300.1-1

METHOD 300.1

 DETERMINATION OF INORGANIC ANIONS IN DRINKING WATER BY ION

CHROMATOGRAPHY

Revision 1.0

John D. Pfaff (USEPA, ORD, NERL) - Method 300.0, (1993)

Daniel P. Hautman (USEPA, Office of Water) and David J. Munch (USEPA, Office of Water)

- Method 300.1, (1997)

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
ERRATA COVER SHEET

ERRATA COVER SHEET TO U.S.EPA METHOD 300.1

April 27, 1999

The following were editorial changes which have been incorporated into U.S.EPA Method 300.1. These

minor clarifications are incorporated into the body of this text as follows:

ERRATA #1 - 

An additional sentence was added to Section 4.1.1 reiterating the analyst’s responsibilities when

incorporating any method change, including modifying eluent strength, or any other method parameter. 

The additional sentence states, 

“...The analyst must verify that these changes do not negatively affect performance by repeating

and passing all the QC criteria in Section 9.” 

On this same theme, section 11.9, was also further clarified and specific precautions were added as

follows,

“...The analysts must verify that this dilution does not negatively affect performance by repeating

and passing all the QC criteria in Section 9. As a specific precaution, upon dilution of the

carbonate eluent, a peak for bicarbonate may be observed within the retention time window for

bromate which will negatively impact the analysis.”

ERRATA #2 - 

An acronym in Section 9.3.2.2 for Laboratory Fortified Blank (LFB) was incorrectly identified as LRB. 

This typographical error was corrected.

ERRATA #3 - 

Clarifications and corrections were made to Section 9.4.1.5, 9.4.3.2 and 9.4.3.3. These clarifications

pertain to data reportability for Laboratory Fortified Sample Matrices (LFM) as well as to analysis

continuation when Duplicate Sample QC acceptance criteria are not met. 

Section 9.4.1.5 clarifies and now specifies how to report data when the LFM recovery falls outside the

established control criteria by stating, 

“...the recovery problem encountered with the LFM is judged to be matrix induced and the results

for that sample and the LFM are reported with a “matrix induced bias” qualifier.”

 

Section 9.4.3.2 required the correction of a typographical reference by removing “%Diff” in the duplicate

sample acceptance criteria and replacing it with the defined RPD, indicating “relative percent difference”.

Section 9.4.3.3, also had a “%Diff” reference corrected with RPD and included clarification regarding

continuation of an analysis set when a duplicate analysis fails to meet the acceptance criteria. This section

now reads,

“If the RPD fails to meet these criteria, the samples must be reported with a qualifier identifying

the sample analysis result as yielding a poor duplicate analysis RPD. This should not be a

chronic problem and if it frequently recurs, (>20% of duplicate analysis) it indicates a problem

with the instrument or individual technique.”
300.1-2

METHOD 300.1

DETERMINATION OF INORGANIC ANIONS IN DRINKING WATER

 BY ION CHROMATOGRAPHY

 

1. SCOPE AND APPLICATION

1.1 This method covers the determination of the following inorganic anions in reagent

water, surface water, ground water, and finished drinking water. As a result of

different specified injection volumes (See conditions in Tables 1A and 1B), these

anions are divided between the common anions listed in Part A and the inorganic

disinfection by-products listed in Part B. These different injection volumes are

required in order to compensate for the relative concentrations of these anions in

drinking water and maintain good chromatographic peak shape throughout the

expected dynamic range of the detector. Bromide is included in both Part A, due

to its importance as a common anion, as well as Part B due to its critical role as a

disinfection by-product precursor.

PART A.-- Common Anions

Bromide Nitrite

Chloride ortho-Phosphate-P

Fluoride Sulfate

Nitrate

PART B.-- Inorganic Disinfection By-products

Bromate Chlorite

Bromide Chlorate

1.2 The single laboratory Method Detection Limits (MDL, defined in Sect. 3.11) for

the above analytes are listed in Tables 1A, 1B and 1C. The MDL for a specific

matrix may differ from those listed, depending upon the nature of the sample and

the specific instrumentation employed. 

1.2.1 In order to achieve comparable detection limits, an ion chromatographic

system must utilize suppressed conductivity detection, be properly

maintained and must be capable of yielding a baseline with no more than

5 nS noise/drift per minute of monitored response over the background

conductivity. 

1.3 This method is recommended for use only by or under the supervision of analysts

experienced in the use of ion chromatography and in the interpretation of the

resulting ion chromatograms. 

1.4 When this method is used to analyze unfamiliar samples for any of the above

anions, anion identification should be supported by the use of a fortified sample

matrix covering the anions of interest. The fortification procedure is described in

Sect. 9.4.1.
300.1-3

1.5 Users of the method data should state the data-quality objectives prior to analysis. 

Users of the method must demonstrate the ability to generate acceptable results

with this method, using the procedures described in Sect. 9.0. 

1.6 Bromide and nitrite react with most oxidants employed as disinfectants. The

utility of measuring these anions in treated water should be considered prior to

conducting the analysis. 

2. SUMMARY OF METHOD

2.1 A small volume of sample, 10 uL for Part A and 50 uL for Part B, is introduced

into an ion chromatograph. The anions of interest are separated and measured,

using a system comprised of a guard column, analytical column, suppressor

device, and conductivity detector. 

2.2 The ONLY difference between Parts A and B is the volume of sample analyzed

by the ion chromatographic system. The separator columns and guard columns as

well as eluent conditions are identical.

3. DEFINITIONS

3.1 ANALYSIS BATCH -- A group of no more than 20 field samples (Field sample

analyses include only those samples derived from a field sample matrix. These

include the initial and duplicate field samples as well as all Laboratory Fortified

Sample Matrices). The analysis batch must include an Initial Calibration Check

Standard, an End Calibration Check Standard, Laboratory Reagent Blank, and a

Laboratory Fortified Blank. Within an ANALYSIS BATCH, for every group of

ten field samples, at least one Laboratory Fortified Matrix (LFM) and either a

Field Duplicate, a Laboratory Duplicate or a duplicate of the LFM must be

analyzed. When more than 10 field samples are analyzed, a Continuing

Calibration Check Standard must be analyzed after the tenth field sample analysis.

3.2 CALIBRATION STANDARD (CAL) -- A solution prepared from the primary

dilution standard solution or stock standard solutions and the surrogate analyte. 

The CAL solutions are used to calibrate the instrument response with respect to

analyte concentration. 

3.2.1 INITIAL CALIBRATION STANDARDS -- A series of CAL solutions

used to initially establish instrument calibration and develop calibration

curves for individual target anions.

3.2.2 INITIAL CALIBRATION CHECK STANDARD -- An individual CAL

solution, analyzed initially, prior to any sample analysis, which verifies

previously established calibration curves.

3.2.3 CONTINUING CALIBRATION CHECK STANDARD -- An individual

CAL solution which is analyzed after every tenth field sample analyses
300.1-4

which verifies the previously established calibration curves and confirms

accurate analyte quantitation for the previous ten field samples analyzed.

3.2.4 END CALIBRATION CHECK STANDARD -- An individual CAL

solution which is analyzed after the last field sample analyses which

verifies the previously established calibration curves and confirms

accurate analyte quantitation for all field samples analyzed since the last

continuing calibration check.

3.3 FIELD DUPLICATES -- Two separate samples collected 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. 

3.4 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) -- A solution of

one or more method analytes, surrogates, or other test substances used to

evaluate the performance of the instrument system with respect to a defined set of

criteria.

3.5 LABORATORY DUPLICATE -- Two sample aliquots, taken in the laboratory

from a single sample bottle, and analyzed separately with identical procedures. 

Analyses of LD1 and LD2 indicate precision associated specifically with the

laboratory procedures, removing any associated variables attributed by sample

collection, preservation, or storage procedures.

3.6 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or

other blank matrices 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 the methodology is in control, and whether the laboratory is

capable of making accurate and precise measurements.

3.7 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.

3.8 LABORATORY REAGENT BLANK (LRB) -- An aliquot of reagent water or

other blank matrices that are treated exactly as a sample including exposure to all

glassware, equipment, solvents, reagents, and surrogates 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 the apparatus.
300.1-5

3.9 LINEAR CALIBRATION RANGE (LCR) -- The concentration range over which

the instrument response is linear.

3.10 MATERIAL SAFETY DATA SHEET (MSDS) -- Written information provided

by vendors concerning a chemical's toxicity, health hazards, physical properties,

fire, and reactivity data including storage, spill, and handling precautions.

3.11 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.

3.12 MINIMUM REPORTING LEVEL (MRL) -- The minimum concentration that

can be reported for an anion in a sample following analysis. This defined

concentration can be no lower than the concentration of the lowest calibration

standard and can only be used if acceptable quality control criteria for this standard

are met.

