ASTM D6363-20

Standard Test Method for Determination of Hydrogen Peroxide and Combined Organic Peroxides in Atmospheric Water Samples by Peroxidase Enzyme Fluorescence Method

SIGNIFICANCE AND USE
5.1 Hydrogen peroxide (formed photochemically in the atmosphere) is a primary oxidizer of dissolved sulfur dioxide in atmospheric water. Detection of H2O2 in atmospheric water is useful for inferring gas-phase H2O2 concentrations and for assessing the relative importance of various acidifying mechanisms under specific atmospheric conditions.  
5.2 Hydroperoxides in samples to be analyzed are unstable in water and can decay rapidly due to bacterial action or chemical reaction with other constituents. The test method includes procedures for sample derivatization and methods for estimating and correcting for hydroperoxide decay.
SCOPE
1.1 This test method covers the determination of hydroperoxides, which include hydrogen peroxide (H2O2) and combined organic peroxides, in samples of atmospheric water by the method of horseradish peroxidase derivatization and fluorescence analysis of the derived dimer.2,3  
1.2 The range of applicable hydrogen peroxide concentrations was determined to be 0.6–176.0 × 10−6 M from independent laboratory tests of the test method.  
1.3 The primary use of the test method is for hydrogen peroxide, but it may also be used to quantitate organic hydroperoxides. Determinations of organic hydroperoxide concentration levels up to 30 × 10−6 M may be adequately obtained by calibration with hydrogen peroxide.2,3 While organic hydroperoxides have not been detected at significant concentration levels in rain or cloud water, their presence may be tested by operation of the test method with the addition of catalase for destruction of H2O2.3  
1.4 Because of the instability of hydroperoxides in atmospheric water samples, proper sample collection, at-collection derivatization, and stringent quality control are essential aspects of the analytical process.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Aug-2020
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: D22 - Air Quality
• Drafting Committee: D22.03 - Ambient Atmospheres and Source Emissions

Relations

• Refers: ASTM D1356-20a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Sep-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D1356-20 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Mar-2020
• Refers: ASTM D1356-15a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Oct-2015
• Refers: ASTM D1356-15 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Jul-2015
• Refers: ASTM D1356-14b - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Dec-2014
• Refers: ASTM D1356-14a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-May-2014
• Refers: ASTM D1356-14 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Jan-2014
• Refers: ASTM D1356-05(2010) - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Apr-2010
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D5012-01(2008) - Standard Guide for Preparation of Materials Used for the Collection and Preservation of Atmospheric Wet Deposition
• Effective Date: 01-Apr-2008
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D5111-99(2006) - Standard Guide for Choosing Locations and Sampling Methods to Monitor Atmospheric Deposition at Non-Urban Locations
• Effective Date: 01-Apr-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006

Overview

ASTM D6363-20 is the internationally recognized standard test method for the determination of hydrogen peroxide (H₂O₂) and combined organic peroxides in atmospheric water samples using the peroxidase enzyme fluorescence method. Developed by ASTM Committee D22 on Air Quality, this method provides a reliable approach for monitoring the concentration of hydroperoxides-specifically hydrogen peroxide and organic peroxides-in rain, snow, fog, cloud water, and other forms of atmospheric water.

Hydrogen peroxide is a critical component in atmospheric chemistry, acting as a primary oxidizer of dissolved sulfur dioxide and playing a significant role in the acidity of atmospheric waters. Accurate measurement of H₂O₂ and organic hydroperoxides supports research into atmospheric processes, environmental monitoring, and regulatory compliance.

Key Topics

• Hydroperoxide Determination: Covers both hydrogen peroxide and combined organic peroxides.
• Peroxidase Enzyme Fluorescence Method: Employs horseradish peroxidase and p-hydroxyphenylacetic acid derivatization, followed by fluorescence detection.
• Sample Collection and Preservation: Stresses the importance of sample integrity, at-collection derivatization, and quality control due to the instability of hydroperoxides.
• Quantitative Range: Suitable for hydrogen peroxide concentrations from 0.6 to 176.0 × 10⁻⁶ M and organic hydroperoxides up to 30 × 10⁻⁶ M.
• Quality Assurance: Requires standards, blanks, and specific calibration procedures for reliable results.
• Interference Management: Reagent formulation counters interferences from other sample constituents, including transition metals and ionic components.

