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: E299 − 25
Standard Test Method for
Trace Amounts of Peroxides In Organic Solvents1
This standard is issued under the fixed designation E299; 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.
This standard has been approved for use by agencies of the U.S. Department of Defense.
1. Scope* Development of International Standards, Guides and Recom-
2,3
1.1 This test method covers organic solvents containing mendations issued by the World Trade Organization Technical
active oxygen in the range from 5 µg ⁄g (ppm) to 80 µg/g (ppm) Barriers to Trade (TBT) Committee.
or higher. By using a special reaction-absorption cell, the test
2. Referenced Documents
method can be extended to cover the range from 0 ppm to 5
ppm. The test method can be used to determine numerous 2.1 ASTM Standards:4
peroxide classes of varying reactivity such as hydroperoxides, D1193 Specification for Reagent Water
diacyl peroxides, diaroyl peroxides, peresters, and ketone D6809 Guide for Quality Control and Quality Assurance
peroxides. The stable di-tert-alkyl peroxides do not react under Procedures for Aromatic Hydrocarbons and Related Ma-
the conditions of analysis. terials
E29 Practice for Using Significant Digits in Test Data to
1.2 Solvents that can be analyzed successfully include
Determine Conformance with Specifications
saturated and aromatic hydrocarbons, alcohols, ethers, ketones,
E180 Practice for Determining the Precision of ASTM
and esters. In addition, the test method is applicable to olefinic
Methods for Analysis and Testing of Industrial and Spe-
solvents and to certain compounds that contain α, β, and
cialty Chemicals (Withdrawn 2009)5
conjugated unsaturation. Solid samples that are soluble in the
E200 Practice for Preparation, Standardization, and Storage
acetic acid-chloroform solvent also can be analyzed.
of Standard and Reagent Solutions for Chemical Analysis
1.3 The values stated in SI units are to be regarded as E691 Practice for Conducting an Interlaboratory Study to
standard. The values given in parentheses are for information Determine the Precision of a Test Method
only. 2.2 Other Document:
1.4 In determining the conformance of the test results using OSHA Regulations, 29 CFR, paragraphs 1910.1000 and
this method to applicable specifications, results shall be 1910.1200 Air Contaminants – Exposure Limits and Haz-
rounded in accordance with the rounding off methods of ard Communication6
Practice E29.
3. Summary of Test Method
1.5 This standard does not purport to address the safety
concerns, if any, associated with its use. It is the responsibility 3.1 A sample is dissolved in a mixture of acetic acid and
of the user of this standard to establish appropriate safety, chloroform. The solution is deaerated and potassium iodide
health, and environmental practices and determine the appli- reagent solution is added. The mixture is allowed to react in the
cability of regulatory limitations prior to use. dark for 1 h, thereby releasing an equivalent amount of iodine.
1.6 This international standard was developed in accor- The absorbance of the solution is measured at 470 nm and the
dance with internationally recognized principles on standard- amount of active oxygen present in the sample is determined
ization established in the Decision on Principles for the by reference to a calibration curve prepared from iodine.
3.2 For samples containing 0 µg ⁄g (ppm) to 5 µg/g (ppm)
active oxygen, a special reaction-absorption cell is employed.
1
This test method is under the jurisdiction of ASTM Committee D16 on
Aromatic, Industrial, Specialty and Related Chemicals and is the direct responsi-
4
bility of Subcommittee D16.12 on Caustics and Peroxides. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved March 15, 2025. Published April 2025. Originally contact ASTM Customer Service at www.astm.org/contact. For Annual Book of
approved in 1966. Last previous edition approved in 2017 as E299 – 17a. DOI: ASTM Standards volume information, refer to the standard’s Document Summary
10.1520/E0299-25. page on the ASTM website.
2 5
Banerjee, D. K., and Budke, C. C., Analytical Chemistry, ANCHAM, Vol 36, The last approved version of this historical standard is referenced on
1964, pp. 792–796. www.astm.org.
3 6
Banerjee, D. K., and Budke, C. C., Analytical Chemistry, ANCHAM, Vol 36, Banerjee, D. K., and Budke, C. C., Analytical Chemistry, ANCHAM, Vol 36,
1964, pp. 2367–2368. 1964, pp. 792–796.
*A Summary of Changes section appears at the end of this standard
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E299 − 25
The sample is de-aerated and the reaction is carried out within where such specifications are available.7 Other grades may be
the cell. Absorbance measurements are made at 410 nm to used, provided it is first ascertained that the reagent is of
increase the sensitivity. sufficiently high purity to permit its use without lessening the
accuracy of the determination.
