METHOD 327.0 DETERMINATION OF CHLORINE DIOXIDE AND CHLORITE ION 

IN DRINKING WATER USING LISSAMINE GREEN B AND 

HORSERADISH PEROXIDASE WITH DETECTION BY 

VISIBLE SPECTROPHOTOMETRY 

EPA 815-B-03-001 

Revision 1.0 

July 2003 

Teri A. Dattilio and Barry V. Pepich, Shaw Environmental, Inc. 

David J. Munch and Patricia S. Fair, US EPA, Office of Ground Water and 

Drinking Water 

Zsolt Körtvélyesi and Gilbert Gordon, Miami University 

TECHNICAL SUPPORT CENTER

OFFICE OF GROUND WATER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

327.0-1
METHOD 327.0

DETERMINATION OF CHLORINE DIOXIDE AND CHLORITE ION IN DRINKING WATER

USING LISSAMINE GREEN B AND HORSERADISH PEROXIDASE WITH DETECTION 

BY VISIBLE SPECTROPHOTOMETRY

1. SCOPE AND APPLICATION 

1.1 This is a spectrophotometric method for the analysis of chlorine dioxide and chlorite* in 

finished drinking waters. It is primarily intended to be used at drinking water utilities in 

conjunction with daily monitoring requirements. 

1.2 The experimentally determined Detection Limits (DLs) for chlorine dioxide and chlorite 

are provided in Section 17, Table 1. The DL is defined as the statistically calculated 

minimum concentration that can be measured with 99% confidence that the reported 

value is greater than zero.(1) Detection Limits are technique and instrument dependent. 

The DL differs from, and is lower than, the Minimum Reporting Level (MRL) 

(Sect. 3.10). Method performance was demonstrated over a combined concentration 

range of 0.2 mg/L to 2.2 mg/L. Precision and accuracy data are presented in Section 17, 

Tables 1-3. 

1.3 Chlorine dioxide is a volatile, reactive gas. Care was taken during method development 

to minimize steps that could result in the loss of chlorine dioxide during analysis. 

Analyst technique can affect method precision and accuracy. 

2. SUMMARY OF METHOD 

2.1 A drinking water sample is collected headspace-free in a 16-mL amber glass vial. A 

second sample is collected and sparged with an inert gas to remove all traces of chlorine 

dioxide and then transferred to a second 16-mL amber vial. A third vial is filled with 

reagent water. A 1.0-mL aliquot of water is removed from each vial, and a 1.0-mL 

aliquot of a concentrated citric acid buffer containing glycine is added. The samples are 

capped and gently mixed, and then a second 1.0-mL aliquot is removed and a 1.0-mL 

aliquot of a Lissamine Green B (LGB)/Horseradish Peroxidase (HRP) reagent is added. 

The HRP catalyzes the conversion of chlorite to chlorine dioxide.(2,3) Chlorine dioxide 

rapidly oxidizes the LGB, reducing its absorption in the red region of the visible spectrum 

in proportion to the chlorine dioxide concentration.(4,5) A visible spectrophotometer is 

used to measure the absorbance of the reagent water blank and sample absorbance at 

633 nm, which is the absorbance maximum for LGB in the citric acid/glycine buffer. The 

absorbance difference between the reagent water blank and the samples is used to 

calculate the concentrations of chlorine dioxide, using an external standard calibration 

*Throughout this document chlorite ion (ClO2

–

) is referred to as ‘chlorite’ for simplicity, 

even though the use of ‘chlorite ion’ is more accurate. 

327.0-2 
curve, determined using chlorite standards. The unsparged sample is used to determine 

the total chlorite and chlorine dioxide concentration and the sparged sample is used to 

determine the chlorite concentration. The chlorine dioxide concentration is the difference 

between these two values. 

3. DEFINITIONS 

3.1 ANALYSIS BATCH – A set of samples that is processed and analyzed together and 

includes no more than 10 Field Samples. An analysis batch must also include at least one 

Method Blank and all required QC samples. The Method Blanks and QC samples do not 

contribute to the maximum Field Sample total of 10. 

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

standard solution and/or stock standard solution. The CAL solutions are used to calibrate 

the instrument response with respect to analyte concentration. 

3.3 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing the 

method analyte(s), which is analyzed periodically to verify the accuracy of the existing 

calibration for those analytes. 

3.4 DETECTION LIMIT (DL) – 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. 9.2.3), and 

accurate quantitation is not expected at this level.(1) 

3.5 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 and storage, as well as laboratory procedures. 

3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – An aliquot of an environmental sample to which known quantities of the method analytes 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. 

3.7 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A second 

aliquot of the field sample used to prepare the LFSMD, 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. 

327.0-3
3.8 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. 

3.9 METHOD BLANK (MB) – An aliquot of reagent water that is free from all method 

analytes and is processed and analyzed with the samples in the analysis batch. The 

absorbance value of the MB is used to calculate the absorbance change caused by the 

reaction of chlorine dioxide with LGB. 

3.10 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 

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

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

standard are met. 

3.11 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. 

3.12 REAGENT WATER – A high-purity water, typically either distilled or deionized, that is 

free from organic and inorganic contaminants and all method analytes. 

3.13 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 Free available chlorine reacts with LGB to discolor the solution. Glycine reacts with the 

free available chlorine to form chloroaminoacetic acid, thereby eliminating the potential 

for this type of interference. 

4.2 Laboratory glassware can potentially exhibit a demand for chlorine dioxide.(5) While this 

was not observed during method development, it could bias the analytical results and lead 

to poor accuracy and/or precision. This potential problem can be avoided if sample 

collection vials are cleaned and reused as described in Section 8.1.2. 

4.3 Chlorite concentrations are calculated after the samples are sparged to remove chlorine 

dioxide. Samples that are not efficiently sparged to remove the chlorine dioxide will 

yield chlorite values that are higher than the true value. 

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5. SAFETY 

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

identified; 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.(6-8) 

5.2 This method does not require the preparation of chlorine dioxide standards, which could 

have inhalation hazards. It also does not require the preparation of chlorite standards 

from a recrystallized solid standard, which can have an explosion hazard. Because of the 

significant potential dangers associated with these materials, they are not recommended 

for use in conjunction with this method. The chlorite standard cited in Section 7.3.1 does 

not pose an explosion hazard. 

6. EQUIPMENT AND SUPPLIES 

6.1 VISIBLE SPECTROPHOTOMETER – A single- or double-beam spectrophotometer that 

uses a monochromator to make accurate absorbance measurements at discrete 

wavelengths (Agilent Model 8453 or equivalent) equipped with a standard, rectangular 

1-cm path length cell is recommended. The spectrophotometer must have a linear 

response and acceptable photometric accuracy up to an absorbance value (A) of 1.6. 

6.1.1 Nondispersive spectrophotometers (e.g., filter-based, light emitting diode-based, 

etc.) with alternate cuvette shapes and pathlengths are allowed as long as they 

meet the method QC requirements. These devices must have filters with 

transmission maximum near 633 nm and must have a linear response and 

acceptable photometric accuracy up to an absorbance value of 1.6. 

6.1.2 Longer pathlength cells may be used for applications that require lower DLs; 

however, the concentration of the Buffered LGB Stock Solution (Sect. 7.2.4) 

must be adjusted accordingly. Shorter pathlengths are not recommended. 

