vials, should be used.
4.3	Sample turbidity and particles are removed by
filtration through a 0.45 |jm non-glass membrane filters
after sample collection.
4.4	This method corrects for refractive index and salt
error interferences which occur if sampler wash solution
and calibration standards are not matched with samples
in salinity.
4.5	Frozen samples should be filled about 3/4 full in
the sample bottles. The expansion of water on freezing
will squeeze some of the brine out of the bottle if the
bottle was overfilled. The overfill of the sample bottle
during freezing will drastically alter the nutrient
concentrations in the sample that remains.
5.0	Safety
5.1	Water samples collected from the estuarine and
coastal environment are generally not hazardous.
However, the individual who collects samples should use
proper technique.
5.2	Good laboratory technique should be used when
preparing reagents. A lab coat, safety goggles, and
gloves should be worn when preparing the sulfuric acid
reagent.
6.0	Equipment and Supplies
6.1	Gas Segmented Continuous Flow
Autoanalyzer Consisting of:
6.1.1 Autosampler.
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6.1.2	Analytical cartridge with reaction coils for silicate
analysis.
6.1.3	Proportioning pump.
6.1.4	Monochromator or spectrophotometer equipped
with a tungsten lamp (380-800 nm) and a low refractive
index flowcell.
6.1.5	Strip chart recorder or computer based data
acquisition system.
6.2 Glassware and Supplies
6.2.1	All labware used in the analysis must be low in
residual silicate to avoid sample or reagent
contamination. Soaking with lab grade detergent, rinsing
with tap water, followed by rinsing with 10% HCI (v/v) and
thoroughly rinsing with reagent water was found to be
effective.
6.2.2	Glassware made of borosilicate glass should be
avoided for storage of solutions for silicate analysis.
Plastic containers are preferable for silicate analysis.
6.2.3	Non-glass membrane filters with 0.45 |jm
nominal pore size. Plastic syringes with syringe filters,
pipets, 60 mL polyethylene bottles, and polyethylene
volumetric flasks, plastic sample vials.
6.2.4	Drying oven, desiccator and analytical balance.
7.0	Reagents and Standards
7.1	Stock Reagent Solutions
7.1.1	Sulfuric Acid Solution (0.05 M) - Cautiously add
2.8 mL of concentrated Analytical Reagent Grade sulfuric
acid (H2S04) to approximately 800 mL of reagent water,
mix then bring up to 1 L with reagent water.
7.1.2	Ammonium Molybdate Solution (10 g/L) -
Dissolve 10 g of ammonium molybdate (VI) tetrahydrate
((NH4)6Mo7024.4H20) in approximately 800 mL of 0.05 M
sulfuric acid solution and dilute to 1000 mL with 0.05 M
sulfuric acid solution. Store in an amber plastic bottle.
This solution is stable for one month. Inspect the solution
before use. If a white precipitation forms on the wall of
container, discard the solution and make a fresh one.
7.1.3	Stock Silicate Solution (100 mg Si/L) -
Quantitatively transfer 0.6696 g of pre-dried (105°C for 2
hours) sodium hexafluorosilicate (Na2SiF6) to a 1000 mL
polypropylene flask containing approximate 800 mL of
reagent water, cover with plastic film and dissolve on a stir
plate using a Teflon-coated stirring bar. Complete
dissolution usually takes 2-24 hours. Dilute the solution to
1000 mL in polyethylene volumetric flask with reagent
water. Store the stock solution in a plastic bottle. This
solution is stable for one year if care is taken to prevent
contamination and evaporation.
7.1.4 Low Nutrient Sea Water (LNSW) - Obtain natural
low nutrient seawater from surface seawater in the Gulf
Stream or Sargasso Sea (salinity 36 %o, < 0.03 mg Si/L)
and filter through 0.45 |jm pore size non-glass membrane
filters. In addition, commercially available low nutrient sea
water ( < 0.03 mg Si/L) with salinity of 35 %o (Ocean
Scientific International, Wormley, U.K.) can be used.
7.2 Working Reagents
7.2.1	Dowfax Start-up Solution - Add 2 mL of Dowfax
2A1 surfactant (Dow Chemical Company) to 1000 mL
reagent water and mix gently.
Note: Dowfax 2A1 contains (w/w) 47% benzene, 1,1-
oxybis, tetrapropylene derivatives, sulfonate, sodium salt,
1% sodium sulfate, 3% sodium chloride and 49% water.
7.2.2	Working Molybdate Reagent - Add 0.5 mL
Dowfax 2A1 to 250 mL of ammonium molybdate solution,
mix gently. Prepare this solution daily. This volume of
solution is sufficient for an 8-hour run.
7.2.3	Ascorbic Acid Solution - Dissolve 4.4 g of
ascorbic acid (C6H806) in 200 mL of reagent water and
12.5 mL of acetone(C3H60), dilute to 250 mL with reagent
water. Store in a plastic container. This solution is stable
for one week if stored at 4°C. Discard the solution if it
turns brown.
7.2.4	Oxalic Acid Solution - Dissolve 50 g of oxalic acid
(C2H204) in approximately 800 mL of reagent water and
dilute to 1000 mL with reagent water. Store in a plastic
container. This solution is stable for approximately 3-
months.
