United States Environment Monitoring and
Environmental Protection Support Laboratory
Agency Cincinnati OH 45268
Research and Development EPA-600/4-84-017 Mar. 1984
Test Method
Technical Addition to
Methods for Chemica
Analysis of Water and
Wastes (EPA-600/4-79-020)
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United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/4-84-017 Mar. 1984
vvEPA
Test Method
The Determination of
Inorganic Anions in
Water by Ion Chromatography
Method 300.0
James W. O'Dell, John D. Pfaff, Morris E. Gales, and Gerald D. McKee
1. Scope and Application
1.1 This method covers the
determination of the following
inorganic anions.
Analyte
Chloride
Fluoride
Nitrate-N
Nitrite-N
Ortho-Phosphate-P
Sulfate
Storet No.
Total Dissolved
00940 —
00951 00950
00620 —
00615 -
— 00671
00945 -
1.2 This is an ion chromatographic
(1C) method applicable to the
determination of the anions listed
above in drinking water, surface
water, and mixed domestic and
industrial wastewater.
1.3 The Method Detection Limit
(MDL, defined in Section 13) for the
above analytes is listed in Table 1.
The MDL for a specific matrix may
differ from those listed, depending
upon the nature of the sample.
1.4 This method is restricted to use
by or under the supervision of
analysts experienced in the use of ion
Chromatography and in the
intrepretation of the resulting ion
chromatogram. Each analyst must
demonstrate the ability to generate
acceptable results with this method,
using the procedure described in
Section 10.2.
1.5 When this method is used to
analyze unfamiliar samples for any of
the above anions, anion identification
should be supported by the addition
of spike solutions covering the anions
of interest. The spike procedure is
described in Section 11.6.
2. Summary of Method
2.1 A small volume of sample,
typically 2 to 3 mL, is introduced into
an ion chromatograph. The anions of
interest are separated and measured,
using a system comprised of a guard
column, separator column, suppressor
column, and conductivity detector.
3. Definitions
3.1 Stock standard solution — a
concentrated solution containing a
certified standard that is a method
analyte. Stock standard solutions are
used to prepare secondary standard
solutions.
3.2 Calibration standards — a
solution of analytes prepared in the
laboratory from stock standard
solutions and diluted as needed to
prepare aqueous calibration solutions.
3.3 Quality control check sample —
a solution containing known
concentrations of analytes, prepared
by a laboratory other than the
laboratory performing the analysis.
The analyzing laboratory uses this
solution to demonstrate that it can
300.0-1
Jan. 1984
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obtain acceptable identifications and
measurements with a method.
3.4 Performance evaluation sample
— a solution of method analytes
distributed by the Quality Assurance
Branch (QAB), Environmental
Monitoring and Support Laboratory
(EMSL-Cincinnati), USEPA, Cincinnati,
Ohio, to multiple laboratories for
analysis. A volume of the solution is
added to a known volume of reagent
water and analyzed with procedures
used for samples. Results of analyses
are used by the QAB to determine
statistically the accuracy and precision
that can be expected when a method
is performed by a competent analyst.
Analyte true values are unknown to
the analyst.
3.5 Laboratory control standards —
a solution of analytes prepared in the
laboratory by adding appropriate
volumes of the stock standard
solutions to reagent water.
3.6 Laboratory duplicates — two
aliquots of the same sample that are
treated exactly the same throughout
laboratory analytical procedures.
Analyses of laboratory duplicates
indicate precision associated with
laboratory procedures but not the
sample collection, preservation, or
storage procedures.
3.7 Field duplicates — two samples
taken at the same time and place
under identical circumstances and
treated exactly the same throughout
field and laboratory procedures.
Analyses of field duplicates indicate the
precision associated with sample
collection, preservation and storage,
as well as with laboratory procedures.
4. Interferences
4.1 Interferences can be caused by
substances with retention times that
are similar to and overlap those of the
anion of interest. Large amounts of an
anion can interfere with the peak
resolution of an adjacent anion.
Sample dilution and/or spiking can be
used to solve most interference
problems.
4.2 The water dip or negative peak
that elutes near and can interfere
with the fluoride peak can be
eliminated by the addition of the
equivalent of 1 mL of concentrated
eluent (7.3 100X) to 100 mL of each
standard and sample.
4.3 Method interferences may be
caused by contaminants in the
reagent water, reagents, glassware,
and other sample processing
apparatus that lead to discrete
artifacts or elevated baseline in ion
chromatograms.
4.4 Samples that contain particles
larger than 0.45 microns and reagent
solutions that contain particles larger
than 0.20 microns require filtration to
prevent damage to instrument
columns and flow systems.