Applications

The ASTM D6363-20 test method is widely applicable across research, industry, and environmental science sectors:

• Environmental Monitoring: Supports the analysis of rainwater, fog, snow, and cloud water to assess atmospheric oxidation processes.
• Air Quality Studies: Valuable for inferring gas-phase hydrogen peroxide concentrations and evaluating the role of various acidifying mechanisms in the atmosphere.
• Regulatory Compliance: Facilitates reporting and monitoring required by environmental regulations concerning atmospheric deposition and acid rain.
• Atmospheric Chemistry Research: Enables detailed studies of aqueous-phase oxidation, sulfur cycles, and the fate of atmospheric pollutants.
• Quality Control in Sample Analysis: Assists laboratories in implementing rigorous procedures for sample derivatization, storage, and analysis, ensuring data accuracy.

Related Standards

ASTM D6363-20 references and aligns with several key standards and guides that support sampling, terminology, and reagent preparation:

• ASTM D1129 – Terminology Relating to Water
• ASTM D1193 – Specification for Reagent Water
• ASTM D1356 – Terminology Relating to Sampling and Analysis of Atmospheres
• ASTM D5012 – Guide for Preparation of Materials Used for the Collection and Preservation of Atmospheric Wet Deposition
• ASTM D5111 – Guide for Choosing Locations and Sampling Methods to Monitor Atmospheric Deposition at Non-Urban Locations
• ASTM E200 – Practice for Preparation, Standardization, and Storage of Standard and Reagent Solutions for Chemical Analysis

Practical Value

By using ASTM D6363-20, laboratories and researchers can achieve:

• Sensitive Detection: Specialized for low-level hydroperoxide detection in diverse atmospheric water matrices.
• Robust Data Accuracy: Built-in protocols for controlling sample degradation and correcting for hydroperoxide decay.
• Confidence in Results: Established international standard ensures comparability and reliability across studies and regulatory frameworks.
• Versatility: Supports analysis of both inorganic and organic hydroperoxides for comprehensive atmospheric water quality assessment.

