ASTM D5412-93(2024)

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: D5412 − 93 (Reapproved 2024)
Standard Test Method for
Quantification of Complex Polycyclic Aromatic Hydrocarbon
Mixtures or Petroleum Oils in Water
This standard is issued under the fixed designation D5412; 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 mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This test method covers a means for quantifying or
characterizing total polycyclic aromatic hydrocarbons (PAHs)
2. Referenced Documents
by fluorescence spectroscopy (Fl) for waterborne samples. The
2.1 ASTM Standards:
characterization step is for the purpose of finding an appropri-
D1129 Terminology Relating to Water
ate calibration standard with similar emission and synchronous
D1193 Specification for Reagent Water
fluorescence spectra.
D2777 Practice for Determination of Precision and Bias of
1.2 This test method is applicable to PAHs resulting from
Applicable Test Methods of Committee D19 on Water
petroleum oils, fuel oils, creosotes, or industrial organic
D3325 Practice for Preservation of Waterborne Oil Samples
mixtures. Samples can be weathered or unweathered, but either
D3326 Practice for Preparation of Samples for Identification
the same material or appropriately characterized site-specific
of Waterborne Oils
PAH or petroleum oil calibration standards with similar fluo-
D3415 Practice for Identification of Waterborne Oils
rescence spectra should be chosen. The degree of spectral
D3650 Test Method for Comparison of Waterborne Petro-
similarity needed will depend on the desired level of quantifi-
leum Oils By Fluorescence Analysis (Withdrawn 2018)
cation and on the required data quality objectives.
D4489 Practices for Sampling of Waterborne Oils
D4657 Test Method for Polynuclear Aromatic Hydrocarbons
1.3 The values stated in SI units are to be regarded as
in Water (Withdrawn 2005)
standard. No other units of measurement are included in this
E131 Terminology Relating to Molecular Spectroscopy
standard.
E169 Practices for General Techniques of Ultraviolet-Visible
1.4 This standard does not purport to address all of the
Quantitative Analysis
safety concerns, if any, associated with its use. It is the
E275 Practice for Describing and Measuring Performance of
responsibility of the user of this standard to establish appro-
Ultraviolet and Visible Spectrophotometers
priate safety, health, and environmental practices and deter-
E388 Test Method for Wavelength Accuracy and Spectral
mine the applicability of regulatory limitations prior to use.
Bandwidth of Fluorescence Spectrometers
1.5 This international standard was developed in accor-
E578 Test Method for Linearity of Fluorescence Measuring
dance with internationally recognized principles on standard-
Systems
ization established in the Decision on Principles for the
E579 Test Method for Limit of Detection of Fluorescence of
Development of International Standards, Guides and Recom-
Quinine Sulfate in Solution
1 2
This test method is under the jurisdiction of ASTM Committee D19 on Water For referenced ASTM standards, visit the ASTM website, www.astm.org, or
and is the direct responsibility of Subcommittee D19.06 on Methods for Analysis for contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Organic Substances in Water. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved April 1, 2024. Published April 2024. Originally the ASTM website.
ε1 3
approved in 1993. Last previous edition approved in 2017 as D5412 – 93 (2017) . The last approved version of this historical standard is referenced on
DOI: 10.1520/D5412-93R24. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5412 − 93 (2024)
3. Terminology required to eliminate these other components. Fortunately, for
the most commonly expected PAH mixtures, such substituted
3.1 Definitions:
PAHs and heterocycles are not major components of the
3.1.1 For definitions of terms used in this standard, refer to
mixtures and do not cause serious errors.
Terminology D1129, Terminology E131, and Practice D3415.
6. Interferences
4. Summary of Test Method
6.1 The fluorescence spectra may be distorted or quantifi-
4.1 This test method consists of fluorescence analysis of
cation may be affected if the sample is contaminated with an
dilute solutions of PAHs or petroleum oils in appropriate
appreciable amount of other fluorescent chemicals that are
solvents (spectroquality solvents such as cyclohexane or other
excited and which fluoresce in the same spectral regions with
appropriate solvents, for example, ethanol, depending on
relatively high fluorescence yields. Usually the fluorescence
polarity considerations of the sample). The test method re-
spectra would be distorted at levels greater than 1 % to 2 % of
quires an initial qualitative characterization step involving both
such impurities before the quantification would be seriously
fluorescence emission and synchronous spectroscopy in order
affected. (Warning—Storage of samples in improper contain-
to select appropriate calibration standards with similar fluores-
ers (for example, plastics other than TFE-fluorocarbon) may
cence spectra as compared to the samples (see Annex A1 for
result in contamination.)