5. Safety
5.1 Normal, accepted laboratory
safety practices should be followed
during reagent preparation and
instrument operation. No known
carcinogenic materials are used in
this method.
6. Apparatus and Materials
6.1 Balance — Analytical, capable of
accurately weighing to the nearest
0.0001 g.
6.2 Ion chromatograph — Analytical
system complete with ion
chromatograph and all required
accessories including syringes,
analytical columns, compressed air,
detector, and stripchart recorder. A
data system is recommended for peak
integration.
6.2.1 Anion guard column: 4 x 50
mm, Dionex P/N 030825, or
equivalent.
6.2.2 Anion separator column: 4 x
250 mm, Dionex P/N 030827, or
equivalent.
6.2.3 Anion suppressor column:
fiber, Dionex P/N 35350, or
equivalent.
6.2.4 Detector — Conductivity cell:
approximately 6 fjL volume, Dionex, or
equivalent.
7. Reagents and
Consumable Materials
7.1 Sample bottles: Glass or
polyethylene of sufficient volume to
allow replicate analyses of anions of
interest.
7.2 Reagent water: Distilled or
deionized water, free of the anions of
interest. Water should contain
particles no larger than 0.20 microns.
7.3 Eluent solution: Sodium
bicarbonate (CAS RN 144-55-8} 0.003
M, sodium carbonate (CAS RN 497-
19-8) 0.0024M. Dissolve 1.0081 g
sodium bicarbonate (NaHCOs) and
1.0176 g of sodium carbonate
(Na2CO3) in reagent water and dilute
to 4 liters.
7.4 Regeneration solution (fiber
suppressor): Sulfuric acid (CAS RN
7664-93-9) 0.025N. Dilute 2.8 mL
cone, sulfuric acid (H2SO,i) to 4 liters
with reagent water.
7.5 Stock standard solutions, 1000
mg/L (1 mg/mL): Stock standard
solutions may be purchased as
certified solutions or prepared from
ACS reagent grade materials (dried
at 105°C for 30 min.) as listed below.
7.5. 7 Chloride (CL~) 1000 mg/L:
Dissolve 1.6485 g sodium chloride
(NaCL, CAS RN 7647-14-5) in reagent
water and dilute to 1 liter.
7.5.2 Fluoride (F~) 1000 mg/L:
Dissolve 2.2100 g sodium fluoride
(NaF, CAS RN 7681-49-4) in reagent
water and dilute to 1 liter.
7.5.3 Nitrate (NOl-N) 1000 mg/L:
Dissolve 6.0679 g sodium nitrate
(NaN03, CAS RN 7631-99-4) in
reagent water and dilute to 1 liter.
7.5.4 Nitrite (NOJT-N) 1000 mg/L:
Dissolve 4.9257 g sodium nitrite
(NaNOa, CAS RN 7632-00-0) in
reagent water and dilute to 1 liter.
7.5.5 Phosphate (PO<-P) 1000 mg/L:
Dissolve 4.3937 g potassium
phosphate (KH2P04, CAS RN 7778-77-
0) in reagent water and dilute to 1
liter.
7.5.6 Sulfate (SOT) 1000 mg/L:
Dissolve 1.8141 g potassium sulfate
(K2S04, CAS RN 7778-80-5) in
reagent water and dilute to 1 liter.
7.5.7 Stability of standards: Stock
standards (7.5) are stable for at least
one month when stored at 4°C. Dilute
working standards should be prepared
weekly, except those that contain
nitrite and phosphate should be
prepared fresh daily.
8. Sample Collection,
Preservation and Storage
8.1 Samples should be collected in
scrupulously clean glass or
polyethylene bottles.
8.2 Sample preservation and holding
times for the anions that can be
determined by this method are as
follows:
noiuiny
Analyte Preservation Time
Chloride None required 28 days
Fluoride None required 28 days
Nitrate-N Cool to 4°C 48 hours
Nitrite-N Cool to 4°C 48 hours
O-Phosphate-P Filter and cool 48 hours
to4°C
Sulfate
Cool to 4°C 28 days
Jan. 1984
300.0-2
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8.3 The method of preservation and
the holding time for samples analyzed
by this method are determined by the
anions of interest. In a given sample,
the anion that requires the most
preservation treatment and the
shortest holding time will determine
the preservation treatment and
holding time for the total sample.
9. Calibration amid
Standardization
9.1 Establish ion chromatographic
operating parameters equivalent to
those indicated in Table 1.
9.2 For each analyte of interest,
prepare calibration standards at a
minimum of three concentration
levels and a blank by adding
accurately measured volumes of one
or more stock standards (7.5) to a
volumetric flask and diluting to
volume with reagent water. If the
working range exceeds the linear
range of the system, a sufficient
number of standards must be
analyzed to allow an accurate
calibration curve to be established.