4. Significance and Use
7.2 Purity of Water—Unless otherwise indicated, references
4.1 Dilute solutions of peroxides in various organic solvents to water shall be understood to mean Type I or Type II reagent
frequently are used as catalysts or reaction initiators. Peroxides water conforming to Specification D1193.
also can be formed through autoxidation in certain classes of
7.3 Acetic Acid-Chloroform Solvent (2 + 1)—Mix 2 vol-
compounds including ethers, acetals, dienes, and alkylaromatic
umes of acetic acid with 1 volume of chloroform.
hydrocarbons and present a potential safety hazard. This test
method provides a procedure for determining the peroxide or 7.4 Acetic Acid-Chloroform Solvent (Containing Approxi-
active oxygen level. mately 4 % Water)—Add 40 mL of water to 1 L of solvent
prepared as described in 7.3.
5. Interferences 7.5 Iodine.
5.1 Oxidizing or reducing substances present in the sample 7.6 Nitrogen Cylinder of gas with 99.999 % purity.
will interfere. Colored solutions can be analyzed if an absor-
bance correction is made. 7.7 Potassium Iodide Solution (50 %)—Dissolve 20 g of
potassium iodide (KI) in 20 mL of de-aerated water. This
6. Apparatus reagent should be freshly prepared just prior to use.
6.1 Spectrophotometer—General spectrophotometer with 7.8 Water, De-aerated—Pass nitrogen through distilled wa-
matched 1 cm cells. ter for several minutes prior to use.
6.2 Special Reaction-Absorption Cell (Fig. 1)—When this 8. Hazards
cell is used, the regular Beckman cell carriage shall be replaced 8.1 Review the current Safety Data Sheets (SDS) for de-
with the attachment provided for measuring the absorbance in tailed information concerning toxicity, first aid practices, and
test tubes. safety precautions.
7. Reagents 8.2 Chloroform is a colorless liquid that quickly evaporates
into vapor. It can harm the eyes, skin, liver, kidneys, and
7.1 Purity of Reagents—Reagent grade chemicals shall be
nervous system. Chloroform can be toxic if inhaled or swal-
used in all tests. Unless otherwise indicated, it is intended that
lowed. Chloroform has been shown to be carcinogenic in
all reagents shall conform to the specifications of the Commit-
animals after oral exposure, resulting in an increase in kidney
tee on Analytical Reagents of the American Chemical Society,
and liver tumors. EPA has classified chloroform as a Group B2,
probable human carcinogen.
9. Procedure
9.1 High Range—0 µg to 400 µg of Active Oxygen:
9.1.1 Preparation of Calibration Curve:
9.1.1.1 Dissolve 0.1270 g of iodine in acetic acid-
chloroform solvent (2 + 1), record weight, and dilute to
100 mL in a volumetric flask. Calculate the concentration.
When this solution contains 1.27 mg of iodine/mL, this is
equivalent to 80.0 µg of active oxygen/mL.
9.1.1.2 Transfer 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL
aliquots of this solution to 25 mL volumetric flasks and dilute
each to volume with the acetic acid-chloroform solvent. Mix
thoroughly.
9.1.1.3 Using a hypodermic needle or glass capillary, sparge
the solution with nitrogen for 1 min to 1.5 min, add 1 mL of
7
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. Pharma-
FIG. 1 Absorption Cell for Low-Active Oxygen copeial Convention, Inc. (USPC), Rockville, MD.
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E299 − 25
freshly prepared KI solution, and continue the nitrogen flow for 9.2.1.6 The absorption tubes shall be matched and provided
1 min. Stopper and mix well. with a glass ear for reproducible positioning before absorbance
9.1.1.4 Measure the absorbance of each solution at 470 nm, measurements are made. Insert the tube into the cell carriage
using 1 cm cells and a water reference. and rotate until the glass ear contacts the side of the tube
9.1.1.5 Subtract the absorbance of the blank and plot the holder. Measure the absorbance of the solution at 410 nm
absorbance of each standard against micrograms of active against water contained in another matched absorption tube.
oxygen per 25 mL. 9.2.1.7 Subtract the absorbance of the blank and plot absor-
bance against micrograms of active oxygen per 25 mL.
NOTE 1—(abs=slope*cell width of 1*conc); slope=rise/run = abs/conc; 9.2.2 Analysis of Sample:
conc = Abs/slope.
9.2.2.1 Transfer a 5.00 mL sample to a 25 mL volumetric
9.1.2 Analysis of Sample: flask and dilute to volume with acetic acid-chloroform solvent
9.1.2.1 Transfer a sample containing up to 400 µg of active (2 + 1) containing 4 % water. Mix well.
oxygen to a 25 mL volumetric flask and dilute to volume with 9.2.2.2 Transfer a portion of the solution to the special
acetic acid-chloroform solvent (2 + 1) (Note 2). Mix thor- absorption cell and develop the color as described in 9.2.1.3,
oughly. 9.2.1.4, and 9.2.1.5.