7.2.5	Refractive Index Matrix Solution - Add 0.5 mL
Dowfax 2A1 to 250 mL of 0.05 M sulfuric acid solution
and mix gently.
7.2.6	Colored SYNC Peak Solution - Add 50 |jL of blue
food coloring solution to 1000 mL reagent water and mix
thoroughly. The solution should give a peak of between
25 to 100 percent full scale, otherwise the volume of food
coloring added needs to be adjusted.
7.2.7	Calibration Standards - Prepare a series of
calibration standards (CAL) by diluting suitable volumes
of Stock Silicate Solution (Section 7.1.3) to 100 mL with
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reagent water or low nutrient seawater. Prepare these
standards daily. The concentration range of calibration
standards should bracket the expected concentrations of
samples and not exceed two orders of magnitude. At
least five calibration standards with equal increments in
concentration should be used to construct the calibration
curve.
When working with samples of a narrow range of
salinities (± 2 %o), it is recommended that the CAL
solutions be prepared in Low Nutrient Seawater (Section
7.1.4) diluted to the salinity of samples, and the Sampler
Wash Solution also be Low Nutrient Seawater (Section
7.1.4) diluted to that salinity. If this procedure is
performed, it is not necessary to perform the salt error
and refractive index corrections outlined in Sections 12.2
and 12.3.
When analyzing samples of varying salinities, it is
recommended that the calibration standard solutions and
sampler wash solution be prepared in reagent water and
corrections for salt error and refractive index be made to
the sample concentrations (Section 12.2 and 12.3).
7.2.8 Saline Silicate Standards - If CAL solutions will
not be prepared to match sample salinity, then saline
silicate standards must be prepared in a series of
salinities in order to quantify the salt error, the change in
the colorimetric response of silicate due to the change in
the ionic strength of the solution. The following dilutions
prepared in 100 mL volumetric flasks, diluted to volume
with reagent water, are suggested.
Salinity Volume of Volume(mL) Cone.
(%0) LNSW(mL) Si stock std mg Si/L
0
0
1.5
1.5
9
25
1.5
1.5
18
50
1.5
1.5
27
75
1.5
1.5
35
98
1.5
1.5
8.0	Sample Collection, Preservation
and Storage
8.1	Sample Collection - Samples collected for
nutrient analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Go-Flo or equivalent) that are attached at fixed
intervals to a hydro wire. These bottles are sent through
the water column open and are closed either
electronically or via a mechanical messenger when the
bottles have reached the desired depth.
8.1.2	In a submersible pump system, a weighted hose
is sent to the desired depth in the water column and water
is pumped from that depth to the deck of the ship for
sample processing.
8.1.3	For collecting surface samples, an acid - cleaned
plastic bucket or a large plastic bottle can be used as
convenient samplers. Wash the sampler three times with
sample water before collecting samples.
8.1.4	Samples must be filtered through a 0.45 |jm non-
glass membrane filters as soon as possible after
collection.
8.1.5	60-mL high density polyethylene bottles are used
for sample storage. Sample bottles should be rinsed 3
times with about 20 mL of sample, shaking with the cap
in place after each rinse. Pour the rinse water into the cap
to dissolve and rinse away salt crusts trapped in the
threads of the cap. Finally, fill the sample bottle about 3/4
full, and screw the cap on firmly. The expansion of water
on freezing will squeeze some of the brine out of the
bottle if the bottle was overfilled.
8.2	Sample Preservation - After collection and
filtration, samples should be analyzed as soon as
possible. If samples will be analyzed within 24 hours then
keep refrigerated in tightly sealed, high density
polyethylene bottles in the dark at 4°C until analysis can
be performed.
8.3	Sample Storage - If samples are to be frozen
for long-term storage ensure that none of the sample
bottles are filled more than 3/4 full and the cap is firmly
screwed on. Place the bottles upright on a rack and store
in the freezer (-20°C).
Before analysis, frozen samples must be taken out of the
freezer and allowed to thaw in a refrigerator at 4°C in the
dark. Thawing times depend upon sample salinities. It
was found that the frozen low salinity estuarine water took
4 days to thaw. After completely thawing, take samples
out of the refrigerator and mix thoroughly. Keep samples
in the dark at room temperature overnight before
analysis.
Effects of thawing conditions on the recoveries of frozen
samples are more pronounced in low salinity estuarine
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waters than high salinity coastal waters as shown in each set of samples as a continuing check on
following results:	performance.
Day
Recoverv
%)
Remark
9.2 Initial Demonstration of Performance
(Mandatory)

