326.0-1

EPA Document # EPA 815-R-03-007

METHOD 326.0 DETERMINATION OF INORGANIC OXYHALIDE DISINFECTION

BY-PRODUCTS IN DRINKING WATER USING ION

CHROMATOGRAPHY INCORPORATING THE ADDITION OF A

SUPPRESSOR ACIDIFIED POSTCOLUMN REAGENT FOR TRACE

BROMATE ANALYSIS

Revision 1.0

June 2002

Herbert P. Wagner and Barry V. Pepich, Shaw Environmental Inc

Daniel P. Hautman and David J. Munch, US EPA, Office of Ground Water and Drinking

Water

E. Salhi and Urs von Gunten, Swiss Federal Institute for Environmental Science and

Technology, EAWAG, CH-8600, Dubendorf, Switzerland

TECHNICAL SUPPORT CENTER

OFFICE OF GROUND WATER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
326.0-2

METHOD 326.0

DETERMINATION OF INORGANIC OXYHALIDE DISINFECTION BY-PRODUCTS 

IN DRINKING WATER USING ION CHROMATOGRAPHY INCORPORATING THE

ADDITION OF A SUPPRESSOR ACIDIFIED POSTCOLUMN REAGENT FOR TRACE

BROMATE ANALYSIS

1. SCOPE AND APPLICATION

1.1 This method covers the determination of inorganic oxyhalide disinfection byproduct anions in reagent water, surface water, ground water, and finished

drinking water. In addition, bromide can be accurately determined in source or

raw water and it has been included due to its critical role as a disinfection byproduct precursor. Bromide concentration in finished water can differ due to

numerous variables which can influence the concentration. Since this method,

prior to the addition of the postcolumn reagent (PCR), employs the same hardware

as EPA Method 300.1 (1), the analysis of the common anions (using EPA Method

300.1, Part A1

) can be performed using this instrument setup with the postcolumn

hardware attached but “off-line” and with the appropriate smaller sample loop. 

Inorganic Disinfection By-products by Conductivity Detection

Analyte Comments

Bromate report values > 15.0 ug/L *

Bromide report values from source and raw waters only

Chorite report values $Minimum Reporting Level in calibration

range

Chloride report values $Minimum Reporting Level in calibration

range

Inorganic Disinfection By-products by Absorbance Detection

Analyte Comments

Bromate report values $ Minimum Reporting Level to 15.0 ug/L *

* the concentrations reported for bromate assume both detectors to be running

simultaneously.
326.0-3

1.2 The single laboratory reagent water Detection Limits (Sect. 3.15) for the above

analytes are listed in Table 1. The Detection Limit is defined as the statistically

calculated minimum concentration that can be measured with 99% confidence that

the reported value is greater than zero.(2) The Detection Limit differs from, and is

lower than the Minimum Reporting Level (MRL) (Sect. 3.16). The Detection

Limit 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 on the conductivity

detector, 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 nanosiemen (nS) noise/drift per

minute of monitored response over the background conductivity. 

1.2.2 In order to achieve acceptable detection limits on the postcolumn

absorbance detector, the postcolumn reagent must be delivered

pneumatically and some form of software signal filtering or smoothing of

the absorbance signal from the absorbance detector must be incorporated.

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

Section 9.8.

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

with this method, using the procedures described in Section 9. 

2. SUMMARY OF METHOD

2.1 The development of this method was based upon the work of several investigators

as summarised elsewhere.(3) A volume of sample, approximately 225 :L (see

Note), is introduced into an ion chromatograph (IC) which includes a guard

column, analytical column, suppressor devices, conductivity detector, a

postcolumn reagent delivery system (pneumatically controlled), a heated

postcolumn reaction coil, and a ultraviolet/visible (UV/Vis) absorbance detector

(see Figure 1). After separation and suppression of the eluent, the oxyhalide

anions chlorite, chlorate, bromate >15.0 :g/L and bromide are measured using

conductivity detection. To facilitate low-level detection of bromate, the

suppressed effluent from the conductivity detector is combined with an acidic
326.0-4

solution of potassium iodide containing a catalytic amount of molybdenum VI. 

The mixture is heated at 80B

 C (to facilitate complete reaction) where the bromate

reacts with iodide to form the tri-iodide ion which is measured by its UV

absorption at 352 nm.

NOTE: A 225 uL sample loop can be made using approximately 111 cm (44

inches) of 0.02 inch i.d. PEEK tubing. The volume should be verified to be

within 5% by weighing the sample loop empty, filling the loop with deionized

water and re-weighing the loop assuming the density of water is 1 mg/uL. 

3. DEFINITIONS

3.1 ANALYSIS BATCH – A sequence of samples, which are analyzed within a 30

hour period and include no more than 20 field samples. An analysis batch must

also include all required QC samples, which do not contribute to the maximum

field sample total of 20. The required QC samples include:

• Laboratory Reagent Blank (LRB)

• Continuing Calibration Check Standards (CCCs)

• Laboratory Fortified Blank (LFB)

• Laboratory Fortified Sample Matrix (LFSM), and 

• Either a Field Duplicate (FD) or a Laboratory Fortified Sample

Martix Duplicate (LFSMD). 

3.2 SURROGATE ANALYTE (SUR) – A pure analyte, which chemically resembles

target analytes and is extremely unlikely to be found in any sample. This analyte

is added to a sample aliquot in known amount(s) before filtration or other

processing and is measured with the same procedures used to measure other

sample components. The purpose of the SUR is to monitor method performance

with each sample.

3.3 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or

other blank matrix that is treated exactly as a sample including exposure to all

glassware, equipment, solvents and reagents, sample preservatives, and

surrogates that are used in the analysis batch. The LRB is used to determine if

method analytes or other interferences are present in the laboratory environment,

the reagents, or the apparatus.

3.4 LABORATORY FORTIFIED BLANK (LFB) – An aliquot of reagent water or

other blank matrix to which known quantities of the method analytes and all the

preservation compounds 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. 
326.0-5

3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – An aliquot of an

environmental sample to which known quantities of the method analytes and all

the preservation compounds are added in the laboratory. The LFSM 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 LFSM corrected for background concentrations.

3.6 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A

second aliquot of the field sample used to prepare the LFSM fortified, processed

and analyzed identically. The LFSMD is used instead of the Field Duplicate to

access method precision when the occurrence of target analytes is low. 

3.7 LABORATORY DUPLICATES (LD1 and LD2) – Two aliquots of the same

sample taken in the laboratory and analyzed separately with identical procedures. 

Analyses of LD1 and LD2 indicate precision associated with laboratory

procedures, but not with sample collection, preservation, or storage procedures.

3.8 FIELD DUPLICATES (FD1 and FD2) – 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 FD1 and FD2 give a

measure of the precision associated with sample collection, preservation, and

storage, as well as laboratory procedures.

3.9 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.

3.10 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A solution

containing the analytes prepared in the laboratory from stock standard solutions

and diluted as needed to prepare calibration solutions and other needed analyte

solutions.

3.11 CALIBRATION STANDARD (CAL) – A solution prepared from the primary

dilution standard solution and/or stock standard solution, and the surrogate

analytes. The CAL solutions are used to calibrate the instrument response with

respect to analyte concentration.

3.12 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.
326.0-6

3.13 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard

containing the method analytes and surrogates (s), which is analyzed periodically

to verify the accuracy of the existing calibration for those analytes.

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

surrogate(s) of known concentrations that is obtained from a source external to the

laboratory and different from the source of calibration standards. It is used to

check standard integrity.

3.15 DETECTION LIMIT – The minimum concentration of an analyte that can be

identified, measured and reported with 99% confidence that the analyte

concentration is greater than zero. This is a statistical determination of precision

(Sect. 9.2.4), and accurate quantitation is not expected at this level. (2)

3.16 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can

be reported as a quantitated value for a target analyte in a sample following

analysis. This defined concentration can be no lower than the concentration of the

lowest continuing calibration standard for that analyte and can only be used if

acceptable quality control criteria for this standard are met.

3.17 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.

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

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 on 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 induce any

negative affects on method performance by repeating and passing all the

QC criteria as described in Section 9.
326.0-7

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 Section 11.2.6. 

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

matrix interferences. With any proposed pretreatment, the analyst must

verify that target analyte(s) are not affected by monitoring recovery after

pretreatment. With advances in analytical separator column technology

which employ higher capacity anion exchange resins, the need for these

cartridges has been greatly reduced.

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

require filtration to prevent damage to instrument columns and flow systems.

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

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

or subsequent analysis. Normally, in this analysis, the elution of sulfate (retention

time of 17.5 min.) indicates the end of a chromatographic run, but in the ozonated

and chlorine dioxide matrices, a small response (200 nS baseline rise) was

observed for a very late eluting unknown peak following the response for sulfate. 

Consequently, a run time of 25 minutes is recommended to allow for the proper

elution of any potentially interfering 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.

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

additional chlorite prior to analysis. If residual chlorine dioxide is suspected in

the sample, the sample must be sparged with an inert gas (helium, argon or

nitrogen) for approximately five minutes. This sparging must be conducted prior

to ethylenediamine preservation and at the time of sample collection.

4.6 The presence of chlorite can interfere with the quantitation of low concentrations

of bromate on the postcolumn UV/Vis absorbance detector. In order to accurately
326.0-8

quantify bromate concentrations in the range 0.5 - 15.0 :g/L in this postcolumn

system, the excess chlorite must be removed prior to analysis as outlined in

Section 11.1.4. 

5. SAFETY

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

precisely defined; each chemical compound should be treated as a potential health

hazard, and exposure to these chemicals should be minimized. The 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 MSDSs should also be made available to all personnel involved in the

chemical analysis. Additional references to laboratory safety are available.(4-7)

5.2 Pure standard materials and stock standards of these compounds should be

handled with suitable protection to skin and eyes. Care should be taken not to

breathe the vapors or ingest the materials.