3.13 PERFORMANCE EVALUATION SAMPLE (PE) -- A certified solution of

method analytes whose concentration is unknown to the analyst. Often, an aliquot

of this solution is added to a known volume of reagent water and analyzed with

procedures used for samples. Results of analyses are used to determine

statistically the accuracy and precision that can be expected when a method is

performed by a competent analyst.

3.14 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of

known concentrations that 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.

3.15 SURROGATE ANALYTE -- An analyte added to a sample, which is unlikely to

be found in any sample at significant concentration, and which is added directly to

a sample aliquot in known amounts before any sample processing procedures are

conducted. It is measured with the same procedures used to measure other sample

components. The purpose of the surrogate analyte is to monitor method

performance with each sample.

3.16 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing

one or more method analytes prepared in the laboratory using assayed reference

materials or purchased from a reputable commercial source.

4. INTERFERENCES

4.1 Interferences can be divided into three different categories: direct

chromatographic coelution, where an analyte response is observed at very nearly

the same retention time as the target anion; concentration dependant coelution,

which is observed when the response of higher than typical concentrations of the
300.1-6

neighboring peak overlap into the retention window of the target anion; and, ionic

character displacement, where retention times may significantly shift due to the

influence of high ionic strength matrices (high mineral content or hardness)

overloading the exchange sites in the column and significantly shortening target

analyte's retention times. 

4.1.1 A direct chromatographic coelution may be solved by changing columns,

eluent strength, modifying the eluent with organic solvents (if compatible

with IC columns), changing the detection systems, or selective removal of

the interference with pretreatment. Sample dilution will have little to no

effect. The analyst must verify that these changes do not negatively affect

performance by repeating and passing all the QC criteria in Section 9. 

4.1.2 Sample dilution may resolve some of the difficulties if the interference is

the result of either concentration dependant coelution or ionic character

displacement, but it must be clarified that sample dilution will alter your

Minimum Reporting Limit (MRL) by a proportion equivalent to that of

the dilution. Therefore, careful consideration of project objectives should

be given prior to performing such a dilution. An alternative to sample

dilution, may be dilution of the eluent as outlined in 11.9. 

4.1.3 Pretreatment cartridges can be effective as a means to eliminate certain

matrix interferences. Prior to using any pretreatment, the analyst should

be aware that all instrument calibration standards must be pretreated in

exactly the same manner as the pretreated unknown field samples. The

need for these cartridges have been greatly reduced with recent advances

in high capacity anion exchange columns.

4.1.3.1 Extreme caution should be exercised in using these

pretreatment cartridges. Artifacts are known to leach from

certain cartridges which can foul the guard and analytical

columns causing loss of column capacity indicated by

shortened retention times and irreproducible results. 

Frequently compare your calibration standard chromatograms

to those of the column test chromatogram (received when the

column was purchased) to insure proper separation and similar

response ratios between the target analytes is observed. 

4.2 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 baselines in an ion chromatogram. These interferences can

lead to false positive results for target analytes as well as reduced detection limits

as a consequence of elevated baseline noise.

4.3 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.
300.1-7

4.4 Any anion that is only weakly retained by the column may elute in the retention

time window of fluoride and potentially interfere. At concentrations of fluoride

above 1.5 mg/L, this interference may not be significant, however, it is the

responsibility of the user to generate precision and accuracy information in each

sample matrix.

 

4.5 Close attention should be given to the potential for carry over peaks from one

analysis which will effect the proper detection of analytes of interest in a second,

subsequent analysis. Normally, the elution of sulfate (retention time of 13.8 min.)

indicates the end of a chromatographic run, but, in the ozonated and chlorine

dioxide matrices, which were included as part of the single operator accuracy and

bias study (See Table 2B), a small response (200 nS baseline rise) was observed

for a very late eluting unknown peak at approximately 23 minutes. Consequently,

a run time of 25 minutes is recommended to allow for the proper elution of any

potentially interferant late peaks. It is the responsibility of the user to confirm

that no late eluting peaks have carried over into a subsequent analysis thereby

compromising the integrity of the analytical results.

4.6 Any residual chlorine dioxide present in the sample will result in the formation of

additional chlorite prior to analysis. If any concentration of chlorine dioxide is

suspected in the sample, the sample must be purged with an inert gas (helium,

argon or nitrogen) for approximately five minutes or until no chlorine dioxide

remains. This sparging must be conducted prior to ethylenediamine preservation

and at time of sample collection.

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method have not been

fully established. Each chemical should be regarded as a potential health hazard

and exposure should be as low as reasonably achievable. Cautions are included

for known extremely hazardous materials or procedures.

5.2 Each laboratory is responsible for maintaining a current awareness file of OSHA

regulations regarding the safe handling of the chemicals specified in this method. 

A reference file of Material Safety Data Sheets (MSDS) should be made available

to all personnel involved in the chemical analysis. The preparation of a formal

safety plan is also advisable.

5.3 The following chemicals have the potential to be highly toxic or hazardous,

consult MSDS.

5.3.1 Sulfuric acid -- When used to prepared a 25 mN sulfuric acid regenerant

solution for chemical suppression using a Dionex Anion Micro Membrane

Suppressor (AMMS).
300.1-8

6. EQUIPMENT AND SUPPLIES

6.1 Ion chromatograph -- Analytical system complete with ion chromatograph and all

required accessories including syringes, analytical columns, compressed gasses

and a conductivity detector. 

6.1.1 Anion guard column: Dionex AG9-HC, 2 mm (P/N 52248), or

equivalent. This column functions as a protector of the separator column. 

If omitted from the system the retention times will be shorter. 

6.1.2 Anion separator column: Dionex AS9-HC column, 2 mm (P/N 52244),

or equivalent. The microbore (2 mm) was selected in the development of

this method as a means to tighten the bromate elution band and thus

reduce the detection limit. An optional column (2 mm or 4 mm) may be

used if comparable resolution of peaks is obtained, and the requirements

of Sect. 9.0 can be met. The AS9-HC, 2 mm column using the conditions

outlined in Table 1A and 1B produced the separation shown in Figures 1

through 4. 

6.1.2.1 If a 4 mm column is employed, the injection volume should be

raised by a factor of four to 40 uL for Part A anions and 200

uL for Part B anions in order to attain comparable detection

limits. A four fold increase in injection volume compensates

for the four fold increase in cross sectional surface area of the

4 mm standard bore column over the 2 mm microbore column. 

6.1.2.2 Comparable results can be attained using the Dionex, AS9-HC,

4 mm column. MDLs for the part B, inorganic disinfection byproducts using this 4 mm column are displayed along with

analysis conditions in Table 1C. 

6.1.3 Anion suppressor device: The data presented in this method were

generated using a Dionex Anion Self Regenerating Suppressor (ASRS,

P/N 43187). An equivalent suppressor device may be utilized provided

comparable detection limits are achieved and adequate baseline stability is

attained as measured by a combined baseline drift/noise of no more than 5

nS per minute over the background conductivity.

6.1.3.1 The ASRS was set to perform electrolytic suppression at a

current setting of 100 mA using an external source DI water

mode. Insufficient baseline stability was observed using the

ASRS in recycle mode.

6.1.4 Detector -- Conductivity cell (Dionex CD20, or equivalent) capable of

providing data as required in Sect. 9.2.
300.1-9

6.2 The Dionex Peaknet Data Chromatography Software was used to generate all the

data in the attached tables. Systems using a strip chart recorder and integrator or

other computer based data system may achieve approximately the same MDL's

but the user should demonstrate this by the procedure outlined in Sect. 9.2. 

6.3 Analytical balance, ±0.1 mg sensitivity. Used to accurately weigh target analyte

salts for stock standard preparation.

6.4 Top loading balance, ±10 mg sensitivity. Used to accurately weigh reagents to

prepare eluents.

6.5 Weigh boats, plastic, disposable - for weighing eluent reagents.

6.6 Syringes, plastic, disposable, 10 mL - used during sample preparation.

6.7 Pipets, Pasteur, plastic or glass, disposable, graduated, 5 mL and 10 mL.

6.8 Bottles, high density polyethylene (HDPE), opaque or glass, amber, 30 mL, 125

mL, 250 mL. For sampling and storage of calibration solutions. Opaque or

amber due to the photoreactivity of chlorite anion.

6.9 Micro beakers, plastic, disposable - used during sample preparation.

7. REAGENTS AND STANDARDS

7.1 Reagent water: Distilled or deionized water, free of the anions of interest. Water

should contain particles no larger than 0.20 microns. 

7.2 Eluent solution : Sodium carbonate (CASRN 497-19-8) 9.0 mM. Dissolve 1.91

g sodium carbonate (Na2CO3) in reagent water and dilute to 2 L.

