ASTM D5412-93(2000)

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An American National Standard
Designation: D 5412 – 93 (Reapproved 2000)
Standard Test Method for
Quantification of Complex Polycyclic Aromatic Hydrocarbon
Mixtures or Petroleum Oils in Water
This standard is issued under the fixed designation D 5412; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope E 131 Terminology Relating to Molecular Spectroscopy
E 169 Practices for General Techniques of Ultraviolet-
1.1 This test method covers a means for quantifying or
Visible Quantitative Analysis
characterizing total polycyclic aromatic hydrocarbons (PAHs)
E 275 Practice for Describing and Measuring Performance
by fluorescence spectroscopy (Fl) for waterborne samples. The
of Ultraviolet, Visible, and New Infrared Spectrophotom-
characterization step is for the purpose of finding an appropri-
eters
ate calibration standard with similiar emission and synchro-
E 388 Test Method for Spectral Bandwidth and Wavelength
nous fluorescence spectra.
Accuracy of Fluorescence Spectrometers
1.2 This test method is applicable to PAHs resulting from
E 578 TestMethodforLinearityofFluorescenceMeasuring
petroleum oils, fuel oils, creosotes, or industrial organic
System
mixtures.Samplescanbeweatheredorunweathered,buteither
E 579 Test Method for Limit of Detection of Fluorescence
the same material or appropriately characterized site-specific
of Quinine Sulfate
PAH or petroleum oil calibration standards with similar fluo-
rescence spectra should be chosen. The degree of spectral
3. Terminology
similarity needed will depend on the desired level of quantifi-
3.1 Definitions—For definitions of terms used in this test
cation and on the required data quality objectives.
method, refer toTerminology D 1129,Terminology E 131, and
1.3 This standard does not purport to address all of the
Practice D 3415.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety and health practices and determine the applica-
4.1 This test method consists of fluorescence analysis of
bility of regulatory limitations prior to use.
dilute solutions of PAHs or petroleum oils in appropriate
solvents (spectroquality solvents such as cyclohexane or other
2. Referenced Documents
appropriate solvents, for example, ethanol, depending on
2.1 ASTM Standards:
2 polarity considerations of the sample). The test method re-
D 1129 Terminology Relating to Water
2 quiresaninitialqualitativecharacterizationstepinvolvingboth
D 1193 Specification for Reagent Water
fluorescence emission and synchronous spectroscopy in order
D 2777 Practice for Determination of Precision and Bias of
2 to select appropriate calibration standards with similar fluores-
Applicable Methods of Committee D-19 on Water
cence spectra as compared to the samples (see Annex A1 for
D 3325 Practice for Preservation of Waterborne Oil
3 the definition of spectral similarity). Intensities of peak
Samples
maxima of suitable emission spectra are then used to develop
D 3326 Practices for Preparation of Samples for Identifica-
3 calibration curves for quantification.
tion of Waterborne Oils
D 3415 Practice for Identification of Waterborne Oils
NOTE 1—Although some sections of the characterization part of this
D 3650 Test Method for Comparison of Waterborne Petro- test method are similar to Test Method D 3650, there are also significant
differences (See Annex A1). Since the purpose and intent of the two test
leum Oils by Fluorescence Analysis
methods are different, one should not be substituted for the other.
D 4489 Practices for Sampling of Waterborne Oils
D 4657 Test Method for Polynuclear Aromatic Hydrocar-
5. Significance and Use
bons in Water
5.1 This test method is useful for characterization and rapid
quantification of PAH mixtures including petroleum oils, fuels,
This test method is under the jurisdiction of ASTM Committee D19 on Water
creosotes,andindustrialorganicmixtures,eitherwaterborneor
andisthedirectresponsibilityofSubcommitteeD19.06onMethodsforAnalysisfor
obtained from tanks.
Organic Substances in Water.
Current edition approved May 15, 1993. Published August 1993.
Annual Book of ASTM Standards, Vol 11.01.
