366.0-1

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
Version 1.0 September 1997 366.0-2

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, H4SiO4, 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 rate , but no significant amount 1

of these large polymers exists in estuarine and coastal

waters. This method is based upon the method of

2,3

Koroleff, adapted to automated gas segmented

4

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. The 8

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, $-

molybdosilicic acid is formed by reaction of the silicate

contained in the sample with molybdate in acidic solution.

The $-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 Fm 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. 
 366.0-3 Version 1.0 September 1997

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 μm 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.
Version 1.0 September 1997 366.0-4

6.1.2 Analytical cartridge with reaction coils for silicate plate using a Teflon-coated stirring bar. Complete

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% HCl (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 μm

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 (H SO ) to approximately 800 mL of reagent water, 2 4

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)6Mo7O24.4H2O) 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 o

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

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 μm 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 (C6H8O6) in 200 mL of reagent water and

12.5 mL of acetone(C H O), dilute to 250 mL with reagent 3 6

water. Store in a plastic container. This solution is stable

for one week if stored at 4 C. Discard the solution if it

o

turns brown.

7.2.4 Oxalic Acid Solution - Dissolve 50 g of oxalic acid

(C2H2O4) 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 μL 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
 366.0-5 Version 1.0 September 1997

reagent water or low nutrient seawater. Prepare these intervals to a hydro wire. These bottles are sent through

standards daily. The concentration range of calibration the water column open and are closed either

standards should bracket the expected concentrations of electronically or via a mechanical messenger when the

samples and not exceed two orders of magnitude. At bottles have reached the desired depth.

least five calibration standards with equal increments in

concentration should be used to construct the calibration 8.1.2 In a submersible pump system, a weighted hose

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) Conc.

(%o) 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

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 μm nonglass 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 o

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). o

Before analysis, frozen samples must be taken out of the

freezer and allowed to thaw in a refrigerator at 4 C in the o

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 
Version 1.0 September 1997 366.0-6

waters than high salinity coastal waters as shown in each set of samples as a continuing check on

following results: performance.

_______________________________________

Day Recovery (%) Remark 9.2 Initial Demonstration of Performance

S=35.85 S=18.07 S=2.86 (Mandatory)

_______________________________________

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

14 98.59 101.06 86.49 a

21 99.51 99.30 83.49 a

27 98.86 a

98.86 91.43 b

35 98.70 b

98.66 92.98 b

42 100.87 b

49 102.44 79.12 c

103.92 79.10 d

99.92 89.68 e

56 103.47 c

104.12 d

99.35 e

84 100.80 91.71 f

99.90 93.81 g

91 100.65 f

100.22 g

______________________________________

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 o

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 o

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

o

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

to characterize instrument performance by determining

the MDL and LDR and laboratory performance by

analyzing quality control samples prior to analysis of

samples using this method.

9.2.2 Method Detection Limits (MDLs) should be

established using a low level seawater sample containing,

or fortified at, approximately 5 times the estimated

detection limit. To determine MDL values, analyze at least

seven replicate aliquots of water which have been

processed through the entire analytical method. Perform

all calculations defined in the method and report

concentration in appropriate units. Calculate the MDL as

follows:

MDL = (t)(S)

where, S = the standard deviation of the 

replicate analyses

 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 yintercept. Incrementally analyze standards of higher

concentration until the measured absorbance response,

R, of a standard no longer yields a calculated

concentration C , that is within 100 ± 10% of the known c

concentration, C, where C = (R-b)/m. That concentration c 
 366.0-7 Version 1.0 September 1997

defines the upper limit of the LDR for the instrument. 9.4 Assessing Analyte Recovery -

Should samples be encountered that have a Laboratory Fortified Sample Matrix 

concentration that is 90% of the upper limit of LDR, then (LFM)

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 greater than the MDL. 

samples. LRB data are used to assess contamination

from the laboratory environment. Should an analyte value 9.4.2 Calculate percent recovery of analyte, corrected

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 S

samples. The LFB must be at a concentration that is

within the daily calibration range. The LFB data are used where, R = percent recovery

to calculate accuracy as percent recovery. If the recovery

of the analyte falls outside the required control limits of C = measured fortified sample

90 -110%, the source of the problem should be identified concentration (background + 

and resolved before continuing the analyses. addition in mg Si/L)

9.3.3 The laboratory must use LFB analyses data to C = sample background concentration

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 required control limits of 90-110%, but the laboratory

limits as follows: performance for that analyte is within the control limits,

Upper Control Limit = x + 3S analyzed. If the result is the same after reanalysis, the