One of the standards should be
representative of a concentration
near, but above, the method detection
limit if the system is operated on an
applicable attenuator range. The other
standards should correspond to the
range of concentrations expected in
the sample or should define the
working range of the detector. Unless
the attenuator range settings are
proven to be linear, each setting must
be calibrated individually.
9.3 Using injections of 0.1 to 1.0 mL
(determined by injection loop volume)
of each calibration standard, tabulate
peak height or area responses against
the concentration. The results are
used to prepare a calibration curve for
each analyte. During this procedure,
retention times must be recorded. The
retention time is inversely
proportional to the concentration.
9.4 The working calibration curve
must be verified on each working day,
or whenever the anion eluent is
changed, and after every 20 samples.
If the response or retention time for
any analyte varies from the expected
values by more than ± 10%, the test
must be repeated, using fresh
calibration standards. If the results
are still more than ± 10%, an entire
new calibration curve must be
prepared for that analyte.
9.5 Nonlinear response can result
when the separator column capacity
is exceeded (overloading). Maximum
column loading (all anions) should not
exceed about 400 ppm.
10. Quality Control
10.1 Each laboratory using this
method should have a formal quality
control program. The minimum
requirements of this program consist
of an initial demonstration of
laboratory capability (10.2) and the
analysis of spiked samples as a
continuing check on performance. The
laboratory should maintain
performance records to define the
quality of data that are generated.
10.1.1 In recognition of the rapid
advances occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications to the method are made,
the analyst is required to repeat the
procedure in Section 10.2
10.1.2 The laboratory should spike
and analyze a minimum of 10% of all
samples to monitor continuing
laboratory performance. Field and
laboratory duplicates should also be
analyzed.
10.2 Before performing any
analyses, the analyst should
demonstrate the ability to generate
acceptable accuracy and precision
with this method, using a laboratory
control standard.
10.2.1 Select a representative
spike concentration for each analyte
to be measured. Using stock
standards, prepare a quality control
check sample concentrate in reagent
water 100 times more concentrated
than the selected concentrations.
10.2.2 Using a pipet, add 1.00 mL
of the check sample concentrate
(10.2.1) to each of a minimum of four
100-mL aliquots of reagent water.
Analyze the aliquots according to the
procedure in Section 11.
10.2.3 Calculate the average
percent recovery (R), and the standard
deviation(s) of the percent recovery, for
the results.
10.2.4 Using the appropriate data
from Table 2, determine the recovery
and single operator precision expected
for the method, and compare these
results to the values calculated in
Section 10.2.3. If the data are not
comparable within control limits
(10.3.1), review potential problem
areas and repeat the test.
10.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration of analyte being
measured.
10.3.1 Calculate upper and lower
control limits for method performance
as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in
Section 10.2.3. The UCL and LCL can
be used to construct control charts
that are useful in observing trends in
performance.
10.4 The laboratory should develop
and maintain separate accuracy
statements of laboratory performance
for water and wastewater samples.
An accuracy statement for the method
is defined as R ± s. The accuracy
statement should be developed by the
analyses of four aliquots of water or
wastewater, as described in Section
10.2.2, followed by the calculation of
R and s.
10.5 Before processing any
samples, the analyst must
demonstrate through the analysis of
an aliquot of reagent water that all
glassware and reagent interferences
are under control. Each time there is
a change in reagents, a laboratory
reagent blank must be processed as a
safeguard against laboratory
contamination.
10.6 It is recommended that the
laboratory adopt additional quality
assurance practices for use with this
method. The specific practices that
are most productive depend upon the
needs of the laboratory and the nature
of the samples. Field duplicates may
be analyzed to monitor the precision
of the sampling technique. When
doubt exists over the identification of
a peak in the chromatogram,
confirmatory techniques such as
sample dilution and spiking, must be
used. Whenever possible, the
laboratory should perform analysis of
quality control check samples and
participate in relevant performance
evaluation sample studies.
11. Procedure
11.1 Table 1 summarizes the
recommended operating conditions for
the ion chromatograph. Included in
this table are estimated retention
times that can be achieved by this
method. Other columns,
chromatographic conditions, or
300.0-3
Jan. 1984
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detectors may be used if the
requirements of Section 10.2 are met.
11.2 Check system calibration daily
and, if required, recalibrate as
described in Section 9.
11.3 Load and inject a fixed amount
of well mixed sample. Flush injection
loop thoroughly, using each new
sample. Use the same size loop for
standards and samples. Record the
resulting peak size in area or peak
height units. An automated constant
volume injection system may also be
used.
