1、Designation: D5412 93 (Reapproved 2011)Standard 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 adopti
2、on 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.1. Scope1.1 This test method covers a means for quantifying orcharacterizing total pol
3、ycyclic 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 fluorescence spectra.1.2 This test method is applicable to PAHs resulting
4、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 similar fluo-rescence spectra should be chosen. The degree of spec
5、tralsimilarity 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 thisstandard.1.4 This standard does not purport to address all of thesa
6、fety 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. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to
7、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 of Samples for Identifica-tion of Waterborne OilsD3415 Practice
8、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 Polynuclear Aromatic Hydrocar-bons in Water3E131 Terminology Relating to Molecular SpectroscopyE169 Practic
9、es for General Techniques of Ultraviolet-Visible Quantitative AnalysisE275 Practice for Describing and Measuring Performanceof Ultraviolet and Visible SpectrophotometersE388 Test Method for Wavelength Accuracy and SpectralBandwidth of Fluorescence SpectrometersE578 Test Method for Linearity of Fluor
10、escence 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, Terminology E131, andPractice D3415.4. Summary of Test Method4.1 This test method co
11、nsists 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 considerations of the sample). The test method re-quires an initial qualitative ch
12、aracterization 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 definition of spectral similarity). Intensities of peakmaxima of suitable emissi
13、on spectra are then used to developcalibration curves for quantification.NOTE 1Although some sections of the characterization part of thistest method are similar to Test Method D3650, there are also significantdifferences (See Annex A1). Since the purpose and intent of the two testmethods are differ
14、ent, one should not be substituted for the other.1This 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
15、lyapproved in 1993. Last previous edition approved in 2005 as D5412 93 (2005).DOI: 10.1520/D5412-93R11.2For 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 standards
16、 Document Summary page onthe ASTM website.3Withdrawn. The last approved version of this historical standard is referencedon www.astm.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5. Significance and Use5.1 This test method is u
17、seful 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 by itsfluorescence emission and synchronous scanning spectra. Then
18、a 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), GC-mass spectrometry (GC-MS), or high performance liquidchroma
19、tography (HPLC). Some suggested independent ana-lytical methods are included in References (17)4and TestMethod D4657. Other analytical methods can be substituted byan experienced analyst depending on the intended data qualityobjectives. Peak maxima intensities of appropriate fluores-cence emission s
20、pectra 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 References (818).5.3 For the purpose of the present test method poly
21、nucleararomatic 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 to PAHs ofsimilar molecular weight ranges. If PAHs in the more clas
22、sicdefinition, 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 substitutedPAHs and heterocycles are not major components of themix
23、tures 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 areexcited and which fluoresce in the same spectral regions withrelativel
24、y 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 containers (forexample, plastics other than TFE-fluorocarbon) may result
25、 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 unopened bottles.NOTE 4This test method is normally used without a mat
26、rix 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 thatfluoresce at longer wavelengths than the emission of the PAH mixture.
27、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 of themixture can be ratioed against the total mixture. Otherwise,s
28、ince 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. Apparatus7.1 Fluorescence SpectrometerAn instrument recordingin the spectral r
29、ange 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 instrument should meet the specifications inTable 1. (Also known as spe
30、ctrofluorometer 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 Method D3650 in many respects,there are differences in the purpose an
31、d 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 quantification. Other differencesbetween the test methods are instrumentation
32、 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). Other continuum sources (eithercontinuous or pulsed) having sufficien
33、t 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 preferred toprevent sample evaporation and contamination.7.4 Data
34、Recording SystemPreferably the instrumentshould be interfaced to a suitable computer system compatiblewith the instrument and with suitable software for spectral 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 Mic
35、ropipet, 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.4The boldface numbers in parentheses refer to the list of references at the end ofthis standard.TABLE 1 Specifications for Fluorescence SpectrometersWav
36、elength ReproducibilityExcitation monochromator 62nmorbetterEmission monochromator 62nmorbetterGratings (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
37、equivalentSpectral ResolutionsExcitation monochromator spectral bandpass of 2.5 nm or lessEmission monochromator spectral bandpass 2.5 nm or lessMaximum bandpasses for both monochromators at least 10 nmD5412 93 (2011)28. Reagents and Materials8.1 Purity of ReagentsUse spectroquality grade reagentsin
38、 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 Water References to water mean Type IVwater conforming to Specification D1193. Since
39、 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, (CH3COCH3).8.4 Cyclohexane, spectroquality or HPLC grade. The fluo-rescence s
40、olvent 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 glasswash bottle, but, for prolonged storage, store cyclohexane onlyin glass
41、.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 in Practice D4489.9. Sampling and Sample Preparation9.1 Collect a r
42、epresentative sample (see Practice 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 surface films or layers onwater) require only dilution in spectroquali
43、ty 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-absorption effects in the solution.Most PAH mixtures and oils have been
44、 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 preconcentrations asrequired. The assumption is that no naturally-occurri
45、ng 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) levels). This usually becomesa problem only at PAH levels in the low g/L r
46、ange. 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 separatory funnel,add 5.0 mL of cyclohexane and shake for 2 min. Vent the
47、separatory 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 extraction.Combine the two extracts and dilute to 10.0 mL with cyclo-hexane.
48、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 Pasteur pipet.It is important to collect most of the top layer to ma
49、ximizepercent 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 alwaysthe case, the analyst shall demonstrate method performancewhen changing