7.2.1 This eluent solution must be purged for 10 minutes with helium prior to

use to remove dissolved gases which may form micro bubbles in the IC

compromising system performance and adversely effecting the integrity of

the data.

7.3 Stock standard solutions, 1000 mg/L (1 mg/mL): Stock standard solutions may

be purchased as certified solutions or prepared from ACS reagent grade,

potassium or sodium salts as listed below, for most analytes. Chlorite requires

careful consideration as outline below in 7.3.5.1.

7.3.1 Bromide (Br-) 1000 mg/L: Dissolve 0.1288 g sodium bromide (NaBr,

CASRN 7647-15-6) in reagent water and dilute to 100 mL in a

volumetric flask.
300.1-10

7.3.2 Bromate (BrO3

-

) 1000 mg/L: Dissolve 0.1180 g of sodium bromate

(NaBrO3, CASRN 7789-38-0) in reagent water and dilute to 100 mL in a

volumetric flask.

7.3.3 Chlorate (C103

-

) 1000 mg/L: Dissolve 0.1275 g of sodium chlorate

(NaC103, CASRN 7775-09-9) in reagent water and dilute to 100 mL in a

volumetric flask.

7.3.4 Chloride (Cl-) 1000 mg/L: Dissolve 0.1649 g sodium chloride (NaCl,

CASRN 7647-14-5) in reagent water and dilute to 100 mL in a

volumetric flask. 

 

7.3.5 Chlorite (C102

-

) 1000 mg/L: Assuming an exact 80.0 % NaC102 is

amperometrically titrated from technical grade NaC102 (See Sect.

7.3.5.1). Dissolve 0.1676 g of sodium chlorite (NaC102, CASRN 7758-

19-2) in reagent water and dilute to 100 mL in a volumetric flask.

7.3.5.1 High purity sodium chlorite (NaClO 2) is not currently

commercially available due to potential explosive instability. 

Recrystallization of the technical grade (approx. 80%) can be

performed but it is labor intensive and time consuming. The

simplest approach is to determine the exact % NaClO 2 using

the iodometric titration procedure (Standard Methods, 19th

Ed., 4500-ClO2.C). Following titration, an individual

component standard of chlorite must be analyzed to determine

if there is any significant contamination (greater than 1% of the

chlorite weight) in the technical grade chlorite standard from

any of the Part B components. These contaminants will place

a high bias on the calibration of the other anions if all four Part

B components are mixed in an combined calibration solution. 

If these other anions are present as contaminants, a separate

chlorite calibration needs to be performed. 

7.3.6 Fluoride (F-) 1000 mg/L: Dissolve 0.2210 g sodium fluoride (NaF,

CASRN 7681-49-4) in reagent water and dilute to 100 mL in a

volumetric flask.

7.3.7 Nitrate (NO3-N) 1000 mg/L: Dissolve 0.6068 g sodium nitrate (NaNO3,

CASRN 7631-99-4) in reagent water and dilute to 100 mL in a

volumetric flask.

 

7.3.8 Nitrite (NO2-N) 1000 mg/L: Dissolve 0.4926 g sodium nitrite (NaNO2,

CASRN 7632-00-0) in reagent water and dilute to 100 mL in a

volumetric flask.

 
300.1-11

7.3.9 Phosphate (PO4

3--P) 1000 mg/L: Dissolve 0.4394 g potassium

dihydrogenphosphate (KH2PO4, CASRN 7778-77-0) in reagent water

and dilute to 100 mL in a volumetric flask. 

 

7.3.10 Sulfate (SO4

2-) 1000 mg/L: Dissolve 0.1814 g potassium sulfate (K2SO4

,

CASRN 7778-80-5) in reagent water and dilute to 100 mL in a

volumetric flask. 

NOTE: Stability of standards: Stock standards (7.3) for most anions are

stable for at least 6 months when stored at 4oC. Except for the

chlorite standard which is only stable for two weeks when stored

protected from light at 4oC, and nitrite and phosphate which are only

stable for 1 month when stored at 4oC. Dilute working standards

should be prepared monthly, except those that contain chlorite, or

nitrite and phosphate which should be prepared fresh daily.

7.4 Ethylenediamine (EDA) preservation solution, 100 mg/mL: Dilute 2.8 mL of

ethylenediamine (99%) (CASRN 107-15-3) to 25 mL with reagent water. 

Prepare fresh monthly.

7.5 Surrogate Solution: 0.50 mg/mL dichloroacetate (DCA) prepared by dissolving

0.065 g dichloroacetic acid, potassium salt (Cl2CHCO2K, CASRN 19559-59-2) in

reagent water and dilute to 100 mL in a volumetric flask. 

7.5.1 Dichloroacetate is potentially present in treated drinking waters as the

acetate of the organic disinfection by product, dichloroacetic acid

(DCAA). Typical concentrations of DCAA rarely exceed 50 ug/L,

which, for this worst case example, would represent only a five percent

increase in the observed response over the fortified concentration of 1.00

mg/L. Consequently, the criteria for acceptable recovery (90% to 115%)

for the surrogate is weighted to 115% to allow for this potential

background.

7.5.2 Prepare this solution fresh every 3 months or sooner if signs of

degradation are present.

8. SAMPLE COLLECTION, PRESERVATION AND STORAGE

8.1 Samples should be collected in plastic or glass bottles. All bottles must be

thoroughly cleaned and rinsed with reagent water. Volume collected should be

sufficient to insure a representative sample, allow for replicate analysis, if

required, and minimize waste disposal.

8.2 Special sampling requirements and precautions for chlorite.

8.2.1 Sample bottles used for chlorite analysis must be opaque to protect the

sample from light.
300.1-12

8.2.2 When preparing the LFM, be aware that chlorite is an oxidant and may

react with the natural organic matter in an untreated drinking water

matrix as a result of oxidative demand. If untreated water is collected for

chlorite analysis, and subsequently used for the LFM, EDA preservation

will not control this demand and reduced chlorite recoveries may be

observed.

8.3 Sample preservation and holding times for the anions that can be determined by

this method are as follows:

PART A : Common Anions

Analyte Preservation Holding Time

Bromide None required 28 days

Chloride None required 28 days

Fluoride None required 28 days

Nitrate-N Cool to 4oC 48 hours

Nitrite-N Cool to 4oC 48 hours 

ortho-Phosphate-P Cool to 4oC 48 hours 

Sulfate Cool to 4oC 28 days 

PART B : Inorganic Disinfection By-products

Analyte Preservation Holding Time

Bromate 50 mg/L EDA 28 days

Bromide None required 28 days

Chlorate 50 mg/L EDA 28 days

Chlorite 50 mg/L EDA, Cool to 4oC 14 days

8.4 When collecting a sample from a treatment plant employing chlorine dioxide, the

sample must be sparged with an inert gas (helium, argon, nitrogen) prior to

addition of the EDA preservative at time of sample collection. 

8.5 All four anions, in Part B, can be analyzed in a sample matrix which has been

preserved with EDA. Add a sufficient volume of the EDA preservation solution

(Sect. 7.4) such that the final concentration is 50 mg/L in the sample. This would

be equivalent to adding 0.5 mL of the EDA preservation solution to 1 L of

sample. 

8.6 EDA is primarily used as a preservative for chlorite. Chlorite is susceptible to

degradation both through catalytic reactions with dissolved iron salts and

reactivity towards free chlorine which exists as hypochlorous acid/hypochlorite

ion in most drinking water as a residual disinfectant. EDA serves a dual purpose

as a preservative for chlorite by chelating iron as well as any other catalytically

destructive metal cations and removing hypochlorous acid/hypochlorite ion by

forming an organochloroamine. EDA preservation of chlorite also preserves the

integrity of chlorate which can increase in unpreserved samples as a result of

chlorite degradation. EDA also preserves the integrity of bromate concentrations

by binding with hypobromous acid/hypobromite which is an intermediate formed
300.1-13

as by-product of the reaction of either ozone or hypochlorous acid/hypochlorite

with bromide ion. If hypobromous acid/hypobromite is not removed from the

matrix further reactions may form bromate ion.

8.7 Degradation of ortho-phosphate has been observed in samples held at room

temperature for over 16 hrs (see table 3A). Therefore, samples to be analyzed for

ortho-phosphate must not be held at room temperature for more than 12

cumulative hours.

9. QUALITY CONTROL

9.1 Each laboratory using this method is required to operate a formal quality control

(QC) program. The requirements of this program consist of an initial

demonstration of laboratory performance, and subsequent analysis in each

analysis batch (Sect. 3.1) of a Laboratory Reagent Blank, Laboratory Fortified

Blank, Instrument Performance Check Standard, calibration check standards,

Laboratory Fortified Sample Matrices (LFM) and either Field, Laboratory or

LFM duplicate sample analyses. This section details the specific requirements for

each of these QC parameters. The laboratory is required to maintain performance

records that define the quality of the data that are generated.