5.3 The following chemical has the potential to be highly toxic or hazardous. The

Material Safety Data Sheet (MSDS) should be consulted.

5.3.1 Sulfuric acid – used to prepare regenerant solution (Sect. 7.1.8) for the

second suppressor (Dionex AMMS or Ultra ASRS-1 used in the chemical

mode) and to for pretreatment of the samples for chlorite removal (Sect.

7.1.7, 11.1.4)

6. EQUIPMENT AND SUPPLIES

6.1 ION CHROMATOGRAPH – Analytical system complete with ion

chromatographic pump and all required accessories including syringes, analytical

columns, compressed gasses, suppressors, conductivity detector, mixing “tee”, 

postcolumn reagent delivery system, reaction coil, reaction coil heater, UV/Vis

absorbance detector (Figure 1) and a computer-based data acquisition and control

system.

NOTE: Because the KI PCR solution is susceptible to oxidation, resulting in a

yellow colored solution, the PCR MUST be flushed from the suppressor, reaction

coil and detector cell with reagent water upon completion of the final analysis and

prevented from draining through the reaction coil by gravity once the system is

shut down. This can be accomplished either manually or by incorporating a

column switching valve in combination with a reagent water flush. 
326.0-9

6.1.1 ANION GUARD COLUMN – Dionex AG9-HC 4 mm (Cat.#: 51791or

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, 4 mm

(Cat.#: 51786 or equivalent, see Note). The AS9-HC, 4-mm column using

the conditions outlined in Table 1 produced the separations shown in

Figures 2 and 3.

NOTE: The use of 2-mm columns is not recommended. A 50-uL sample

loop would be required with the 2-mm columns. This reduced injection

volume would decrease the “on-column” bromate and negatively affect

PCR reactivity and the subsequent absorbance response. As well, the 2-

mm columns require a flow rate approximately 4 times less than the 4-mm

columns. At the lower flow rates, band broading may become an issue and

it would be difficult to accurately maintain the appropriate reduced flow

rate for the PCR.

6.1.3 ANION SUPPRESSOR DEVICES – The data presented in this method

were generated using a Dionex Ultra-1 Anion Self Regenerating

Suppressor (4 mm ASRS, Cat.#: 53946) for electrolytic suppression of the

eluent and a second Ultra -1 ASRS was used in the chemical mode to

acidify the PCR just prior to addition to the mixing tee. Equivalent

suppressor devices may be utilized providing comparable conductivity

detection limits are achieved and adequate baseline stability is attained as

measured by a combined baseline drift/noise of no more than 10 nS per

minute over the background conductivity. Alternative suppressor

evaluations subsequent to this method development work have indicated

that improved detection limits and precision and accuracy can be obtained

for bromate by conductivity detection (8). If conductivity analytes below

10 ug/L are to be reported, the combined baseline drift/noise will be

required to be no greater than 5 nS per minute over the background

conductivity. The suppressor must be able to withstand approximately 80

-120 psi back pressure which results from connecting the postcolumn

hardware to the “eluent out” port of the suppressor. The suppressor used

to acidify the PCR must be capable of continuous operation using 150 mN

sulfuric acid as the regenerant.

6.1.3.1 – The conductivity suppressor was set to perform electrolytic

suppression at a current setting of 100 mA using the external water mode. 

Insufficient baseline stability was observed on the conductivity detector

using an ASRS in recycle mode.
326.0-10

6.1.4 CONDUCTIVITY DETECTOR – Conductivity cell (Dionex CD20 or

equivalent) capable of providing data as required in Section 9.2.

6.1.5 ABSORBANCE DETECTOR – Absorbance detector (Dionex AD20 or

equivalent with 10-mm cell pathlength, equipped with a deuterium source

bulb, or equivalent and capable of measuring absorbance at 352 nm)

capable of providing data as required in Section 9.2.

6.1.6 POSTCOLUMN REAGENT DELIVERY SYSTEM –Delivery system

(Dionex PC-10 or equivalent) capable of pneumatically delivering the

postcolumn reagent to the “eluent in” port of the suppressor to acidify the

PCR prior to entering the mixing tee (see Note). The pressure settings will

need to be established on an individual basis for each specific instrument

configuration and at a level which yields the prescribed PCR flow rates. 

NOTE: Since KI is photosensitive, the KI/Mo PCR solution was observed

to develop a light yellow color with time, even when stored under helium

in the opaque plastic PC-10 delivery container inside the PC-10

pressurization vessel. Purging the KI/Mo solution with helium

immediately after preparation to remove all oxygen did not completely

eliminate the problem. Consequently, in order to facilitate overnight (24

hours) operation, the external wall of the PC-10 plastic pressurization

vessel was wrapped with an opaque tape, or other light impervious

material, to prevent any light exposure to the KI/Mo PCR (care must be

exercised to leave about 1/16th of an inch at both the top and bottom of the

vessel free of tape to allow for proper sealing of the top and bottom. The

generation of the tri-iodide ion is pH dependant and the second suppressor

is used to acidify the PCR just before entering the reaction coil. Prior to

initiating any analysis batch, to ensure that the pH of the reaction mixture

is below 2, the effluent from the absorbance detector should be monitored

using pH test strips.

6.1.7 REACTION COIL – 500-uL internal volume, knitted, potted and 

configured to fit securely in the postcolumn reaction coil heater. (Dionex

Cat.#: 39349 or equivalent).

6.1.8 POSTCOLUMN REACTION COIL HEATER – Capable of maintaining a

temperature of 80 °C (Dionex PCH-2 or equivalent).

6.2 DATA SYSTEM – The Dionex Peaknet Data Chromatography Software was used

to generate all the data in the attached tables. Other computer-based data systems

may achieve approximately the same Detection Limits, but the user must

demonstrate this by the procedure outlined in Section 9.2. 
326.0-11

6.3 ANALYTICAL BALANCE – Used to accurately weigh target analyte salts for

stock standard preparation (±0.1 mg sensitivity).

6.4 TOP LOADING BALANCE – Used to accurately weigh reagents to prepare

eluents (±10 mg sensitivity).

6.5 WEIGH BOATS – Plastic, disposable, used to weigh 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 – Opaque, high density polyethylene (HDPE) or amber glass, 30 mL,

125 mL, 250 mL - used for sample collection and storage of calibration solutions. 

Opaque bottles are required due to the photoreactivity of the chlorite anion.

6.9 MICRO BEAKERS – Plastic, disposable, used during sample preparation.

6.10 PARTICULATE FILTERS – Gelman ion chromatography Acrodisc 0.45 um

(Cat.#: 4485 or equivalent) syringe filters. These cartridges are used to remove

particulates and [Fe(OH)3(s)] which are formed during the oxidation-reduction

reaction between Fe (II) and ClO2

-

 (see Sect. 11.1.4). 

6.11 HYDROGEN CARTRIDGES – Dionex OnGuard-H cartridges (Cat.#: 039596 or

equivalent). These cartridges are conditioned according to the manufacturer’s

directions and are used to protect the analytical column and the suppressor

membrane by removing excess ferrous iron [Fe (II)]. The ferrous iron is added to

field samples to reduce chlorite levels prior to analysis of chlorine dioxide

disinfected water samples for trace levels of bromate (see Sect. 11.1.4).

7. REAGENTS AND STANDARDS

7.1 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be

used. Unless otherwise indicated, it is intended that all reagents shall conform to 

the specifications of the Committee on Analytical Reagents of the American

Chemical Society, where such specifications are available. Other grades may be

used, provided it is first determined that the reagent is of sufficiently high purity

to permit its use without lessening the quality of the determination.

7.1.1 REAGENT WATER – Distilled or deionized water 18 M S or better, free

of the anions of interest. Water should contain particles no larger than

0.20 microns. 
326.0-12

7.1.2 ELUENT SOLUTION – Sodium carbonate (CAS#: 497-19-8) 9.0 mM. 

Dissolve 1.91 g sodium carbonate (Na2CO3

) in reagent water and dilute to

2 L.

7.12.1 This eluent solution must be sparged 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. Alternatively, an in-line degas

apparatus may be employed.

7.1.3 ETHYLENEDIAMINE (EDA) PRESERVATION SOLUTION (100

mg/mL) – Dilute 2.8 mL of ethylenediamine (99%) (CAS#: 107-15-3) to

25 mL with reagent water. Prepare fresh monthly.

7.1.4 AMMONIUM MOLYBDATE SOLUTION – A 2.0-mM solution of

ammonium molybdate tetrahydrate [(NH 4)6Mo7O24·4H2O, CAS#:

12027667, Fluka Cat.#: 09878 or equivalent] is prepared by dissolving

0.247 g in 100 mL of reagent water. This reagent is stored in an opaque

plastic storage bottle and prepared fresh monthly.

7.1.5 POSTCOLUMN REAGENT (0.26 M KI, 43 uM ammonium molybdate

heptahydrate) –The postcolumn reagent is prepared by adding 43.1 g of

potassium iodide (KI, CAS#: 7681110, Fluka Cat.#: 60400 or equivalent)

to a 1-L volumetric flask containing about 500 mL of reagent water. Two

hundred and fifteen μL of the ammonium molybdate solution (Sect. 7.1.4)

is added to the volumetric flask and diluted to volume with reagent water. 

The PCR is sparged with helium for 20 minutes to remove all traces of

dissolved oxygen and immediately placed in the PC-10 delivery vessel and

pressurized with helium. The reagent is stable for 24 hours if properly

protected from light (Sect. 6.1.6).