Keywords: hydrogen peroxide measurement, atmospheric water analysis, hydroperoxide determination, peroxidase enzyme fluorescence, ASTM D6363-20, environmental monitoring, acid rain analysis, atmospheric chemistry.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D6363 − 20
Standard Test Method for
Determination of Hydrogen Peroxide and Combined Organic
Peroxides in Atmospheric Water Samples by Peroxidase
Enzyme Fluorescence Method
This standard is issued under the fixed designation D6363; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.1 This test method covers the determination of
1.7 This international standard was developed in accor-
hydroperoxides, which include hydrogen peroxide (H O ) and
2 2
dance with internationally recognized principles on standard-
combined organic peroxides, in samples of atmospheric water
ization established in the Decision on Principles for the
by the method of horseradish peroxidase derivatization and
2,3 Development of International Standards, Guides and Recom-
fluorescence analysis of the derived dimer.
mendations issued by the World Trade Organization Technical
1.2 The range of applicable hydrogen peroxide concentra-
Barriers to Trade (TBT) Committee.
−6
tions was determined to be 0.6–176.0 × 10 M from indepen-
dent laboratory tests of the test method. 2. Referenced Documents
1.3 The primary use of the test method is for hydrogen
2.1 ASTM Standards:
peroxide, but it may also be used to quantitate organic D1129Terminology Relating to Water
hydroperoxides.Determinationsoforganichydroperoxidecon- D1193Specification for Reagent Water
−6
centration levels up to 30 × 10 M may be adequately D1356Terminology Relating to Sampling and Analysis of
2,3
obtained by calibration with hydrogen peroxide. While Atmospheres
organic hydroperoxides have not been detected at significant D5012Guide for Preparation of Materials Used for the
concentration levels in rain or cloud water, their presence may Collection and Preservation of Atmospheric Wet Deposi-
be tested by operation of the test method with the addition of tion
catalase for destruction of H O . D5111Guide for Choosing Locations and Sampling Meth-
2 2
ods to Monitor Atmospheric Deposition at Non-Urban
1.4 Because of the instability of hydroperoxides in atmo-
Locations
spheric water samples, proper sample collection, at-collection
E200Practice for Preparation, Standardization, and Storage
derivatization, and stringent quality control are essential as-
of Standard and Reagent Solutions for ChemicalAnalysis
pects of the analytical process.
1.5 The values stated in SI units are to be regarded as
3. Terminology
standard. No other units of measurement are included in this
3.1 Definitions—For definitions of terms used in this test
standard.
method, refer to Terminologies D1129 and D1356 and Guide
1.6 This standard does not purport to address all of the
D5111.
safety concerns, if any, associated with its use. It is the
3.2 Definitions of Terms Specific to This Standard:
responsibility of the user of this standard to establish appro-
3.2.1 atmospheric water, n—liquidorsolidwatersuspended
in the atmosphere or deposited from the atmosphere. Forms of
atmospheric water include rain, snow, fog, cloud water, dew,
This test method is under the jurisdiction of ASTM Committee D22 on Air
and frost.
Quality and is the direct responsibility of Subcommittee D22.03 on Ambient
Atmospheres and Source Emissions.
3.2.2 derivatization, n—formation of the
Current edition approved Sept. 1, 2020. Published September 2020. Originally
p-hydroxyphenylacetic acidic dimer by combination of
approved in 1998. Last previous edition approved in 2013 as D6363–98 (2013).
p-hydroxyphenylacetic acid, horseradish peroxidase reagent,
DOI: 10.1520/D6363-20.
Lazrus, A. L., Kok, G. L., Gitlin, S. N., and Lind, J. A., “Automated
Fluorometric Method for Hydrogen Peroxide in Atmospheric Precipitation,” Ana-
lytical Chemistry, Vol 57, 1985, pp. 917–922. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Kok,G.L.,Thompson,K.,andLazrus,A.L.,“DerivatizationTechniqueforthe contactASTM Customer Service at [e-mail]. ForAnnual Book ofASTM
Determination of Peroxides in Precipitation,” Analytical Chemistry, Vol 58, 1986, Standards volume information, refer to the standard’s Document Summary page on
pp. 1192–1194. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6363 − 20
and hydroperoxide(s). Also the procedure of addition of the Sulfate, Nitrate, Chloride, Formate)—Potentialinterferenceby
derivatizing reagent to samples. transition metals is overcome by the formation of ethylenedi-
aminetetraacetic acid (EDTA) complexes. Tests of simulated
3.2.3 hydroperoxides, n—hydrogen peroxide and organic
rain samples containing transition metals and common ionic