9.2 INITIAL DEMONSTRATION OF PERFORMANCE 

9.2.1 The initial demonstration of performance is used to characterize

instrument performance (determination of accuracy through the analysis

of the QCS) and laboratory performance (determination of MDLs) prior

to performing analyses by this method.

9.2.2 Quality Control Sample (QCS) -- When beginning the use of this method,

on a quarterly basis or as required to meet data-quality needs, verify the

calibration standards and acceptable instrument performance with the

preparation and analyses of a QCS. If the determined concentrations are

not within ± 15% of the stated values, performance of the determinative

step of the method is unacceptable. The source of the problem must be

identified and corrected before either proceeding with the initial

determination of MDLs or continuing with on-going analyses.

9.2.3 Method Detection Limit (MDL) -- MDLs must be established for all

analytes, using reagent water (blank) fortified at a concentration of three

to five times the estimated instrument detection limit.(6) To determine

MDL values, take seven replicate aliquots of the fortified reagent water

and process through the entire analytical method over at least three

separate days. Perform all calculations defined in the method and report

the concentration values in the appropriate units. Calculate the MDL as

follows:

MDL = (t) x (S)
300.1-14

where, t = Student's t value for a 99% confidence level and a

standard deviation estimate with n-1 degrees of freedom

[t = 3.14 for seven replicates].

S = standard deviation of the replicate analyses.

9.2.3.1 MDLs should be determined every 6 months, when a new

operator begins work or whenever there is a significant change

in the background, or instrument response.

9.3 ASSESSING LABORATORY PERFORMANCE 

9.3.1 Laboratory Reagent Blank (LRB) -- The laboratory must analyze at least

one LRB with each analysis batch (defined Sect 3.1). Data produced are

used to assess contamination from the laboratory environment. Values

that exceed the MDL indicate laboratory or reagent contamination should

be suspected and corrective actions must be taken before continuing the

analysis.

9.3.1.1 If conducting analysis for the Part B anions, EDA must be

added to the LRB at 50 mg/L. By including EDA in the LRB,

any bias as a consequence of the EDA which may be observed

in the field samples, particularly in terms of background

contamination, will be identified.

9.3.2 Laboratory Fortified Blank (LFB) -- The LFB should be prepared at

concentrations similar to those expected in the field samples and ideally at

the same concentration used to prepare the LFM. Calculate accuracy as

percent recovery (Sect. 9.4.1.3). If the recovery of any analyte falls

outside the required concentration dependant control limits (Sect.

9.3.2.2), that analyte is judged out of control, and the source of the

problem should be identified and resolved before continuing analyses.

9.3.2.1 If conducting analysis for the Part B anions, EDA must be

added to the LFB at 50 mg/L. The addition of EDA to all

reagent water prepared calibration and quality control samples

is required not as a preservative but rather as a means to

normalize any bias attributed by the presence of EDA in the

field samples.

9.3.2.2 Control Limits for the LFB

Concentration range Percent Recovery Limits

 MRL to 10xMRL 75 - 125 %

10xMRL to highest calibration level 85 - 115 % 
300.1-15

9.3.2.2.1 These control limits only apply if the MRL is established within

a factor of 10 times the MDL. Otherwise, the limits are set at

85% to 115%.

9.3.2.3 The laboratory must use the LFB to assess laboratory performance against the required control limits listed in 9.3.2.2. 

When sufficient internal performance data become available

(usually a minimum of 20-30 analyses), optional control limits

can be developed from the percent mean recovery (x) and the

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

those listed in 9.3.2.2. After each five to ten new recovery

measurements, new control limits can be calculated using only

the most recent 20-30 data points. Also, the standard

deviation (S) data should be used to establish an on-going

precision statement for the level of concentrations monitored. 

These data must be kept on file and be available for review.

9.3.3 Instrument Performance Check Solution (IPC) -- The Initial Calibration

Check Standard is to be evaluated as the instrument performance check

solution in order to confirm proper instrument performance. Proper

chromatographic performance must be demonstrated by calculating the

Peak Gaussian Factor (PGF), which is a means to measure peak

symmetry and monitoring retention time drift in the surrogate peak over

time. Critically evaluate the surrogate peak in the initial calibration check

standard, and calculate the PGF as follows,

 1.83 x W(1/2)

PGF = ----------------------- 

 W(1/10)

 where: W(1/2) is the peak width at half height 

W(1/10) is the peak width at tenth height

9.3.3.1 The PGF must fall between 0.80 and 1.15 in order to

demonstrate proper instrument performance.

9.3.3.2 The retention time for the surrogate in the IPC must be closely

monitored on each day of analysis and throughout the lifetime of

the analytical column. Small variations in retention time can be

anticipated when a new solution of eluent is prepared but if

shifts of more than 2% are observed in the surrogate retention

time, some type of instrument problem is present. Potential
300.1-16

problems include improperly prepared eluent, erroneous method

parameters programmed such as flow rate or some other system

problem. The chromatographic profile (elution order) of the

target anions following an ion chromatographic analysis should

closely replicate the profile displayed in the test chromatogram

that was shipped when the column was purchased. As a column

ages, it is normal to see a gradual shift and shortening of

retention times, but if after several years of use, extensive use

over less than a year, or use with harsh samples, this retention

time has noticeably shifted to any less than 80% of the original

recorded value, the column may require cleaning or

replacement. Particularly if resolution problems are beginning

to become common between previously resolved peaks. A

laboratory must retain a historic record of retention times for

the surrogate and all the target anions to provide evidence of an

analytical columns vitality.

9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY

9.4.1 Laboratory Fortified Sample Matrix (LFM) -- The laboratory must add a

known amount of analyte to a minimum of 10% of the field samples

within an analysis batch. The LFM sample must be prepared from a

sample matrix which has been analyzed prior to fortification. The analyte

concentration must be high enough to be detected above the original

sample and should adhere to the requirement of 9.4.1.2. It is

recommended that the solutions used to fortify the LFM be prepared

from the same stocks used to prepare the calibration standards and not

from external source stocks. This will remove the bias contributed by an

externally prepared stock and focus on any potential bias introduced by

the field sample matrix.

9.4.1.1 If the fortified concentration is less than the observed 

background concentration of the unfortified matrix, the

recovery should not be calculated. This is due to the difficulty

in calculating accurate recoveries of the fortified concentration

when the native sample concentration is so high.

9.4.1.2 The LFM should be prepared at concentrations no greater than

five times the highest concentration observed in any field

sample. If no analyte is observed in any field sample, the LFM

must be fortified no greater than five times the lowest

calibration level which as outlined in 12.2 is the minimum

reported level (MRL). For example, if bromate is not detected

in any field samples above the lowest calibrations standard

concentration of 5.00 ug/L, the highest LFM fortified

concentration allowed is 25.0 ug/L.
300.1-17

9.4.1.3 Calculate the percent recovery for each analyte, corrected for

concentrations measured in the unfortified sample. Percent

recovery should be calculated using the following equation:

 Cs - C

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

 s

where, R = percent recovery.

Cs = fortified sample concentration

C = sample background concentration

s = concentration equivalent of analyte added to

sample.

9.4.1.4 Until sufficient data becomes available (usually a minimum of 20

to 30 analysis), assess laboratory performance against recovery

limits of 75 to 125%. When sufficient internal performance data

becomes available develop control limits from percent mean

recovery and the standard deviation of the mean recovery. The

optional control limits must be equal to or better than the

required control limits of 75-125%.

9.4.1.5 If the recovery of any analyte falls outside the designated LFM

recovery range and the laboratory performance for that analyte

is shown to be in control (Sect. 9.3), the recovery problem

encountered with the LFM is judged to be matrix induced and

the results for that sample and the LFM are reported with a

“matrix induced bias” qualifier.

9.4.2 SURROGATE RECOVERY -- Calculate the surrogate recovery from all

analyses using the following formula

 SRC

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

 SFC

where, R = percent recovery.

SRC = Surrogate Recovered Concentration

SFC = Surrogate Fortified Concentration

9.4.2.1 Surrogate recoveries must fall between 90-115% for proper

instrument performance and analyst technique to be verified. The

recovery of the surrogate is slightly bias to 115% to allow for the

potential contribution of trace levels of dichloroacetate as the

halogenated organic disinfection by-product (DBP) dichloroacetic

acid (DCAA) Background levels of this organic DBP are rarely

observed above 50 ug/L (0.05 mg/L) which constitutes only 5%

of the 1.00 mg/L recommended fortified concentration.

9.4.2.2 If the surrogate recovery falls outside the 90-115% recovery

window, a analysis error is evident and sample reanalysis is
300.1-18

required. Poor recoveries could be the result of imprecise sample

injection or analyst fortification errors. 