7.1.6 FERROUS IRON SOLUTION [1000 mg/L Fe (II)] – Dissolve 0.124 g

ferrous sulfate heptahydrate (FeSO4.7H2O, CAS#: 7782630, Sigma Cat. #:

F-7002 or equivalent) in approximately 15 mL reagent water containing 

6 uL of concentrated nitric acid and dilute to 25 mL with reagent water in

a volumetric flask (final pH -2). The Fe (II) solution must be prepared

fresh every two days.

7.1.7 SULFURIC ACID (0.5 N) – Dilute 1.4 mL of concentrated sulfuric acid

(Fisher Scientific Certified ACS Plus, A 300-500) to 100 mL.

7.1.8 SULFURIC ACID (0.15 N) – Dilute 8.5 mL of concentrated sulfuric acid

(Fisher Scientific Certified ACS Plus, A 300-500) to 2000 mL.
326.0-13

7.2 STANDARD SOLUTIONS – Standard Solutions may be prepared from certified,

commercially available solutions or from solid compounds. Compounds used to

prepare solutions must be 96% pure or greater and the weight may be used

without correction for purity to calculate the concentration of the stock standard. 

Solution concentrations listed in this section were used to develop this method

and are included as an example. Even though stability times for standard

solutions are suggested in the following sections, laboratories should use

standard QC practices to determine when Standard Solutions described in

this section need to be replaced.

7.2.1 ANALYTE STANDARD SOLUTIONS (1000 mg/L) – 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. Certified chlorite standard solutions are commercially available. 

If these are not used, chlorite requires careful consideration as outlined

below in Section 7.2.1.1.4.

7.2.1.1 ANALYTE STOCK STANDARD SOLUTIONS – Individual

Analyte Stock Standards Solutions are prepared as described

below. 

7.2.1.1.1 Bromide (Br-) 1000 mg/L – Dissolve 0.1288 g sodium

bromide (NaBr, CAS#: 7647-15-6) in reagent water and

dilute to 100 mL in a volumetric flask.

7.2.1.1.2 Bromate (BrO3

-

) 1000 mg/L – Dissolve 0.1180 g of

sodium bromate (NaBrO3, CAS#: 7789-38-0) in reagent

water and dilute to 100 mL in a volumetric flask.

7.2.1.1.3 Chlorate (C1O3

-

) 1000 mg/L – Dissolve 0.1275 g of

sodium chlorate (NaC1O3, CAS#: 7775-09-9) in

reagent water and dilute to 100 mL in a volumetric

flask. 

 

7.2.1.1.4 Chlorite (C1O2

-

) 1000 mg/L – Prepare from

commercially available standards or as described below. 

If the amperometric titration of the technical grade

sodium chlorite (NaC1O2), as specified in the Note

below, had indicated the purity of the salt to be 80.0 %

NaC1O2, the analyst would dissolve 0.1676 g of sodium

chlorite (NaC1O2

, CAS#: 7758-19-2) in reagent water

and dilute to 100 mL in a volumetric flask.
326.0-14

Note: High purity sodium chlorite (NaClO 2) is not

currently commercially available due to its 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 purity of the NaClO 2 using the

iodometric titration procedure. (9) 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)

from chlorate, bromate or bromide (as other method

target anions) in the technical grade chlorite standard. 

7.2.1.2 ANALYTE PRIMARY DILUTION STANDARD (Analyte PDS)

SOLUTION – Prepare two Analyte PDSs by diluting the Analyte

Stock Standard Solutions with reagent water containing EDA (at a

final concentration of 50 mg/L) in volumetric glassware. The

dilutions used to prepare these solutions during the method

development studies are provided below as an example. Prior to

using mixed standards for calibration or spiking solutions, ensure

that the individual Analyte Stock Standard Solutions do not contain

any appreciable concentrations of the other target analytes. 

Dilutions of these Analyte PDSs, referred to as Solution A and B

below, are used to prepare the calibration solutions (Sect. 7.2.3)

and the continuing calibration check solutions (Sect. 10.3) for both

detectors. 

Analyte PDS Solution A 

Analyte Initial Conc.

(mg/L)

Volume

(mL) 

Final Volume

(mL)

Final Conc.

(mg/L)

Chlorite 1000 2.5 25 100

Bromide 10000 0.25 25 100

Chlorate 1000 2.5 25 100

 Analyte PDS Solution B

Analyte Initial Conc.

(mg/L)

Volume

(mL) 

Final Volume

(mL)

Final Conc.

(mg/L)

Bromate 1000 1.0 100 10
326.0-15

7.2.2 SURROGATE ANALYTE (SUR) SOLUTION, DICHLOROACETATE

(DCA, CAS#: 19559-59-2 ) 

7.2.2.1 SURROGATE STOCK SOLUTION (0.50 mg/mL) – Prepare a

surrogate stock solution by dissolving 0.065 g of dichloroacetic

acid, potassium salt (Cl2CHCO2K) in reagent water and diluting to

100 mL in a volumetric flask. This solution is used to fortify all

field samples, QC samples and calibration standards by adding a

20-uL aliquot of the Surrogate Stock Solution to 10 mL of the

sample. This solution must be prepared fresh every 3 months or

sooner if signs of degradation are present.

7.2.2.1.1 Dichloroacetate is potentially present in treated drinking

waters as the acetate of the organic disinfection

byproduct, dichloroacetic acid (DCAA). Typical

concentrations of DCAA rarely exceed 50 :g/L, which

would represent only a five percent increase in the

observed response over the fortified concentration of

1.00 mg/L. Consequently, the upper recovery limit for

the surrogate (90% to 115%) has been increased to

allow for this potential background.

7.2.2.1.2 If the analyst is exclusively interested in monitoring

trace bromate using the PCR and the UV/VIS

absorbance detector, suppression of the eluent prior to

reaction with the PCR MUST be incorporated. In

addition, the surrogate must also be included and meet

the QC requirements as outlined in Section 9.7.1. 

7.2.2.2 SURROGATE PRIMARY DILUTION STANDARD – A

Surrogate PDS is not prepared since the Surrogate Stock (Sect

7.2.2.1) is used to fortify samples. 

7.2.3 CALIBRATION STANDARDS (CAL) – At least 5 calibration 

concentrations are required to prepare the initial calibration curve (Sect.

10.2) for each detector. Prepare the calibration standards over the

concentration range of interest from dilutions of the Analyte PDSs A and

B in reagent water containing EDA (50 mg/L). The lowest concentration 

calibration standard must be at or below the MRL, which may depend

upon system sensitivity. The calibration standards for the development of

this method were prepared by adding aliquots of the two Analyte PDSs

(Analyte PDS Solution A and B described above in Sect. 7.2.1.2) as shown

in the tables below to a volumetric flask, containing the listed volume of
326.0-16

EDA solution, and diluting to volume with reagent water. These standards

may be also be used as CCCs. 

Conductivity Detector CAL and CCC Standards

Cal

Std.

Stock A

(μL)

Stock B 

(μL) 

EDA

Volume

(uL)

Final

Volume

(mL)

ClO2

, Br, ClO3

Final Conc.

(μg/L)

Bromate

Final Conc.

(μg/L)

 1* 10 100 50 100 10 10

2 25 250 50 100 25 25

 3* 75 500 50 100 75 50

4 200 750 50 100 200 75

 5* 500 1000 50 100 500 100

*Prepared in larger volume and used as CCCs

Absorbance Detector CAL and CCC Standards

Cal

Std.

Stock B

(μL) 

EDA

Volume

(uL)

Final 

Volume

(mL)

Bromate

Final Conc. 

(μg/L)

 1* 5 50 100 0.5

2 10 50 100 1.0

3 20 50 100 2.0

 4* 50 50 100 5.0

5 100 50 100 10.0

 6* 150 50 100 15.0

*Prepared in larger volume and used as CCCs

7.2.3.1 Fortify each CAL or CCC standard by adding a 20 uL aliquot of

the Surrogate Stock Standard Solution (Sect. 7.2.2.1) to a 20 mL

disposable plastic micro beaker containing 10.0 mL of the

calibration standard (or CCC) and mix. These volumes may be

adjusted to meet specific laboratory autosampler volume

requirements.
326.0-17

8. SAMPLE COLLECTION, PRESERVATION AND STORAGE

8.1 SAMPLE COLLECTION 

8.1.1 Samples should be collected in opaque plastic or amber glass bottles. All

bottles must be thoroughly cleaned and the volume collected should be

sufficient to ensure a representative sample, allow for replicate analysis

and laboratory fortified matrix analysis, if required, while minimizing

waste disposal.

8.1.2 When collecting a field sample from a treatment plant employing chlorine

dioxide, the field sample must be sparged with an inert gas (helium or

nitrogen) prior to addition of the EDA preservative at time of sample

collection. The sample should be collected in a clean wide mouth flask

(such as an Erlenmeyer flask). The sparging gas can be obtained by using

a lecture bottle of nitrogen or helium fitted with a regulator and connected

to a disposable glass Pasteur pipette with PVC tubing. The gas flow

should be adjusted to produce a steady flow of bubbles. After 10-15

minutes of sparging, all traces of chlorine dioxide should be removed from

the sample. It can then be poured from the flask into the sample bottle that

contains the ethylenediamine (EDA) preservative. In order to eliminate

potential cross contamination problems, it is recommended that a clean

Erlenmeyer flask and a new disposable pipette be used at each sampling

point. 

8.1.3 Add a sufficient volume of the EDA preservation solution (Sect. 7.1.3)

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. 

8.2 SPECIAL SAMPLING REQUIREMENTS AND PRECAUTIONS FOR

CHLORITE

8.2.1 Sample bottles used for chlorite analysis must be opaque plastic or amber

glass to protect the sample from light.