6.2 CUVETTES – A matched set of 1-cm glass cuvettes with polytetrafluoroethylene (PTFE) 

stoppers (Fisher Cat. #: 22200-350 or equivalent). Alternate path lengths and/or cell 

shapes are allowed as long as the method QC criteria are met. 

6.3 ANALYTICAL BALANCE – Used to accurately weigh analytical standard materials 

(±0.1 mg sensitivity). 

6.4 TOP LOADING BALANCE – Used to accurately weigh reagents (±10 mg sensitivity). 

327.0-5
6.5 WEIGHING BOATS – Plastic, disposable - used to weigh reagents. 

6.6 WEIGHING FUNNEL – Used to weigh and transfer LGB and HRP (Fisher Cat. #: 

19958-3 or equivalent). 

6.7 PIPETTES – Use 0.5-mL and 1.0-mL Class A, volumetric pipettes (Fisher Cat. #: 13-

651-11 or equivalent) and/or variable volume single channel pipettes with disposable 

plastic tips. 

6.7.1 Use a variable volume, single channel pipette (Brinkman Eppendorf Research 

Pro Pipette, Fisher Cat. #:21-378-84 or equivalent) with a volume of 500 to 

5000 μL to deliver volumes of 1.0 mL or larger. 

6.7.2 Use a variable volume, single channel pipette (Brinkman Eppendorf Research 

Pro Pipette, Fisher Cat. #:21-378-83 or equivalent) with a volume of 50 to 

1000 μL to deliver volumes less than 1.0 mL. 

6.8 VIALS – 16-mL amber glass vials with PTFE-lined screw caps (Fisher Cat. #: 03-391-8E 

or equivalent) used to collect samples. 

6.9 ERLENMEYER FLASKS – 500 mL and 250 mL glass, used to prepare reagents. 

6.10 BOTTLES – Amber glass bottles with PTFE-lined screw caps or opaque, high-density 

polyethylene (HDPE) bottles of various sizes to store reagents and standards. 

6.11 BEAKERS – 100 mL (Fisher Cat.#: 02-540H or equivalent), used to collect and sparge 

samples for chlorite analyses. 

6.12 VOLUMETRIC FLASKS – Class A, of varying sizes. 

6.13 SPARGING APPARATUS – A cylinder of nitrogen or helium equipped with a regulator 

that is attached to an appropriate length of Tygon tubing and a disposable, Pasteur 

pipette. Glass gas-dispersion tubes with fritted cylinders or disks may also be used as 

long as they are rinsed between uses. 

7. REAGENTS AND STANDARDS 

7.1 INTENDED USE OF REAGENTS AND STANDARD SOLUTIONS – Sections 7.2 and 

7.3 describe reagents, solvents and standards that are compatible with a 1-cm rectangular 

cuvette. In this configuration, and with these reagents, this method has a linear range of 

0.2 - 2.2 mg/L total chlorite and chlorine dioxide. If longer pathlength cells are used to 

obtain concentration data at lower levels, the Buffered LGB Stock Solution (Sect. 7.2.4) 

must be adjusted accordingly. 

327.0-6
7.2 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be used. 

Unless otherwise indicated, all reagents should conform to the specifications of the 

Committee on Analytical Reagents of the American Chemical Society(9), 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. 

7.2.1 REAGENT WATER – Purified water (typically either deionized or distilled) 

which does not contain any chlorine dioxide or chlorite or compounds that 

absorb light at 633 nm. The absorbance of the reagent water at 633 nm for a 

1-cm pathlength (against a cuvette containing air) should be 5 milli-absorbance 

units (mA) or less. 

7.2.2 CONCENTRATED CITRIC ACID/GLYCINE BUFFER – Add 9.0 g of 

trisodium citrate dihydrate (C6O7H5Na3•2H2O) (Sigma Cat. #: C3434 or 

equivalent), 5.0 g disodium hydrogencitrate sesquihydrate (C6O7H6Na2•1.5H2O) 

(Sigma Cat. #: 35908-4 or equivalent) and 1.0 g glycine (Sigma Cat. #: G7162 

or equivalent) to a 250-mL Erlenmeyer flask. Add 125 mL of reagent water and 

mix thoroughly. Add one drop of chloroform (or about 50 μL) (Aldrich HPLC 

Grade, Cat. #: 36,692-7 or equivalent) and store this solution at # 6 °C. 

7.2.2.1 Care must be taken to ensure that all crystalline material is completely 

dissolved either by using a stir bar or by swirling the contents of the 

flask manually for several minutes. 

7.2.2.2 The pH of the concentrated buffer stock solution should be confirmed 

with a pH electrode to be in the pH range of 6.0 - 6.2 upon dilution by 

adding a 1.0-mL aliquot of the buffer to 15 mL of reagent water. The 

pH of the concentrated stock solution may be adjusted, if necessary, by 

adding small amounts of the C6O7H6Na2•1.5H2O to lower the pH value 

or C6O7H5Na3•2H2O to increase the pH value. 

7.2.2.3 Chloroform acts as an antimicrobial agent. If the buffer solution turns 

cloudy, which is indicative of microbial growth, it must be discarded. 

7.2.3 LISSAMINE GREEN B (LGB) CONCENTRATED STOCK SOLUTION IN 

REAGENT WATER – Accurately weigh 240 mg of LGB (Aldrich Cat.#: 

19958-3 or equivalent) in a glass weighing funnel (Sect. 6.6). Transfer the LGB 

to a 250-mL wide neck volumetric flask with reagent water and dilute to the 

mark using reagent water. Add a small PTFE stirbar and allow the solution to 

stir overnight. Transfer the solution to a 500-mL amber glass bottle which has a 

PTFE-lined cap and store this solution at # 6 °C. If stored properly, this 

solution should be stable for 2 months. 

327.0-7
7.2.3.1 The minimum acceptable purity for the LGB solid is 70%. Higher 

purity is recommended. Although Aldrich specifies that their LGB has 

a minimum purity of 60%, the lot of LGB used in this work was 73% 

pure according to the manufacturer. This information can be obtained 

from Aldrich by contacting a technical service representative. 

7.2.3.2 Confirm that the LGB solid is at least 70% pure as follows. Add 6 mL 

of the Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) to a 

100-mL volumetric flask. Fill the flask about half full with reagent 

water and then add a 1.0-mL aliquot of the LGB Concentrated Stock 

Solution. Fill the flask to the mark with reagent water and mix 

thoroughly. Allow the flask to equilibrate at room temperature for 15 -

20 minutes, and then mix again. Transfer an aliquot of this solution to 

a cuvette with a 1-cm pathlength. The absorbance of this solution 

must be $ 0.99. 

7.2.4 BUFFERED LGB STOCK SOLUTION – Prepare a dilution of the LGB 

Concentrated Stock Solution in the Concentrated Citric Acid/Glycine Buffer. 

The concentration of this solution should be confirmed prior to mixing it 

with the Horseradish Peroxidase (HRP) Stock Solution to prepare the 

Combined LGB/HRP Reagent (Sect. 7.2.6). This step will eliminate waste 

of the HRP enzyme, which is the most expensive reagent in the method. 

The procedure below is used to determine that the Buffered LGB Stock Solution 

has been prepared at the appropriate concentration so that when it is used in the 

procedure it will yield a Method Blank absorbance in the required absorbance 

range of 1.5 ± 0.1. 