peroxides dissolved in water.
components of precipitation have demonstrated both the gen-
3.2.4 intrinsic hydroperoxides, n—hydroperoxides con-
eral applicability of this test method to samples containing
tained in reagent water used for the method.
commoncontaminantsandthestabilityofderivatizedsolutions
3.2.5 post-derivatization, n—addition of the derivatizing
stored at 4°C for more than five days.
reagent to the sample after collection.
7. Apparatus
3.2.6 pre-derivatization, n—addition of the derivatizing re-
agent to the sample collection container prior to sample
7.1 Flow System, consisting of the following:
collection.
7.1.1 Automatic sampler or injection valve.
7.1.2 Automated wet chemistry (peristaltic) pump.
3.2.7 systems blank, n—afieldblankofreagentwaterthatis
subjected to a similar or identical environment and derivatiza- 7.1.3 Reagent manifold.
tion time as a collected atmospheric water sample. 7.1.4 Mixing coil, 5-turn, 2-mm inner diameter.
7.1.5 Fluorometer,excitationat320nmandmeasurementof
3.2.8 systems standard, n—aH O calibration standard
2 2
the fluorescence signal at 400 nm, flow-through fluorescence
solution subjected to a similar or identical environment and
cell.
derivatization time as a collected atmospheric water sample.
7.1.6 Recorder.
4. Summary of Test Method
7.2 Sample and Standards Containers—All containers used
4.1 Theperoxidaseenzymefluorescencemethodisbasedon for sample collection and sample transport, for storage and
analysis of samples and standards, and for reagents should be
the reaction of hydroperoxides, horseradish peroxidase, and
p-hydroxyphenylacetic (PHOPAA) acid, forming a fluorescent high density polyethylene, TFE-fluorocarbon, or borosilicate
glass, cleaned in accordance with procedures established for
dimer of the latter. This dimer is detected using a fluorometric
technique,andthehydroperoxidesarequantifiedbycalibration analyses of common inorganic ions (see Guide D5012).
with hydrogen peroxide. The formation of the dimer (deriva-
7.3 PipetteswithDisposableTips—Solutionpreparationand
tization) shall be accomplished soon after sample collection to
sample fixing operations are generally conducted using auto-
minimize H O decay. In addition, strict quality assurance
2 2
matic pipettes. Solution volumes delivered by these devices
practices are part of the method, including use of systems
should be verified to confirm consistent and accurate perfor-
standards and systems blanks to estimate hydroperoxide loss 5
mance.
and to assess derivatizing solution effectiveness.
7.4 Reagent Bottles—Allcontainersusedforthepreparation
and storage of derivatizing and other reagent solutions shall be
5. Significance and Use
dedicated for hydroperoxides. Containers for solutions of
5.1 Hydrogen peroxide (formed photochemically in the
catalase shall not be used for non-catalase solutions.
atmosphere)isaprimaryoxidizerofdissolvedsulfurdioxidein
atmospheric water. Detection of H O in atmospheric water is
2 2
8. Reagents and Materials
useful for inferring gas-phase H O concentrations and for
2 2
8.1 Purity of Reagents—Unless otherwise noted, reagent
assessing the relative importance of various acidifying mecha-
grade chemicals shall be used.
nisms under specific atmospheric conditions.
8.2 Purity of Water—Unless otherwise indicated, references
5.2 Hydroperoxides in samples to be analyzed are unstable
to water shall be understood to mean reagent water as defined
in water and can decay rapidly due to bacterial action or
by Type I of Specification D1193, with the added stipulation
chemical reaction with other constituents. The test method
that the total organic carbon content be less than 20 µg/L. A
includes procedures for sample derivatization and methods for
Type I water system equipped with an organic extraction
estimating and correcting for hydroperoxide decay.
cartridge and a 0.2 µm filter is an acceptable water source.
6. Interferences
Water to be used for reagents, standard solutions, and analyti-
cal rinsing should be stored in borosilicate glass.
6.1 The derivatizing reagent is formulated to counteract the
effects of the following potentially interfering species.
6.2 Hydroxymethane Sulfonate (HMSA)—The addition of
formaldehyde (HCHO) to the derivatizing reagent will sup-
press the negative interference of HMSA. In the absence of
Schwartz, L.M., “Calibration of Pipets: A Statistical View,” Analytical
added HCHO, the PHOPAA dimer in a derivatized simulated
−5 −4 Chemistry, Vol 61, 1989, pp. 1080–1083.
rain sample, containing 1.2 × 10 MH O and 1.0 × 10 M
2 2 6
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
HMSA, displayed a fluorescence signal 5% lower than that
Standard-Grade Reference Materials, American Chemical Society, Washington,