3 4
Annual Book of ASTM Standards, Vol 11.02. Annual Book of ASTM Standards, Vol 14.01.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 5412 – 93 (2000)
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
asuitablesite-specificcalibrationstandardwithsimilarspectral 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
7. Apparatus
independent methods such as gas chromatography (GC), GC-
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 D 4657. Other analytical methods can be substituted both monochromators simultaneously at a constant speed with
by an experienced analyst depending on the intended data
a constant wavelength offset between them for synchronous
quality objectives. Peak maxima intensities of appropriate scanning. The instrument should meet the specifications in
fluorescence 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
NOTE 5—Although the characterization section of this test method
be found in References (8–18).
(given in Annex A1) is similar to Test Method D 3650 in many respects,
5.3 For the purpose of the present test method polynuclear
there are differences in the purpose and intents of the two test methods.
aromatic hydrocarbons are defined to include substituted poly-
The purpose of the characterization step of this test method is to find an
cyclic aromatic hydrocarbons with functional groups such as oil with similar fluorescence properties as the sample in order to serve as
an appropriate calibration standard for quantification. Other differences
carboxyl acid, hydroxy, carbonyl and amino groups, and
between the test methods are instrumentation requirements and the use of
heterocycles giving similar fluorescence responses to PAHs of
synchronous spectra as well as emission spectra for this test method.
similar molecular weight ranges. If PAHs in the more classic
7.2 Excitation Source—A high-pressure xenon lamp (a
definition, that is, unsubstituted PAHs, are desired, chemical
150-W continuous xenon lamp or a 10-W pulsed xenon lamp
reactions, extractions, or chromatographic procedures may be
has been proven acceptable). Other continuum sources (either
required to eliminate these other components. Fortunately, for
continuousorpulsed)havingsufficientintensitythroughoutthe
the most commonly expected PAH mixtures, such substituted
ultraviolet and visible regions may also be used.
PAHs and heterocycles are not major components of the
7.3 Fluorescence Cells—Standard cells made from
mixtures and do not cause serious errors.
fluorescence-free fused silica with a path length of 10 mm and
6. Interferences
a height of at least 45 mm. Stoppered cells may be preferred to
prevent sample evaporation and contamination.
6.1 The fluorescence spectra may be distorted or quantifi-
7.4 Data Recording System—Preferably the instrument
cation may be affected if the sample is contaminated with an
should be interfaced to a suitable computer system compatible
appreciable amount of other fluorescent chemicals that are
with the instrument and with suitable software for spectral data
excited and which fluoresce in the same spectral regions with
manipulation. Use of a strip chart or X-Y recorder with a
relatively high fluorescence yields. Usually the fluorescence
response time of less than 1 s for full-scale deflection is
spectra would be distorted at levels greater than 1 to 2 % of
acceptable.
such impurities before the quantification would be seriously
7.5 Micropipet, glass, 10 to 50-µL capacity.
affected.
7.6 WeighingPans,5to7-mmdiameter,18-mmthick,made
NOTE 2—Caution: Storage of samples in improper containers (for
of aluminum or equivalent. Check pans for contamination.
example, plastics other than TFE-fluorocarbon) may result in contamina-
tion.
8. Reagents and Materials
NOTE 3—Spectroquality solvents may not have low enough fluores-
cence background to be used as solvent blanks. Solvent lots vary in the 8.1 Purity of Reagents—Use spectroquality grade reagents
content of fluorescent impurities that may increase with storage time even
in all instances unless otherwise stated. Since the goal is to
for unopened bottles.
NOTE 4—This test method is normally used without a matrix spike due
TABLE 1 Specifications for Fluorescence Spectrometers
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
Wavelength Reproducibility
quantification process. Compounds that could be used are dyes that
Excitation monochromator 62nmorbetter
Emission monochromator 62nmorbetter
fluoresce at longer wavelengths than the emission of the PAH mixture.