7.2.4.1 For LGB with a purity of 73%, transfer 40 mL of the LGB 

Concentrated Stock Solution (Sect. 7.2.3) to a wide neck 100-mL 

volumetric flask. Add 6 mL of the Concentrated Citric Acid/Glycine 

Buffer (Sect. 7.2.2), dilute to the mark with reagent water, and mix 

thoroughly. For LGB that has lower purity, the volume of the 

Concentrated Stock Solution can be estimated using the equation 

where VLGB is the volume of the LGB Concentrated Stock Solution 

required, and PLGB is the purity of the LGB (obtained from the 

manufacturer) expressed as a whole number. 

7.2.4.2 Confirm that the Buffered LGB Stock Solution concentration is in the 

correct range as follows. Fill a 16-mL vial with reagent water, remove 

327.0-8
1.0 mL of the water and add 1.0 mL of the of the Concentrated Citric 

Acid/Glycine Buffer (Sect. 7.2.2). Cap the vial and shake. Remove 

0.5 mL of the buffered reagent water and add 0.5 mL of the Buffered 

LGB Stock Solution. Recap the vial and mix the contents thoroughly 

by shaking. Transfer an aliquot of this solution to a 1-cm cuvette and 

determine the absorbance at 633 nm. The absorbance of this solution 

at 633 nm must be in the range of 1.5 ± 0.1. If not, the Buffered LGB 

Stock Solution should be reprepared from the LGB Concentrated 

Stock Solution. The appropriate volume of the LGB Concentrated 

Stock Solution can be calculated using the equation 

where Vreq is the volume of the LGB Concentrated Stock Solution that 

is required to prepare the new solution, Vadded is the volume of the LGB 

Concentrated Stock Solution originally added (40 mL in Sect. 7.2.4.1), 

and Ameas is the measured absorbance. 

7.2.4.3 If the analyst is using the method to determine concentrations 

< 0.2 mg/L, a longer pathlength cuvette is required. The Buffered 

LGB Stock Solution concentration must be adjusted accordingly so 

that the absorbance in the longer pathlength cuvette, when checked as 

described in Section 7.2.4.2, is in the absorbance range of 1.5 ± 0.1. 

To calculate the amount of LGB Concentrated Stock Solution required 

for the solution, simply divide the recommended volume in Section 

7.2.4.1 by the pathlength. 

7.2.4.4 This stock solution is used to prepare the Combined LGB/HRP 

Reagent and is not preserved with chloroform. This solution should 

not be kept or stored for more than one day. 

7.2.5 BUFFERED HORSERADISH PEROXIDASE (HRP) STOCK SOLUTION – 

Weigh out 60 - 70 mg of Type II HRP (Sigma Cat. #: P6782 or equivalent) 

using a glass weighing funnel (Sect. 6.6) and an analytical balance. Transfer the 

HRP to a wide-neck, 50-mL volumetric flask by washing it from the funnel with 

reagent water. The HRP is a lyophalized powder that can be lost during transfer 

if care is not taken to minimize air currents. Carefully rinse the walls of the 

funnel with reagent water to ensure complete transfer. Add 6 mL of the 

Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) and then dilute the flask 

contents to the mark with reagent water and mix thoroughly. 

7.2.5.1 The HRP must have a minimum activity of 150 units per mg of solid 

using the purpurogallin method.(10) The Type II HRP sold by Sigma 

meets this criterion. According to Sigma, their HRP is stable for up to 

327.0-9
1 year if stored as a solid at # -20 °C.(11) Other sources of HRP may 

also be suitable, but they have not been evaluated. 

7.2.5.2 This stock solution is used to prepare the Combined LGB/HRP 

Reagent and is not preserved with chloroform, and therefore should 

not be kept or stored for more than one day. 

7.2.6 COMBINED LGB/HRP REAGENT – Confirm the concentration of the 

Buffered LGB Stock Solution according to Section 7.2.4.2 prior to performing 

this step. This will prevent wasting the Buffered HRP Stock Solution, which is 

the most expensive reagent in this method. Combine 50 mL of the Buffered 

LGB Stock Solution (Sect. 7.2.4) with the entire volume of Buffered HRP Stock 

Solution (Sect. 7.2.5) in an amber glass bottle with a PTFE-lined cap. Do not 

rinse the 50-mL volumetric flask containing the Buffered HRP Stock Solution 

to transfer the remaining droplets as quantitative transfer of this reagent is not 

necessary. The volume of the combined reagent is sufficient for 

approximately100 tests (e.g., blanks, samples, calibrations, etc.). Add one drop 

of chloroform (or about 50 μL). This solution may be used for 14 days if stored 

at # 6 °C. 

7.2.7 HELIUM OR NITROGEN ($99.995%) – UHP Grade or purer, used to sparge 

samples to remove chlorine dioxide. 

7.3 STANDARD SOLUTIONS – Because chlorite is converted to chlorine dioxide using the 

HRP reagent, this method uses chlorite standards to calibrate for both chlorine dioxide 

and chlorite (Sect. 2.1). Chlorite standard solutions may be prepared from certified, 

commercially available solutions or from solid compounds. Preparation of chlorite 

standards from a recrystallized solid of high purity (95%+) is not recommended as this 

form of chlorite can pose an explosion hazard. 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 should be replaced. 

7.3.1 CHLORITE STOCK STANDARD SOLUTION (1000 mg/L) – Certified 

chlorite standard solutions are commercially available (Absolute Standards, Cat. 

# 54106 or equivalent). Commercial standards should be stored at # 6 °C in an 

amber glass bottle or high density polyethylene (HDPE) container to prolong 

standard life. If stored properly, this solution should be stable for 6 months. 

7.3.2 CHLORITE PRIMARY DILUTION STANDARD (PDS) (10 mg/L) – This 

solution is used to prepare Calibration Standards and Continuing Calibration 

Check Standards. Partially fill a 500-mL volumetric flask with reagent water. 

Add 5.0 mL of the Chlorite Stock Standard Solution (Sect. 7.3.1) using a pipette 

and fill the flask to the mark using reagent water. Invert and shake the flask 

327.0-10
several times to ensure that the solution is completely mixed and then transfer 

the flask contents to an amber bottle with a PTFE-lined cap or an HDPE 

container. This solution should be stored at # 6 °C. If stored properly, this 

solution should be stable for 3 months. 

7.3.3 CALIBRATION STANDARDS – At least three Calibration Standards are 

required to establish an initial calibration curve. Five Calibration Standards are 

recommended. The concentration of the Calibration Standards must span the 

intended reporting range. An example of the dilutions that were used to prepare 

the standards used in the development of this method is included in the table 

below. All Calibration Standards are to be prepared from the Chlorite Primary 

Dilution Standard (Sect. 7.3.2) using Class A volumetric or variable-volume 

pipettes and volumetric flasks. Transfer an appropriate aliquot of the Chlorite 

PDS (10 mg/L) (Sect. 7.3.2) to the appropriate flask and dilute to the mark with 

reagent water. Mix the contents of the flask thoroughly and transfer to an amber 

glass bottle or opaque plastic container. These solutions should be stored at 

# 6 °C. If stored properly, these solutions should be stable for 3 months. 

Chlorite PDS 

Volume 

(mL) 

Final 

Volume 

(mL) 

Calibration 

Std. Conc. 