9.4.3 FIELD OR LABORATORY DUPLICATES -- The laboratory must

analyze either a field or a laboratory duplicate for a minimum of 10% of the

collected field samples or at least one with every analysis batch, whichever

is greater. The sample matrix selected for this duplicate analysis must

contain measurable concentrations of the target anions in order to establish

the precision of the analysis set and insure the quality of the data. If none

of the samples within an analysis batch have measurable concentrations, the

LFM should be employed as a laboratory duplicate.

9.4.3.1 Calculate the relative percent difference (RPD) of the initial

quantitated concentration (IC) and duplicate quantitated

concentration (Dc) using the following formula,

 (IC - Dc)

RPD = -------------- X 100

 ([IC + Dc]/2)

9.4.3.2 Duplicate analysis acceptance criteria

Concentration range RPD Limits

 MRL to 10xMRL +/- 20 %

 10xMRL to highest calibration level +/- 10 % 

9.4.3.3 If the RPD fails to meet these criteria, the samples must be

reported with a qualifier identifying the sample analysis result as

yielding a poor duplicate analysis RPD. This should not be a

chronic problem and if it frequently recurs (>20% of duplicate

analyses) it indicates a problem with the instrument or individual

technique. 

9.4.4 Where reference materials are available, they should be analyzed to provide

additional performance data. The analysis of reference samples is a

valuable tool for demonstrating the ability to perform the method

acceptably. 

9.4.5 In recognition of the rapid advances occurring in chromatography, the

analyst is permitted certain options, such as the use of different columns,

injection volumes, and/or eluents, 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 Sect. 9.2 and

adhere to the condition of baseline stability found in Sect. 1.2.1. 

9.4.6 It is recommended that the laboratory adopt additional quality assurance

practices for use with this method. The specific practices that are most
300.1-19

productive depend upon the needs of the laboratory and the nature of the

samples. Whenever possible, the laboratory should perform analysis of

quality control check samples and participate in relevant performance

evaluation sample studies.

10. CALIBRATION AND STANDARDIZATION

10.1 Establish ion chromatographic operating parameters equivalent to those indicated

in Tables 1A or 1B if employing a 2 mm column, Table 1C if employing a 4 mm

column. 

10.2 Estimate the Linear Calibration Range (LCR) -- The LCR should cover the

expected concentration range of the field samples and should not extend over

more than 2 orders of magnitude in concentration (For example, if quantitating

nitrate in the expected range of 1.0 mg/L to 10 mg/L, 2 orders of magnitude

would permit the minimum and maximum calibration standards of 0.20 mg/L and

20 mg/L, respectively.) The restriction of 2 orders of magnitude is prescribed

since beyond this it is difficult to maintain linearity throughout the entire

calibration range. 

10.2.1 If quantification is desired over a larger range, then two separate

calibration curves should be prepared. 

10.2.2 For an individual calibration curve, a minimum of three calibration

standards are required for a curve that extends over a single order of

magnitude and a minimum of five calibration standards are required if the

curve covers two orders of magnitude. (For example, using the nitrate

example cited above in section 10.2, but in this case limit the curve to

extend only from 1.0 mg/L to 10 mg/L or a single order of magnitude. A

third standard is required somewhere in the middle of the range. For the

calibration range of 0.20 mg/L to 20 mg/L, over two orders of

magnitude, five calibrations standards should be employed, one each at

the lower and upper concentration ranges and the other three

proportionally divided throughout the middle of the curve.) 

10.3 Prepare the calibration standards by carefully adding measured volumes of one or

more stock standards (7.3) to a volumetric flask and diluting to volume with

reagent water. 

 

10.3.1 For the Part B anions, EDA must be added to the calibration standards at

50 mg/L. The addition of EDA to all reagent water prepared calibration

and quality control samples is required not as a preservative but rather as

a means to normalize any bias attributed by the presence of EDA in the

field samples.

10.3.2 Prepare a 10.0 mL aliquot of surrogate fortified calibration solution

which can be held for direct manual injection or used to fill an

autosampler vial. Add 20 uL of the surrogate solution (7.5) to a 20 mL

disposable plastic micro beaker. Using a 10.0 mL disposable pipet, place
300.1-20

exactly 10.0 mL of calibration standard into the micro beaker and mix. 

The calibration standard is now ready for analysis. The same surrogate

solution that has been employed for the standards should also be used in

the section 11.3.2 for the field samples.

10.4 Using a 2 mm column, inject 10 uL (Part A) or 50 uL (Part B) of each calibration

standard. Using a 4 mm column, inject 50 uL (Part A) or 200 uL (Part B) of each

calibration standard. Tabulate peak area responses against the concentration. 

The results are used to prepare calibration curves using a linear least squares fit

for each analyte. Acceptable calibration curves are confirmed after reviewing the

curves for linearity and passing the criteria for the initial calibration check

standard in section 10.5.1. Alternately, if the ratio of response to concentration

(response factor) is constant over the LCR (indicated by < 15% relative standard

deviation (RSD), linearity through the origin can be assumed and the average

ratio or calibration factor can be used in place of a calibration curve, 

10.4.1 Peak areas are strongly recommended since they have been found to be

more consistent, in terms of quantitation, than peak heights. Peak height

can tend to be suppressed as a result of high levels of common anions in a

given matrix which can compete for exchange sites. Using peak areas, it

is the analyst responsibility to review all chromatograms to insure

accurate baseline integration of target analyte peaks since poorly drawn

baselines will more significantly influence peak areas than peak heights.

10.5 Once the calibration curves have been established they must be verified prior to

conducting any sample analysis using an initial calibration check standard (3.2.2). 

This verification must be performed on each analysis day or whenever fresh eluent

has been prepared. A continuing calibration check standard (3.2.3) must be

analyzed after every tenth sample and at the end of the analysis set as an end

calibration check standard (3.2.4). The response for the initial, continuing and end

calibration check must satisfy the criteria listed in 10.5.1. If during the analysis set,

the response differs by more than the calibration verification criteria shown in

10.5.1., or the retention times shift more than ± 5% from the expected values for

any analyte, the test must be repeated, using fresh calibration standards. If the

results are still outside these criteria, sample analysis must be discontinued, the

cause determined and/or in the case of drift, the instrument recalibrated. All

samples following the last acceptable calibration check standard must be

reanalyzed. 

10.5.1 Control limits for calibration verification

Concentration range Percent Recovery Limits

 MRL to 10xMRL 75 - 125 %

10xMRL to highest calibration level 85 - 115 % 

 10.5.1.1 These control limits only apply if the MRL is established within

a factor of 10 times the MDL. Otherwise, the limits are set at

85% to 115%.
300.1-21

10.5.2 CALIBRATION VERIFICATION REQUIREMENT FOR PART B

As a mandatory requirement of calibration verification, the laboratory

MUST verify calibration using the lowest calibration standard as the

initial calibration check standard.

10.5.3 After satisfying the requirement of 10.5.2, the levels selected for the other

calibration check standards should be varied between a middle calibration

level and the highest calibration level.

11. PROCEDURE

11.1 Tables 1A and 1B summarize the recommended operating conditions for the ion

chromatograph. Included in these tables are estimated retention times that can be

achieved by this method. Other columns, chromatographic conditions, or

detectors may be used if the requirements of Sect. 9.2 are met. 

11.2 Check system calibration daily and, if required, recalibrate as described in Sect.

10. 

11.3 Sample Preparation

11.3.1 For refrigerated or samples arriving to the laboratory cold, ensure the

samples have come to room temperature prior to conducting sample

analysis by allowing the samples to warm on the bench for at least 1 hour.

11.3.2 Prepare a 10.0 mL aliquot of surrogate fortified sample which can be held

for direct manual injection or used to fill an autosampler vial. Add 20 uL

of the surrogate solution (7.5) to a 20 mL disposable plastic micro beaker. 

Using a 10.0 mL disposable pipet, place exactly 10.0 mL of sample into

the micro beaker and mix. Sample is now ready for analysis.

11.3.2.1 The less than 1% dilution error introduced by the addition of the

surrogate is considered insignificant.

11.4 Using a Luer lock, plastic 10 mL syringe, withdraw the sample from the micro

beaker and attach a 0.45 um particulate filter (demonstrated to be free of ionic

contaminants) directly to the syringe. Filter the sample into an autosampler vial

(If vial is not designed to automatically filter) or manually load the injection loop

injecting a fixed amount of well mixed sample. If using a manually loaded

injection loop, flush the loop thoroughly between sample analysis using sufficient

volumes of each new sample matrix. 

11.5 Using a 2 mm column, inject 10 uL (Part A) or 50 uL (Part B) of each sample. 

Using a 4 mm column, inject 40 uL (Part A) or 200 uL (Part B) of each sample. 