Gratings (Typical Values)
6.2 If the PAH mixture to be analyzed is a complex mixture
Excitation monochromator minimum of 600 lines/mm
blazed at 300 nm
such as an oil or creosote, it is assumed that a well-
Emission monochromator minimum of 600 lines/mm
characterized sample of the same or similar material is avail-
blazed at 300 nm or 500 nm
able as a calibration standard so the fluorescent fraction of the
Photomultiplier Tube
S-20 or S-5 response or equivalent
mixture can be ratioed against the total mixture. Otherwise,
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 the list of references at the end of Maximum bandpasses for both monochromators at least 10 nm
this standard.
D 5412 – 93 (2000)
have as low a fluorescence blank as possible, and since the sample in the bottle and add 5.0 mLof cyclohexane, shake
different brands and lots of spectroquality solvent may vary, for 2 min and collect most of the top layer with a Pasteur pipet.
check reagents frequently. It is important to collect most of the top layer to maximize
8.2 Purity of Water— References to water mean Type IV percentrecovery(tilttheflasktoseetheseparationbetweenthe
water conforming to Specification D 1193. Since fluorescent two layers more easily). Add 5.0 mL of cyclohexane to the
organic impurities in the water may introduce an interference, aqueous layer and perform a second extraction. Combine the
check the purity of the water by analyzing a water blank using two cyclohexane extracts and dilute to 10.0 mL with cyclo-
the same instrumental conditions as for the solvent blank. hexane.
8.3 Acetone, spectroquality, (CH COCH ). 9.4.1.3 See 12.6 to check extraction recoveries. Other ex-
3 3
8.4 Cyclohexane, spectroquality or HPLC grade. The fluo- tractionmethodscanbeusedatthediscretionoftheanalyst,by
rescence solvent blank must be as low as possible and less than adding an appropriate solvent exchange step to cyclohexane
5 % of the intensity of the maximum emission peak for the and by checking for recoveries and interferences.As is always
lowest concentration of PAHs analyzed. Dispense cyclohexane the case, the analyst shall demonstrate method performance
during the procedure from either a TFE-fluorocarbon or glass when changing the method. At the mg/L level or above, the
wash bottle, but, for prolonged storage, store cyclohexane only PAH mixture might not be totally in solution. If the PAH
in glass. mixture is emulsified in water, is sparingly soluble in water, or
8.5 Nitric Acid (1 + 1)—Carefully add one volume of con- if the concentration of the unknown must be known more
centrated HNO (sp gr 1.42) to one volume of water. accurately, it may be necessary to evaporate the solution to
8.6 TFE-Fluorocarbon Strips, 25 mm by 75 mm, 0.25-mm dryness or to extract the PAH mixture into a suitable solvent,
thickness. Use TFE strips when sampling neat PAH films on followed by evaporation, weighing, and redissolving in cyclo-
water as described in Practice D 4489. hexane.
9.4.1.4 At the mg/L level or above, the PAH mixture in
9. Sampling and Sample Preparation
water might not be totally in solution.
9.1 Collect a representative sample (see Practice D 4489 for
9.5 Sample bottles must be made of glass, precleaned with
water samples).
dilute nitric acid (1 + 1) and sealed with plastic screw caps
9.2 Preserve samples in containers as specified in Practice
having TFE-fluorocarbon liners. Solutions must be prepared in
D 3325. Do not cool samples below 5°C to avoid dewaxing of
precleaned volumetric flasks. Because many aromatics are
oil or creosote samples.
subject to photodegradation, flasks must be low-actinic (am-
9.3 Neat PAH samples (including surface films or layers on
ber) or covered with aluminum foil. Volumetric flasks and
water) require only dilution in spectroquality cyclohexane.
fluorescence cells must be cleaned with dilute nitric acid
Prepare initial concentration for the unknown at 100 µg/mLfor
followed by rinsing with water and then air-drying them. To
a check of the fluorescence signal. Further dilutions down to 1
remove the water more quickly, use a triple rinse with
µ/mL may be needed to bring the fluorescence signal into the
spectroquality acetone. As a final step, triple rinse glassware
linear range and to avoid self-absorption effects in the solution.
and cells with the solvent used for analysis, usually cyclohex-
Most PAH mixtures and oils have been found to be soluble in
ane.
cyclohexane at the concentrations listed. Alternative solvents
10. Preparation o
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