ASTM D5412-93(2017)e1
(Test Method)Standard Test Method for Quantification of Complex Polycyclic Aromatic Hydrocarbon Mixtures or Petroleum Oils in Water
Standard Test Method for Quantification of Complex Polycyclic Aromatic Hydrocarbon Mixtures or Petroleum Oils in Water
SIGNIFICANCE AND USE
5.1 This test method is useful for characterization and rapid quantification of PAH mixtures including petroleum oils, fuels, creosotes, and industrial organic mixtures, either waterborne or obtained from tanks.
5.2 The unknown PAH mixture is first characterized by its fluorescence emission and synchronous scanning spectra. Then a suitable site-specific calibration standard with similar spectral characteristics is selected as described in Annex A1. This calibration standard may also be well-characterized by other independent methods such as gas chromatography (GC), GC-mass spectrometry (GC-MS), or high performance liquid chromatography (HPLC). Some suggested independent analytical methods are included in References (1-7)4 and Test Method D4657. Other analytical methods can be substituted by an experienced analyst depending on the intended data quality objectives. Peak maxima intensities of appropriate fluorescence emission spectra are then used to set up suitable calibration curves as a function of concentration. Further discussion of fluorescence techniques as applied to the characterization and quantification of PAHs and petroleum oils can be found in References (8-18).
5.3 For the purpose of the present test method polynuclear aromatic hydrocarbons are defined to include substituted polycyclic aromatic hydrocarbons with functional groups such as carboxyl acid, hydroxy, carbonyl and amino groups, and heterocycles giving similar fluorescence responses to PAHs of similar molecular weight ranges. If PAHs in the more classic definition, that is, unsubstituted PAHs, are desired, chemical reactions, extractions, or chromatographic procedures may be required to eliminate these other components. Fortunately, for the most commonly expected PAH mixtures, such substituted PAHs and heterocycles are not major components of the mixtures and do not cause serious errors.
SCOPE
1.1 This test method covers a means for quantifying or characterizing total polycyclic aromatic hydrocarbons (PAHs) by fluorescence spectroscopy (Fl) for waterborne samples. The characterization step is for the purpose of finding an appropriate calibration standard with similar emission and synchronous fluorescence spectra.
1.2 This test method is applicable to PAHs resulting from petroleum oils, fuel oils, creosotes, or industrial organic mixtures. Samples can be weathered or unweathered, but either the same material or appropriately characterized site-specific PAH or petroleum oil calibration standards with similar fluorescence spectra should be chosen. The degree of spectral similarity needed will depend on the desired level of quantification and on the required data quality objectives.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
1.5 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.
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Buy Standard
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.
´1
Designation:D5412 −93 (Reapproved 2017)
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.
ε NOTE—Warning statements were editorially corrected throughout in December 2017.
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.Samplescanbeweatheredorunweathered,buteither
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
cation and on the required data quality objectives.
D4489 Practices for Sampling of Waterborne Oils
D4657 TestMethodforPolynuclearAromaticHydrocarbons
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 PracticesforGeneralTechniquesofUltraviolet-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 PracticeforDescribingandMeasuringPerformanceof
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
andisthedirectresponsibilityofSubcommitteeD19.06onMethodsforAnalysisfor 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 Dec. 15, 2017. Published January 2018. Originally the ASTM website.
ε1 3
approved in 1993. Last previous edition approved in 2011 as D5412 – 93 (2011) . The last approved version of this historical standard is referenced on
DOI: 10.1520/D5412-93R17E01. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
D5412−93 (2017)
3. Terminology the most commonly expected PAH mixtures, such substituted
PAHs and heterocycles are not major components of the
3.1 Definitions:
mixtures and do not cause serious errors.
3.1.1 For definitions of terms used in this standard, refer to
Terminology D1129, Terminology E131, and Practice D3415.