the definition of spectral similarity). Intensities of peak
NOTE 2—Spectroquality solvents may not have low enough fluores-
maxima of suitable emission spectra are then used to develop
cence background to be used as solvent blanks. Solvent lots vary in the
calibration curves for quantification.
content of fluorescent impurities that may increase with storage time even
NOTE 1—Although some sections of the characterization part of this test for unopened bottles.
NOTE 3—This test method is normally used without a matrix spike due
method are similar to Test Method D3650, there are also significant
differences (see Annex A1). Since the purpose and intent of the two test to possible fluorescence interference by the spike. If a spike is to be used,
it must fluoresce in a spectral region where it will not interfere with the
methods are different, one should not be substituted for the other.
quantification process. Compounds that could be used are dyes that
fluoresce at longer wavelengths than the emission of the PAH mixture.
5. Significance and Use
6.2 If the PAH mixture to be analyzed is a complex mixture
5.1 This test method is useful for characterization and rapid
such as an oil or creosote, it is assumed that a well-
quantification of PAH mixtures including petroleum oils, fuels,
characterized sample of the same or similar material is avail-
creosotes, and industrial organic mixtures, either waterborne or
able as a calibration standard so the fluorescent fraction of the
obtained from tanks.
mixture can be ratioed against the total mixture. Otherwise,
5.2 The unknown PAH mixture is first characterized by its
since the samples and standards are weighed, the nonfluores-
fluorescence emission and synchronous scanning spectra. Then
cent portion of the mixture would bias the quantification
a suitable site-specific calibration standard with similar spectral
although the characterization portion of the test method for
characteristics is selected as described in Annex A1. This
PAHs given in Annex A1 would be unaffected.
calibration standard may also be well-characterized by other
independent methods such as gas chromatography (GC), GC-
7. Apparatus
mass spectrometry (GC-MS), or high performance liquid
7.1 Fluorescence Spectrometer—An instrument recording
chromatography (HPLC). Some suggested independent ana-
in the spectral range of 250 nm to at least 600 nm for both
lytical methods are included in References (1-7) and Test
excitation and emission responses and capable of scanning
Method D4657. Other analytical methods can be substituted by
both monochromators simultaneously at a constant speed with
an experienced analyst depending on the intended data quality
a constant wavelength offset between them for synchronous
objectives. Peak maxima intensities of appropriate fluores-
scanning. The instrument should meet the specifications in
cence emission spectra are then used to set up suitable
Table 1. (Also known as spectrofluorometer or fluorescence
calibration curves as a function of concentration. Further
spectrophotometer.) Consult manufacturer’s instrument manu-
discussion of fluorescence techniques as applied to the char-
als for specific operating instructions.
acterization and quantification of PAHs and petroleum oils can
be found in References (8-18).
TABLE 1 Specifications for Fluorescence Spectrometers
Wavelength Reproducibility
5.3 For the purpose of the present test method polynuclear
Excitation monochromator ±2 nm or better
aromatic hydrocarbons are defined to include substituted poly-
Emission monochromator ±2 nm or better
cyclic aromatic hydrocarbons with functional groups such as
Gratings (Typical Values)
Excitation monochromator minimum of 600 lines/mm blazed at
carboxyl acid, hydroxy, carbonyl and amino groups, and
300 nm
heterocycles giving similar fluorescence responses to PAHs of
Emission monochromator minimum of 600 lines/mm blazed at
similar molecular weight ranges. If PAHs in the more classic
300 nm or 500 nm
Photomultiplier Tube
definition, that is, unsubstituted PAHs, are desired, chemical
S-20 or S-5 response or equivalent
reactions, extractions, or chromatographic procedures may be
Spectral Resolutions
Excitation monochromator spectral bandpass of 2.5 nm or less
Emission monochromator spectral bandpass 2.5 nm or less
The boldface numbers in parentheses refer to a list of references at the end of Maximum bandpasses for both monochromators at least 10 nm
this standard.