(mg/L) 

10 500 0.20

25 500 0.50 

50 500 1.00 

75 500 1.50 

100 500 2.00 

7.3.4 CONTINUING CALIBRATION CHECK (CCC) STANDARDS – Calibration 

Standards prepared above may also be used as CCC Standards. 

7.3.5 LFSM FORTIFICATION SOLUTION (100 mg/L) – This solution is used to 

fortify Field Samples for the Laboratory Fortified Sample Matrix requirement 

(Sect. 9.5). Partially fill a 100-mL volumetric flask with reagent water. Add 

10.0 mL of the Chlorite Stock Standard Solution (Sect. 7.3.1) using a pipette, 

and then fill the flask to the mark using reagent water. Invert and shake the 

flask several times to ensure the solution is completely mixed and then transfer 

the contents of the flask to an amber bottle with a PTFE-lined cap or an HDPE 

container. This solution should be stored at #6 °C. If stored properly, this 

solution should be stable for 3 months. 

327.0-11 
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE 

8.1 SAMPLE VIAL PREPARATION 

8.1.1 VIAL SELECTION – The procedure and reagent concentrations were 

developed using the vials listed in Section 6.8. The manufacturer lists the 

volume of these vials to be 16 mL. The procedure used to select appropriate 

vials is described below. If alternate vials are used that have an average volume 

that differs from 16 mL by more than 10%, reagent concentrations and volumes 

must be adjusted accordingly. 

8.1.1.1 Select an appropriate number of vials based on anticipated sample load 

requirements. Vials that do not have appropriate volumes will be 

rejected during this process, so begin the selection process with 10-

15% more vials than needed. Vials may be reused after cleaning. 

Method development was conducted with approximately 30 vials. 

8.1.1.2 Label and cap each 16-mL vial and determine the tare weight with the 

PTFE-lined cap. Fill the vial with water and recap. Dry the external 

walls of the vial and confirm that there is no headspace by inverting 

the vial and looking for an air bubble. Weigh the full vial and record 

the water weight. Convert the water weight to volume of the vial by 

assuming 1.0 g of water is equal to 1.0 mL. 

8.1.1.3 Determine the average volume for the vials tested, and select the vials 

that are closest in volume. Discard any vial that has a volume that is 

greater than 1% above or below the average value. During 

development of this method, the average volume was 16.4 mL and 

vials were accepted that were in the volume range of 16.4 ± 0.1 (or 

within 0.63%). Approximately 10% of the vials were rejected. 

8.1.2 VIAL CLEANING – Vials may be reused. Prior to reuse they should be 

thoroughly cleaned with soap and water and rinsed several times with reagent 

water. Vials (without caps) can be dried in an oven at 140 °C. Reusing vials 

may help avoid any potential for bias associated with chlorine dioxide 

demand of the glassware (Sect. 4.2). 

8.2 SAMPLE COLLECTION 

8.2.1 Prior to sample collection, remove the aerator (if present) to minimize loss of 

chlorine dioxide during sample collection. Open the tap and allow the system to 

flush until the water temperature has stabilized (usually 3-5 minutes). Reduce 

the tap flow just before collecting the samples. 

327.0-12
8.2.2 Two aliquots must be collected at each sampling point if both chlorine dioxide 

and chlorite are to be determined. Both aliquots require sample processing to 

halt the continued formation and/or reproportionation of the method analytes. 

Two pipette tips (or pipettes) will be required for this procedure. Tips can be 

used repeatedly to perform the same functions with each Field Sample. This is 

not expected to cause significant contamination due to carryover. However, 

care should be taken to ensure that fluid is not retained in (or on) the pipette tip 

prior to performing the next transfer. These pipette tips (or pipettes) are given 

numbers for clarity. The pipette tips are summarized in the table below with 

their function. These tips may be saved for use in Section 11. 

Pipette Tip # Used to 

1 Remove sample and remove buffered sample 

2 Transfer citric acid/glycine buffer 

8.2.3 FIELD SAMPLE COLLECTION TO DETERMINE CHLORINE DIOXIDE 

AND CHLORITE – Fill the 16-mL vial completely so that no headspace 

remains after capping the vial. This is most easily done by slightly overfilling 

the vial. Invert the capped vial to ensure that the vial does not contain an air 

bubble (or headspace). Recollect the sample if a bubble is noted. Remove a 

1.0-mL aliquot of the Field Sample from the vial using pipette tip 1 and add a 

1.0-mL aliquot of the Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) 

using pipette tip 2. Free available chlorine is removed in this step. Gently mix 

the vial by inverting it three times. Clearly label the sample. This aliquot will 

be used to determine the total chlorine dioxide and chlorite concentration. 

Note: When removing the 1.0-mL aliquot from the vial, carefully place the 

pipette tip into the vial in such a way that the sample does not overflow out of 

the vial. As sample is withdrawn into the pipette, the tip should be gently 

lowered so that it is always below the surface of the sample. 

8.2.4 FIELD SAMPLE COLLECTION TO DETERMINE CHLORITE – Collect a 

second aliquot (approximately 40 mL) in an open container with a large surface 

area such as a 100-mL beaker. Containers like bottles are not efficiently 

sparged even over long times and should not be used. This aliquot will be used 

to determine the chlorite concentration and must be sparged immediately after 

collection. Field Samples that are not completely sparged will yield chlorite 

concentrations that are high. 

8.2.4.1 Sparge the sample using a constant flow of nitrogen or helium gas 

until the chlorine dioxide is removed. This can be accomplished using 

327.0-13
a lecture bottle or gas cylinder with a regulator equipped with a 

suitable length of Tygon tubing attached to a Pasteur pipette or a gasdispersion tube with a fritted cylinder or disk (Sect. 6.13). A new 

pipette should be used for each sample. Glass gas-dispersion tubes 

should be rinsed thoroughly between samples. 

8.2.4.1.1 Sparging a sample contained in a beaker with nitrogen at a 

flow rate of 250 mL/minute for 15 minutes using a Pasteur 

pipette removed over 97% of the chlorine dioxide. 

8.2.4.1.2 Laboratories should confirm that their procedure used to 

sparge chlorine dioxide from Field Samples is sufficient by 

analyzing a sample known to contain high levels of 

chlorine dioxide ( > 2 mg/L) and evaluating its removal at 

various sparge times. 

8.2.4.2 Transfer an aliquot of the sparged Field Sample to a 16-mL vial. 

Invert the capped vial to ensure that it is completely filled. If an air 

bubble is noted, uncap the vial and add additional sparged sample as 

necessary. Remove a 1.0-mL aliquot of the sparged Field Sample 

from the vial using pipette tip 1 and add a 1.0-mL aliquot of the 

Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) using pipette tip 

2. This step removes any free available chlorine from the sample. 

Gently mix the vial by inverting it three times. 

8.2.5 QC SAMPLES – Field Duplicates require collection of an extra 16-mL vial as 

per Section 8.2.3 and an additional sparged sample as per Section 8.2.4. A 

portion of the Field Sample sparged in Section 8.2.4 should be used for the 

Laboratory Fortified Sample Matrix. 

8.3 SAMPLE STORAGE – Field Samples and Field Duplicates should be analyzed as soon 

as possible. Samples may be held for up to 4 hours if collected properly and stored in a 

cooler at #10 °C. 

