1、Designation: D5412 93 (Reapproved 2011)1Standard Test Method forQuantification of Complex Polycyclic Aromatic HydrocarbonMixtures or Petroleum Oils in Water1This standard is issued under the fixed designation D5412; the number immediately following the designation indicates the year oforiginal adopt
2、ion or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1NOTEEditorial corrections were made throughout in March 2014.1. Scope1.1 This test m
3、ethod covers a means for quantifying orcharacterizing total polycyclic aromatic hydrocarbons (PAHs)by fluorescence spectroscopy (Fl) for waterborne samples. Thecharacterization step is for the purpose of finding an appropri-ate calibration standard with similiar emission and synchro-nous fluorescenc
4、e spectra.1.2 This test method is applicable to PAHs resulting frompetroleum oils, fuel oils, creosotes, or industrial organicmixtures. Samples can be weathered or unweathered, but eitherthe same material or appropriately characterized site-specificPAH or petroleum oil calibration standards with sim
5、ilar fluo-rescence spectra should be chosen. The degree of spectralsimilarity needed will depend on the desired level of quantifi-cation and on the required data quality objectives.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstan
6、dard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referen
7、ced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD2777 Practice for Determination of Precision and Bias ofApplicable Test Methods of Committee D19 on WaterD3325 Practice for Preservation of Waterborne Oil SamplesD3326 Practice for Preparation o
8、f Samples for Identificationof Waterborne OilsD3415 Practice for Identification of Waterborne OilsD3650 Test Method for Comparison of Waterborne Petro-leum Oils By Fluorescence AnalysisD4489 Practices for Sampling of Waterborne OilsD4657 Test Method for PolynuclearAromatic Hydrocarbonsin Water (With
9、drawn 2005)3E131 Terminology Relating to Molecular SpectroscopyE169 Practices for General Techniques of Ultraviolet-VisibleQuantitative AnalysisE275 Practice for Describing and Measuring Performance ofUltraviolet and Visible SpectrophotometersE388 Test Method for Wavelength Accuracy and SpectralBand
10、width of Fluorescence SpectrometersE578 Test Method for Linearity of Fluorescence MeasuringSystemsE579 Test Method for Limit of Detection of Fluorescence ofQuinine Sulfate in Solution3. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminology D1129, Terminolog
11、y E131, andPractice D3415.4. Summary of Test Method4.1 This test method consists of fluorescence analysis ofdilute solutions of PAHs or petroleum oils in appropriatesolvents (spectroquality solvents such as cyclohexane or otherappropriate solvents, for example, ethanol, depending onpolarity consider
12、ations of the sample). The test method re-quires an initial qualitative characterization step involving bothfluorescence emission and synchronous spectroscopy in orderto select appropriate calibration standards with similar fluores-cence spectra as compared to the samples (see Annex A1 forthe defini
13、tion of spectral similarity). Intensities of peak1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.06 on Methods forAnalysis forOrganic Substances in Water.Current edition approved May 1, 2011. Published June 2011. Original
14、lyapproved in 1993. Last previous edition approved in 2005 as D5412 93 (2005).DOI: 10.1520/D5412-93R11E01.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standa
15、rds Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1maxima of suitable emission spectra are then used to deve
16、lopcalibration curves for quantification.NOTE 1Although some sections of the characterization part of this testmethod are similar to Test Method D3650, there are also significantdifferences (see Annex A1). Since the purpose and intent of the two testmethods are different, one should not be substitut
17、ed for the other.5. Significance and Use5.1 This test method is useful for characterization and rapidquantification of PAH mixtures including petroleum oils, fuels,creosotes, and industrial organic mixtures, either waterborne orobtained from tanks.5.2 The unknown PAH mixture is first characterized b
18、y itsfluorescence emission and synchronous scanning spectra. Thena suitable site-specific calibration standard with similar spectralcharacteristics is selected as described in Annex A1. Thiscalibration standard may also be well-characterized by otherindependent methods such as gas chromatography (GC