D5412 − 93 (2024)
NOTE 4—Although the characterization section of this test method
9.2 Preserve samples in containers as specified in Practice
(given in Annex A1) is similar to Test Method D3650 in many respects,
D3325. Do not cool samples below 5 °C to avoid dewaxing of
there are differences in the purpose and intents of the two test methods.
oil or creosote samples.
The purpose of the characterization step of this test method is to find an
oil with similar fluorescence properties as the sample in order to serve as
9.3 Neat PAH samples (including surface films or layers on
an appropriate calibration standard for quantification. Other differences
water) require only dilution in spectroquality cyclohexane.
between the test methods are instrumentation requirements and the use of
Prepare initial concentration for the unknown at 100 μg/mL for
synchronous spectra as well as emission spectra for this test method.
a check of the fluorescence signal. Further dilutions down to
7.2 Excitation Source—A high-pressure xenon lamp (a
1 μ ⁄mL may be needed to bring the fluorescence signal into the
150 W continuous xenon lamp or a 10 W pulsed xenon lamp
linear range and to avoid self-absorption effects in the solution.
has been proven acceptable). Other continuum sources (either
Most PAH mixtures and oils have been found to be soluble in
continuous or pulsed) having sufficient intensity throughout the
cyclohexane at the concentrations listed. Alternative solvents
ultraviolet and visible regions may also be used.
can be substituted with appropriate tests.
7.3 Fluorescence Cells—Standard cells made from
9.4 If any unknown PAH mixture is dissolved in water, test
fluorescence-free fused silica with a path length of 10 mm and
the mixture with appropriate dilutions or preconcentrations as
a height of at least 45 mm. Stoppered cells may be preferred to
required. The assumption is that no naturally-occurring fluo-
prevent sample evaporation and contamination.
rescent materials such as humic or fulvic acids are present at
7.4 Data Recording System—Preferably the instrument
levels interfering with the determination (refer to Fig. A2.5 and
should be interfaced to a suitable computer system compatible
Fig. A2.6 to show that humic acid does not interfere with the
with the instrument and with suitable software for spectral data
test method even at high (μg/L) levels). This usually becomes
manipulation. Use of a strip chart or X-Y recorder with a
a problem only at PAH levels in the low μg/L range. Extraction
response time of less than 1 s for full-scale deflection is
methods (or separation by column chromatography) are listed
acceptable.
in Practice D3326.
7.5 Micropipet, glass, 10 μL to 50 μL capacity.
9.4.1 An extraction method that proved satisfactory for the
collaborative test is as follows:
7.6 Weighing Pans, 5 mm to 7 mm diameter, 18 mm thick,
made of aluminum or equivalent. Check pans for contamina- 9.4.1.1 Pour 50.0 mL of the sample into a separatory funnel,
tion. add 5.0 mL of cyclohexane and shake for 2 min. Vent the
separatory funnel occasionally. Withdraw the aqueous layer
8. Reagents and Materials (keep this for a second extraction). Collect the cyclohexane
extract in a 10 mL volumetric flask. Add 5.0 mL of cyclo-
8.1 Purity of Reagents—Use spectroquality grade reagents
hexane to the aqueous layer and perform a second extraction.
in all instances unless otherwise stated. Since the goal is to
Combine the two extracts and dilute to 10.0 mL with cyclo-
have as low a fluorescence blank as possible, and since
hexane.
different brands and lots of spectroquality solvent may vary,
9.4.1.2 For field use, it has proven satisfactory to use a
check reagents frequently.
reagent bottle instead of a separatory funnel. Pour 50.0 mL of
8.2 Purity of Water—References to water mean Type IV
the sample in the bottle and add 5.0 mL of cyclohexane, shake
water conforming to Specification D1193. Since fluorescent
for 2 min and collect most of the top layer with a Pasteur pipet.
organic impurities in the water may introduce an interference,
It is important to collect most of the top layer to maximize
check the purity of the water by analyzing a water blank using
percent recovery (tilt the flask to see the separation between the
the same instrumental conditions as for the solvent blank.
two layers more easily). Add 5.0 mL of cyclohexane to the
8.3 Acetone, spectroquality, (CH COCH ).
aqueous layer and perform a second extraction. Combine the
3 3
two cyclohexane extracts and dilute to 10.0 mL with cyclo-
8.4
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