S=35.85
S=18.07
S=2.86

0
100.00
100.00
100.00

9.2.1 The Initial demonstration of performance is used
7
102.44
102.65
89.37
a
to characterize instrument performance by determining
14
98.59
101.06
86.49
a
the MDL and LDR and laboratory performance by
21
99.51
99.30
83.49
a
analyzing quality control samples prior to analysis of
27
98.86


a
samples using this method.


98.86
91.43
b

35
98.70


b
9.2.2 Method Detection Limits (MDLs) should be


98.66
92.98
b
established using a low level seawater sample containing,
42
100.87


b
or fortified at, approximately 5 times the estimated
49

102.44
79.12
c
detection limit. To determine MDL values, analyze at least


103.92
79.10
d
seven replicate aliquots of water which have been


99.92
89.68
e
processed through the entire analytical method. Perform
56
103.47


c
all calculations defined in the method and report

104.12


d
concentration in appropriate units. Calculate the MDL as

99.35


e
follows:
84

100.80
91.71
f



99.90
93.81
g
MDL = (t)(S)
91
100.65


f


100.22


g
where, S = the standard deviation of the
replicate analyses
S = Salinity
a,	overnight thawing at room temperature
b,	20 hours thawing at room temperature
c,	24 hours thawing at room temperature
d,	8 hours thawing at room temperature then
heating at 80°C for 16 hours
e,	24 hours thawing at room temperature in the dark
f,	4 days thawing at room temperature in the dark
g,	4 days thawing at 4°C in a refrigerator in the dark
To ensure a slow process of depolymerization of
polysilicate to occur, thawing the frozen samples in the
dark at 4°C for 4 days is critical condition for obtaining
high recoveries of silicate in frozen samples. A maximum
holding time for frozen estuarine and coastal waters is
two months.9"11
9.0	Quality Control
9.1	Each laboratory using this method is required to
implement a formal quality control(QC) program. The
minimum requirements of this program consists of an
initial demonstration of performance, continued analysis
of Laboratory Reagent Blanks (LRB), laboratory
duplicates and Laboratory Fortified Blanks (LFB) with
t = Student's t value for n-1 degrees of
freedom at the 99% confidence
limit; t = 3.143 for six degrees of
freedom.
MDLs should be determined every 6-months or whenever
a significant change in background or instrument
response occurs or a new matrix is encountered.
9.2.3 The LDR should be determined by analyzing a
minimum of eight calibration standards ranging from 0.03
to 5.00 mg Si/L across all sensitivity settings (Absorbance
Units Full Scale) of the detector. Standards and sampler
wash solutions should be prepared in low nutrient
seawater with salinities similar to that of samples,
therefore a correction factor for salt error, or refractive
index, will not be necessary. Normalize responses by
multiplying the response by the Absorbance Units Full
Scale output range setting. Perform the linear regression
of normalized response vs. concentration and obtain the
constants m and b, where m is the slope and b is the y-
intercept. Incrementally analyze standards of higher
concentration until the measured absorbance response,
R, of a standard no longer yields a calculated
concentration Cc, that is within 100 ± 10% of the known
concentration, C, where Cc = (R-b)/m. That concentration
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defines the upper limit of the LDR for the instrument.
Should samples be encountered that have a
concentration that is 90% of the upper limit of LDR, then
these samples must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1	Laboratory Reagent Blank (LRB) - A laboratory
should analyze at least one LRB with each set of
samples. LRB data are used to assess contamination
from the laboratory environment. Should an analyte value
in the LRB exceed the MDL, then laboratory or reagent
contamination should be suspected. When the LRB value
constitutes 10% or more of the analyte concentration
determined for a sample, duplicates of the sample must
be prepared and analyzed again after the source of
contamination has been corrected and acceptable LRB
values have been obtained.
9.3.2	Laboratory Fortified Blank (LFB) - A laboratory
should analyze at least one LFB with each set of