6. Interferences
6.1 The fluorescence spectra may be distorted or quantifi-
4. Summary of Test Method
cation may be affected if the sample is contaminated with an
4.1 This test method consists of fluorescence analysis of
appreciable amount of other fluorescent chemicals that are
dilute solutions of PAHs or petroleum oils in appropriate
excited and which fluoresce in the same spectral regions with
solvents (spectroquality solvents such as cyclohexane or other
relatively high fluorescence yields. Usually the fluorescence
appropriate solvents, for example, ethanol, depending on
spectra would be distorted at levels greater than 1 to 2 % of
polarity considerations of the sample). The test method re-
such impurities before the quantification would be seriously
quiresaninitialqualitativecharacterizationstepinvolvingboth
affected. (Warning—Storage of samples in improper contain-
fluorescence emission and synchronous spectroscopy in order
ers (for example, plastics other than TFE-fluorocarbon) may
to select appropriate calibration standards with similar fluores-
result in contamination.)
cence spectra as compared to the samples (see Annex A1 for
the definition of spectral similarity). Intensities of peak NOTE 2—Spectroquality solvents may not have low enough fluores-
cence background to be used as solvent blanks. Solvent lots vary in the
maxima of suitable emission spectra are then used to develop
content of fluorescent impurities that may increase with storage time even
calibration curves for quantification.
for unopened bottles.
NOTE 3—This test method is normally used without a matrix spike due
NOTE1—Althoughsomesectionsofthecharacterizationpartofthistest
to possible fluorescence interference by the spike. If a spike is to be used,
method are similar to Test Method D3650, there are also significant
it must fluoresce in a spectral region where it will not interfere with the
differences (see Annex A1). Since the purpose and intent of the two test
quantification process. Compounds that could be used are dyes that
methods are different, one should not be substituted for the other.
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
such as an oil or creosote, it is assumed that a well-
5.1 This test method is useful for characterization and rapid
characterized sample of the same or similar material is avail-
quantification of PAH mixtures including petroleum oils, fuels,
able as a calibration standard so the fluorescent fraction of the
creosotes,andindustrialorganicmixtures,eitherwaterborneor
mixture can be ratioed against the total mixture. Otherwise,
obtained from tanks.
since the samples and standards are weighed, the nonfluores-
5.2 The unknown PAH mixture is first characterized by its
cent portion of the mixture would bias the quantification
fluorescence emission and synchronous scanning spectra.Then
although the characterization portion of the test method for
asuitablesite-specificcalibrationstandardwithsimilarspectral
PAHs given in Annex A1 would be unaffected.
characteristics is selected as described in Annex A1. This
calibration standard may also be well-characterized by other
7. Apparatus
independent methods such as gas chromatography (GC), GC-
7.1 Fluorescence Spectrometer—An instrument recording
mass spectrometry (GC-MS), or high performance liquid
in the spectral range of 250 nm to at least 600 nm for both
chromatography (HPLC). Some suggested independent ana-
excitation and emission responses and capable of scanning
lytical methods are included in References (1-7) and Test
both monochromators simultaneously at a constant speed with
MethodD4657.Otheranalyticalmethodscanbesubstitutedby
a constant wavelength offset between them for synchronous
an experienced analyst depending on the intended data quality
scanning. The instrument should meet the specifications in
objectives. Peak maxima intensities of appropriate fluores-
Table 1. (Also known as spectrofluorometer or fluorescence
cence emission spectra are then used to set up suitable
spectrophotometer.) Consult manufacturer’s instrument manu-
calibration curves as a function of concentration. Further
als for specific operating instructions.
discussion of fluorescence techniques as applied to the char-
acterization and quantification of PAHs and petroleum oils can NOTE 4—Although the characterization section of this test method
be found in References (8-18).
5.3 For the purpose of the present test method polynuclear TABLE 1 Specifications for Fluorescence Spectrometers
aromatic hydrocarbons are defined to include substituted poly-
Wavelength Reproducibility
Excitation monochromator ±2 nm or better
cyclic aromatic hydrocarbons with functional groups such as
Emission monochromator ±2 nm or better
carboxyl acid, hydroxy, carbonyl and amino groups, and
Gratings (Typical Values)
heterocycles giving similar fluorescence responses to PAHs of
Excitation monochromator minimum of 600 lines/mm blazed at
300 nm
similar molecular weight ranges. If PAHs in the more classic
Emission monochromator minimum of 600 lines/mm blazed at
definition, that is, unsubstituted PAHs, are desired, chemical
300 nm or 500 nm
reactions, extractions, or chromatographic procedures may be
Photomultiplier Tube
S-20 or S-5 response or equivalent
required to eliminate these other components. Fortunately, for
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.