Lower Control Limit = x - 3S recovery problem encountered with the fortified sample is

The optional control limits must be equal to or better than be flagged.

the required control limits of 90-110%. After each 5 to 10

new recovery measurements, new control limits can be 10.0 Calibration and Standardization

calculated using only the most recent 20 to 30 data

points. Also the standard deviation (S) data should be 10.1 At least five calibration standards should be

used to establish an ongoing precision statement for the prepared daily for system calibration. 

level of concentrations included in the LFB. These data

must be kept on file and be available for review. 10.2 A calibration curve should be constructed for

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

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:

(C - C) s

R = --------------- x 100

s 

 (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

the fortified sample should be prepared again and

judged to be matrix related and the sample data should

each run by analyzing a set of calibration standard

solutions. A run should contain no more than 60 samples.
Version 1.0 September 1997 366.0-8

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, P = the peak height of the high high

silicate standard

P = the peak height of the first b1 

blank sample

P = the peak height of the second b2 

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,

P . i-1

CO = kxPi-1

To correct a given peak height reading, Pi, one subtracts

the carry over correction.12,13

Pi,c = Pi - CO

where P is corrected peak height. The correction for i,c

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 o

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
 366.0-9 Version 1.0 September 1997

sample line with reagent water, 1 N HCl solution, reagent standards and samples. The peak heights obtained from

water, 1N NaOH solution for a few minutes each at the these measurements must be subtracted from the peak

end of the daily analysis. Make sure to rinse the system heights of samples analyzed with color developing

well with reagent water after pumping NaOH solution to reagent pumping through the system. If a low refractive

prevent precipitation of Mg(OH)2 when seawater is index flowcell is used, the correction for refractive index is

introduced into the system. Keep the reagents and negligible. This procedure is optional.

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% HCl 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

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
Version 1.0 September 1997 366.0-10

through the system. Salinity is entered as the independent _______________________________________

variable and the apparent silicate concentration due to Salinty Peak height of Uncorrected Si 

refractive index in that detector is entered as the (% ) 1.50 mg Si/L conc.calculated 

dependent variable. The resulting regression allows the from standards

operator to calculate apparent silicate concentration due in reagent water

to refractive index when the salinity is known. Thus, the _______________________________________

operator would not be required to obtain refractive index 0 2503 1.50

peak heights for all samples. 9 2376 1.32

12.2.4 Refractive index correction can be minimized by 27 2250 1.25

using a low refractive index flowcell. An example of typical

results using a low refractive index flowcell follows:

______________________________________

Salinity Apparent silicate conc. due 

(% ) to refractive index (mg Si/L) salt error is: o

_______________________________________ 

4.5 0.0003 Uncorrected mg Si/L

9.0 0.0005 Corrected mg Si/L = ------------------------------------

18.0 0.0016 1 - 0.02186x/S

27.0 0.0027

36.0 0.0042 where S is salinity.

_______________________________________

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:

o

18 2282 1.27

36 2202 1.23

______________________________________

12.3.3 An example of a typical equation to correct for

12.3.4 Results of sample analyses should be reported

in mg Si/L or in μg Si/L.

mg Si/L = ppm (parts per million)

μg 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

(% ) (μg/L) (%) (μg/L) o

_____________________________________

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
 366.0-11 Version 1.0 September 1997

each sample were processed and analyzed with salinities choice. Whenever feasible, laboratory personnel should

ranging from 2.86 to 35.85. The results were as follows: 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

Sample Salinity Concentration RSD best option.

 (% ) (mg Si/L) (%) o

______________________________________ 14.2 For information about pollution prevention that

1 35.85 0.097 1.2 institutions, consult Less is Better: Laboratory Chemical

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:

_______________________________________

Concentration

Salinity (mg Si/L) RSD Recovery

(% ) Ambient Fortified (%) (%) o

______________________________________

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

may be applicable to laboratories and research

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. 
Version 1.0 September 1997 366.0-12

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.
1

2

3

4

5

6

7

8

9

10

0.41

0.41

0.41

0.32

0.25

0.41

1.57

Pump

ml/min

Debubbler

Detector

660nm

Manifold

Ascorbic Acid 

Oxalic Acid 

Sample

Air

Molybdate 

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Coil

Sample:Wash = 20":40"

Coil3

Coil1

Coil2

 366.0-13 Version 1.0 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Figure 1. Manifold Configuration for Silicate Analysis.