samples. The LFB must be at a concentration that is
within the daily calibration range. The LFB data are used
to calculate accuracy as percent recovery. If the recovery
of the analyte falls outside the required control limits of
90 -110%, the source of the problem should be identified
and resolved before continuing the analyses.
9.3.3	The laboratory must use LFB analyses data to
assess laboratory performance against the required
control limits of 90 -110%. When sufficient internal
performance data become available (usually a minimum
of 20 to 30 analyses), optional control limits can be
developed from the percent mean recovery (x) and
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery -
Laboratory Fortified Sample Matrix
(LFM)
9.4.1	A laboratory should add a known amount of
analyte to a minimum of 5% of the total number of
samples or one sample per sample set, whichever is
greater. The analyte added should be 2-4 times the
ambient concentration and should be at least four times
greater than the MDL.
9.4.2	Calculate percent recovery of analyte, corrected
for background concentration measured in a separate
unfortified sample. These values should be compared
with the values obtained from the LFBs. Percent
recoveries may be calculated using the following
equation:
(Cs-C)
R =	X 100
S
where, R = percent recovery
Cs = measured fortified sample
concentration (background +
addition in mg Si/L)
C = sample background concentration
(mg Si/L)
S = concentration in mg Si/L added to the
environmental sample.
9.4.3 If the recovery of the analyte falls outside the
required control limits of 90-110%, but the laboratory
performance for that analyte is within the control limits,
the fortified sample should be prepared again and
analyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample is
judged to be matrix related and the sample data should
be flagged.
10.0	Calibration and Standardization
10.1	At least five calibration standards should be
prepared daily for system calibration.
10.2	A calibration curve should be constructed for
each run by analyzing a set of calibration standard
solutions. A run should contain no more than 60 samples.
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It is suggested that a large set of samples be analyzed in
several sets with individual calibration curves.
10.3	Place the calibration standards before samples
for each run. All the calibration solutions should be
analyzed in duplicate.
10.4	The calibration curve containing five data points
or more should have a correlation coefficient 0.995.
10.5	Place a high standard solution cup and follow by
two blank cups to quantify the carry-over of the system.
The difference in peak heights between two blank cups is
due to the carry over from the high standard cup. The
carry-over coefficient, k, is calculated as follows:
Pb1 " Pb2
k =	X 100
Phigh
where, Phigh = the peak height of the high
silicate standard
Pb1 = the peak height of the first
blank sample
Pb2 = the peak height of the second
blank sample.
The carry over coefficient, k, for a system should be
measured in seven replicates in order to obtain a
statistically significant number. The k should be
remeasured when a change in the plumbing of the
manifold or replacement of pump tube occur.
The carry over correction (CO) on a given peak, i, is
proportional to the peak height of the preceding sample,
Pm-
CO = kxPM
To correct a given peak height reading, Ph one subtracts
the carry over correction.1213
Pi c = P; - CO
where Pic is corrected peak height. The correction for
carry over should be applied to all the peak heights
throughout a run. The carry over should be less than 2%.
10.6	Place a high standard solution at the end of a run
to check sensitivity drift. The sensitivity drift should be ±
5% during the run.
11.0	Procedure
11.1	If samples are frozen, thaw the sample at 4°C in
the dark as outlined in Section 8.3. Mix samples
thoroughly prior to analyses.
11.2	Turn on the continuous flow analyzer and PC
components and warm up at least 30 minutes.
11.3	Set up the cartridge and pump tubes as shown in
Figure 1.
Note: Fluctuation of ambient temperature can cause