´1
D5412−93 (2017)
(given in Annex A1) is similar to Test Method D3650 in many respects,
9.2 Preserve samples in containers as specified in Practice
there are differences in the purpose and intents of the two test methods.
D3325. Do not cool samples below 5°C to avoid dewaxing of
The purpose of the characterization step of this test method is to find an
oil or creosote samples.
oil with similar fluorescence properties as the sample in order to serve as
an appropriate calibration standard for quantification. Other differences
9.3 Neat PAH samples (including surface films or layers on
between the test methods are instrumentation requirements and the use of
water) require only dilution in spectroquality cyclohexane.
synchronous spectra as well as emission spectra for this test method.
Prepare initial concentration for the unknown at 100 µg/mLfor
7.2 Excitation Source—A high-pressure xenon lamp (a
a check of the fluorescence signal. Further dilutions down to 1
150-W continuous xenon lamp or a 10-W pulsed xenon lamp
µ/mL may be needed to bring the fluorescence signal into the
has been proven acceptable). Other continuum sources (either
linear range and to avoid self-absorption effects in the solution.
continuousorpulsed)havingsufficientintensitythroughoutthe
Most PAH mixtures and oils have been found to be soluble in
ultraviolet and visible regions may also be used.
cyclohexane at the concentrations listed. Alternative solvents
can be substituted with appropriate tests.
7.3 Fluorescence Cells—Standard cells made from
fluorescence-free fused silica with a path length of 10 mm and
9.4 If any unknown PAH mixture is dissolved in water, test
a height of at least 45 mm. Stoppered cells may be preferred to
the mixture with appropriate dilutions or preconcentrations as
prevent sample evaporation and contamination.
required. The assumption is that no naturally-occurring fluo-
rescent materials such as humic or fulvic acids are present at
7.4 Data Recording System—Preferably the instrument
levelsinterferingwiththedetermination(refertoFig.A2.5and
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/Lrange. 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 to 50-µL capacity.
9.4.1 An extraction method that proved satisfactory for the
collaborative test is as follows:
7.6 Weighing Pans,5to7-mmdiameter,18-mmthick,made
of aluminum or equivalent. Check pans for contamination.
9.4.1.1 Pour50.0mLofthesampleintoaseparatoryfunnel,
add 5.0 mL of cyclohexane and shake for 2 min. Vent the
8. Reagents and Materials separatory funnel occasionally. Withdraw the aqueous layer
(keep this for a second extraction). Collect the cyclohexane
8.1 Purity of Reagents—Use spectroquality grade reagents
extract in a 10-mL volumetric flask. Add 5.0 mL of cyclo-
in all instances unless otherwise stated. Since the goal is to
hexane to the aqueous layer and perform a second extraction.
have as low a fluorescence blank as possible, and since
Combine the two extracts and dilute to 10.0 mL with cyclo-
different brands and lots of spectroquality solvent may vary,
hexane.
check reagents frequently.
9.4.1.2 For field use, it has proven satisfactory to use a
8.2 Purity of Water—References to water mean Type IV
reagent bottle instead of a separatory funnel. Pour 50.0 mL of
water conforming to Specification D1193. Since fluorescent
the sample in the bottle and add 5.0 mLof cyclohexane, shake
organic impurities in the water may introduce an interference,
for 2 min and collect most of the top layer with a Pasteur pipet.
check the purity of the water by analyzing a water blank using
It is important to collect most of the top layer to maximize
the same instrumental conditions as for the solvent blank.
percentrecovery(tilttheflasktoseetheseparationbetweenthe
two layers more easily). Add 5.0 mL of cyclohexane to the
8.3 Acetone, spectroquality, (CH COCH ).
3 3
aqueous layer and perform a second extraction. Combine the
8.4 Cyclohexane, spectroquality or HPLC grade. The fluo-
two cyclohexane extracts and dilute to 10.0 mL with cyclo-
rescence
...