8.2.2 When preparing the LFSM, 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 LFSM, EDA preservation will not

control this demand and reduced chlorite recoveries may be observed.
326.0-18

8.2.3 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.(10) 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

organochloramine. 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 ion which

is an intermediate formed as a by-product of the reaction of either ozone or

hypochlorous acid/hypochlorite ion with bromide ion. If hypobromous

acid/hypobromite ion is not removed from the matrix, further reactions

may form bromate ion.

8.3 SAMPLE SHIPMENT AND STORAGE – All samples must be chilled during

shipment and must not exceed 10 oC during the first 48 hours after collection. 

Samples must be confirmed to be at or below 10 oC when they are received at the

laboratory. Samples stored in the lab must be held at or below 6 oC and protected

from light until analysis. Samples should not be frozen. Sample preservation and

holding times for the anions are as follows:

Analyte Preservation Holding Time 

Bromate 50 mg/L EDA, store at < 6 oC 28 days

Chlorate 50 mg/L EDA, store at < 6 oC 28 days

Chlorite 50 mg/L EDA, store at <6

 oC 14 days

Bromide* 50 mg/L EDA, store at <6

 oC 28 days

*Source and raw water only.

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 capability (IDC), and subsequent analysis in each

Analysis Batch (Sect. 3.1) of a Laboratory Reagent Blank (LRB), Continuing

Calibration Check Standards (CCCs), Laboratory Fortified Blank (LFB),

Instrument Performance Check Standard (IPC), Laboratory Fortified Sample

Matrix (LFSM) and either Laboratory Fortified Sample Matrix Duplicate

(LFSMD) or a Field Duplicate (FD) Sample. This section details the specific
326.0-19

requirements for each of these QC parameters for both the conductivity and

absorbance detectors used in this application. Although the Detection Limits and

MRLs may differ, the QC requirements and acceptance criteria are the same for

both detectors. The QC criteria discussed in the following sections are

summarized in Section 17, Tables 4 and 5. These criteria are considered the

minimum acceptable QC criteria, and laboratories are encouraged to institute

additional QC practices to meet their specific needs.

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – Requirements for the

Initial Demonstration of Capability are described in the following sections and

summarized in Section 17, Table 4.

9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND –

Before any field samples are analyzed, and any time a new set of reagents

is used, it must be demonstrated that a laboratory reagent blank is

reasonably free of contamination and that the criteria in Section 9.4 are

met.

9.2.2 INITIAL DEMONSTRATION OF ACCURACY – Prior to the analysis of

the IDC samples, verify calibration accuracy with the preparation and

analysis of a mid-level QCS as defined in Section 9.11. If the analyte

recovery is not + 15% of the true value, the accuracy of the method is

unacceptable. The source of the problem must be identified and corrected. 

After the accuracy of the calibration has been verified, prepare and analyze 

7 replicate LFBs fortified at a recommended concentration of 20 ug/L for

the conductivity detector or near the mid-range of the initial calibration

curve. For the absorbance detector, prepare 7 replicate LFBs fortified at a

recommended concentration of 2.0 ug/L bromate. Sample preservatives as

described in Section 8.1.3 must be added to all LFBs. The average

recovery of the replicate values must be within ± 15% of the true value. 

9.2.3 INITIAL DEMONSTRATION OF PRECISION – Using the same set of

replicate data generated for Section 9.2.2, calculate the standard deviation

and percent relative standard deviation of the replicate recoveries. The

percent relative standard deviation (%RSD) of the results of the replicate

analyses must be # 20%.

9.2.4 DETECTION LIMIT DETERMINATION – Prepare and analyze at least 7

replicate LFBs at a concentration estimated to be near the Detection Limit

over at least 3 days using the procedure described in Section 11. This

fortification level may be estimated by selecting a concentration with a

signal of 2 to 5 times the noise level. The appropriate concentration will

be dependent upon the sensitivity of the IC system being used. Sample
326.0-20

preservatives as described in Section 8.1.3 must be added to these

samples. Calculate the Detection Limit using the equation

Detection Limit = St( n - 1, 1 - alpha = 0.99)

where

t

(n-1,1-alpha = 0.99) = Student's t value for the 99% confidence level with n-1

degrees of freedom,

n = number of replicates, and

S = standard deviation of replicate analyses.

NOTE: Calculated Detection Limits need only be less than 1/

3

 of the

laboratory’s MRL to be considered acceptable. Do not subtract blank

values when performing Detection Limit calculations. The Detection

Limit is a statistical determination of precision only.(2) No precision and

accuracy criteria are specified. 

9.3 MINIMUM REPORTING LEVEL (MRL) – The MRL is the threshold

concentration of an analyte that a laboratory can expect to accurately quantitate in

an unknown sample. The MRL should not be established at an analyte

concentration that is less than either three times the Detection Limit or a

concentration which would yield a response less than a signal-to-noise (S/N) ratio

of five. Depending upon the study’s data quality objectives it may be set at a

higher concentration. The lowest calibration standard must be at or below the

MRL and therefore, the MRL must never be established at a concentration

lower than the lowest calibration standard. 

9.4 LABORATORY REAGENT BLANK (LRB) – A LRB is required with each

Analysis Batch (Sect. 3.1) of samples to determine any background system

contamination. If within the retention time window of any analyte, the LRB

produces a peak that would prevent the determination of that analyte, determine

the source of contamination and eliminate the interference before processing

samples. Background contamination must be reduced to an acceptable level

before proceeding. Background from method analytes or contaminants that

interfere with the measurement of method analytes must be below 1/3 the MRL. 

If the target analytes are detected in the LRB at concentrations equal to or greater

than this level, then all data for the problem analyte(s) must be considered invalid

for all samples in the analysis batch. 

9.4.1. EDA must be added to the LRB at 50 mg/L. By including EDA in the

LRB, any potential background contamination from the EDA will be

identified.
326.0-21

9.4.2 When the PCR method is used for low level bromate analysis on samples

from public water systems (PWSs) which employ chlorine dioxide

disinfection, the matrix must be pretreated to remove the potentially

interfering chlorite anion (Sect. 11.1.4). When these types of pretreated

samples, or any type of pretreatment is applied to field samples included as

part of an analysis batch, a second LRB must be prepared, pretreated and

analyzed to confirm no background effects of the pretreatment are present. 

If the analysis batch contains only pretreated samples, then only a

pretreated LRB is required.

9.5 CONTINUING CALIBRATION CHECK (CCC) – CCCs are prepared in the

same manner as the Calibration Standards (Sect. 7.2.3), using reagent water and

EDA as described in Section 8.1.3. They are analyzed during an analysis batch at

a required frequency to confirm that the instrument meets initial calibration

criteria. See Section 10.3 for concentration requirements, frequency requirements, 

and acceptance criteria. 

9.6 LABORATORY FORTIFIED BLANK (LFB) - A LFB is required with each

analysis batch to confirm acceptable method accuracy. Since calibration solutions

are prepared in large volumes and can be used over an extended period of time,

the integrity of the concentration of the solution used to fortify the LFSM is

checked by preparing the LFB using the same Analyte Stock Standard Solutions

used to prepare the LFSM fortification solution. The fortified concentration of the

LFB should be rotated between, low, medium, and high concentrations from batch

to batch. The low concentration LFB must be as near as practical to, but no more

than two times the MRL. Similarly, the high concentration should be near the

high end of the calibration range established during the initial calibration (Sect.

10.2). The recovery of all analytes fortified at the low concentration must be 75-

125% of the true value, and 85-115% when fortified at the medium and high

concentrations. If the LFB recovery for an analysis batch does not meet these

recovery criteria, the data are considered invalid, and the source of the problem

must be identified and resolved before continuing with analyses. 

LFB Fortified Concentration Range LFB Recovery Limits

MRL to 2 x MRL 75 - 125 %

2 x MRL to highest calibration level 85 - 115 % 

9.7 SURROGATE RECOVERY – The surrogate standard is fortified into all samples,

blanks, CCCs, QDCSs, LRBs, and LFSMs and LFSMDs prior to analysis. It is
326.0-22

also added to the calibration curve and calibration check standards. The surrogate

is a means of assessing chromatographic method performance.

9.7.1 Surrogate recoveries must fall between 90-115% for proper instrument

performance and analyst technique to be verified. The recovery range for

the surrogate is extended to 115% to allow for the potential contribution of

trace levels of dichloroacetate as a halogenated organic disinfection byproduct (DBP) of dichloroacetic acid (DCAA). Background levels of this

organic DBP are rarely observed above 50 :g/L (0.05 mg/L) which

constitutes only 5% of the 1.00 mg/L recommended fortified

concentration.

9.7.2 When surrogate recovery from a sample, blank, or CCC is less than 90%

or greater than 115%, check (1) calculations to locate possible errors, (2)

standard solutions for degradation, (3) contamination, and (4) instrument

performance. If those steps do not reveal the cause of the problem,

reanalyze the sample.

9.7.3 If the reanalysis meets the surrogate recovery criteria, report only data for

the reanalyzed sample.

9.7.4 If the sample reanalysis fails the 90-115% surrogate recovery criteria, the

analyst should check the calibration by re-injecting the most recently

acceptable calibration standard. If the calibration standard fails the criteria

of Section 10.3, recalibrate as described in Section 10.2. If the calibration

standard is acceptable, preparation and analysis of the sample should be

repeated provided the sample is still within the holding time. If this

sample reanalysis also fails the recovery criteria, report all data for that

sample as suspect due to surrogate recovery. 

9.7.5 If a laboratory chooses to monitor exclusively for trace bromate using PCR

and the UV/VIS absorbance detector, suppression of the eluent MUST be

used and the surrogate added and monitored on the conductivity detector

and the appropriate QC criteria for the surrogate as outlined in Section

9.7.1 must be met.