19、), GC-mass spectrometry (GC-MS), or high performance liquidchromatography (HPLC). Some suggested independent ana-lytical methods are included in References (1-7)4and TestMethod D4657. Other analytical methods can be substituted byan experienced analyst depending on the intended data qualityobjective
20、s. Peak maxima intensities of appropriate fluores-cence emission spectra are then used to set up suitablecalibration curves as a function of concentration. Furtherdiscussion of fluorescence techniques as applied to the char-acterization and quantification of PAHs and petroleum oils canbe found in Re
21、ferences (8-18).5.3 For the purpose of the present test method polynucleararomatic hydrocarbons are defined to include substituted poly-cyclic aromatic hydrocarbons with functional groups such ascarboxyl acid, hydroxy, carbonyl and amino groups, andheterocycles giving similar fluorescence responses
22、to PAHs ofsimilar molecular weight ranges. If PAHs in the more classicdefinition, that is, unsubstituted PAHs, are desired, chemicalreactions, extractions, or chromatographic procedures may berequired to eliminate these other components. Fortunately, forthe most commonly expected PAH mixtures, such
23、substitutedPAHs and heterocycles are not major components of themixtures and do not cause serious errors.6. Interferences6.1 The fluorescence spectra may be distorted or quantifi-cation may be affected if the sample is contaminated with anappreciable amount of other fluorescent chemicals that areexc
24、ited and which fluoresce in the same spectral regions withrelatively high fluorescence yields. Usually the fluorescencespectra would be distorted at levels greater than 1 to 2 % ofsuch impurities before the quantification would be seriouslyaffected.NOTE 2Caution: Storage of samples in improper conta
25、iners (forexample, plastics other than TFE-fluorocarbon) may result in contamina-tion.NOTE 3Spectroquality solvents may not have low enough fluores-cence background to be used as solvent blanks. Solvent lots vary in thecontent of fluorescent impurities that may increase with storage time evenfor uno
26、pened bottles.NOTE 4This test method is normally used without a matrix spike dueto 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 thequantification process. Compounds that could be used are dyes thatfl
27、uoresce at longer wavelengths than the emission of the PAH mixture.6.2 If the PAH mixture to be analyzed is a complex mixturesuch as an oil or creosote, it is assumed that a well-characterized sample of the same or similar material is avail-able as a calibration standard so the fluorescent fraction
28、of themixture can be ratioed against the total mixture. Otherwise,since the samples and standards are weighed, the nonfluores-cent portion of the mixture would bias the quantificationalthough the characterization portion of the test method forPAHs given in Annex A1 would be unaffected.7. Apparatus7.
29、1 Fluorescence SpectrometerAn instrument recordingin the spectral range of 250 nm to at least 600 nm for bothexcitation and emission responses and capable of scanningboth monochromators simultaneously at a constant speed witha constant wavelength offset between them for synchronousscanning. The inst
30、rument should meet the specifications inTable 1. (Also known as spectrofluorometer or fluorescencespectrophotometer.) Consult manufacturers instrument manu-als for specific operating instructions.NOTE 5Although the characterization section of this test method(given in Annex A1) is similar to Test Me
31、thod 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 anoil with similar fluorescence properties as the sample in order to serve asan appropriate calibration standard for quantifica
32、tion. Other differencesbetween the test methods are instrumentation requirements and the use ofsynchronous spectra as well as emission spectra for this test method.7.2 Excitation SourceA high-pressure xenon lamp (a150-W continuous xenon lamp or a 10-W pulsed xenon lamphas been proven acceptable). Ot
33、her continuum sources (eithercontinuous or pulsed) having sufficient intensity throughout theultraviolet and visible regions may also be used.7.3 Fluorescence CellsStandard cells made fromfluorescence-free fused silica with a path length of 10 mm anda height of at least 45 mm. Stoppered cells may be
34、 preferred toprevent sample evaporation and contamination.4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.TABLE 1 Specifications for Fluorescence SpectrometersWavelength ReproducibilityExcitation monochromator 2 nm or betterEmission monochromator 2 nm or
35、 betterGratings (Typical Values)Excitation monochromator minimum of 600 lines/mmblazed at 300 nmEmission monochromator minimum of 600 lines/mmblazed at 300 nm or 500 nmPhotomultiplier TubeS-20 or S-5 response or equivalentSpectral ResolutionsExcitation monochromator spectral bandpass of 2.5 nm or le