9. QUALITY CONTROL 

9.1 Each laboratory using this method is required to operate a Quality Control (QC) program. 

The requirements of this program consist of an Initial Demonstration of Capability (IDC), 

and subsequent analysis in each analysis batch of a Continuing Calibration Check 

Standard (CCC). A Laboratory Fortified Sample Matrix (LFSM) and a Field Duplicate 

Sample are recommended weekly. This section details the specific requirements for each 

QC parameter. The QC criteria discussed in the following sections are summarized in 

Section 17, Tables 5 and 6. These criteria are considered the minimum acceptable QC 

327.0-14
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. The full IDC must 

be conducted prior to the analysis of any Field Samples each time a new analyst uses the 

method. Sections 9.2.1 and 9.2.2 must be repeated each time a new LGB Concentrated 

Stock Solution (Sect. 7.2.3) is prepared. An acceptable Initial Calibration (Sect. 10.2) 

must be conducted prior to performing the IDC. 

9.2.1 INITIAL DEMONSTRATION OF ACCURACY – Process and analyze five 

highest-level Calibration Standards and five reagent water Method Blanks 

through the procedure described in Section 11.2. Calculate the chlorite 

concentration for each Calibration Standard as described in Section 12. The 

average concentration for the five replicates must be ± 30% of the fortified 

value. 

9.2.2 INITIAL DEMONSTRATION OF PRECISION – Using the same set of 

replicate data generated for Section 9.2.1, calculate the standard deviation and 

relative standard deviation (RSD) of the replicate values. The RSD is calculated 

using the equation 

where 

S is the standard deviation for the replicate values, 

and is the average value for the replicates. 

The RSD of the results of the replicate analyses must be less than 20%. 

9.2.3 DETECTION LIMIT (DL) DETERMINATION – Prepare and analyze at least 

seven replicate CCCs at a chlorite concentration estimated to be near the DL 

using the procedure described in Section 11.2 for total chlorite and chlorine 

dioxide. For a 1-cm pathlength, the CCCs must be fortified with chlorite in the 

concentration range of 0.20 to 0.35 mg/L or at a concentration no more than 3-

to 5-times the calculated DL. Calculate the DL using the equation 

DL = S×t(n-1,1-"=0.99) 

where 

t(n - 1, 1 - " = 0.99) is the Student’s t value for the 99% confidence level with 

n-1 degrees of freedom, 

n is the number of replicates, 

and S is the standard deviation of replicate analyses. 

327.0-15
 Selected t values can be found in the table below. 

Number of Replicates 7 8 9 10 11 

Student’s t value 3.143 2.998 2.896 2.821 2.764 

9.2.3.1 The DL for this method is a function of pathlength and analyte 

concentration. Longer pathlengths will result in improved sensitivity 

and should decrease the DLs for chlorine dioxide and chlorite. DLs 

for a 1-cm pathlength calculated by fortifying reagent water with 

chlorite in the concentration range of 0.20 to 0.35 mg/L should be #

0.25 mg/L. 

9.2.3.2 Detection Limit is a statistical determination of precision only.(1) If the 

DL replicates are fortified at a low enough concentration, they may 

result in a calculated DL that is higher than the fortified concentration. 

Therefore no precision and accuracy are specified for the DL. 

9.3 CONTINUING CALIBRATION CHECK (CCC) STANDARDS – A mid-level CCC is 

prepared as described in Section 7.3.4 and analyzed with each analysis batch or each day 

samples are analyzed. For a 1-cm pathlength, this CCC should have a chlorite 

concentration near 1.0 mg/L. The calculated chlorite concentration for the CCC 

(Sect.12.5) must be ± 30% of the expected value. If this criterion is not met, the Field 

Sample(s) should be re-collected and reanalyzed after establishing acceptable CCC 

recovery (Sect. 10.3.2). 

9.4 FIELD DUPLICATE (FD) – A Field Duplicate is recommended weekly for both chlorine 

dioxide and chlorite. Calculate the relative percent difference (RPD) between the Field 

Sample (FD1) and the Field Duplicate (FD2) as shown below. The RPD for samples 

with concentrations greater than the lowest calibration standard should not exceed 30%. 

The RPD at concentrations at or near the lowest calibration standard (at or below 0.30 

mg/L for a 1-cm pathlength) should not exceed 50%. 

If the RPD for the Field Sample and the Field Duplicate falls outside the designated 

range, and the laboratory performance for that analyte meets the QC performance 

criterion in the CCC, the recovery is judged to be matrix biased. The result for that 

analyte in the Field Sample is labeled “suspect/matrix” to inform the data user that the 

results are suspect due to matrix effects. 

327.0-16
9.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – A Laboratory Fortified 

Sample Matrix is recommended weekly. 

9.5.1 Collect a Field Sample for the LFSM as described in Section 8.2.4 and process 

it as described in Section 11.4. Fortify the Field Sample using the LFSM 

Fortification Solution (Sect. 7.3.5) with a chlorite concentration that is greater 

than the expected concentration based on historical levels, being careful not to 

exceed the upper limit of the initial calibration curve. This should be done in a 

manner that does not increase the sample volume by more than 1%. 

9.5.2 Calculate the recovery (R) for each analyte using the equation 

where A is the measured concentration in the fortified sample, B is the measured 

concentration in the unfortified sample, and C is the fortification concentration. 

9.5.3 Recoveries from samples fortified at or above their native concentration should 

range between 70 and 130% unless the samples were fortified at low levels 

(e.g., total concentrations at or below 0.30 mg/L) where 50 to 150% recoveries 

are acceptable. If the accuracy of the LFSM falls outside the designated range, 

and the laboratory performance for chlorite is shown to be in control (e.g., the 

QC criterion for the CCC was met), 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. 

10. CALIBRATION 

10.1 An acceptable initial calibration must be established before Field or QC Samples are 

analyzed. After initial calibration is successful, a Continuing Calibration Check (CCC) is 

required during each analysis batch or each day samples are analyzed. 

10.2 INITIAL CALIBRATION – An initial calibration curve must be generated prior to 

conducting the IDC and prior to analyzing any Field Samples. An initial calibration is 

also required after preparing new Combined LGB/HRP Reagent as described in Section 

7.2.6 (at least every 2 weeks). An initial calibration curve is established by plotting the 

absorbance differences ()A) between the Method Blank and a number of Calibration 

Standards versus chlorite concentrations in the Calibration Standards. Studies conducted 

during method development indicated that calibration curves generated for chlorite and 

chlorine dioxide using the Combined LGB/HRP Reagent (Sect. 7.2.6) were similar 

enough to allow the calibration for both analytes using chlorite. This procedure is 

described below. 

327.0-17
10.2.1 Prepare at least three Chlorite Calibration Standards that span the intended 

reporting range as described in Section 7.3.3. Five Calibration Standards are 

recommended. 

10.2.1.1 The high-level calibration standard should not exceed a concentration 

of 2.2 mg/L. At higher concentrations, the response becomes 

nonlinear. 

10.2.2 Fill a 16-mL sample vial with each Calibration Standard and process each vial 

along with at least one Method Blank (MB) according to the procedure outlined 

in Section 11.2. Two MBs are recommended. 

10.2.3 Calculate )A at 633 nm between the MB and each Calibration Standard. If two 

MBs are used, use the average MB absorbance value for this calculation. 