9.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysis of LFSMs

are required in each analysis batch and are used to determine that the sample

matrix does not adversely affect method accuracy. Additional LFSM

requirements, as described in Section 9.8.4, apply when the PCR system is used

for low level bromate in waters disinfected with chlorine dioxide. If the

occurrence of target analytes in the samples is infrequent, or if historical trends are
326.0-23

unavailable, a second LFSM, or LFSMD (Sect. 9.9), must be prepared, and

analyzed from a duplicate of the field sample used to prepare the LFSM to assess

method precision. Analytical batches that contain LFSMDs will not require the

analysis of a Field Duplicate (Sect. 9.9). If a variety of different sample matrices

are analyzed regularly, for example, drinking water from groundwater and surface

water sources, method performance should be established for each. Over time,

LFSM data should be documented for all routine sample sources for the

laboratory. 

9.8.1 Within each analysis batch, a minimum of one field sample is fortified as a

LFSM for every 20 samples processed. The LFSM is prepared by spiking

a sample with an appropriate amount of the appropriate Analyte PDS

(Sect. 7.2.1.2). Select a spiking concentration at least twice the matrix

background concentration, if known. Use historical data or rotate through

a range of concentrations when selecting a fortifying concentration. 

Selecting a duplicate bottle of a sample that has already been analyzed aids

in the selection of appropriate spiking levels.

9.8.2 Calculate the percent recovery (%R) for each analyte using the equation

where

A = measured concentration in the fortified sample

B = measured concentration in the unfortified sample, and

C = fortification concentration.

9.8.3 Analyte recoveries may exhibit a matrix bias. For samples fortified at or

above their native concentration, recoveries should range between 75 -

125%. If the accuracy of any analyte falls outside the designated range,

and the CCC performance for that analyte is shown to meet the acceptance

criteria, the recovery is judged to be matrix-biased. The result for that

analyte in the unfortified sample is labeled suspect/matrix to inform the

data user that the results are suspect due to matrix effects. 

9.8.4 When the PCR method is used for low level bromate analysis on field

samples from PWSs which employ chlorine dioxide disinfection and

consequently contain chlorite, a LFSM must be prepared, exclusively for

trace bromate, for each of these field samples. Initially, the field sample is

analyzed and chlorite, chlorate and bromide levels are determined. Then,

a second aliquot of field sample is pretreated to remove chlorite, as
326.0-24

described in Section 11.1.4, and analyzed to determine native bromate

concentration. A third aliquot of the field sample then must be fortified

with bromate, pretreated as described in Section 11.1.4 to remove chlorite,

and analyzed to assess bromate recovery from that matrix. This additional

QC is required to rule out matrix effects and to confirm that the laboratory

performed the chlorite removal step (Sect. 11.1.4) appropriately. This

LFSM should be fortified with bromate at concentrations close to but

greater than the level determined in the native sample. Recoveries are

determined as described above (Sect. 9.8.2). Samples that fail the LFSM

percent recovery criteria of 75 - 125% must be reported as suspect/matrix.

9.9 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX

DUPLICATE (FD or LFSMD) – Within each analysis batch, a minimum of one

Field Duplicate (FD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD)

must be analyzed. Duplicates check the precision associated with sample

collection, preservation, storage, and laboratory procedures. If target analytes are

not routinely observed in field samples, a LFSMD should be analyzed rather than

a FD. 

9.9.1 Calculate the relative percent difference (RPD) for duplicate

measurements (FD1 and FD2) using the equation

9.9.2 If a LFSMD is analyzed instead of a Field Duplicate, calculate the relative

percent difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using

the equation

9.9.3 RPDs for FDs and duplicate LFSMs should fall in the range presented in

the table below for samples fortified at or above their native concentration.

Greater variability may be observed when LFSMs are spiked near the

MRL. 
326.0-25

Concentration Range RPD Limits

MRL to 5 x MRL ± 20 %

5 x MRL to highest calibration level ± 10 % 

If the accuracy of any analyte falls outside the designated range, and the

laboratory performance for that analyte is shown to meet the acceptance

criteria in the LFB, the recovery for that analyte is judged to be matrix

biased. The result for that analyte in the unfortified sample is labeled

suspect/matrix to inform the data user that the results are suspect due to

matrix effects

9.10 INSTRUMENT PERFORMANCE CHECK – The low-level CCC Standard is 

evaluated in each analytical batch in order to confirm proper instrument

performance (Sect. 10.3). This analysis confirms the MRL and demonstrates

proper chromatographic performance at the beginning of each analysis batch. 

Chromatographic performance is judged by calculating the Peak Gaussian Factor

(PGF), which is a means to measure peak symmetry and monitor retention time

drift in the surrogate peak over time. The PGF, as determined below, must fall

between 0.80 and 1.15, and the retention time for the surrogate must be at least

80% of the initial retention time when the IC column was new. If these criteria

are not met, corrective action must be performed prior to analyzing additional

samples. Major maintenance such as replacing columns requires repeating the

IDC determination (Sect. 9.2). 

9.10.1 The PGF is calculated using the equation

where

W(1⁄2) is the peak width at half height, and 

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

NOTE: Values for W(1⁄2) and W (1/

10) for each peak can be attained

through most data acquisition software packages.

9.10.2 Small variations in retention time can be anticipated when a new solution

of eluent is prepared but if sudden shifts of more than 5% are observed in

the surrogate retention time, some type of instrument problem should be
326.0-26

suspected. Potential problems include improperly prepared eluent,

erroneous method parameters 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 resemble 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 requires cleaning or replacement, especially if

resolution problems are beginning to occur between previously resolved

peaks. A laboratory should retain a historic record of retention times for

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

column’s efficiency.

9.10.3 If a laboratory chooses to monitor exclusively for trace bromate using PCR

and the UV/VIS absorbance detector, suppression of the eluent MUST be

used and the surrogate added and monitored on the conductivity detector

and the appropriate QC criteria for the surrogate as outlined in Section

9.7.1 must be met. 

9.11 QUALITY CONTROL SAMPLE (QCS) – Each time new Calibration Standards

(Sect. 7.2.3) are prepared, or at least quarterly, analyze a QCS from a source

different from the source of the calibration standards. The acceptance criteria for

the QCS is 85-115% of the true value. If measured analyte concentrations are not

of acceptable accuracy, check the entire analytical procedure to locate and correct

the problem source.

10. CALIBRATION AND STANDARDIZATION

10.1 Demonstration of acceptable initial calibration is required prior to performing the

IDC and before any samples are analyzed. It is also required intermittently

throughout sample analysis to meet required QC performance criteria summarized

in Tables 4 and 5. Initial calibration verification is performed using a QCS (Sect.

9.11) as well as with each analysis batch using Continuing Calibration Check

Standards. The procedure for establishing the initial calibration curve is

described in Section 10.2. The procedure to verify the calibration with each

analysis batch is described in Section 10.3. 

10.2 INITIAL CALIBRATION 

10.2.1 Establish ion chromatographic configuration and operating parameters

equivalent to those indicated in Table 1 and Figure 1.
326.0-27

10.2.2 Estimate the calibration range over which the instrument response is

linear. On the conductivity detector for the four target analytes (chlorite,

bromate, bromide and chlorate) the linear range should cover the expected

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

than two orders of magnitude in concentration. The method development

data were collected on single linear calibrations that spanned 5 to 500 ug/L

for chlorite, bromide and chlorate and 5 to 100 ug/L bromate for the

conductivity detector and 0.5 to 15.0 ug/L for bromate on the absorbance

detector.

10.2.2.1 If quantification is desired over a larger range, then two or more

separate calibration curves must be prepared. 

10.2.2.2 A minimum of five Calibration Standards (Sect. 7.2.3) should be

prepared for each calibration. It is recommended that at least

four of the Calibration Standards are at a concentration $ the

MRL. Because high concentrations of chlorite can interfere with

the postcolumn analysis of low levels of bromate, the

conductivity and absorbance detectors must be calibrated

separately. 

10.2.2.3 When quantitated using the initial calibration curve, each

calibration point, except the lowest point, for each analyte should

calculate to be 85-115% of its true value. The lowest point

should calculate to be 75-125% of its true value. Failure to meet

this criteria may indicate future difficulty in meeting CCC QC

requirements during the analysis batch.

10.2.2.4 Since the concentration ranges in actual field samples by

conductivity detection for chlorite, bromide and chlorate are

expected to cover two orders of magnitude, the use of calibration

standards in the range 5 - 500 :g/L is recommended. 

10.2.2.5 Bromate concentrations are expected to be significantly lower. It

is suggested that the conductivity detector be calibrated using 

bromate calibration standard levels in the range 5 - 100 :g/L. 

Additionally, report values for bromate by conductivity ONLY

when they are measured by the PCR above 15.0 ug/L. The

conductivity detector may exhibit a response for bromate at

concentrations below 15.0 ug/L, but these should not be reported. 

When using both detectors, PCR results for bromate in this range

(5 - 15 ug/L) will have far better precision and accuracy. 
326.0-28

10.2.3 Prepare a set of at least 5 calibration standards as described in Section

7.2.3. The lowest concentration calibration standard must be at or below

the MRL, which may depend on system sensitivity. 

10.2.4 Inject 225 :L of each calibration standard and tabulate peak area responses

against the concentration for the four target analytes, the surrogate from

the conductivity detector, and bromate from the postcolumn absorbance

detector. Prepare calibration curves using linear regression analysis for

each analyte on the conductivity detector and using a quadratic polynomial

function for bromate on the absorbance detector. 