erratic results due to the effect of temperature on kinetics
of color development. The laboratory temperature should
be maintained as close to a constant temperature as
possible. The cartridge should be away from the direct
path of air flow from a heater or air conditioner. In cases
such as on a ship where the fluctuation of temperature
can be extreme, the temperature effect can be minimized
by increasing the length of mixing coil 1 (Figure 1) to bring
the formation of silicomolybdic acid reaction to
completion.
11.4	Set the wavelength at 660 nm on the
spectrometer/monochrometer.
Note: The absorption spectra of silicomolybdeum blue
complex has two maxima at 820 nm and 660 nm with 820
nm higher than 660 nm. This method measures
absorbance at 660 nm because the detector works in the
range of 380 to 800 nm. The sensitivity of the method is
satisfactory at 660 nm. The sensitivity, however, can be
improved by using 820 nm if this wavelength is available
on the detector.
11.5	On the monochromator, set the Absorbance Unit
Full Scale at an appropriate setting according to the
highest concentration of silicate in the samples. The
highest setting used in this method was 0.2 for 6 mg Si/L.
11.6	Prepare all reagents and standards.
11.7	Begin pumping the Dowfax start-up solution
(Section 7.2.1) through the system and obtain a steady
baseline. Place the reagents on-line. The reagent
baseline will be higher than the start-up solution baseline.
After the reagent baseline has stabilized, reset the
baseline.
NOTE: To minimize the noise in the reagent baseline,
clean the flow system by sequentially pumping the
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sample line with reagent water, 1 N HCI solution, reagent
water, 1N NaOH solution for a few minutes each at the
end of the daily analysis. Make sure to rinse the system
well with reagent water after pumping NaOH solution to
prevent precipitation of Mg(OH)2 when seawater is
introduced into the system. Keep the reagents and
samples free of particulate. Filter the reagents and
samples if necessary.
If the baseline drifts upward, pinch the waste line for a few
seconds to increase back pressure. If absorbance drops
down rapidly when back pressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach
a syringe at the waste outlet of the flowcell. Air bubbles
in the flowcell can often be eliminated by simply attaching
a syringe for a few minutes or, if not, dislodged by
pumping the syringe pistion. Alternatively, flushing the
flowcell with alcohol was found to be effective in removing
air bubbles from the flowcell.
For analysis of samples with a narrow range of salinities
(± 2 %o), it is recommended that the wash water in the
sampler be prepared in Low Nutrient Seawater diluted to
the salinity of samples in place of reagent water. For
samples with varying salinities, it is suggested that reagent
waters and procedures in Sections 12.2 and 12.3 be
employed.
11.8	A good sampling rate is approximately 60
samples per hour with 40 seconds of sample time and 20
seconds of wash time.
11.9	Use 10% HCI followed by reagent water to rinse
sample cups. Place CAL solutions and saline standards
(optional) in sampler. Complete filling the sampler tray
with samples, laboratory reagent blanks, laboratory
fortified blanks, laboratory fortified sample matrices, and
QC samples. Place a blank every ten samples and
between samples of high and low concentrations.
11.10	Commence analysis.
11.11	If the reagent water is used as wash solution
instead of Low Nutrient Seawater and an operator wants
to quantify the refractive index correction due to the
difference in salinities between sample and wash solution,
the following procedures are used. Replace ammonium
molybdate solution (Section 7.1.2) with refractive index
matrix solution (Section 7.2.5). All other reagents remain
the same. Replace the synchronization cup with the
colored SYNC peak solution (Section 7.2.6). Commence
analysis and obtain a second set of peak heights for all
standards and samples. The peak heights obtained from
these measurements must be subtracted from the peak
heights of samples analyzed with color developing
reagent pumping through the system. If a low refractive