10.2.4 Plot )A for each calibration standard as a function of chlorite concentration (or 

chlorine dioxide concentration, since chlorite is converted to chlorine dioxide 

prior to reacting with LGB). This is a procedural calibration technique, where 

the calibration standards are processed identically to the Field and QC Samples. 

This eliminates the need to adjust the Calibration Standard concentrations to 

account for dilutions made during the procedure. 

10.2.5 Fit the data using a linear regression and determine the slope and y intercept 

using the equation 

)A = Slope[ClO2

–

] + Int. 

where )A is the absorbance difference between the blank and the sample, 

[ClO2

–

] is the chlorite concentration in mg/L, and Slope and Int. are the slope 

and y intercept for the linear fit, respectively. 

10.2.6 The r2 value for the linear fit should be 0.98 or better. Use the equation for the 

line to calculate chlorine dioxide and/or chlorite concentration. 

10.2.7 Check the Initial Calibration by calculating the concentrations of the Calibration 

Standards that were used to create the calibration curve. Use the linear 

regression fit formula for the calculation. Each calculated concentration, except 

the calculated concentration for the lowest Calibration Standard, should 

calculate to be 70-130% of its expected value. The lowest Calibration Standard 

should calculate to be 50-150% of its expected value. Laboratories that have 

difficulty achieving these criteria will have trouble meeting the QC 

requirements summarized in Section 9. 

10.3 CONTINUING CALIBRATION CHECK (CCC) – A Continuing Calibration Check is 

required with each Analysis Batch or each day samples are analyzed. This QC sample is 

327.0-18
processed with the Method Blank(s) and Field Sample(s) in the Analysis Batch to ensure 

that the data collected in the Analysis Batch are valid. 

10.3.1 Process a CCC in an identical manner to the Field Samples as described in 

Section 11.2. The CCC concentration should be near the mid-point of the initial 

calibration range. Calibration Standards used to prepare the initial calibration 

curve may be used for this purpose. For a 1-cm pathlength, the CCC should 

have a chlorite concentration at or near 1.0 mg/L. 

10.3.2 Calculate the CCC concentration using the initial calibration curve generated 

above (Sect. 10.2) as described in Section 12. The CCC response must be in the 

range of ± 30% of the expected value. If this criterion is not met, then all data 

for the Field Samples in the Analysis Batch are invalid. Remedial action should 

be taken which could require establishing a new initial calibration and may 

require the preparation of new reagents (the HRP and LGB solutions have the 

shortest shelf lives). Field Samples should be re-collected and reanalyzed after 

re-establishing acceptable QC performance. 

11. PROCEDURE 

11.1 As noted in Section 2.1, this procedure involves a two-step process for the determination 

of chlorine dioxide and chlorite in drinking waters. The first step, described in Section 

11.2, uses the Combined LGB/HRP Reagent to convert chlorite to chlorine dioxide to 

measure the total concentration of both analytes. This procedure is used to process Field 

Samples, Field Duplicates, and CCCs. In the second step, described in Section 11.3, 

samples are sparged to remove the chlorine dioxide, and are then analyzed to determine 

the chlorite concentration. Chlorite is not removed during sparging. This procedure is 

used to process Field Samples and Field Duplicates. (The chlorine dioxide concentration 

is calculated by subtracting the chlorite concentration determined in the second step from 

the total concentration determined in the first step.) Section 11.4 describes the processing 

of LFSMs, which require a slight modification to the procedure described in Section 11.3. 

11.1.1 This is a headspace-free procedure. Care taken to minimize headspace will help 

the analyst to achieve the QC performance criteria summarized in Section 9. 

11.2 TOTAL CHLORINE DIOXIDE AND CHLORITE PROCEDURE USED TO PROCESS 

FIELD SAMPLES, FIELD DUPLICATES AND CCCs 

11.2.1 The unsparged samples collected in the 16-mL vials are prepared and analyzed 

using the procedure described in this section. If samples have been stored (Sect. 

8.3), they should be allowed to equilibrate to room temperature. 

327.0-19
11.2.2 Fill a 16-mL vial with reagent water and label it as the Method Blank. One MB 

is required in each Analysis Batch (# 10 Field Samples). Two MBs are 

recommended. 

11.2.3 Fill a 16-mL vial with a CCC solution prepared according to the procedure 

described in Section 7.3.4. 

11.2.4 This procedure requires three separate pipette tips (or pipettes). Each tip will be 

used repeatedly to perform the same function with each Field and QC sample. 

This is not expected to cause significant contamination due to carryover. 

However, care should be taken to ensure that fluid is not retained in (or on) the 

pipette tip prior to performing the next transfer. These pipette tips (or pipettes) 

are given numbers for clarity. The pipette tips are summarized in the table 

below with their function. 

Pipette Tip # Used to 

1 Remove sample and remove buffered sample 

2 Transfer citric acid/glycine buffer 

3 Transfer HRP/LGB mixed reagent 

11.2.5 Remove a 1.0-mL aliquot from the CCC and MBs using pipette tip 1. Add a 

1.0-mL aliquot of the Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) to 

the CCC and MBs using pipette tip 2. 

Note: Field Samples and Field Duplicates are buffered with the Concentrated 

Citric Acid/Glycine Buffer at the time of collection to remove the free available 

chlorine. 

11.2.6 Cap the CCC and MB vials and gently mix their contents by inverting them 

three times. This step buffers the samples to a uniform pH. 

11.2.7 Process all Field Samples, Field Duplicates, CCCs and MBs at the same time 

through the remainder of the procedure described in this section. Be careful not 

to agitate the samples any more than necessary. Uncap the vial and remove an 

additional 1.0-mL aliquot using pipette tip 1. Immediately add 1.0-mL of the 

Combined LGB/HRP Reagent (Sect. 7.2.6) using pipette tip 3. Cap and mix the 

contents of the vial thoroughly. 

11.2.8 Check the vial for headspace by inverting it and looking for an air bubble. If the 

vial contains air bubbles, the sample must be re-collected (or reprepared in the 

case of a CCC or MB). 

327.0-20
11.2.9 Allow the samples to react at room temperature for at least 20 minutes, but 

preferably not longer than 40 minutes. Loss of color during this period is 

normal and is a result of the reaction between LGB and chlorine dioxide. 

11.2.10 Thoroughly mix the contents of each vial and then transfer an aliquot to a 1-cm 

cuvette. The cuvette should first be rinsed with reagent water or an aliquot of the 

sample. Wipe the outside of the cuvette to remove water droplets and/or finger 

prints. Use the same cuvette, to measure the visible absorbance at 633 nm for 

the MB(s), Field Sample(s), Field Duplicate (if processed), and CCC 

minimizing the time between measurements. 

11.3 CHLORITE PROCEDURE USED TO PROCESS FIELD SAMPLES AND FIELD 

DUPLICATES 

11.3.1 This procedure may be used as a stand-alone procedure for the determination of 

chlorite or in conjunction with the procedure in Section 11.2 for the 

determination of chlorine dioxide as well. The sparged samples are analyzed 

using the procedure described in this section. 

11.3.2 Collect, sparge, and preserve each FS or FD to remove chlorine dioxide and free 

available chlorine as described in Section 8.2.4. 

11.3.3 If samples have been stored (Sect. 8.3), they should be allowed to equilibrate to 

room temperature. 