10.2.4.1 Use of 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 leading to peak broadening.

However, poorly drawn baselines can have a more significant

influence on peak areas than peak heights. It is the analyst’s

responsibility to review all chromatograms to ensure accurate

baseline integration of target analyte peaks.

10.2.5 After establishing (or re-establishing) calibration curves, the accuracy of

this calibration must be verified through the analysis of a QCS or an

externally prepared second source standard. The QCS should be prepared

at a concentration near the middle of the calibration range. As specified in

Section 9.11, determined concentrations must fall within ± 15% of the

stated values.

10.3 CONTINUING CALIBRATION CHECK (CCC) – Initial calibrations may be

stable for extended periods of time. Once the calibration curves have been

established for both the conductivity and absorbance detectors, they must be

verified for each analysis batch prior to conducting any field sample analyses

using CCCs. The first CCC each day must be at or below the MRL in order to

verify instrument sensitivity prior to any analyses. Subsequent CCCs must be run

after every 10 field samples and should alternate between a mid- and high-level

CCC. LRBs, CCCs, LFSMs and LFSMDs are not counted as field samples. 

10.3.1 A low-level CCC must be determined to be valid each day prior to

analyzing any samples by injecting an aliquot of the appropriate CCC

under the same instrumental conditions used to collect the initial

calibration. Since two detectors are incorporated in this method, this must

be accomplished by using a mixed calibration check standard for the four

conductivity analytes and a separate low-level bromate CCC for the
326.0-29

absorbance detector. The low-level CCC for both detectors must be at or

below the MRL. Percent recovery for the low-level CCC must be in the

range of 75 - 125% before the analyst is allowed to analyze samples. 

10.3.2 Additional CCC standards must be analyzed after every tenth field sample

and at the end of the analysis batch. If more than 10 field samples are

included in an analysis batch, the analyst should alternate between the

mid- and high-level CCC Standards. Percent recovery for the mid- and

high-level CCCs must be in the range of 85 - 115%. 

10.3.3 If the calibration verification criteria listed above are not met, or the

retention times shift more than ± 2% from the last acceptable initial or

continuing calibration check standard for any analyte, then all samples

analyzed after the last acceptable calibration check standard are considered

invalid and must be reanalyzed. The source of the problem must be

identified and resolved before reanalyzing the samples or continuing with

the analyses. 

10.3.2.1 In the case where the end calibration failed to meet performance

criteria, but the initial and middle calibration check standards

were acceptable, the samples bracketed by the acceptable

calibration check standards may be reported. However, all field

samples between the middle and end calibration check standards

must be reanalyzed.

11. PROCEDURE

11.1 SAMPLE PREPARATION

11.1.1 For refrigerated or field samples arriving at 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.

11.1.2 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. This is done

by adding 20 :L of the surrogate solution (Sect. 7.2.2) to a 20-mL

disposable plastic micro beaker. Next, place a 10.0-mL aliquot of sample

in the micro beaker and mix. These volumes may be adjusted to meet

specific laboratory autosampler volume requirements provided the

fortified surrogate concentration is at the prescribed concentration of 1.0

mg/L. The sample is now ready for analysis.
326.0-30

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

surrogate is considered insignificant. If a laboratory chooses to monitor

exclusively for trace bromate using PCR and the UV/VIS absorbance

detector, suppression of the eluent MUST be used and the surrogate added

and monitored on the conductivity detector and the appropriate QC criteria

for the surrogate as outlined in Section 9.7.1 must be met.

11.1.3 Using a Luer lock, plastic 10-mL syringe, withdraw the sample from the 

micro beaker and attach a 0.45-:m 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 filtered, 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. 

11.1.4 CHLORINE DIOXIDE - TREATED WATERS CONTAINING

CHLORITE – Treatment plants that use chlorine dioxide as part of their

treatment process can produce high levels of chlorite in samples. Since

chlorite can interfere with the postcolumn quantitation of low levels of

bromate as described in Section 4.6, chlorite must be removed from these

samples prior to analysis.(11) The oxidation-reduction reaction between

ferrous iron and chlorite(12) is used to remove chlorite without any adverse

affects on the bromate concentration.

(13) 

 

11.1.4.1 Place a 10-mL aliquot of sample in a 20-mL micro beaker and add

35 uL of 0.5 N sulfuric acid (Sect. 7.1.7). After mixing, verify the

pH is between 5 and 6 using pH test strips, add 40 uL of ferrous

iron solution (Sect. 7.1.6), mix and allow to react for 10 minutes. 

Filter the reaction mixture using a 0.45 micron particulate filter

(Sect. 6.10) attached to a 10-mL syringe into the barrel of a second

syringe to which a pre-conditioned hydrogen cartridge (Sect. 6.11)

is attached. Pass the solution through a hydrogen cartridge at a

flow rate of approximately 2 mL per minute. Discard the first 3

mL, and collect an appropriate volume (depending on autosampler

vial size) for analysis. Add the respective volume of surrogate

solution, depending on the volume collected. The sample is ready

for analysis (Sect. 11.2). 

NOTE: Pretreated samples can be held for no more than 30 hours

after initial pretreatment. If this time has expired, the pretreatment

steps must be repeated on a second aliquot of both the field sample

matrix and the respective LFSM.
326.0-31

11.1.4.2 In order to ensure data quality, all samples from PWSs which

utilize chlorine dioxide which have been pretreated to remove

chlorite, MUST also be used to prepare a pretreated LFSM specific

to trace bromate. This LFSM should be fortified with bromate at

concentrations close to but greater than the level determined in the

native sample. Initially, the field sample is analyzed and chlorite,

chlorate and bromide levels are determined. Then, a second

aliquot of field sample is pretreated to remove chlorite, as

described above and analyzed to determine native bromate

concentrations. A third aliquot of the field sample then must be

fortified with bromate, pretreated to remove chlorite, and analyzed

to assess bromate recovery from that matrix. This additional QC

is required to rule out matrix effects and to confirm that the

laboratory performed the chlorite removal step appropriately. If

the bromate recovery falls outside the acceptance range of 75 -

125% (Sect. 9.8), that particular sample should be reported as

suspect/matrix.

11.1.4.3 All samples from PWSs that utilize chlorine dioxide, which have

been pretreated to remove chlorite, MUST also include an

additional pretreated LRB in the analytical batch (Sect. 9.4.2). 

11.1.4.4 Suppressor devices which have had long term exposure to iron

cations may have reduced method performance in other

applications, such as the determination of certain common

inorganic anions. If reduced peak response is observed,

particularly for fluoride or phosphate, the suppressor should be

cleaned according to the manufacturer’s recommendations.

11.2 SAMPLE ANALYSIS

11.2.1 Table 1 summarizes the recommended operating conditions for the ion

chromatograph and delivery of the postcolumn reagent. Included in this

table are the actual retention times and Detection Limits that were

determined during the development of this method. Other columns or

chromatographic conditions may be used if the requirements of Section 9 are

met. 

11.2.2 Establish a valid initial calibration as described in Section 10.2 and complete

the IDC (Sect. 9.2). Check system calibration by analyzing a low-level

CCC (Sect. 10.3.1) as part of the initial QC for the analysis batch and, if

required, recalibrate as described in Section 10.2. 
326.0-32

11.2.3 Inject 225 :L of each sample. Use the same size loop for standards and

samples. An automated constant volume injection system may also be used. 

11.2.4 The width of the retention time window used to make identifications should

be based upon measurements of actual retention time variations of standards

measured over several days. Three times the standard deviation of 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. 

11.2.5 If the response of a sample analyte exceeds the calibration range, the sample

must be diluted with an appropriate amount of EDA fortified reagent water

and reanalyzed.

11.2.6 Should more complete resolution be needed between any two coeluting

peaks, the eluent (Sect. 7.1.2) can be diluted. This will extend the run,

however, and will cause late eluting anions to be retained even longer. The

analyst must verify that this dilution does not negatively affect performance

by repeating the IDC (Sect. 9.2), and by reestablishing a valid initial

calibration curve (Sect. 10.2). As a specific precaution, upon dilution of the

carbonate eluent, a peak for bicarbonate may be observed on the 

conductivity detector within the retention time window for bromate which

will negatively impact the analysis.

11.2.6.1 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

Detection Limit for each analyte.

11.3 AUTOMATED ANALYSIS WITH METHOD 326.0 

11.3.1 Laboratories conducting analyses on large numbers of samples often prepare

large analysis batches that are run in an automated manner. When

conducting automated analyses, careful attention must be paid to all

reservoirs to be certain sufficient volumes are available to sustain extended

operation. Laboratories must ensure that all QC performance criteria are

met as described in preceding sections to ensure their data are of acceptable

quality.

11.3.1.1 Special attention must be paid when the PCR reservoir is refilled.

The PCR is stable for only 24 hours and consequently the reservoir

must be regularly filled with freshly prepared reagent. Since this is

a pneumatically driven system, the baseline will require a
326.0-33

minimum of ten minutes to restabilize after the reservoir has been

refilled and the bottle repressurized.

11.3.2 Because this method has two detectors that require independent calibration, 

analysis sequences must be carefully constructed to meet required QC

specifications and frequency (Sect. 17, Table 5). To help with this task, an

acceptable sequence for a sample analysis batch, with all the methodrequired QC, is shown in Table 6. This schedule is included only as an

example of a hypothetical analysis batch where the analyst desires to collect

data using both detectors. Within the analysis batch, references to exact

concentrations for the CCCs are for illustrative purposes only. The analyses

for sample #14 provides an example of the QC requirements for a complete

conductivity and trace bromate PCR analysis of a sample from a PWS

employing chlorine dioxide disinfection. 