Tabulate peak area responses against the concentration. During this procedure,

retention times must be recorded. Use the same size loop for standards and
300.1-22

samples. Record the resulting peak size in area units. An automated constant

volume injection system may also be used. 

11.6 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 each analyte. However, the

experience of the analyst should weigh heavily in the interpretation of

chromatograms. 

11.7 If the response of a sample analyte exceeds the calibration range, the sample may

be diluted with an appropriate amount of reagent water and reanalyzed. If this is

not possible then three new calibration concentrations must be employed to create

a separate high concentration curve, one standard near the estimated

concentration and the other two bracketing around an interval equivalent to ±

25% the estimated concentration. The latter procedure involves significantly

more time than a simple sample dilution therefore, it is advisable to collect

sufficient sample to allow for sample dilution or sample reanalysis, if required.

11.8 Shifts in retention time are inversely proportional to concentration. Nitrate,

phosphate and sulfate will exhibit the greatest degree of change, although all

anions can be affected. In some cases this peak migration may produce poor

resolution or make peak identification difficult. 

11.9 Should more complete resolution be needed between any two coeluting peaks, the

eluent (7.2) can be diluted. This will spread out the run, however, and will cause

late eluting anions to be retained even longer. The analysts must verify that this

dilution does not negatively affect performance by repeating and passing all the QC

criteria in Section 9. As a specific precaution, upon dilution of the carbonate

eluent, a peak for bicarbonate may be observed within the retention time window

for bromate which will negatively impact the analysis.

11.9.1 Eluent dilution will reduce the overall response of an anion due to

chromatographic band broadening which will be evident by shortened

and broadened peaks. This will adversely effect the MDLs for each

analyte.

12. DATA ANALYSIS AND CALCULATIONS

12.1 Prepare a calibration curve for each analyte by plotting instrument response, as

peak area, against standard concentration. Compute sample concentration by

comparing sample response with the standard curve. If a sample has been

diluted, multiply the response by the appropriate dilution factor.

12.2 Report ONLY those values that fall between the lowest and the highest

calibration standards. Samples with target analyte responses exceeding the

highest standard should be diluted and reanalyzed. Samples with target analytes

identified but quantitated below the concentration established by the lowest
300.1-23

calibration standard should be reported as below the minimum reporting limit

(MRL).

12.3 Report results for Part A anions in mg/L and for Part B anions in ug/L. 

12.4 Report NO2

-

 as N

NO3

-

 as N

HPO4

=

 as P

Br- in mg/L when reported with Part A 

Br- in ug/L when reported with Part B 

13. METHODS PERFORMANCE

13.1 Tables 1A, 1B, and 1C give the single laboratory (OW OGWDW TSCCincinnati) retention times, standard conditions and MDL determined for each

anion included in the method. MDLs for the Part A anions were determined in

reagent water on the 2 mm column (Table 1A). MDLs for the Part B anions were

conducted not only in reagent water but also a simulated high ionic strength water

(HIW) on the 2 mm column (Table 1B) and in reagent water on the 4 mm column

(Table 1C). HIW is designed to simulate a high ionic strength field sample. It

was prepared from reagent water which was fortified with the common anions of

chloride at 100 mg/L, carbonate at 100 mg/L, nitrate at 10.0 mg/L as nitrogen,

phosphate at 10.0 mg/L as phosphorous, and sulfate at 100 mg/L.

 13.2 Tables 2A and 2B give the single laboratory (OW OGWDW TSC-Cincinnati)

standard deviation for each anion included in the method in a variety of waters for

the standard conditions identified in Table 1A and 1B, respectively.

13.3 Tables 3A and 3B shown stability data for the Part A and B anions, respectively. 

Each data point in these tables represent the mean percent recovery following

triplicate analysis. These data were used to formulate the holding times shown in

Sect. 8.3.

14. 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 EPA 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 Quantity of the chemicals purchased should be based on expected usage during its

shelf life and disposal cost of unused material. Actual reagent preparation

volumes should reflect anticipated usage and reagent stability.
300.1-24

14.3 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's Department of Government Regulations and Science Policy, 1155 16th

Street N.W., Washington D.C. 20036, 

(202) 872-4477. 

15. WASTE MANAGEMENT

15.1 The Environmental Protection Agency requires that laboratory waste

management practices be conducted consistent with all applicable rules and

regulations. Excess reagents, samples and method process wastes should be

characterized and disposed of in an acceptable manner. 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 waste discharge permit 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 Sect. 14.3.

16. REFERENCES 

1. "Determination of Inorganic Disinfection By-Products by Ion Chromatography",

J. Pfaff, C. Brockhoff. J. Am. Water Works Assoc., Vol 82, No. 4, pg 192. 

2. Standard Methods for the Examination of Water and Wastewater, Method

4110B, "Anions by Ion Chromatography", 18th Edition of Standard Methods

(1992). 

3. Dionex, System DX500 Operation and Maintenance Manual, Dionex Corp.,

Sunnyvale, California 94086, 1996. 

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, page 1426,

December, 1981.

5. American Society for Testing and Materials. Test Method for Anions in Water by

Chemically-Suppressed Ion Chromatography D4327-91. Annual Book of

Standards, Vol 11.01 (1993). 

6. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.

7. Hautman, D.P. & Bolyard, M. Analysis of Oxyhalide Disinfection By-products

and other Anions of Interest in Drinking Water by Ion Chromatography. Jour. of

Chromatog., 602, (1992), 65-74.
300.1-25

8. Standard Methods for the Examination of Water and Wastewater, Method 4500-

ClO 2,C "Amperometric Method I" (for the determination of Chlorine Dioxide),

19th Edition of Standard Methods (1995). 
300.1-26

17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA

TABLE 1A. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION

 LIMITS IN REAGENT WATER FOR THE COMMON ANIONS (PART

A).

MDL DETERMINATION

ANALYTE PEAK # (1)

RETENTION TIME

(MIN.)

Fort Conc,

mg/L

Number 

of

Replicates

DI

MDL

mg/L

Fluoride 1 2.53 0.020 7 0.009

Chloride 2 4.67 0.020 7 0.004

Nitrite-N 3 6.01 0.010 7 0.001

Surrogate: DCA 4 7.03

Bromide 5 8.21 0.040 7 0.014

Nitrate-N 6 9.84 0.010 7 0.008

ortho-Phosphate-P 7 11.98 0.040 7 0.019

Sulfate 8 13.49 0.040 7 0.019

Standard Conditions:

Ion Chromatograph: Dionex DX500

Columns : Dionex AG9-HC / AS9-HC, 2 mm

Detector: Suppressed Conductivity Detector, Dionex CD20 

Suppressor: ASRS-I, external source electrolytic mode, 100 mA current

Eluent: 9.0 mM Na2CO3

Eluent Flow: 0.40 mL/min

Sample Loop: 10 uL

System Backpressure: 2800 psi

Background Conductivity: 22 uS

Recommended method total analysis time: 25 minutes

(1) See Figure 1
300.1-27

TABLE 1B. CHROMATOGRAPHIC CONDITIONS AND METHOD

DETECTION LIMITS IN BOTH REAGENT WATER AND HIGH

IONIC STRENGTH WATER FOR THE INORGANIC

DISINFECTION BY-PRODUCTS (PART B).

MDL DETERMINATION

ANALYTE PEAK # (1)

RETENTION

TIME

(MIN.)

Fort

Conc,

ug/L

Number 

of

Replicates

DI

MDL

ug/L

HIW(2)

MDL

ug/L

Chlorite 1 3.63 2.00 7 0.89 0.45

Bromate 2 4.19 2.00 7 1.44 1.28

Surrogate:

DCA

4 7.28

Bromide 5 8.48 2.00 7 1.44 2.51

Chlorate 6 9.28 2.00 7 1.31 0.78

Standard Conditions:

Ion Chromatograph: Dionex DX500

Columns : Dionex AG9-HC / AS9-HC, 2 mm

Detector: Suppressed Conductivity Detector, Dionex CD20 

Suppressor: ASRS-I, external source electrolytic mode, 100 mA current

Eluent: 9.0 mM Na2CO3

Eluent Flow: 0.40 mL/min

Sample Loop: 50 uL

System Backpressure: 2800 psi

Background Conductivity: 22 uS

Recommended method total analysis time: 25 minutes

(1) See Figure 2 and 3

(2) HIW indicates High Ionic Strength Water which is a simulated drinking water prepared

from reagent water and fortified with chloride at 100 mg/L, carbonate at 100 mg/L,

nitrate at 10.0 mg/L as nitrogen, phosphate at 10.0 mg/L as phosphorous, and sulfate at

100 mg/L.
300.1-28

TABLE 1C. CHROMATOGRAPHIC CONDITIONS AND METHOD

DETECTION LIMITS IN REAGENT WATER FOR THE

INORGANIC DISINFECTION BY-PRODUCTS USING AN

ALTERNATE 4 mm AS9-HC COLUMN (PART B).