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
´1
Designation: D5412 − 93 (Reapproved 2017)
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.
ε NOTE—Warning statements were editorially corrected throughout in December 2017.
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
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 Dec. 15, 2017. Published January 2018. Originally the ASTM website.
ε1 3
approved in 1993. Last previous edition approved in 2011 as D5412 – 93 (2011) . The last approved version of this historical standard is referenced on
DOI: 10.1520/D5412-93R17E01. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
D5412 − 93 (2017)
3. Terminology the most commonly expected PAH mixtures, such substituted
PAHs and heterocycles are not major components of the
3.1 Definitions:
mixtures and do not cause serious errors.
3.1.1 For definitions of terms used in this standard, refer to
Terminology D1129, Terminology E131, and Practice D3415.
6. Interferences
6.1 The fluorescence spectra may be distorted or quantifi-
4. Summary of Test Method
cation may be affected if the sample is contaminated with an
4.1 This test method consists of fluorescence analysis of
appreciable amount of other fluorescent chemicals that are
dilute solutions of PAHs or petroleum oils in appropriate
excited and which fluoresce in the same spectral regions with
solvents (spectroquality solvents such as cyclohexane or other
relatively high fluorescence yields. Usually the fluorescence
appropriate solvents, for example, ethanol, depending on
spectra would be distorted at levels greater than 1 to 2 % of
polarity considerations of the sample). The test method re-
such impurities before the quantification would be seriously
quires an initial qualitative characterization step involving both
affected. (Warning—Storage of samples in improper contain-
fluorescence emission and synchronous spectroscopy in order
ers (for example, plastics other than TFE-fluorocarbon) may
to select appropriate calibration standards with similar fluores-
result in contamination.)
cence spectra as compared to the samples (see Annex A1 for
the definition of spectral similarity). Intensities of peak NOTE 2—Spectroquality solvents may not have low enough fluores-
cence background to be used as solvent blanks. Solvent lots vary in the
maxima of suitable emission spectra are then used to develop
content of fluorescent impurities that may increase with storage time even
calibration curves for quantification.
for unopened bottles.
NOTE 3—This test method is normally used without a matrix spike due
NOTE 1—Although some sections of the characterization part of this test
to possible fluorescence interference by the spike. If a spike is to be used,
method are similar to Test Method D3650, there are also significant
it must fluoresce in a spectral region where it will not interfere with the
differences (see Annex A1). Since the purpose and intent of the two test
quantification process. Compounds that could be used are dyes that
methods are different, one should not be substituted for the other.
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
such as an oil or creosote, it is assumed that a well-
5.1 This test method is useful for characterization and rapid
characterized sample of the same or similar material is avail-
quantification of PAH mixtures including petroleum oils, fuels,
able as a calibration standard so the fluorescent fraction of the
creosotes, and industrial organic mixtures, either waterborne or
mixture can be ratioed against the total mixture. Otherwise,
obtained from tanks.
since the samples and standards are weighed, the nonfluores-
5.2 The unknown PAH mixture is first characterized by its
cent portion of the mixture would bias the quantification
fluorescence emission and synchronous scanning spectra. Then
although the characterization portion of the test method for
a suitable site-specific calibration standard with similar spectral
PAHs given in Annex A1 would be unaffected.