12. DATA ANALYSIS AND CALCULATIONS

12.1 Identify the method analytes in the sample chromatogram by comparing the

retention time of the suspected analyte peak to the retention time of a known analyte

peak in a calibration standard. If analyte retention times have shifted (generally

towards shorter times) since the initial calibration, but are still within acceptance

criteria and are reproducible during the analysis batch, the analyst should use the

retention time in the daily calibrations to confirm the presence or absence of target

analytes.

12.2 Compute sample concentration using the initial calibration curve generated in

Section 10.2. 

12.3 Report ONLY those values that fall between the MRL and the highest calibration

standard. Samples with target analyte responses exceeding the highest standard

must be diluted and reanalyzed. When this is not possible the alternate calibration

procedures described in Section 11.2.5 must be followed. 

12.3.1 Report bromate concentrations using the postcolumn UV/Vis absorbance

detector when they fall between the MRL and 15.0 ug/L. When bromate

concentrations exceed 15.0 ug/L, as detected by UV/Vis absorbance, either

report by conductivity, calibrate the postcolumn UV/Vis absorbance detector

to a higher bromate concentration, or dilute the sample.

12.4 Report analyte concentrations in :g/L (usually with two significant figures). 

12.5 Software filtering of the postcolumn UV/Vis absorbance signal is recommended to

improve the precision of peak measurements, minimize non-random noise and
326.0-34

improve peak appearance, ensuring that all QC requirements for the method are

met. Olympic smoothing (25 points, 5 seconds with 1 iteration) was chosen using

peak area for quantitation because it was determined to have minimal effect on peak

height and/or area.

(14) The use of alternate smoothing routines is acceptable

providing all QC criteria are met. 

13. METHOD PERFORMANCE

13.1 Table 1 lists the standard conditions, typical retention times and single laboratory

Detection Limits in reagent water, as determined for each of the inorganic oxyhalide

DBPs and bromide. 

13.2 Table 2 shows the precision and accuracy of the trace bromate measurement,

evaluated on both detectors, at two fortified concentrations, in reagent water (RW),

a simulated high ionic strength water (HIW) and a simulated high organic (HOW)

content water. The mean recovered bromate concentration (accuracy relative to the

fortified level) and the precision (expressed as %RSD of the replicate analyses) are

tabulated. The HIW was designed to simulate a high ionic strength field sample and

the HOW designed to simulate a high organic content field sample. The HIW 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.(1) The HOW was

prepared from reagent water fortified with 1.0 mg/L humic acid.(1)

13.3 Table 3 summarizes the single laboratory accuracy (%Recovery) and precision (%

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

conditions identified in Table 1.

14. POLLUTION PREVENTION

14.1 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 analytical procedures described in this method generate relatively small

amounts of waste since only small amounts of reagents are used. The matrices of

concern are finished drinking water. However, the Agency requires that laboratory

waste management practices be conducted consistent with all applicable rules and

regulations, and that laboratories protect the air, water, and land by minimizing and
326.0-35

controlling all releases from fume hoods and bench operations. Also, compliance is

required with any sewage discharge permits and 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” also available from the American Chemical Society at the

address in Section 14.1.

16. REFERENCES 

1. U.S. EPA Method 300.1. “Determination of Inorganic Anions in Drinking Water by

Ion Chromatography”. EPA Document number: EPA/600/R-98/118. NTIS number

PB98-169196 INZ.

2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace

Analyses for Wastewaters," Environ. Sci. Technol. 1981, 15, 1426-1435. 

3. Wagner, H..P., Pepich, B.V., Hautman, D.P. and Munch, D.J. “US Environmental

Protection Agency Method 326.0, a New Method for Monitoring Inorganic

Oxyhalides and Optimization of the Postcolumn Derivatization for the Selective

Determination of Trace Levels of Bromate.” J. Chro. A, 2002, 956, 93-101.

4. “OSHA Safety and Health Standards, General Industry,” (29CFR1910).

Occupational Safety and Health Administration, OSHA 2206, (Revised, Jan. 1976).

5. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, “Standard

Practice for Sampling Water,” American Society for Testing and Materials,

Philadelphia, PA, 1986.

6. “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of

Health, Education, and Welfare, Public Health Service, Center for Disease Control,

National Institute of Occupational Safety and Health, Atlanta, Georgia, August

1977.

7. “Safety In Academic Chemistry Laboratories,” 3rd Edition, American Chemical

Society Publication, Committee on Chemical Safety, Washington, D.C., 1979.

8. Wagner, H..P., Pepich, B.V., Hautman, D.P. and Munch, D.J. “Improving the

Performance of EPA Method 300.1 for Drinking Water Compliance Monitoring.” 

J. Chrom. A. Special Edition of the 2002 International Ion Chromatography

Smyposium (in press).
326.0-36

9. 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). 

10. Hautman, D.P. & Bolyard, M. “Analysis of Oxyhalide Disinfection By-products and

other Anions of Interest in Drinking Water by Ion Chromatography.” J. Chrom. A,

1992, 602, 65-74.

11. Wagner, H..P., Pepich, B.V., Hautman, D.P. and Munch, D.J. “Analysis of 500 ppt

Levels of Bromate in Drinking Waters Using Direct Injection Suppressed Ion

Chromatography with a Single, Pneumatically Delivered Postcolumn Reagent.” J.

Chrom. A, 1999, 850, 119-129.

12. Iatrou, A. and Knocke, W.R. “Removing Chlorite by the Addition of Ferrous Iron”. 

Journal of the AWWA, Research and Technology, (November, 1992), 63-68.

13. Wagner, H..P., Pepich, B.V., Hautman, D.P. and Munch, D.J. “Eliminating the

Chlorite Interference in US Environmental Protection Agency Method 317.0 Permits

the Analysis of Trace Bromate Levels in all Drinking Water Matrices.” J. Chrom..

A, 2000, 882, 309-319.

14. Schibler, J.A., “ Improving Precicion and Accuracy with Software-based Signal

Filtering”. American Laboratory, (December, 1997), 63-64.
326.0-37

17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA

TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS IN

REAGENT WATER FOR THE INORGANIC OXYHALIDE DISINFECTION BYPRODUCTS AND BROMIDE.

Standard Conditions and Equipment(a):

Ion Chromatograph: Dionex DX500

Sample Loop: 225 :L

Eluent: 9.0 mM Na2CO3

Eluent Flow: 1.3 mL/min

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

Typical System Backpressure: 2300 psi

Conductivity Suppressor: ASRS-1, external water mode, 100 mA current for conductivity

PCR Suppressor: ASRS-1 used with sulfuric acid regenrant to acidify the PCR 

Detectors: Dionex CD20 suppressed conductivity detector, background

conductivity: 24 :S

Dionex AD20 Absorbance Detector, 10 mm cell path length, set at

352 nm (deuterium lamp)

Postcolumn Reagent Flow: 0.4 mL/min 

Postcolumn Reactor Coil: knitted, potted for heater, 500 uL internal volume

Postcolumn Heater: 80 ° C

Postcolumn Regenerant 150 mN H2SO4, 2.5 mL/min, effluent pH < 2

Total analysis time: 25 minutes

Analyte Retention Times and Detection Limits :

Analyte Retention Time (b)

(min.)

Fortified

Conc.

(:g/L)

# of 

Reps.

Detection

Limit

(:g/L)

Chlorite 4.11 5.0 8 2.0

Bromate (c)

Bromate (d) 

4.73

5.28

5.0

0.50

8

8

1.2

0.17

Surrogate: DCA 8.01

Bromide 9.18 5.0 8 1.7

Chlorate 10.07 5.0 8 1.7

(a) Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use. 

(b) Refer to chromatograms in Figures 2 and 3.

(c) Bromate by conductivity detection

(d) Bromate by absorbance detection
326.0-38

TABLE 2. SINGLE LABORATORY PRECISION IN VARIOUS MATRICES FOR

BROMATE BY ABSORBANCE DETECTION.

PRECISION

Matrix Detection

Fortified

Conc.

(:g/L)

# of Reps.

Mean

(:g/L)

%

RSD

Reagent 

Water

Absorbance 1.0 8 1.1 4.4

Absorbance 5.0 8 5.2 2.1

High Ionic

Water

Absorbance 1.0 8 1.1 4.2

Absorbance 5.0 8 5.2 2.0

High 

Organic

Water

Absorbance 1.0 7 1.1 3.4

Absorbance 5.0 8 5.2 3.2

Standard Conditions: Same as listed in Table 1.
326.0-39

TABLE 3. SINGLE-LABORATORY PRECISION AND RECOVERY FOR THE

INORGANIC DISINFECTION BY-PRODUCTS, BROMIDE AND

SURROGATE.

Analyte Matrix

Fortified

Conc.

(ug/L)

# of

Replicates

Mean

% Recovery %RSD

Chlorite RW 100

500

8

8

107

108

3.0

1.2

HIW 100

500

8

8

102

106

2.0

0.71

HOW 100

500

8

8

99.3

107

2.7

0.49

Bromate by

Conductivity

RW 10.0

25.0

8

8

102

99.8

4.6

4.3

HIW 10.0

25.0

8

8

103

92.9

3.8

11

HOW 10.0

25.0

8

8

101

97.6

8.1

5.9

Bromide RW 10.0

25.0

8

8

97.5

104

5.3

5.1

HIW 10.0

25.0

8

8

108

104

4.8

5.0

HOW 10.0

25.0

8

8

104

99.7

6.1

3.7

Chlorate RW 100

500

8

8

111

104

1.7

0.97

HIW 100

500

8

8

99.0

100

2.3

0.66

HOW 100

500

8

8

101

105

2.8

1.1

Surrogate RW 1.00 8

8

108

106

6.1

4.7

HIW 1.00 8

8

103

105

1.3

2.1

HOW 1.00 8

8

108

108

4.4

4.0

RW = Reagent Water; HIW = High Ionic Strength Water; HOW = High Organic Water
326.0-40

TABLE 4. INITIAL DEMONSTRATION OF CAPABILITY QC REQUIREMENTS.