MDL DETERMINATION

ANALYTE PEAK #

RETENTION

TIME

(MIN.)

Fort

Conc,

ug/L

Number 

of

Replicates

DI

MDL

ug/L

Chlorite 1 4.43 2.00 7 1.44

Bromate 2 5.10 2.00 7 1.32

Surrogate:

DCA

4 8.82

Bromide 5 10.11 2.00 7 0.98

Chlorate 6 10.94 2.00 7 2.55

Standard Conditions:

Ion Chromatograph: Dionex DX500

Columns : Dionex AG9-HC / AS9-HC, 4 mm

Detector: Suppressed Conductivity Detector, Dionex CD20 

Suppressor: ASRS-I, external source electrolytic mode, 300 mA current 

Eluent: 9.0 mM Na2CO3

Eluent Flow: 1.25 mL/min

Sample Loop: 200 uL

System Backpressure: 1900 psi

Background Conductivity: 21 uS

Recommended method total analysis time: 25 minutes
300.1-29

TABLE 2A. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE

COMMON

 ANIONS (PART A).

ANALYTE MATRIX

UNFORT

MATRIX

CONC.,

mg/L

FORT

CONC

mg/L

#

OF

REPLC

MEAN

mg/L

MEAN

%REC SD(n-1) %RSD

Fluoride RW <MDL(1) 2.00 9 1.79 89.7 0.02 1.18

SW 0.139 2.00 9 1.75 80.4 0.01 0.56

GW 0.280 2.00 9 1.97 84.3 0.02 0.85

CDW 0.807 2.00 9 2.59 89.0 0.01 0.46

Chloride RW 0.029 50.0 9 49.4 98.7 0.03 0.10

SW 12.1 50.0 9 58.7 93.3 0.04 0.10

GW 56.6 50.0 9 100. ---

(2) 0.22 0.22

CDW 16.0 50.0 9 64.9 97.8 0.11 0.16

Nitrite-N RW <MDL 1.00 9 0.851 85.1 0.00 0.51

SW <MDL 1.00 9 0.780 78.0 0.00 0.40

GW 0.013 1.00 9 0.879 86.6 0.01 0.77

CDW <MDL 1.00 9 0.720 72.0 0.00 0.55

Bromide RW <MDL 0.500 9 0.480 96.1 0.00 0.92

SW 0.028 0.500 9 0.469 88.1 0.00 0.94

GW 0.153 0.500 9 0.634 96.3 0.00 0.52

CDW <MDL 0.500 9 0.431 86.2 0.01 1.28

Nitrate-N RW <MDL 10.0 9 9.50 95.0 0.01 0.14

SW 2.12 10.0 9 10.9 87.7 0.03 0.30

GW 0.016 10.0 9 9.64 96.3 0.03 0.27

CDW 1.64 10.0 9 10.9 92.4 0.04 0.41

Phosphate-P RW <MDL 10.0 9 9.62 96.2 0.01 0.14

SW <MDL 10.0 9 8.70 87.0 0.02 0.18

GW <MDL 10.0 9 6.12 61.2 0.28 4.66

CDW <MDL 10.0 9 9.15 91.5 0.04 0.42

Sulfate RW <MDL 50.0 9 44.8 89.5 0.05 0.11

SW 47.8 50.0 9 92.1 88.6 0.21 0.23

GW 105 50.0 9 154 ---

(2) 0.60 0.39

CDW 57.8 50.0 9 105 ---

(2) 0.33 0.32

Surrogate: RW ---- 5.00 9 5.12 102.3 0.50 0.49

SW ---- 5.00 9 5.09 102.3 1.12 1.09

GW ---- 5.00 9 5.16 101.8 0.67 0.66

CDW ---- 5.00 9 5.17 103.1 1.36 1.32

RW = Reagent Water GW = Ground Water

SW = Surface Water CDW = chlorine dioxide treated finished drinking water 

(1) <MDL indicates less than method detection limit.

(2) Not calculated since amount fortified was less than unfortified native matrix concentration

(See 9.4.1.1.).
300.1-30

TABLE 2B. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE

INORGANIC DISINFECTION BY-PRODUCTS (PART B).

ANALYTE MATRIX

UNFORT

CONC.

ug/L

FORT

CONC

ug/L

#

OF

REPLC

MEAN

ug/L

MEAN

%REC SD(n-1) %RSD

Chlorite RW <MDL(1) 100 9 96.2 96.2 0.95 0.99

500 9 523 105 3.13 0.60

HIW <MDL 100 9 102 102 2.19 2.15

500 9 520 104 3.64 0.70

SW <MDL 100 9 91.4 91.4 1.22 1.33

500 9 495 99.0 7.54 1.52

GW <MDL 100 9 92.9 92.9 1.65 1.77

500 9 490 98.1 3.40 0.69

ClW <MDL 100 9 87.4 87.4 0.59 0.68

500 9 485 97.1 6.36 1.31

CDW 292 100 9 396 ---

(2) 1.64 0.41

500 9 811 104 4.00 0.49

O3W <MDL 100 9 84.4 84.4 0.46 0.54

500 9 481 96.1 3.24 0.67

Bromate RW <MDL 5.00 9 5.04 101 0.45 8.86

25.0 9 26.5 106 1.71 6.47

HIW <MDL 5.00 9 4.88 97.5 0.95 19.5

25.0 9 25.6 102 1.37 5.37

SW <MDL 5.00 9 4.46 89.2 0.58 13.0

25.0 9 26.3 105 1.10 4.18

GW <MDL 5.00 9 5.10 102 0.50 9.75

25.0 9 22.2 88.9 1.29 5.81

ClW <MDL 5.00 9 4.63 92.6 0.77 16.7

25.0 9 25.1 100 1.64 6.55

CDW <MDL 5.00 9 4.14 82.7 0.62 15.1

25.0 9 25.1 101 1.28 5.09

O3W 1.45 5.00 9 5.49 80.9 0.61 11.1

25.0 9 24.1 90.6 1.13 4.69

 RW = Reagent Water GW = Groundwater

HIW = High Ionic strength Water ClW = Chlorinated drinking water 

 [see note (2) in Table 1B] CDW = Chlorine dioxide treated drinking water

SW = Surface Water O3W = Ozonated drinking water

(1) <MDL indicates less than method detection limit.

(2) Not calculated since amount fortified was less than unfortified native matrix concentration (See

9.4.1.1.).
300.1-31

TABLE 2B. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE INORGANIC

 DISINFECTION BY-PRODUCTS (PART B) (contd.).

ANALYTE MATRIX

UNFORT

CONC.

ug/L

FORT

CONC

ug/L

#

OF

REPLC

MEAN

ug/L

MEAN

%REC SD(n-1) %RSD

Bromide RW <MDL(1) 20.0 9 20.9 104 0.80 3.82

100 9 107 107 0.60 0.56

HIW 3.24 20.0 9 21.8 92.5 0.79 3.63

100 9 105 102 1.05 1

SW 31.0 20.0 9 51.3 ---

(2) 0.97 1.9

100 9 140. 109 1.88 1.35

GW 151 20.0 9 172 ---

(2) 0.78 0.45

100 9 265 ---

(2) 2.18 0.82

ClW 16.3 20.0 9 39.3 115 0.64 1.62

100 9 125 109 2.00 1.6

CDW 11.5 20.0 9 34.4 115 0.76 2.22

100 9 125 113 1.24 0.99

O3W 39.8 20.0 9 65.4 ---

(2) 3.67 5.61

100 9 153 113 1.00 0.65

Chlorate RW <MDL 100 9 98.3 98.3 0.80 0.82

500 9 520 104 4.15 0.8

HIW <MDL 100 9 86.1 86.1 1.47 1.7

500 9 502 100. 4.52 0.9

SW 3.18 100 9 102 98.3 1.57 1.55

500 9 513 102 7.11 1.39

GW <MDL 100 9 93.5 93.5 2.00 2.14

500 9 510 102 3.84 0.75

ClW 34.4 100 9 136 102 1.01 0.74

500 9 549 103 3.11 0.57

CDW 121 100 9 223 ---

(2) 3.20 1.44

500 9 651 106 3.50 0.54

O3W 6.15 100 9 106 100 1.20 1.13

500 9 523 103 2.45 0.47

RW = Reagent Water GW = Groundwater

HIW = High Ionic strength Water ClW = Chlorinated drinking water 

 [see note (2) in Table 1B] CDW = Chlorine dioxide treated drinking water

SW = Surface Water O3W = Ozonated drinking water

(1) <MDL indicates less than method detection limit.

(2) Not calculated since amount fortified was less than unfortified native matrix concentration (See

9.4.1.1.).
300.1-32

TABLE 2B. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE INORGANIC

DISINFECTION BY-PRODUCTS (PART B)(contd.).