9.2.2.3 Allow the sample to stand in the dark for 1 h.
NOTE 2—A sample volume up to 15 mL may be used provided it is
miscible with the amount of acetic acid-chloroform solvent required to 9.2.2.4 Measure the absorbance of the solution at 410 nm
dilute the sample to 25 mL. against water contained in the other matched absorption tube.
9.2.2.5 Subtract the absorbance obtained for a blank carried
9.1.2.2 Sparge the solution with nitrogen for 1 min to 1.5
through the entire procedure, and obtain the micrograms of
min, add 1 mL of freshly prepared KI solution, and continue
active oxygen present in the sample by reference to the
the nitrogen flow for an additional 1 min.
calibration curve.
9.1.2.3 Stopper, mix well, and allow the solution to stand in
the dark for 1 h. 10. Calculation
NOTE 3—Very reactive peroxides react within less than 10 min, while 10.1 Calculate the active oxygen content of the sample as
less reactive peroxides require up to 1 h for complete reaction. A general follows:
reaction time for 1 h is therefore specified. A
active oxygen, µg/g ~ ppm! 5 (1)
9.1.2.4 Measure the absorbance of the solution at 470 nm BC
using 1 cm cells and a water reference.
where:
NOTE 4—Depending on the amount and type of sample present, some A = active oxygen found, µg,
precipitation of KI may occur. However, the KI crystals readily settle to B = sample used, mL, and
the bottom in absorbance measurement. C = density, g/mL.
9.1.2.5 Subtract the absorbance of a blank carried through 10.2 If a specific peroxide is known to be present, convert
the entire procedure, and obtain the micrograms of active the micrograms per gram (parts per million) of active oxygen
oxygen present in the sample by reference to the calibration to peroxide by using the appropriate conversion factor.
curve.
Peroxide X, µg/g ~ ppm! 5 active oxygen in sample, µg/g ~ ppm! × F
9.2 Low Range—0 µg to 40 µg of Active Oxygen: (2)
9.2.1 Preparation of Calibration Curve:
where F = conversion factor for peroxide X.
9.2.1.1 Dissolve 0.0634 g of iodine in acetic acid-
10.2.1 Conversion factors for some common peroxides are
chloroform solvent (2 + 1) and dilute to 100 mL. Transfer a
as follows:
10 mL aliquot to another 100 mL volumetric flask and dilute to
volume with acetic acid-chloroform solvent. This solution Cumene hydroperoxide 9.5125
Benzoyl peroxide 15.1400
contains 63.4 µg of iodine/mL which is equivalent to 4.0 µg of t-butyl hydroperoxide 5.6328
active oxygen/mL. Lauroyle peroxide 24.9150
9.2.1.2 Transfer 0 mL, 1 mL, 3 mL, 5 mL, 8 mL, and 10 mL 11. Report
aliquots to 25 mL volumetric flasks and dilute to volume with
the acetic acid-chloroform solvent containing 4 % water. Mix 11.1 High Range—Report the concentration of the peroxide
well. to the nearest 1 µg/g (ppm).
9.2.1.3 Transfer a portion of each standard to the special 11.2 Low Range—Report the concentration of the peroxide
absorption cell (Fig. 1). Admit a flow of nitrogen through the to the nearest 0.1 µg/g (ppm).
side arm and purge the solution for 3 min. 12. Precision and Bias8
9.2.1.4 Add 5 drops of freshly prepared de-aerated KI
12.1 Precision—High Range—The following criteria shall
solution and replace the stopper loosely. Continue purging with
be used for judging the acceptability of results (Note 5):
nitrogen for an additional 3 min.
9.2.1.5 Tighten the stopper and close the stopcock on the 8
Supporting data have been filed at ASTM International Headquarters and may
inlet tube so that the solution is under a slightly positive be obtained by requesting Research Report RR:E15-1002. Contact ASTM Customer
nitrogen pressure. Service at www.astm.org/contact.
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E299 − 25
12.1.1 Repeatability (Single Analyst)—The standard devia- Number No. of Average
Sample Std. Dev. R
tion for a single determination has been estimated to be Labs analysis Result
1 6 24 47.63 5.48 15.34
0.7 µg ⁄g (ppm) at 36 df. The 95 % limit for the difference 2 6 24 75.67 3.94 11.04
between two such determinations is 2 µg/g (ppm). 3 6 24 22.66 3.65 10.21
12.1.2 Laboratory Precision (Within-Laboratory, Between- 4 5 20 4.02 0.52 1.47
5 5 20 8.83 0.48 1.35
Days Variability)—The standard deviation of results, each the 6 5 20 2.08 0.29 0.81
average of duplicates, obtained by the same analyst on different
days, has been estimated to be 2.9 µg/g (ppm) at 14 df. The 13. Quality Guidelines
95 % limit for the difference between two such averages is 13.1 Laboratories shall have a quality control system in
8.1 µg ⁄g (ppm). place.