36、ssEmission monochromator spectral bandpass 2.5 nm or lessMaximum bandpasses for both monochromators at least 10 nmD5412 93 (2011)127.4 Data Recording SystemPreferably the instrumentshould be interfaced to a suitable computer system compatiblewith the instrument and with suitable software for spectra
37、l datamanipulation. Use of a strip chart or X-Y recorder with aresponse time of less than 1 s for full-scale deflection isacceptable.7.5 Micropipet, glass, 10 to 50-L capacity.7.6 Weighing Pans, 5 to 7-mm diameter, 18-mm thick, madeof aluminum or equivalent. Check pans for contamination.8. Reagents
38、and Materials8.1 Purity of ReagentsUse spectroquality grade reagentsin all instances unless otherwise stated. Since the goal is tohave as low a fluorescence blank as possible, and sincedifferent brands and lots of spectroquality solvent may vary,check reagents frequently.8.2 Purity of WaterReference
39、s to water mean Type IVwater conforming to Specification D1193. Since fluorescentorganic impurities in the water may introduce an interference,check the purity of the water by analyzing a water blank usingthe same instrumental conditions as for the solvent blank.8.3 Acetone, spectroquality, (CH3COCH
40、3).8.4 Cyclohexane, spectroquality or HPLC grade. The fluo-rescence solvent blank must be as low as possible and less than5 % of the intensity of the maximum emission peak for thelowest concentration of PAHs analyzed. Dispense cyclohexaneduring the procedure from either a TFE-fluorocarbon or glasswa
41、sh bottle, but, for prolonged storage, store cyclohexane onlyin glass.8.5 Nitric Acid (1+1)Carefully add one volume of con-centrated HNO3(sp gr 1.42) to one volume of water8.6 TFE-Fluorocarbon Strips, 25 mm by 75 mm, 0.25-mmthickness. Use TFE strips when sampling neat PAH films onwater as described
42、in Practices D4489.9. Sampling and Sample Preparation9.1 Collect a representative sample (see Practices D4489 forwater samples).9.2 Preserve samples in containers as specified in PracticeD3325. Do not cool samples below 5C to avoid dewaxing ofoil or creosote samples.9.3 Neat PAH samples (including s
43、urface films or layers onwater) require only dilution in spectroquality cyclohexane.Prepare initial concentration for the unknown at 100 g/mL fora check of the fluorescence signal. Further dilutions down to 1/mL may be needed to bring the fluorescence signal into thelinear range and to avoid self-ab
44、sorption effects in the solution.Most PAH mixtures and oils have been found to be soluble incyclohexane at the concentrations listed. Alternative solventscan be substituted with appropriate tests.9.4 If any unknown PAH mixture is dissolved in water, testthe mixture with appropriate dilutions or prec
45、oncentrations asrequired. The assumption is that no naturally-occurring fluo-rescent materials such as humic or fulvic acids are present atlevels interfering with the determination (refer to Fig.A2.5 andFig. A2.6 to show that humic acid does not interfere with thetest method even at high (g/L) level
46、s). This usually becomesa problem only at PAH levels in the low g/L range. Extractionmethods (or separation by column chromatography) are listedin Practice D3326.9.4.1 An extraction method that proved satisfactory for thecollaborative test is as follows:9.4.1.1 Pour 50.0 mL of the sample into a sepa
47、ratory funnel,add 5.0 mL of cyclohexane and shake for 2 min. Vent theseparatory funnel occasionally. Withdraw the aqueous layer(keep this for a second extraction). Collect the cyclohexaneextract in a 10-mL volumetric flask. Add 5.0 mL of cyclo-hexane to the aqueous layer and perform a second extract
48、ion.Combine the two extracts and dilute to 10.0 mL with cyclo-hexane.9.4.1.2 For field use, it has proven satisfactory to use areagent bottle instead of a separatory funnel. Pour 50.0 mL ofthe sample in the bottle and add 5.0 mL of cyclohexane, shakefor 2 min and collect most of the top layer with a
49、 Pasteur pipet.It is important to collect most of the top layer to maximizepercent recovery (tilt the flask to see the separation between thetwo layers more easily). Add 5.0 mL of cyclohexane to theaqueous layer and perform a second extraction. Combine thetwo cyclohexane extracts and dilute to 10.0 mL with cyclo-hexane.9.4.1.3 See 12.6 to check extraction recoveries. Other ex-traction methods can be used at the discretion of the analyst, byadding an appropriate solvent exchange step to cyclohexaneand by checking for recoveries and interferences. As is alway