observed when HCHO was added to the derivatizing reagent. DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
6.3 Trace Transition Metals and Common Ionic Compo-
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
nents of Atmospheric Water (Sodium, Ammonium, Hydrogen, copeial Convention, Inc. (USPC), Rockville, MD.
D6363 − 20
6 7
8.3 Catalase Enzyme (1.7 × 10 units/mL) —The enzyme 8.6.2 Peroxide Solution, Systems Blank—Water combined
catalase may be used for the destruction of H O in atmo- with dilute derivatizing reagent to the ratio 1:1. Prepare in
2 2
spheric water samples. Its addition to the sample before peroxide calibration standard vials or in sample collection
additionofthederivatizingreagentremovesH O ,butorganic containers, depending on the derivatization method (see Sec-
2 2
hydroperoxides are preserved. Subsequent addition of the tion 10).
derivatizing reagent results in dimer formation by way of 8.6.3 Peroxide Solution, Systems Standard—See 11.3. Pre-
reactionwithperoxidesotherthanH O .Resultsofanalysesof pare in vials used for peroxide calibration standards or in
2 2
catalase-treated samples may be compared with the measure- sample collection containers, depending on the derivatization
ment of peroxides in samples without catalase to determine method (see Section 10).
H O by difference.
2 2
8.7 Potassium Permanganate (KMnO ) Solution, Standard
8.3.1 Catalase,1+49—Dilute 1 mL of catalase enzyme to
(0.01M)—Dissolve1.58gKMnO in100mLwater,anddilute
a final volume of 50 mL with water. Before pipetting the
the solution with water to 1 L. Seal tightly, and store in an
concentrated solution, ensure that all the solid material is
amberborosilicateglassbottleinthedark.Standardizefollow-
completely suspended by shaking or stirring the bottle of
ing the procedure in Practice E200, Sections 64–68; adjust
concentrate. Allow the dilute solution to stand at least 4 h
chemical proportions according to 9.1 of that Practice.
before use. The solution can be stored for up to 48 h at 4°C.
8.8 Sodium Hydroxide (NaOH) (0.1 M)—Dissolve 4.0 g of
8.4 Derivatizing Reagent, Concentrated—Dissolve 12.11 g
sodium hydroxide in water and dilute to 1 L. Prepare weekly.
of Tris(hydroxymethyl)aminomethane, 0.38 g of EDTA, tetra-
8.9 Sulfuric Acid (H SO ), 5% (3.6 M)—Add 5 mL con-
2 4
sodium salt, 4.57 g of PHOPAA, 300 units of horseradish
centrated H SO to water in a volumetric flask, and dilute to
2 4
peroxidase, and 1 mLconcentrated hydrochloric acid in water,
100 mL.
anddiluteto200mLinavolumetricflask.ThefinalpHofthis
solution should be 9.0. If greater than 9.5 or less than 8.5,
9. Sample Collection
remake. Prepare every four days and store at 4°C. Measure-
9.1 Select sampling locations and sampling methods in
ment of peroxides in aqueous atmospheric samples is based on
accordance with Guide D5111. Additional considerations spe-
the fluorescence of the PHOPAA dimer produced by reaction
cific to sampling for aqueous-phase hydrogen peroxide are
ofhydroperoxideswithPHOPAA.Thefluorescenceofsamples
provided in 9.3 and 9.4.
derivatizedatthetimeofcollectionprovidesameasureoftotal
hydroperoxide (organic and H O ) content of the sample. 9.2 Methods of preparation of sample containers for
2 2
8.4.1 Derivatizing Reagent, 4 + 96—Dilute 4.0 mL of the collection, transport, and storage shall be those detailed in
concentrated derivatizing reagent to 100 mL with water. Guide D5012 under inorganic ionic species (see 8.1 and 8.2 of
Prepare daily as needed, and keep tightly sealed at 4°C. Guide D5012).
9.3 Control procedures designed to ensure sample integrity
NOTE 1—The dilute derivatizing reagent is normally added to samples
to be analyzed in the reagent:sample ratio of 1:1. Other concentrations of in the field (see Section 10) are difficult to perform adequately
dilute derivatizing reagent may be used as long as the final ratio entering
if buckets or other high atmospheric-exposure collectors are
theanalyticalsystemis1:1.Underspecialcircumstances,otherratiosmay
used. Therefore, sampling for rain should be conducted using
be dictated by sampling conditions (see 10.6 and 10.7).
funnel-and-bottle type, or narrow-necked, collectors.
8.5 Hydrochloric Acid (HCl), (1 M)—Add 8.3 mL concen-
9.4 The requirements for controlled derivatization of hy-
trated HCl to water in a volumetric flask and dilute to 100 mL.
droperoxides and timely analysis (see Section 10) dictate that
8.6 Peroxide Solution, Standard Stock (1%)—Dilute com-
sampling for wet deposition be conducted on a daily or more
mercially available (pharmaceutical grade is acceptable) H O
2 2
frequent basis.
solution (30%) approximately1+29 with water in a volu-
10. Derivatization
metric flask. Add sodium stannate (Na SnO ) to a concentra-
2 3