ANALYTE MATRIX

FORT

CONC

mg/L

#

OF

REPLC

MEAN

mg/L

MEAN

%REC SD(n-1) %RSD

Surrogate: DCA RW 5.00 9 5.11 102 0.93 0.91

(see NOTE below) 4.98 99.5 0.69 0.69

HIW 5.00 9 5.00 100 0.79 0.79

4.96 99.2 1.76 1.78

SW 5.00 9 4.95 98.9 0.70 0.7

4.99 99.8 1.60 1.61

GW 5.00 9 5.12 102 0.50 0.49

5.13 103 0.50 0.49

ClW 5.00 9 5.15 103 1.73 1.68

5.13 103 1.12 1.09

CDW 5.00 9 5.01 100 1.02 1.02

5.04 101 1.08 1.07

O3W 5.00 9 4.99 99.8 0.70 0.7

5.11 101 0.53 0.52

 RW = Reagent Water GW = Groundwater

HIW = High Ionic strength Water ClW = Chlorinated drinking water 

[see note (2) in Table 1B] CDW = Chlorine dioxide treated drinking water

SW = Surface Water O3W = Ozonated drinking water

NOTE: The surrogate DCA was fortified at 5 mg/L but due to concerns about measuring trace

concentrations of bromide with such high concentration of the neighboring surrogate

peak, the recommended fortified concentration for the surrogate has been reduced to

1.00 mg/L.
300.1-33

TABLE 3A. STABILITY STUDY RESULTS FOR THE COMMON ANIONS (PART A).

ANALYTE Preservative Matrix

UNFORT

CONC.

mg/L

FORT

CONC

mg/L

Analyte % Recovery

Day

0

Day

14

Day

28

See

Note

Fluoride None RW <MDL 2.00 89.8 88.3 88.4

SW 0.140 2.00 79.9 80.2 80.0

GW 0.280 2.00 84.7 87.8 87.0

CDW 0.929 2.00 82.9 83.6 81.6

Chloride None RW <MDL 50.0 98.8 99.1 98.1

SW 12.0 50.0 93.4 93.5 92.8

GW 56.6 50.0 87.6 87.6 86.5

CDW 16.0 50.0 97.9 98.4 97.8

Nitrite-N None RW <MDL 1.00 85.2 85.5 83.6

SW <MDL 1.00 77.8 76.6 11.9 (1)

GW <MDL 1.00 88.2 85.4 56.1 (1)

CDW <MDL 1.00 71.9 71.7 73.9 (2)

Bromide None RW <MDL 0.500 95.5 97.0 96.2

SW 0.028 0.500 87.5 88.3 86.7

GW 0.153 0.500 96.9 96.0 96.1

CDW <MDL 0.500 85.7 87.1 89.2 (2)

Nitrate-N None RW <MDL 10.0 94.9 94.7 94.2

SW 2.12 10.0 87.6 87.0 88.7

GW <MDL 10.0 96.5 96.5 95.5

CDW 1.64 10.0 92.3 93.3 91.9

Phosphate-P None RW <MDL 10.0 96.3 95.8 95.2

SW <MDL 10.0 86.9 86.4 85.1

GW <MDL 10.0 62.8 93.1 89.5 (3)

CDW <MDL 10.0 91.6 91.4 90.8

Sulfate None RW <MDL 50.0 89.6 89.3 89.1

SW 47.8 50.0 89.0 89.0 88.1

GW 105 50.0 97.5 97.3 96.5

CDW 57.8 50.0 94.3 94.9 93.8

NOTES:

(1) Degradation apparent.

(2) Analyte recovery will be adversely effected by reactions with free chlorine.

(3) Phosphate recovery on day 0 is believed to have been adversely effected by biological

degradation since the sample sat in the autosampler for 18 hrs prior to analysis
300.1-34

TABLE 3B STABILITY STUDY RESULTS FOR THE INORGANIC DISINFECTION BYPRODUCTS (PART B).

ANALYTE Preservative Matrix

UNFORT

CONC.

ug/L

FORT

CONC

ug/L

Analyte % Recovery

Day

0

Day

3

Day

10

Day

30

See

Note

Chlorite None RW <MDL 500 99.8 100 104 94.3

HIW <MDL 500 99.3 98.5 106 89.3

SW <MDL 500 92 88.5 82. 75.1 (1)

GW <MDL 500 93.9 94.5 96. 91.7

ClW <MDL 500 93.7 NA(1) 90. 84.7 (2,3)

CDW 286 500 98.6 101 91. 77.5 (1,3)

O3W <MDL 500 10 NA 82. 90.5 (2)

Chlorite EDA RW <MDL 500 101 101 104 95.3

HIW <MDL 500 98.4 98.7 104 95.4

SW <MDL 500 98.3 97.3 97. 92.7

GW <MDL 500 97.7 97.1 97. 92.6

ClW <MDL 500 98.9 NA 96. 92.6 (2)

CDW 297 500 103 107 102 94.5

O3W <MDL 500 105 NA 96. 91.9 (2)

Bromate None RW <MDL 25.0 93.6 94.1 110 96.1

HIW <MDL 25.0 100 86.0 105 87.7

SW <MDL 25.0 98.7 95.1 105 102

GW <MDL 25.0 79.4 92.4 77. 82.2

ClW <MDL 25.0 102 NA 101 103 (2)

CDW <MDL 25.0 104 96.8 98. 92.1

O3W 2.27 25.0 87.3 NA 84. 99.9 (2)

Bromate EDA RW <MDL 25.0 97.3 95.3 99. 102

HIW <MDL 25.0 86.9 86.1 107 91.2

SW <MDL 25.0 100 104 103 94.9

GW <MDL 25.0 83.2 101 88. 88.3

ClW <MDL 25.0 105 NA 101 102 (2)

CDW <MDL 25.0 117 97.3 98. 83.9

O3W 2.32 25.0 92.6 NA 84. 88.9 (2)

See bottom of next page for explanation of notes
300.1-35

TABLE 3B. STABILITY STUDY RESULTS FOR THE INORGANIC DISINFECTION

BY-PRODUCTS (PART B)(contd.)

ANALYTE Preservative Matrix

UNFORT

CONC.

ug/L

FORT

CONC

ug/L

Analyte % Recovery

Day

0

Day

3

Day

10

Day

30

See

Note

Bromide None RW <MDL 100 99.4 97.2 107 101

HIW <MDL 100 102 103 105 105

SW 30.6 100 102 97.1 107 99.1

GW 149 100 97.7 95.3 109 100

ClW 4.73 100 8.9 NA(1) 37. 11.4 (2,3)

CDW <MDL 100 5.78 23.1 38. 51.3 (3)

O3W 30.4 100 98.3 NA 120 108 (2)

Bromide EDA RW <MDL 100 98.4 98.6 107 100

HIW <MDL 100 104 103 106 105

SW 30.5 100 99.5 98.2 107 100

GW 149 100 100 97 114 97.7

ClW 11.9 100 101 NA 115 97.4 (2,3)

CDW 6.14 100 101 96.5 119 110 (3)

O3W 31.0 100 97.3 NA 122 102 (2)

Chlorate None RW <MDL 500 102 102 105 97.4

HIW <MDL 500 96.5 97.8 101 95.4

SW 5.84 500 99.8 97.8 100 96

GW <MDL 500 99.5 98.7 101 99.8

ClW 37.8 500 102 NA 104 98.2 (2)

CDW 125 500 102 99.9 104 99.6

O3W 8.34 500 100 NA 103 97.3 (2)

Chlorate EDA RW <MDL 500 104 98.6 103 97.3

HIW <MDL 500 97.3 103 100 95

SW 6.70 500 99.7 98.2 99. 95.6

GW <MDL 500 102 97 101 99.3

ClW 38.2 500 101 NA 102 96.1 (2)

CDW 123 500 102 96.5 105 97.7

O3W 8.62 500 98.4 NA 103 96.4 (2)

NOTES:

(1) Degradation in the unpreserved matrix is apparent.

(2) NA indicates "NOT ANALYZED" 

(3) Analyte recovery will be adversely effected by reactions with free chlorine.
300.1-36

Figure 1. Chromatogram showing separation of the Part A common anions on the AS9-HC column. 

See Table 1A for analysis conditions.
300.1-37

Figure 2 Chromatogram showing separation of the Part B inorganic DBPs and bromide on the AS9-HC

column. See Table 1B for analysis conditions.
300.1-38

Figure 3. Chromatogram of the inorganic DBPs and bromide (Part B) during the MDL determination in

reagent water. See Table 1B for analysis conditions.
300.1-39

Figure 4. Chromatogram of the inorganic DBPs and bromide (Part B) in high ionic strength water (HIW). See

Table 1B for analysis conditions.