11.4 The width of the retention time
window used to make identifications
should be based upon measurements
of actual retention time variations of
standards over the course of a day.
Three times the standard deviation of
a retention time can be used to
calculate a suggested window size for
a compound. However, the experience
of the analyst should weigh heavily in
the interpretation of chromatograms.
11.5 If the response for the peak
exceeds the working range of the
system, dilute the sample with an
appropriate amount of reagent water
and reanalyze.
11.6 If the resulting chromatogram
fails to produce adequate resolution,
or if identification of specific anions is
questionable, spike the sample with
an appropriate amount of standard
and reanalyze.
Note: Retention time is inversely
proportional to concentration. Nitrate
and sulfate exhibit the greatest
amount of change, although all anions
are affected to some degree. In some
cases, this peak migration can
produce poor resolution or
misidentification.
12. Calculation
12.1 Prepare separate calibration
curves for each anion of interest by
plotting peak size in area, or peak
height units of standards against
concentration values. Compute
sample concentration by comparing
sample peak response with the
standard curve.
12.2 Report results in mg/L.
zero. The MDL concentrations listed in
Table 1 were obtained using reagent
water.
13.2 Single-operator accuracy and
precision for reagent, drinking and
surface water, and mixed domestic
and industrial wastewater are listed in
Table 2.
14. References
14.1 Annual Book of ASTM
Standards, Part 31 Water, proposed
test method for "Anions in Water by
Ion Chromatography," p. 1485-1492
(1982).
14.2 Standard Methods for the
Examination of Water and
Wastewater, Method 400Z, "Anions
by Ion Chromatography" proposed for
the 16th Edition of Standard Methods.
14.3 Dionex, 1C 16 operation and
maintenance manual, PN 30579,
Dionex Corp., Sunnyvale, California
94086.
14.4 Method detection limit (MDL)
as described in "Trace Analyses for
Wastewater," J. Glaser, D. Foerst,
G. McKee, S. Quave, W. Budde,
Environmental Science and
Technology, Vol. 15, Number 12, p.
1426, December 1981.
13. Precision and Accuracy
— Method Detection Limit
13.1 The method detection limit
(MDL) is defined as the minimum
concentration of a substance that can
be measured and reported with 99%
confidence that the value is above
Jan. 1984
300.0-4
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Table 1. Chromatographic Conditions and Method Detection Limits in Reagent
Water
Retention' Relative Method2
Time Retention Detection Limit
Analyte (Mini Time mq/L
Fluoride
Chloride
Nitrite -N
0-Phosphate-P
Nitrate-N
Sulfate
1.2
3.4
4.5
9.0
11.3
21.4
1.0
2.8
3.8
7.5
9.4
17.8
0.005
0.015
0.004
0.061
0.013
0.206
Sample Loop — 100 (jL
Pump Volume — 2.30 mL/Min
Standard Conditions:
Columns — As specified in 6.2
Detector — As specified in 6.2
Eluent — As specified in 7.3
1 Concentrations of mixed standard (mg/L)
Fluoride 3.0 0-Phosphate-P 9.0
Chloride 4.0 Nitrate-N 30.0
Nitrite-N 10.0 Sulfate 50.0
2' MDL calculated from data obtained using an attenuator setting of 1 fjMHO full
scale. Other settings would produce an MDL proportional to their value.
Table 2. Single-Operator Accuracy and Precision
Analyte
Chloride
Fluoride
Nitrate-N
Nitrite-N
0-Phosphate-P
Sulfate
Sample
Type
RW
DW
SW
WW
RW
DW
SW
WW
RW
DW
SW
WW
RW
DW
SW
WW
RW
DW
SW
WW
RW
DW
SW
WW
Spike
(ma/L)
0.050
10.0
1.0
7.5
0.24
9.3
0.50
1.0
0.10
31.0
0.50
4.0
0.10
19.6
0.51
0.52
0.50
45.7
0.51
4.0
1.02
98.5
10.0
12.5
Number
of
Replicates
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
Mean
Recovery
%
97.7
98.2
105.0
82.7
103.1
87.7
74.0
92.0
100.9
100.7
100.0
94.3
97.7
103.3
88.2
100.0
100.4
102.5
94.1
97.3
102.1
104.3
111.6
134.9
Standard
Deviation
fmg/U
0.0047
0.289
0.139
0.445
0.0009
0.075
0.0038
0.011
0.0041
0.356
0.0058
0.058
0.0014
0.150
0.0053
0.018
0.019
0.386
0.020
0.04
0.066
1.475
0.709
0.466
RW = Reagent Water
DW - Drinking Water
SW = Surface Water
WW = Wastewater
300.0-5
Jan. 198'
*USGPO:1984-759-102-862
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