characteristics is selected as described in Annex A1. This
calibration standard may also be well-characterized by other
7. Apparatus
independent methods such as gas chromatography (GC), GC-
7.1 Fluorescence Spectrometer—An instrument recording
mass spectrometry (GC-MS), or high performance liquid
in the spectral range of 250 nm to at least 600 nm for both
chromatography (HPLC). Some suggested independent ana-
4 excitation and emission responses and capable of scanning
lytical methods are included in References (1-7) and Test
both monochromators simultaneously at a constant speed with
Method D4657. Other analytical methods can be substituted by
a constant wavelength offset between them for synchronous
an experienced analyst depending on the intended data quality
scanning. The instrument should meet the specifications in
objectives. Peak maxima intensities of appropriate fluores-
Table 1. (Also known as spectrofluorometer or fluorescence
cence emission spectra are then used to set up suitable
spectrophotometer.) Consult manufacturer’s instrument manu-
calibration curves as a function of concentration. Further
als for specific operating instructions.
discussion of fluorescence techniques as applied to the char-
acterization and quantification of PAHs and petroleum oils can NOTE 4—Although the characterization section of this test method
be found in References (8-18).
5.3 For the purpose of the present test method polynuclear TABLE 1 Specifications for Fluorescence Spectrometers
aromatic hydrocarbons are defined to include substituted poly-
Wavelength Reproducibility
Excitation monochromator ±2 nm or better
cyclic aromatic hydrocarbons with functional groups such as
Emission monochromator ±2 nm or better
carboxyl acid, hydroxy, carbonyl and amino groups, and
Gratings (Typical Values)
heterocycles giving similar fluorescence responses to PAHs of
Excitation monochromator minimum of 600 lines/mm blazed at
300 nm
similar molecular weight ranges. If PAHs in the more classic
Emission monochromator minimum of 600 lines/mm blazed at
definition, that is, unsubstituted PAHs, are desired, chemical
300 nm or 500 nm
reactions, extractions, or chromatographic procedures may be
Photomultiplier Tube
S-20 or S-5 response or equivalent
required to eliminate these other components. Fortunately, for
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.
´1
D5412 − 93 (2017)
(given in Annex A1) is similar to Test Method D3650 in many respects,
9.2 Preserve samples in containers as specified in Practice
there are differences in the purpose and intents of the two test methods.
D3325. Do not cool samples below 5°C to avoid dewaxing of
The purpose of the characterization step of this test method is to find an
oil or creosote samples.
oil with similar fluorescence properties as the sample in order to serve as
an appropriate calibration standard for quantification. Other differences
9.3 Neat PAH samples (including surface films or layers on
between the test methods are instrumentation requirements and the use of
water) require only dilution in spectroquality cyclohexane.
synchronous spectra as well as emission spectra for this test method.
Prepare initial concentration for the unknown at 100 µg/mL for
7.2 Excitation Source—A high-pressure xenon lamp (a
a check of the fluorescence signal. Further dilutions down to 1
150-W continuous xenon lamp or a 10-W pulsed xenon lamp
µ/mL may be needed to bring the fluorescence signal into the
has been proven acceptable). Other continuum sources (either
linear range and to avoid self-absorption effects in the solution.
continuous or pulsed) having sufficient intensity throughout the
Most PAH mixtures and oils have been found to be soluble in
ultraviolet and visible regions may also be used.
cyclohexane at the concentrations listed. Alternative solvents
can be substituted with appropriate tests.
7.3 Fluorescence Cells—Standard cells made from
fluorescence-free fused silica with a path length of 10 mm and
9.4 If any unknown PAH mixture is dissolved in water, test
a height of at least 45 mm. Stoppered cells may be preferred to
the mixture with appropriate dilutions or preconcentrations as
prevent sample evaporation and contamination.
required. The assumption is that no naturally-occurring fluo-
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 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 to 7-mm diameter, 18-mm thick, made
of aluminum or equivalent. Check pans for contamination.
9.4.1.1 Pour 50.0 mL of the sample into a separatory funnel,
add 5.0 mL of cyclohexane and shake for 2 min. Vent the
8. Reagents and Materials
separatory funnel occasionally. Withdraw the aqueous layer
(keep this for a second extraction). Collect the cyclohexane
8.1 Purity of Reagents—Use spectroquality grade reagents
extract in a 10-mL volumetric flask. Add 5.0 mL of cyclo-
in all instances unless otherwise stated. Since the goal is to
hexane to the aqueous layer and perform a second extraction.
have as low a fluorescence blank as possible, and since
Combine the two extracts and dilute to 10.0 mL with cyclo-
different brands and lots of spectroquality solvent may vary,
hexane.
check reagents frequently.