Reference Requirement Specification and Frequency Acceptance Criteria

Sect. 9.2.1

and 9.4

 

Initial

Demonstration

of Low System

Background

Analyze a method blank (LRB) and

determine that all target analytes are

below 1⁄2 of the proposed MRL prior to

performing the IDC

The LRB

concentration must be

#1/3 of the proposed

MRL

Sect. 9.2.2 Initial

Demonstration

of Accuracy

(IDA)

Run mid-level QCS and determine

recovery.

Conductivity: analyze 7 replicate LFBs

recommend fortify at 20 ug/L 

Absorbance: analyze 7 replicate LFBs

recommend fortify with bromate at 2.0

ug/L Calculate average recovery of IDA

replicates

QCS recovery must be

± 15% of true value.

Mean % recovery for

IDA replicates must

be ± 15% of true

value.

Sect. 9.2.3 Initial

Demonstration

of Precision

(IDP)

Calculate the %RSD of the IDA

replicates.

%RSD must be #20%

Sect. 9.2.6 Detection Limit

Determination

Select a fortifying level at 3-5 times the

estimated instrument detection limit at or

lower than the MRL. Analyze 7

replicate LFBs

Calculate over at least 3 days using

equation in Section 9.2.6 - do not

subtract blank

Detection Limit must

be < 1/3 the MRL
326.0-41

TABLE 5. QUALITY CONTROL REQUIREMENTS (SUMMARY).

Reference Requirement Specification and Frequency Acceptance Criteria

Sect. 8.3 Sample Holding

Time /

Preservation

Bromate 28 days, refrig. at <6 oC / 

 EDA Preservation

Bromide 28 days, EDA Permitted

Chlorate 28 days, refrig. at <6 oC /

EDA Preservation

Chlorite 14 days, refrig. at <6 oC /

EDA Preservation

Holding time and

temperature must not be

exceeded. EDA added to all

samples

Sect. 9.4 Laboratory

Reagent Blank

(LRB)

Include LRB with every analysis

batch (up to 20 samples)

Analyze prior to analyzing field

samples

All analytes must be 

# 1/3 MRL

Sect. 9.4.2

(specific

to PCR)

PRETREATED

Laboratory

Reagent Blank 

REQUIRED in any analysis batch

which includes samples which have

been pretreated to remove chlorite

prior to PCR measurement of trace

bromate.

PCR measured bromate 

< 1/3 MRL

Sect. 9.6 Laboratory

Fortified Blank

(LFB)

Laboratory must analyze LFB in

each analysis batch following the

first CCC. Calculate %REC prior to

analyzing samples

LFB recovery fortified at:

 $MRL to 5X MRL 

 = 75 - 125% 

 $5X MRL to highest CCC 

 = 85 - 115%

Sample results from batches

that fail LFB are invalid

Sect. 10.2 Initial

Calibration

Conductivity: generate calibration

curve using at least 5 standards

Absorbance: generate calibration

curve using at least 5 bromate

standards

The lowest calibration

standard MUST be at or

below the MRL

4 CAL standards should be

above the MRL

Sect. 9.5

and Sect.

10.3

Continuing

Calibration

Check (CCC)

Verify initial calibration by

analyzing a low level CCC prior to

analyzing samples. CCCs are then

injected after every 10 samples and

after the last sample, rotating

concentrations to cover the

calibrated range of the instrument. 

Recovery for each analyte

must be 85-115% of the true

value for all but the lowest

level of calibration. The

lowest calibration level CCC

must be 75-125% of the true

value

All acceptable data MUST

be bracketed by valid CCCs
326.0-42

TABLE 5. QUALITY CONTROL REQUIREMENTS (SUMMARY CONTINUED).

Reference Requirement Specification and Frequency Acceptance Criteria

Sect. 9.7 Surrogate Dichloroacetate is added to all blanks,

samples and standards

Surrogate recovery must

be 90 - 115%.

Samples that fail

surrogate recovery must

be reanalyzed. If second

analysis fails label result

as suspect/matrix 

Sect. 9.8

Sect.

11.1.4.3

Laboratory

Fortified

Sample Matrix

(LFSM)

Must add known amount of each target

analyte to a minimum of 5% of field

samples or at least one within each

analysis batch for both detectors 

LFSM must be fortified above the

native level and at no greater than 5 x

the highest field sample concentration 

Calculate target analyte recovery using

formula (Sect. 9.8.2)

When field samples from chlorine

dioxide plants which contain chlorite

are pretreated prior to the PCR

measurement of trace bromate, an

additional LFSM must be prepared for

each pretreated field sample (Sect.

9.8.4) 

Recovery should be 

75 - 125%

If fortified sample fails

the recovery criteria,

label both as

suspect/matrix.

Sect. 9.9 Field Duplicate

(FD) 

or 

Laboratory

Fortified

Sample Matrix

Duplicate

(LFSMD)

Analyze either a FD or LFSMD for a

minimum of 5% of field samples or at

least one within each analysis batch for

both detectors.

Calculate the relative percent difference

(RPD) using formula in Section 9.9.1

The RPD for

concentrations at MRL

to 5 x MRL should be ±

20% on both detectors,

and ± 10% on both

detectors for

concentrations at 5 x

MRL to highest CCCs. If

this range is exceeded, 

label both as

suspect/matrix 

Sect. 9.10 Instrument

Performance

Check (IPC)

Calculate Peak Gaussian Factor (PGF)

using equation (Sect. 9.10.1) and

monitor retention time for surrogate in

the initial CCC each day

PGF must fall between

0.80 and 1.15

Ret. Time (RT) for

surrogate must remain

80% of initial RT when

column was new
326.0-43

TABLE 6. EXAMPLE SAMPLE ANALYSIS BATCH WITH QUALITY CONTROL

REQUIREMENTS

Injection 

#

Sample

 Description

Acceptance 

Criteria 

1 Laboratory reagent blank (LRB) # 1/3 MRL

2 ICCS conductivity detector (5.0 μg/L) 3.75 to 6.25 μg/L

3 ICCS absorbance detector (0.5 μg/L) 0.375 to 0.625 μg/L

4 Laboratory Fortified Blank (LFB) - 

conductivity detector

 ± 25 % fortified level

5 LFB - absorbance detector ± 25 % fortified level

6 Field sample 1

7 Field sample 1 - Laboratory Duplicate (LD) (a) ± 15 % RPD

8 Field sample 2

9 Field sample 2 - Laboratory Fortified Sample Matrix

(LFSM) (a) at concentrations specific for conductivity

detector 

 ± 25% fortified level

10 Field sample 2 - LFSM specific for trace bromate on the

absorbance detector

 ± 25% fortified level

11 Field sample 3

12 Field sample 4

13 Field sample 5

14 Field sample 6

15 Field sample 7

16 Field sample 8

17 Field sample 9

18 Field sample 10

19 CCCS conductivity detector (75.0 μg/L) 63.8 to 86.3 μg/L

20 CCCS absorbance detector (5.0 μg/L) 4.25 to 5.75 μg/L

21 Field sample 11
326.0-44

22 Field sample 12

23 Field sample 13

24 Field sample 14 - (finished water from PWS using chlorine

dioxide)

25 Pretreate (Sect. 9.3.1.2) using the acid/Fe(II) chlorite

removal procedure (Sect. 11.1.4)

# 1/3 MRL

26 Field sample 14 (b) - (finished water from PWS using

chlorine dioxide) pretreated with acid/Fe(II) (Sect. 11.1.4)

27 Field sample 14 - (finished water from PWS using chlorine

dioxide) LFSM specific for trace bromate on the

absorbance detector, pretreated with acid/Fe(II) (Sect.

11.1.4.2) 

 ± 25% fortified level

28 Field sample 15

29 Field sample 16

30 Field sample 17

31 Field sample 18

32 Field sample 19(b)

33 ECCS conductivity detector (500.0 μg/L) 425 to 575 μg/L

34 ECCS absorbance detector (15.0 μg/L) 12.8 to 17.3 μg/L

(a) If no analytes are observed above the MRL for a sample, an alternate sample which contains

reportable values should be selected as the laboratory duplicate. Alternately, the LFSM can be

selected and reanalyzed as the laboratory matrix duplicate ensuring the collection of QC data for

precision.

(b) Field sample #19 was the final field sample permitted in this batch but 20 total field samples were

analyzed. 

Field sample #14 was analyzed both initially and as a acid/Fe (II) pretreated sample, therefore, it

accounted for two “field sample analyses” toward the maximum of twenty in an analysis batch (Sect.

3.1).
326.0-45

Figure 1: Schematic detailing the configuration of postcolumn hardware addition to an ion

chromatograph. Mention of trade names or commercial products does not constitute

endorsement or recommendation for use. If the requirements found in Section 9 are

met, equivalent products or hardware can be employed.

NOTE: In a typical Method 300.1 hardware configuration, a backpressure coil is

included after the conductivity cell as part of the waste stream when this

manufacture’s equipment is used. These backpressure coils are not required when the

Method 326.0 instrument configuration is employed since the additional PCR system

components, placed in-line, function in the same capacity and provide sufficient

backpressure.
326.0-46

 Figure 2: Reaent water fortified with bromate at 10 ug/L on both detectors.
326.0-47

.

Figure 3: Reagent water fortified with inorganic oxyhalide disinfection by-products and

bromide at 20.0 ug/L and bromate at 10 ug/L on both detectors.