tion of 10.65 mg/Land store at 4°C, and store in a borosilicate
10.1 The following procedures shall be in addition to those
glass bottle. Determine the peroxide concentration by titration
specified for preservation of inorganic anions and cations in
with standard permanganate solution (see 11.2) approximately
Guide D5012 (see Table 1 of Guide D5012).
24 h after preparation. U
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D6363 − 98 (Reapproved 2013) D6363 − 20
Standard Test Method for
Determination of Hydrogen Peroxide and Combined Organic
Peroxides in Atmospheric Water Samples by Peroxidase
Enzyme Fluorescence Method
This standard is issued under the fixed designation D6363; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method covers the determination of hydroperoxides, which include hydrogen peroxide (H O ) and combined organic
2 2
peroxides, in samples of atmospheric water by the method of horseradish peroxidase derivatization and fluorescence analysis of
2,3
the derived dimer.
−6
1.2 The range of applicable hydrogen peroxide concentrations was determined to be 0.6–176.0 × 10 M from independent
laboratory tests of the test method.
1.3 The primary use of the test method is for hydrogen peroxide, but it may also be used to quantitate organic hydroperoxides.
−6
Determinations of organic hydroperoxide concentration levels up to 30 × 10 M may be adequately obtained by calibration with
2,3
hydrogen peroxide. While organic hydroperoxides have not been detected at significant concentration levels in rain or cloud
water, their presence may be tested by operation of the test method with the addition of catalase for destruction of H O . .
2 2
1.4 Because of the instability of hydroperoxides in atmospheric water samples, proper sample collection, at-collection
derivatization, and stringent quality control are essential aspects of the analytical process.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.7 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
This guide test method is under the jurisdiction of ASTM Committee D22 on Air Quality and is the direct responsibility of Subcommittee D22.03 on Ambient
Atmospheres and Source Emissions.
Current edition approved Oct. 1, 2013Sept. 1, 2020. Published October 2013September 2020. Originally approved in 1998. Last previous edition approved in 20092013
ε1
as D6363 – 98 (2009)(2013). . DOI: 10.1520/D6363-98R13.10.1520/D6363-20.
Lazrus, A. L., Kok, G. L., Gitlin, S. N., and Lind, J. A., “Automated Fluorometric Method for Hydrogen Peroxide in Atmospheric Precipitation,” Anal. Chem., 57, 1985,
pp. 917–922.Lazrus, A. L., Kok, G. L., Gitlin, S. N., and Lind, J. A., “Automated Fluorometric Method for Hydrogen Peroxide in Atmospheric Precipitation,” Analytical
Chemistry, Vol 57, 1985, pp. 917–922.
Kok, G. L., Thompson, K., and Lazrus, A. L., “Derivatization Technique for the Determination of Peroxides in Precipitation,” Anal. Chem., 58, 1986, pp. 1192–1194.Kok,
G. L., Thompson, K., and Lazrus, A. L., “Derivatization Technique for the Determination of Peroxides in Precipitation,” Analytical Chemistry, Vol 58, 1986, pp. 1192–1194.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6363 − 20
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D1356 Terminology Relating to Sampling and Analysis of Atmospheres
D5012 Guide for Preparation of Materials Used for the Collection and Preservation of Atmospheric Wet Deposition
D5111 Guide for Choosing Locations and Sampling Methods to Monitor Atmospheric Deposition at Non-Urban Locations
E200 Practice for Preparation, Standardization, and Storage of Standard and Reagent Solutions for Chemical Analysis
3. Terminology
3.1 Definitions—For definitions of terms used in this test method, refer to Terminologies D1129 and D1356 and Guide D5111.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 atmospheric water, n—liquid or solid water suspended in the atmosphere or deposited from the atmosphere. Forms of
atmospheric water include rain, snow, fog, cloud water, dew, and frost.
3.2.2 derivatization, n—formation of the p-hydroxyphenylacetic acidic dimer by combination of p-hydroxyphenylacetic acid,
horseradish peroxidase reagent, and hydroperoxide(s). Also the procedure of addition of the derivatizing reagent to samples.
3.2.3 hydroperoxides, n—hydrogen peroxide and organic peroxides dissolved in water.
3.2.4 intrinsic hydroperoxides, n—hydroperoxides contained in reagent water used for the method.
3.2.5 post-derivatization, n—addition of the derivatizing reagent to the sample after collection.
3.2.6 pre-derivatization, n—addition of the derivatizing reagent to the sample collection container prior to sample collection.
3.2.7 systems blank, n—a field blank of reagent water that is subjected to a similar or identical environment and derivatization time