9.4.1.2 For field use, it has proven satisfactory to use a
8.2 Purity of Water—References to water mean Type IV
reagent bottle instead of a separatory funnel. Pour 50.0 mL of
water conforming to Specification D1193. Since fluorescent
the sample in the bottle and add 5.0 mL of cyclohexane, shake
organic impurities in the water may introduce an interference,
for 2 min and collect most of the top layer with a Pasteur pipet.
check the purity of the water by analyzing a water blank using
It is important to collect most of the top layer to maximize
the same instrumental conditions as for the solvent blank.
percent recovery (tilt the flask to see the separation between the
two layers more easily). Add 5.0 mL of cyclohexane to the
8.3 Acetone, spectroquality, (CH COCH ).
3 3
aqueous layer and perform a second extraction. Combine the
8.4 Cyclohexane, spectroquality or HPLC grade. The fluo-
two cyclohexane extracts an
...
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.
´1 ´1
Designation: D5412 − 93 (Reapproved 2011) D5412 − 93 (Reapproved 2017)
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.
ε NOTE—Editorial corrections were made Warning statements were editorially corrected throughout in March 2014.De-
cember 2017.
1. Scope
1.1 This test method covers a means for quantifying or characterizing total polycyclic aromatic hydrocarbons (PAHs) by
fluorescence spectroscopy (Fl) for waterborne samples. The characterization step is for the purpose of finding an appropriate
calibration standard with similiarsimilar emission and synchronous fluorescence spectra.
1.2 This test method is applicable to PAHs resulting from petroleum oils, fuel oils, creosotes, or industrial organic mixtures.
Samples can be weathered or unweathered, but either the same material or appropriately characterized site-specific PAH or
petroleum oil calibration standards with similar fluorescence spectra should be chosen. The degree of spectral similarity needed
will depend on the desired level of quantification and on the required data quality objectives.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
1.5 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.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
D3325 Practice for Preservation of Waterborne Oil Samples
D3326 Practice for Preparation of Samples for Identification of Waterborne Oils
D3415 Practice for Identification of Waterborne Oils
D3650 Test Method for Comparison of Waterborne Petroleum Oils By Fluorescence Analysis
D4489 Practices for Sampling of Waterborne Oils
D4657 Test Method for Polynuclear Aromatic Hydrocarbons in Water (Withdrawn 2005)
E131 Terminology Relating to Molecular Spectroscopy
E169 Practices for General Techniques of Ultraviolet-Visible Quantitative Analysis
E275 Practice for Describing and Measuring Performance of Ultraviolet and Visible Spectrophotometers
E388 Test Method for Wavelength Accuracy and Spectral Bandwidth of Fluorescence Spectrometers
E578 Test Method for Linearity of Fluorescence Measuring Systems
E579 Test Method for Limit of Detection of Fluorescence of Quinine Sulfate in Solution
This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.06 on Methods for Analysis for
Organic Substances in Water.
Current edition approved May 1, 2011. Published June 2011January 2018. Originally approved in 1993. Last previous edition approved in 20052011 as D5412 – 93
ε1
(2005).(2011) . DOI: 10.1520/D5412-93R11E01.10.1520/D5412-93R17E01.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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D5412 − 93 (2017)
3. Terminology
3.1 Definitions—Definitions: For definitions of terms used in this test method, refer to Terminology D1129, Terminology E131,
and Practice D3415.
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129, Terminology E131, and Practice D3415.
4. Summary of Test Method
4.1 This test method consists of fluorescence analysis of dilute solutions of PAHs or petroleum oils in appropriate solvents
(spectroquality solvents such as cyclohexane or other appropriate solvents, for example, ethanol, depending on polarity
considerations of the sample). The test method requires an initial qualitative characterization step involving both fluorescence
emission and synchronous spectroscopy in order to select appropriate calibration standards with similar fluorescence spectra as
compared to the samples (see Annex A1 for the definition of spectral similarity). Intensities of peak maxima of suitable emission
spectra are then used to develop calibration curves for quantification.