index flowcell is used, the correction for refractive index is
negligible. This procedure is optional.
12.0	Data Analysis and Calculations
12.1	Concentrations of silicate are calculated from the
linear regression, obtained from the standard curve in
which the concentrations of the calibration standards are
entered as the independent variable, and their
corresponding peak heights are the dependent variable.
12.2	Refractive Index Correction for Estuarine and
Coastal Samples (optional)
12.2.1	Obtain a second set of peak heights for all
standards and samples with refractive index matrix
solution being pumped through the system in place of
color reagent (ammonium molybdate solution). All other
reagents remain the same. The peak heights for the
refractive index correction must be obtained at the same
Absorbance Unit Full Scale range setting and on the
same monochromator as the corresponding samples and
standards.
12.2.2	Subtract the refractive index peak heights from
the peak heights obtained from the silicate determination.
12.2.3	An alternative approach is to measure the
relationship between the sample salinity and refractive
index on a particular detector.
First analyze a set of silicate standards in reagent water
with color reagent and obtain a linear regression from the
standard curve.
Prepare a set of different salinity samples with LNSW.
Analyze these samples with refractive index matrix
solution being pumped through the system in place of
color reagent (ammonium molybdate solution). All other
reagents remain the same. The peak heights for the
refractive index correction must be obtained at the same
Absorbance Unit Full Scale setting and on the same
monochromator as the corresponding standards.
For each sample, the apparent silicate concentration due
to refractive index is then calculated from its peak height
obtained with refractive index reagent and the regression
of silicate standards obtained with color reagent pumping
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through the system. Salinity is entered as the independent
variable and the apparent silicate concentration due to
refractive index in that detector is entered as the
dependent variable. The resulting regression allows the
operator to calculate apparent silicate concentration due
to refractive index when the salinity is known. Thus, the
operator would not be required to obtain refractive index
peak heights for all samples.
12.2.4 Refractive index correction can be minimized by
using a low refractive index flowcell. An example of typical
results using a low refractive index flowcell follows:
Salinity
(%o)
Apparent silicate conc. due
to refractive index (mg Si/L)
4.5
0.0003
9.0
0.0005
18.0
0.0016
27.0
0.0027
36.0
0.0042
12.2.5 An example of a typical equation is:
Apparent silicate (mg Si/L) = 0.00001953*S1 5
where S is sample salinity. The form of fitted equation
might vary as the design of flowcell, so the operators are
advised to obtain the appropriate equation which has the
best fit of their own data with the least fitting coefficients.
12.3 Correction for Salt Error in Estuarine and
Coastal Samples
12.3.1	When calculating concentrations of samples of
varying salinities from standards and wash solution
prepared in reagent water, it is usual to first correct for
refractive index errors, then correct for the change in color
development due to the differences in ionic strength
between samples and standards (salt error). The
refractive index correction is negligible, so is optional, but
correction for salt error is necessary.
12.3.2	Plot the salinity of the saline standards (Section
7.2.8) as the independent variable, and the apparent
concentration of silicate (mg Si/L) from the peak height
(corrected for refractive index) calculated from the
regression of standards in reagent water, as the
dependent variable for all 1.50 mg Si/L standards. The
resulting regression equation allows the operator to
correct the concentrations of samples of known salinity
for the color suppression due to salinity effect, e.g., salt
error. An example of typical results follows:
Salinty
Peak height of
Uncorrected Si
(%o)
1.50 mg Si/L
conc.calculated