11.3.4 Process the Field Samples and Field Duplicates through all steps described in 

Sections 11.2.7 through 11.2.10. 

11.4 PROCESSING LABORATORY FORTIFIED SAMPLE MATRICES (LFSMs) 

11.4.1 This procedure is used for the processing of Laboratory Fortified Sample 

Matrices. 

11.4.2 Collect, sparge, and preserve an FS as described in Section 8.2.4. The sparged 

sample should be poured into two 16-mL vials. One vial is analyzed for chlorite 

using the procedure described in Section 11.3 and the other vial is fortified and 

analyzed according to the procedure described below. 

11.4.3 Fortify the Field Sample with the LFSM Fortification Solution (Sect. 7.3.5) that 

is greater than the expected chlorite concentration based on historical 

information. This will require a fourth pipette tip that must not be used for 

other purposes. Small amounts of the LFSM Fortification Solution can 

contaminate other Field or QC samples from carryover because this 

solution is highly concentrated. 

327.0-21
11.4.3.1 Be careful not to exceed the upper concentration of the initial 

calibration curve when calculating the fortification level. Fortification 

levels should be in the range of 1 - 2 times the native level so that the 

chlorite concentration will be within the linear range of the initial 

calibration curve. 

11.4.3.2 As an example, to fortify a Field Sample in a 16.4-mL vial with 

1.0 mg/L of chlorite, one would need to add 0.0164 mg (1.0 mg/L x 

0.0164 L). This would require a 164-μL aliquot of the LFSM 

Fortification Solution ( 0.0164 mg/0.100 mg/mL = 0.164 mL or 

164 μL). 

11.4.3.3 If small volumes of the LFSM Fortification Solution are used (1% of 

the vial volume or less), this aliquot can be delivered without first 

removing a same-size aliquot to make room for reagents. If this 

technique is used the analyst must be careful to insert the pipette tip 

beneath the surface of the vial during delivery of the aliquot and to 

check the pipette tip after delivery of the aliquot to ensure that the 

entire volume has been delivered. Cap the vial once the fortification is 

complete and mix thoroughly. 

11.4.4 Process the LFSM according to the steps described in Sections 11.2.7 through 

11.2.10. 

12. DATA ANALYSIS AND CALCULATION 

12.1 Chlorine dioxide and chlorite concentrations are calculated using an initial calibration 

curve that is generated using chlorite standards. Slopes for both analytes are similar and, 

therefore, no conversion factor is required in order to calculate the chlorine dioxide 

concentrations. 

12.2 Generate an initial calibration curve according to Section 10.2 by processing at least 

three, but preferably five, initial calibration standards through the entire procedure 

outlined in Section 10.2. 

12.3 Establish an Initial Calibration curve as described in Section 10.2. Plot )A against the 

undiluted concentration of the calibration standards. 

12.3.1 Samples processed according to the method generated initial calibration curves 

with slopes in the range of 0.33 - 0.38. Different vial volumes and/or reagent 

volumes will cause the slopes to differ from this value. 

– 12.4 ClO2 AND ClO2 CALCULATION: Determine the absorbance difference for each Field 

and QC Sample processed according to Section 11.2, and calculate the total chlorine 

327.0-22
dioxide and chlorite concentration using the slope and intercept determined in Section 

10.2. If more than one MB is prepared and analyzed in the Analysis Batch, use the 

average MB absorbance when determining the absorbance difference for each sample. 

– 12.5 ClO2 CALCULATION: Determine the absorbance difference for each Field and QC 

Sample processed according to Sections 11.3 and 11.4, and calculate the chlorite 

concentration using the slope and intercept determined in Section 10.2. If more than one 

MB is prepared and analyzed in the Analysis Batch, use the average MB absorbance 

when determining the absorbance difference for each sample. 

12.6 ClO2 CALCULATION: Calculate the chlorine dioxide concentration for each Field and 

QC sample by subtracting the chlorite concentration (Sect. 12.5) from the total chlorite 

plus chlorine dioxide concentration (Sect. 12.4). 

12.7 Quantitate only those values for which the combined chlorine dioxide and chlorite 

concentration falls within the concentration range defined by the lowest and highest 

initial calibration standards. 

12.7.1 Pathlength and reagent concentrations were carefully optimized during method 

development. Analyte concentrations above 2.2 mg/L are in a concentration 

range that is not linear if the reagent concentrations specified in this method for 

a 1-cm pathlength are used. For this reason, analysts must not extrapolate 

beyond the calibration range nor should the initial calibration range be extended 

beyond this concentration. 

12.7.2 Field Samples that exceed this concentration (2.2 mg/L) should be re-collected 

and carefully diluted by a factor of 2 being careful to minimize unnecessary 

sample agitation that will result in the loss of chlorine dioxide. 

12.7.3 Analytical results obtained from dilutions should be considered estimated 

values. 

12.8 Analyte concentrations are reported in mg/L usually to two significant figures. 

13. METHOD PERFORMANCE 

13.1 PRECISION, ACCURACY, AND DETECTION LIMITS – Detection Limits for chlorite 

and chlorine dioxide are presented in Section 17, Table 1 together with method precision 

and accuracy in reagent water. Single laboratory precision and accuracy are presented in 

Table 2 for a chlorinated surface water and in Table 3 for a chlorinated ground water. 

13.2 SAMPLE STORAGE STABILITY STUDIES – A storage stability study was conducted 

by fortifying 0.8 mg/L of chlorine dioxide and 1.0 mg/L of chlorite into a chlorinated 

drinking water sample and analyzing at 0, 2, and 4 hours after storage at 10 °C. The data 

327.0-23
in Table 4 illustrate that chlorite concentrations are stable over a 4-hour time period when 

glycine is present to eliminate the free available chlorine residual. 

14. POLLUTION PREVENTION 

14.1 For information about pollution prevention that may be applicable to laboratory 

operations, consult “Less is Better: Laboratory Chemical Management for Waste 

Reduction” available from the American Chemical Society’s Department of Government 

Relations and Science Policy, 1155 16th Street NW, Washington, D.C., 20036. 

15. WASTE MANAGEMENT 

15.1 The analytical procedures described in this method generate relatively small amounts of 

waste since only small amounts of reagents and solvents 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 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. Glaser, J.A.; Foerst, D.L.; McKee, G.D.; Quave, S.A.; Budde, W.L. "Trace Analyses for 

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

2. Hollenberg, P. F.; Rand-Meir, T.; Hager, L. P. “The Reaction of Chlorite with 

Horseradish Peroxidase and Chloroperoxidase,” J. Biol. Chem.1974, 249, 5816-5825. 

3. Hewson, W. D.; Hager, L. P. “Mechanism of the Chlorination Reaction Catalyzed by 

Horseradish Peroxidase with Chlorite,” J. Biol. Chem.1979, 254, 3175-3181. 

4. Chiswell,B.; O’Hallaran,K.R. “Use of Lissamine Green B as a Spectrophotometric 

Reagent for the Determination of Low Residuals of Chlorine Dioxide” The Analyst, 1991, 

116, 657-661. 

5. Hoffman, R.; Andrews, R.C.;Ye, Q. “Comparison of Spectrophotometric Methods for 

Measuring Chlorine Dioxide in Drinking Water,” Environmental Technology, 1998, 19, 

761-773. 

327.0-24
6. “OSHA Safety and Health Standards, General Industry,” (29CFR1910). Occupational 

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

7. “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. 