NOTE 1—Although some sections of the characterization part of this test method are similar to Test Method D3650, there are also significant differences
(see Annex A1). Since the purpose and intent of the two test methods are different, one should not be substituted for the other.
5. Significance and Use
5.1 This test method is useful for characterization and rapid quantification of PAH mixtures including petroleum oils, fuels,
creosotes, and industrial organic mixtures, either waterborne or obtained from tanks.
5.2 The unknown PAH mixture is first characterized by its fluorescence emission and synchronous scanning spectra. Then a
suitable site-specific calibration standard with similar spectral characteristics is selected as described in Annex A1. This calibration
standard may also be well-characterized by other independent methods such as gas chromatography (GC), GC-mass spectrometry
(GC-MS), or high performance liquid chromatography (HPLC). Some suggested independent analytical methods are included in
References (1-7) and Test Method D4657. Other analytical methods can be substituted by an experienced analyst depending on
the intended data quality objectives. Peak maxima intensities of appropriate fluorescence emission spectra are then used to set up
suitable calibration curves as a function of concentration. Further discussion of fluorescence techniques as applied to the
characterization and quantification of PAHs and petroleum oils can be found in References (8-18).
5.3 For the purpose of the present test method polynuclear aromatic hydrocarbons are defined to include substituted polycyclic
aromatic hydrocarbons with functional groups such as carboxyl acid, hydroxy, carbonyl and amino groups, and heterocycles giving
similar fluorescence responses to PAHs of similar molecular weight ranges. If PAHs in the more classic definition, that is,
unsubstituted PAHs, are desired, chemical reactions, extractions, or chromatographic procedures may be required to eliminate these
other components. Fortunately, for the most commonly expected PAH mixtures, such substituted PAHs and heterocycles are not
major components of the mixtures and do not cause serious errors.
6. Interferences
6.1 The fluorescence spectra may be distorted or quantification may be affected if the sample is contaminated with an
appreciable amount of other fluorescent chemicals that are excited and which fluoresce in the same spectral regions with relatively
high fluorescence yields. Usually the fluorescence spectra would be distorted at levels greater than 1 to 2 % of such impurities
before the quantification would be seriously affected. (Warning—Storage of samples in improper containers (for example, plastics
other than TFE-fluorocarbon) may result in contamination.)
NOTE 2—Caution: Storage of samples in improper containers (for example, plastics other than TFE-fluorocarbon) may result in contamination.
NOTE 2—Spectroquality solvents may not have low enough fluorescence background to be used as solvent blanks. Solvent lots vary in the content of
fluorescent impurities that may increase with storage time even for unopened bottles.
NOTE 3—This test method is normally used without a matrix spike due 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 quantification process. Compounds that could be used are dyes that fluoresce at
longer wavelengths than the emission of the PAH mixture.
6.2 If the PAH mixture to be analyzed is a complex mixture such as an oil or creosote, it is assumed that a well-characterized
sample of the same or similar material is available as a calibration standard so the fluorescent fraction of the mixture can be ratioed
against the total mixture. Otherwise, since the samples and standards are weighed, the nonfluorescent portion of the mixture would
bias the quantification although the characterization portion of the test method for PAHs given in Annex A1 would be unaffected.
7. Apparatus
7.1 Fluorescence Spectrometer—An instrument recording in the spectral range of 250 nm to at least 600 nm for both excitation
and emission responses and capable of scanning both monochromators simultaneously at a constant speed with a constant
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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D5412 − 93 (2017)
wavelength offset between them for synchronous scanning. The instrument should meet the specifications in Table 1. (Also known
as spectrofluorometer or fluorescence spectrophotometer.) Consult manufacturer’s instrument manuals for specific operating
instructions.