12.1.3 Reproducibility (Multilaboratory)—The standard de- 13.1.1 Confirm the performance of the test instrument or
viation of results, each the average of duplicates, obtained by test method by analyzing a quality control sample following
analysts in different laboratories has been estimated to be the guidelines of standard statistical quality control practices.
4.6 µg ⁄g (ppm) at 5 df. The 95 % limit for the difference 13.1.2 A quality control sample is a stable material isolated
between two such averages is 13 µg/g (ppm). from the production process and representative of the sample
NOTE 5—The above precision estimates are based on an interlaboratory being analyzed.
study on three samples containing 30 µg ⁄g (ppm) to 90 µg/g (ppm) of 13.1.3 When QA/QC protocols are already established in
active oxygen. One analyst in each of six laboratories performed duplicate the testing facility, these protocols are acceptable when they
determinations and repeated one day later, for a total of 72 determinations. confirm the validity of test results.
Practice E180 was used in developing these precision estimates. When
comparing results with another lab, the average of duplicates should be 13.1.4 When there are no QA/QC protocols established in
used. the testing facility, use the guidelines described in Guide
12.2 Bias—The bias of this test method has not been D6809 or similar statistical quality control practices.
determined due to the lack of suitable reference materials or 13.2 Interlaboratory Testing:
methodology. 13.2.1 A program that includes multiple laboratories ana-
lyzing the same samples is strongly encouraged. This program
12.3 Precision—Low Range—The following criteria shall
should allow labs to compare their results with other labora-
be used for judging the acceptability of results (Note 6):
tories. This is particularly important when a plant is selling the
12.3.1 Repeatability (Single Analyst)—The standard devia-
product to customers or the laboratory is analyzing the prod-
tion for a single determination has been estimated to be
ucts for acceptance. Producers and customers need to have
0.07 µg ⁄g (ppm) at 24 df. The 95 % limit for the difference
confidence that result from different producers are comparable.
between two such determinations is 0.2 µg/g (ppm).
12.3.2 Laboratory Precision (Within-Laboratory, Between-
Days Variability)—The standard deviation of results, each the
average of duplicates, obtained by the same analyst on different
days, has been estimated to be 0.11 µg/g (ppm) at 13 df. The
95 % limit for the difference between two such averages is
0.31 µg ⁄g (ppm).
12.3.3 Reproducibility (Multilaboratory)—The standard de-
viation of results, each the average of duplicates, obtained by
analysts in different laboratories has been estimated to be
0.49 µg ⁄g (ppm) at 4 df. The 95 % limit for the difference FIG. 2 Peroxides
between two such averages is 1.4 µg/g (ppm).
NOTE 6—The above precision estimates are based on an interlaboratory
study on three samples containing 3 µg ⁄g (ppm) to 10 µg/g (ppm) of active
oxygen. One analyst in each of five laboratories performed duplicate
determinations and repeated one day later, for a total of 60 determinations.
Practice E180 was used in developing these precision estimates. When
comparing within lab results or results between labs, duplicate results
should be used.
12.4 Bias—The bias of this test method has not been
CAS 64-19-7
determined due to the lack of suitable reference materials or
methodology. FIG. 3 Acetic Acid
12.5 Reproducibility, R—calculated from Research Report
RR:E15-1002 data without using duplicate averages.
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E299 − 25
CAS 7681-11-0
FIG. 4 Potassium Iodide
CAS 94-36-0
FIG. 7 Benzoyl Peroxide
CAS 67-66-3
Trichloromenthane
FIG. 5 Chloroform CAS 75-91-2
FIG. 8 t-butyl Hydroperoxide
CAS 80-15-9 CAS 105-74-8
FIG. 6 Cumene Hydroperoxide FIG. 9 Lauroyle Peroxide
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E299 − 25
14. Keywords
14.1 assay; organic; peroxides; spectrophotometric
SUMMARY OF CHANGES
Subcommittee D16.15 has identified the location of selected changes to this standard since the last issue
(E299 – 17a) that may impact the use of this standard. (Approved March 15, 2025.)
(1) Added 1.4 to Scope. (8) Added Interlaboratory Testing to 13.
(2) Added Standards E29, E200, E691, and OSHA reference to (9) Revised Note 5 and Note 6 to include “When comparing
Section 2. within lab results or results between labs, duplicate results
(3) Added Section 8. should be used”.
(4) Added picture and CAS numbers at the end of the standard. (10) Added 12.5 to include Research Report to recalculate
(5) Revised 8.2 to add “vapor” and remove “gas”. Reproducibility values without duplicates technique.
(6) Revised 7.6 to add “gas” and suggested a purity of 99.999+ (11) Added Note 1.
minimum purity for Nitrogen.
(7) Revised 9.1.1.1 to add “record weight” and “calculate the
concentration”.
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