as a collected atmospheric water sample.
3.2.8 systems standard, n—a H O calibration standard solution subjected to a similar or identical environment and derivatization
2 2
time as a collected atmospheric water sample.
4. Summary of Test Method
4.1 The peroxidase enzyme fluorescence method is based on the reaction of hydroperoxides, horseradish peroxidase, and
p-hydroxyphenylacetic (PHOPAA) acid, forming a fluorescent dimer of the latter. This dimer is detected using a fluorometric
technique, and the hydroperoxides are quantified by calibration with hydrogen peroxide. The formation of the dimer
(derivatization) shall be accomplished soon after sample collection to minimize H O decay. In addition, strict quality assurance
2 2
practices are part of the method, including use of systems standards and systems blanks to estimate hydroperoxide loss and to
assess derivatizing solution effectiveness.
5. Significance and Use
5.1 Hydrogen peroxide (formed photochemically in the atmosphere) is a primary oxidizer of dissolved sulfur dioxide in
atmospheric water. Detection of H O in atmospheric water is useful for inferring gas-phase H O concentrations and for assessing
2 2 2 2
the relative importance of various acidifying mechanisms under specific atmospheric conditions.
5.2 Hydroperoxides in samples to be analyzed are unstable in water and can decay rapidly due to bacterial action or chemical
reaction with other constituents. The test method includes procedures for sample derivatization and methods for estimating and
correcting for hydroperoxide decay.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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6. Interferences
6.1 The derivatizing reagent is formulated to counteract the effects of the following potentially interfering species.
6.2 Hydroxymethane Sulfonate (HMSA)—The addition of formaldehyde (HCHO) to the derivatizing reagent will suppress the
negative interference of HMSA. In the absence of added HCHO, the PHOPAA dimer in a derivatized simulated rain sample,
−5 −4
containing 1.2 × 10 M H O and 1.0 × 10 M HMSA, displayed a fluorescence signal 5 % lower than that observed when HCHO
2 2
was added to the derivatizing reagent.
6.3 Trace Transition Metals and Common Ionic Components of Atmospheric Water (Sodium, Ammonium, Hydrogen, Sulfate,
Nitrate, Chloride, Formate)—Potential interference by transition metals is overcome by the formation of ethylenediaminetet-
raacetic acid (EDTA) complexes. Tests of simulated rain samples containing transition metals and common ionic components of
precipitation have demonstrated both the general applicability of this test method to samples containing common contaminants and
the stability of derivatized solutions stored at 4°C for more than five days.
7. Apparatus
7.1 Flow System, consisting of the following:
7.1.1 Automatic sampler or injection valve.
7.1.2 Automated wet chemistry (peristaltic) pump.
7.1.3 Reagent manifold.
7.1.4 Mixing coil, 5-turn, 2-mm inner diameter.
7.1.5 Fluorometer, excitation at 320 nm and measurement of the fluorescence signal at 400 nm, flow-through fluorescence cell.
7.1.6 Recorder.
7.2 Sample and Standards Containers—All containers used for sample collection and sample transport, for storage and analysis
of samples and standards, and for reagents should be high density polyethylene, TFE-fluorocarbon, or borosilicate glass, cleaned
in accordance with procedures established for analyses of common inorganic ions (see Guide D5012).
7.3 Pipettes with Disposable Tips—Solution preparation and sample fixing operations are generally conducted using automatic
pipettes. Solution volumes delivered by these devices should be verified to confirm consistent and accurate performance.
7.4 Reagent Bottles—All containers used for the preparation and storage of derivatizing and other reagent solutions shall be
dedicated for hydroperoxides. Containers for solutions of catalase shall not be used for non-catalase solutions.
8. Reagents and Materials
8.1 Purity of Reagents—Unless otherwise noted, reagent grade chemicals shall be used.
8.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type
I of Specification D1193, with the added stipulation that the total organic carbon content be less than 20 μg/L. A Type I water
system equipped with an organic extraction cartridge and a 0.2 μm filter is an acceptable water source. Water to be used for
reagents, standard solutions, and analytical rinsing should be stored in borosilicate glass.
Schwartz, L.M., “Calibration of Pipets: A Statistical View,” Analytical Chemistry, Vol. 61, 1989, pp. 1080–1083. Schwartz, L.M., “Calibration of Pipets: A Statistical
View,” Analytical Chemistry, Vol 61, 1989, pp. 1080–1083.