NOTE 4—Although the characterization section of this test method (given in Annex A1) is similar to Test Method D3650 in many respects, there are
differences in the purpose and intents of the two test methods. 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 an appropriate calibration standard for quantification. Other differences between the test methods
are instrumentation requirements and the use of synchronous spectra as well as emission spectra for this test method.
7.2 Excitation Source—A high-pressure xenon lamp (a 150-W continuous xenon lamp or a 10-W pulsed xenon lamp has been
proven acceptable). Other continuum sources (either continuous or pulsed) having sufficient intensity throughout the ultraviolet and
visible regions may also be used.
7.3 Fluorescence Cells—Standard cells made from fluorescence-free fused silica with a path length of 10 mm and a height of
at least 45 mm. Stoppered cells may be preferred to prevent sample evaporation and contamination.
7.4 Data Recording System—Preferably the instrument should be interfaced to a suitable computer system compatible with the
instrument and with suitable software for spectral data manipulation. Use of a strip chart or X-Y recorder with a response time of
less than 1 s for full-scale deflection is acceptable.
7.5 Micropipet, glass, 10 to 50-μL capacity.
7.6 Weighing Pans, 5 to 7-mm diameter, 18-mm thick, made of aluminum or equivalent. Check pans for contamination.
8. Reagents and Materials
8.1 Purity of Reagents—Use spectroquality grade reagents in all instances unless otherwise stated. Since the goal is to have as
low a fluorescence blank as possible, and since different brands and lots of spectroquality solvent may vary, check reagents
frequently.
8.2 Purity of Water—References to water mean Type IV water conforming to Specification D1193. Since fluorescent organic
impurities in the water may introduce an interference, check the purity of the water by analyzing a water blank using the same
instrumental conditions as for the solvent blank.
8.3 Acetone, spectroquality, (CH COCH ).
3 3
8.4 Cyclohexane, spectroquality or HPLC grade. The fluorescence solvent blank must be as low as possible and less than 5 %
of the intensity of the maximum emission peak for the lowest concentration of PAHs analyzed. Dispense cyclohexane during the
procedure from either a TFE-fluorocarbon or glass wash bottle, but, for prolonged storage, store cyclohexane only in glass.
8.5 Nitric Acid (1 + 1)—Carefully add one volume of concentrated HNO (sp gr 1.42) to one volume of water.
8.6 TFE-Fluorocarbon Strips, 25 mm by 75 mm, 0.25-mm thickness. Use TFE strips when sampling neat PAH films on water
as described in Practices D4489.
9. Sampling and Sample Preparation
9.1 Collect a representative sample (see Practices D4489 for water samples).
9.2 Preserve samples in containers as specified in Practice D3325. Do not cool samples below 5°C to avoid dewaxing of oil or
creosote samples.
9.3 Neat PAH samples (including surface films or layers on water) require only dilution in spectroquality cyclohexane. Prepare
initial concentration for the unknown at 100 μg/mL for a check of the fluorescence signal. Further dilutions down to 1 μ/mL may
TABLE 1 Specifications for Fluorescence Spectrometers
Wavelength Reproducibility
Excitation monochromator ±2 nm or better
Emission monochromator ±2 nm or better
Gratings (Typical Values)
Excitation monochromator minimum of 600 lines/mm
blazed at 300 nm
Emission monochromator minimum of 600 lines/mm
blazed at 300 nm or 500 nm
Photomultiplier Tube
S-20 or S-5 response or equivalent
Spectral Resolutions
Excitation monochromator spectral bandpass of 2.5 nm or less
Emission monochromator spectral bandpass 2.5 nm or less
Maximum bandpasses for both monochromators at least 10 nm
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D5412 − 93 (2017)
be needed to bring the fluorescence signal into the linear range and to avoid self-absorption effects in the solution. Most PAH
mixtures and oils have been found to be soluble in cyclohexane at the concentrations listed. Alternative solvents can be substituted
with appropriate tests.
9.4 If any unknown PAH mixture is dissolved in water, test the mixture with appropriate dilutions or preconcentrations as
required. The assumption is that no naturally-occurring fluorescent materials such as humic or fulvic acids are present at levels
interfering with the determi
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