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

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

9. “Reagent Chemicals,” Sixth Edition, American Chemical Society Specifications, Official 

from Jan. 1, 1982, American Chemical Society, S, Washington, D. C., 1981. 

10. Peroxidase description, Sigma catalog 2002-2003, page 1635 and references contained 

therein. 

11. Personal communication with Technical Representative at Sigma (1-800-521-8956). 

327.0-25
17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 

TABLE 1: PRECISION AND ACCURACY AND DETECTION LIMITS FOR CHLORINE 

DIOXIDE AND CHLORITE IN REAGENT WATER 

Fortification 

Concentration Chlorite Chlorine Dioxide 

ClO2 

– 

(mg/L) 

ClO2 

(mg/L) 

Recovery 

(%) 

RSD 

(%) 

DLa 

(mg/L) 

Recovery 

(%) 

RSD 

(%) 

DLa 

(mg/L) 

0.25 

not 

fortified 112 12 0.11 

not 

fortified 

not 

fortified 

not 

fortified 

0.25 0.95 118 8.5 0.078 116 3.6 

not 

calculated 

not 

fortified 0.26 

not 

fortified 

not 

fortified 

not 

calculated 102 5.1 0.042 

1.0 0.26 103 2.9 

not 

calculated 124 16 0.16 

1.0 0.94 98.5 3.2 

not 

calculated 111 4.8 

not 

calculated 

a

Based on seven replicates analyzed over one day. Detection Limits were calculated as follows: 

DL = S×t(n-1,1-"=0.99) 

where 

t(n-1,1-" = 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. 

327.0-26
TABLE 2: PRECISION AND ACCURACY FOR CHLORITE AND CHLORINE DIOXIDE 

IN A CHLORINATED SURFACE WATERa 

Fortification Level Chlorite Chlorine Dioxide 

ClO2 

– 

(mg/L) 

ClO2 

(mg/L) 

Recovery 

(%) 

RSD 

(%) 

Recovery 

(%) 

RSD 

(%) 

1.0 not fortified 107 4.2 not fortified not fortified 

not fortified 0.82 not fortified not fortified 100 1.9 

1.0 0.82 109 3.7 91.3 8.9 

2.0 not fortified 105 1.4 not fortified not fortified 

not fortified 1.94 not fortified not fortified 96.9 1.0 

a

The drinking water used in these studies had a free available chlorine concentration of 0.9 mg/L. 

327.0-27
TABLE 3: PRECISION AND ACCURACY FOR CHLORITE AND CHLORINE DIOXIDE 

IN A CHLORINATED GROUND WATERa 

Fortification Level Chlorite Chlorine Dioxide 

ClO2 

– 

(mg/L) 

ClO2 

(mg/L) 

Recovery 

(%) 

RSD 

(%) 

Recovery 

(%) 

RSD 

(%) 

1.0 not fortified 110 4.4 not fortified not fortified 

not fortified 0.90 not fortified not fortified 92.0 3.3 

1.0 0.90 107 1.6 93.8 3.0 

2.0 not fortified 100 2.7 not fortified not fortified 

not fortified 2.0 not fortified not fortified 110 1.7 

a

The drinking water used in these studies had a free available chlorine concentration of 

0.8 mg/L and a hardness of 325 mg/L (CaCO3). 

327.0-28
TABLE 4: STORAGE STABILITY FOR CHLORITE AND CHLORINE DIOXIDE IN A 

CHLORINATED SURFACE WATER SAMPLEa 

Holding 

Time 

(Hours) 

Chlorite Fortified 

at 1.0 mg/L 

Chlorine Dioxide and 

Chlorite Fortified at a Total 

Concentration of 1.8 mg/Lb 

Recovery 

(%) 

RSD 

(%) 

Recovery 

(%) 

RSD 

(%) 

0 83.3 2.5 97.3 1.4 

2 100 6.6 98.1 1.4 

4 99.3 2.4 98.1 2.8 

a The drinking water used in these studies had a free available chlorine concentration of 0.9 mg/L. 

b Chlorine dioxide and chlorite were fortified into the chlorinated drinking water sample using 

concentrated standards just prior to preserving the samples so that the concentrations in the 

surface water were 0.80 and 1.0 mg/L, respectively. 

327.0-29
TABLE 5: INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS

Method 

Reference Requirement Specification and Frequency Acceptance Criteria 

Initial Demonstration 

of Accuracy 

Section 

9.2.2 

Initial Demonstration 

of Precision 

Section 

9.2.3 

Detection Limit 

Determination 

Section 

9.2.1 

Prior to the analysis of any 

Field Samples by a new 

analyst and each time a new 

LGB Concentrated Stock 

Solution is prepared, process 

five highest-level calibration 

standards. Calculate the 

average concentration for 

five standards. 

Using the same data from 

Section 9.2.1, calculate the 

RSD. 

Prior to the analysis of any 

Field Samples by a new 

analyst, prepare and analyze 

a minimum of seven 

replicate CCCs fortified at a 

chlorite concentration of 

0.20 - 0.35 mg/L if using the 

1-cm pathlength procedure. 

Calculate the DL using the 

equation in Section 9.2.3. 

Mean concentration must be 

within ± 30% of the fortified 

value. 

RSD must be < 20%. 

DL should be # 0.25 mg/L 

when using the 1-cm 

pathlength procedure. 

327.0-30
TABLE 6: QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method 

Reference Requirement Specification and Frequency Acceptance Criteria 

Section 8.3 Sample Analyze samples as soon as Sample results are valid only if 

Storage possible. Samples may be stored at samples are analyzed within 

# 10 °C for up to 4 hours holding time. 

Section 

10.2 

Sections 

9.3 and 

10.3 

Initial 

Calibration 

Continuing 

Calibration 

Check (CCC) 

Calibrate with at least three 

calibration standards prior to the 

IDC and each time a new 

Combined LGB/HRP Reagent is 

prepared. 

Five calibration standards are 

recommended. 

Highest level for the 1-cm 

pathlength procedure must not 

exceed 2.2 mg/L. 

Verify initial calibration by 

analyzing a mid-level CCC each 

day samples are analyzed. 

The CCC should be at or near 1.0 

mg/L for the 1-cm pathlength 

procedure. 

When each calibration standard 

is calculated as an unknown 

using the calibration curve, the 

result should be 70-130% of the 

expected value for all except 

the lowest standard, which 

should be 50-150% of the 

expected value. 

The calculated chlorite 

concentration must be ± 30% of 

the expected value. 

Section 9.4 Field 

Duplicate (FD) 

Section 9.5 Laboratory 

Fortified 

Sample Matrix 

(LFSM) 

An LFSM is recommended weekly. 

Calculate Recovery rding to 

Section 9.5. 

acco

An FD is recommended weekly. 

Calculate RPD according to 

Section 9.4. 

RPD should not exceed 30% 

for concentrations above the 

low-level calibration standard. 

RPD should not exceed 50% 

for concentrations at or near the 

low-level calibration standard 

(at or below 0.3 mg/L for the 1-

cm pathlength procedure). 

Recovery should be 70 - 130% 

except for samples fortified at 

low levels (total concentrations 

at or below 0.3 mg/L for the 1-

cm pathlength procedure), 

where the Recovery should be 

50 - 150%. 

327.0-31