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for
Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC),
Rockville, MD.
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6 7
8.3 Catalase Enzyme (1.7 × 10 units/mL) —The enzyme catalase may be used for the destruction of H O in atmospheric water
2 2
samples. Its addition to the sample before addition of the derivatizing reagent removes H O , but organic hydroperoxides are
2 2
preserved. Subsequent addition of the derivatizing reagent results in dimer formation by way of reaction with peroxides other than
H O . Results of analyses of catalase-treated samples may be compared with the measurement of peroxides in samples without
2 2
catalase to determine H O by difference.
2 2
8.3.1 Catalase, 1 + 49—Dilute 1 mL of catalase enzyme to a final volume of 50 mL with water. Before pipetting the concentrated
solution, ensure that all the solid material is completely suspended by shaking or stirring the bottle of concentrate. Allow the dilute
solution to stand at least 4 h before use. The solution can be stored for up to 48 h at 4°C.
8.4 Derivatizing Reagent, Concentrated—Dissolve 12.11 g of Tris(hydroxymethyl)aminomethane, 0.38 g of EDTA, tetrasodium
salt, 4.57 g of PHOPAA, 300 units of horseradish peroxidase, and 1 mL concentrated hydrochloric acid in water, and dilute to 200
mL in a volumetric flask. The final pH of this solution should be 9.0. If greater than 9.5 or less than 8.5, remake. Prepare every
four days and store at 4°C. Measurement of peroxides in aqueous atmospheric samples is based on the fluorescence of the
PHOPAA dimer produced by reaction of hydroperoxides with PHOPAA. The fluorescence of samples derivatized at the time of
collection provides a measure of total hydroperoxide (organic and H O ) content of the sample.
2 2
8.4.1 Derivatizing Reagent, 4 + 96—Dilute 4.0 mL of the concentrated derivatizing reagent to 100 mL with water. Prepare daily
as needed, and keep tightly sealed at 4°C.
NOTE 1—The dilute derivatizing reagent is normally added to samples to be analyzed in the reagent:sample ratio of 1:1. Other concentrations of dilute
derivatizing reagent may be used as long as the final ratio entering the analytical system is 1:1. Under special circumstances, other ratios may be dictated
by sampling conditions (see 10.6 and 10.7).
8.5 Hydrochloric Acid (HCl), (1 M)—Add 8.3 mL concentrated HCl to water in a volumetric flask and dilute to 100 mL.
8.6 Peroxide Solution, Standard Stock (1 %)—Dilute commercially available (pharmaceutical grade is acceptable) H O solution
2 2
(30 %) approximately 1 + 29 with water in a volumetric flask. Add sodium stannate (Na SnO ) to a concentration of 10.65 mg/L
2 3
and store at 4°C, and store in a borosilicate glass bottle. Determine the peroxide concentration by titration with standard
permanganate solution (see 11.2) approximately 24 h after preparation. Update the concentration determination by titration at one
month intervals.
8.6.1 Peroxide Solution, 1 + 199—Dilute 500 μL of the standard stock (1 %) solution to 100 mL with water in a volumetric flask.
The approximate H O concentration of the resulting solution is 1500 μM (50 mg/L). Calibration standards are prepared
2 2
immediately before sample analysis by diluting aliquots of this solution (see 11.3).
8.6.2 Peroxide Solution, Systems Blank—Water combined with dilute derivatizing reagent to the ratio 1:1. Prepare in peroxide
calibration standard vials or in sample collection containers, depending on the derivatization method (see Section 10).
8.6.3 Peroxide Solution, Systems Standard—See 11.3. Prepare in vials used for peroxide calibration standards or in sample
collection containers, depending on the derivatization method (see Section 10).
8.7 Potassium Permanganate (KMnO )Solution, Standard (0.01 M)—Dissolve 1.58 g KMnO in 100 mL water, and dilute the
4 4
solution with water to 1 L. Seal tightly, and store in an amber borosilicate glass bottle in the dark. Standardize following the
procedure in Practice E200, Sections 64–68; adjust chemical proportions according to 9.1 of that Practice.
8.8 Sodium Hydroxide (NaOH) (0.1 M)—Dissolve 4.0 g of sodium hydroxide in water and dilute to 1 L. Prepare weekly.
8.9 Sulfuric Acid (H SO ), 5 % (3.6 M)—Add 5 mL concentrated H SO to water in a volumetric flask, and dilute to 100 mL.
2 4 2 4
Catalase enzyme, 1.7 × 10 units/mL, has been found satisfactory for this purpose. Available through Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178.
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9. Sample Collection
9.1 Select sampling locations and sampling methods in accordance with Guide D5111. Additional considera
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