from standards


in reagent water
0
2503
1.50
9
2376
1.32
18
2282
1.27
27
2250
1.25
36
2202
1.23
12.3.3	An example of a typical equation to correct for
salt error is:
Uncorrected mg Si/L
Corrected mg Si/L =	
1 - 0.02186x/S
where S is salinity.
12.3.4	Results of sample analyses should be reported
in mg Si/L or in |jg Si/L.
mg Si/L = ppm (parts per million)
|jg Si/L = ppb (part per billion)
13.0	Method Performance
13.1	Single Laboratory Validation
13.1.1 Method Detection Limit - A method detection limit
(MDL) of 0.0012 mg Si/L has been determined by one
laboratory in seawaters of three different salinities.
Salinity
SD
Recovery
MDL
(%o)
(MQ/L)
(%)
(MQ/L)
36
0.3924
105
1.233
36
0.4980
107
1.565
27
0.2649
104
0.832
27
0.3362
104
1.056
27
0.4671
100
1.468
18
0.3441
101
1.081
18
0.2809
105
0.883
18
0.2432
104
0.764
3
0.3441
101
1.081
3
0.2331
102
0.733
3
0.1963
98
0.617
3
0.2809
99
0.883
13.1.2 Single Analyst Precision - A single laboratory
analyzed three samples collected from the Miami River
and Biscayne Bay areas of Florida. Seven replicates of
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each sample were processed and analyzed with salinities
ranging from 2.86 to 35.85. The results were as follows:
Sample Salinity Concentration RSD
(%0)	(mg Si/L)	(%)
1	35.85	0.097	1.2
2	18.07	1.725	1.4
3	2.86	3.322	0.9
13.1.3 Laboratory Fortified Sample Matrix - Laboratory
fortified sample matrixes were processed in three different
salinities ranging from 2.86 to 35.85 and ambient
concentrations from 0.095 to 3.322 mg Si/L with three
fortified levels at each salinity. Seven replicates of each
sample were analyzed and the results were as follows:
Salinity
(%o)
Concentration
(ma Si/L")
RSD
(%)
Recovery
(%)
Ambient
Fortified
35.85
0.095
0.1647
0.82
99.37
35.85
0.095
0.2196
1.34
100.61
35.85
0.095
0.2747
1.74
99.62
18.07
1.725
0.5517
1.11
107.18
18.07
1.725
1.1008
0.77
104.69
18.07
1.725
1.6508
0.98
103.62
2.86
3.322
0.5421
0.99
101.03
2.86
3.322
1.0801
1.26
103.22
2.86
3.322
1.6188
0.98
100.59
13.2 Multi-Laboratory Validation
Multi-laboratory validation has not been conducted for this
method and, therefore, multi-laboratory data is currently
unavailable.
14.0	Pollution Prevention
14.1	Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the next
best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society, Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0	Waste Management
15.1	The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in Section 14.2.
16.0 References
1 Chow, D. T-W., and Robinson, R.J. 1953, Forms of
silicate available for colorimetric determination.
Analytical Chemistry. 25, 646-648.
2. Burton, J. D., T.M. Leatherland and P.S. Liss, 1970.
The reactivity of dissolved silicon in some natural
waters. Limnology and Oceanography, 15, 473-
476.
3 Isshiki, K., Sohrin, Y, and Nakayama, E., 1991.
Form of dissolved silicon in seawater. Marine
Chemistry, 32, 1-8.
4. Koroleff, F. 1983, Determination of silicon, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp174 -187.
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5.	Grasshoff, K. 1965. On the automatic
determination of silicate, phosphate and fluoride in
seawater. I.C.E.S. Hydrographic Committee
Report, No. 129, Rome. (Mimeographed).
6.	Brewer P. G. and J. P. Riley. 1966. The automatic
determination of silicate-silicon in natural water with
special reference to sea water. Anal. Chim. Acta,
35, 514-519.
7.	Hansen, H.P., K.Grasshoff, Statham and P.J.LeB.
Williams. 1983, Automated chemical analysis, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp374 -395.
8.	40 CFR, 136 Appendix B. Definition and Procedure
for the Determination of Method Detection Limit.
Revision 1.11.
9.	MacDonald, R.W. and F.A. McLaughlin. 1982. The
effect of storage by freezing on dissolved inorganic
phosphate, nitrate, and reactive silicate for samples
from coastal and estuarine waters. Water
Research, 16:95-104.
10.	MacDonald, R.W. , F.A. McLaughlin and C. S.
Wong. 1986. The storage of reactive silicate
samples by freezing. Limnol. Oceanogr.,
31 (5): 1139-1142.
11.	Salley, B.A., J.G. Bradshaw, and B.J. Neilson.
1987. Results of comparative studies of
preservation techniques for nutrient analysis on
water samples. Virginia Institute of Marine Science,
Gloucester Point, VA 23062. USEPA, CBP/TRS
6/87, 32pp.
12.	Angelova, S, and H.W.Holy. 1983. Optimal speed
as a function of system performance for continuous
flow analyzers. Analytica Chimica Acta, 145:51-58.
13.	Zhang, J.-Z. 1997. Distinction and quantification of
carry-over and sample interaction in gas
segmented continuous flow analysis. J. Automatic
Chemistry, 19(6):205-212.
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Debubbler
T o Waste —
0'
Detector
660nm
\ /
T o Waste
Coil2
o
10
O-
° • "1
8 O (3
6

a:
o 3
2 a
1
Coil3

Manifold
Wash To Sampler
0.41
0.41
0.32
0.41
0.25
0.41
1.57
Ascorbic Acid
Oxalic Acid
Sample
Air
Molybdate
Reagent Water
or Low Nutrient Seawater
Pump
ml/min
Sample:Wash = 20":40"
Figure 1. Manifold Configuration for Silicate Analysis.
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