1、Designation: D 5412 93 (Reapproved 2005)Standard Test Method forQuantification of Complex Polycyclic Aromatic HydrocarbonMixtures or Petroleum Oils in Water1This standard is issued under the fixed designation D 5412; the number immediately following the designation indicates the year oforiginal adop
2、tion or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a means for quantifying orcharacterizing total
3、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 fluorescence spectra.1.2 This test method is applicable to PAHs resulti
4、ng 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 s
5、pectralsimilarity needed will depend on the desired level of quantifi-cation and on the required data quality objectives.1.3 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-p
6、riate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 2777 Practice for Determination of Precision and Bias ofApplicable Test Met
7、hods of Committee D19 on WaterD 3325 Practice for Preservation of Waterborne OilSamplesD 3326 Practice for Preparation of Samples for Identifica-tion of Waterborne OilsD 3415 Practice for Identification of Waterborne OilsD 3650 Test Method for Comparison of Waterborne Petro-leum Oils By Fluorescence
8、 AnalysisD 4489 Practices for Sampling of Waterborne OilsD 4657 Test Method for Polynuclear Aromatic Hydrocar-bons in Water3E 131 Terminology Relating to Molecular SpectroscopyE 169 Practices for General Techniques of Ultraviolet-Visible Quantitative AnalysisE 275 Practice for Describing and Measuri
9、ng Performanceof Ultraviolet, Visible, and Near-Infrared Spectrophotom-etersE 388 Test Method for Wavelength Accuracy of SpectralBandwidth of Fluorescence SpectrometersE 578 Test Method for Linearity of Fluorescence MeasuringSystemsE 579 Test Method for Limit of Detection of Fluorescenceof Quinine S
10、ulfate in Solution3. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminology D 1129, Terminology E 131, andPractice D 3415.4. Summary of Test Method4.1 This test method consists of fluorescence analysis ofdilute solutions of PAHs or petroleum oils in appropri
11、atesolvents (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 characterization step involving bothfluorescence emission and synchronous spectroscopy in
12、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 emission spectra are then used to developcalibration curves for quantification.NOTE 1Although
13、some sections of the characterization part of thistest method are similar to Test Method D 3650, there are also significant1This 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
14、 Water.Current edition approved Dec. 1, 2005. Published January 2006. Originallyapproved in 1993. Last previous edition approved in 2000 as D5412 93 (2000).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of AST
15、MStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.differences (See Annex A1). Since the purpose and intent of the two testmethods
16、 are different, one should not be substituted 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 u
17、nknown PAH mixture is first characterized by 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 otherindepen
18、dent methods such as gas chromatography (GC), GC-mass spectrometry (GC-MS), or high performance liquidchromatography (HPLC). Some suggested independent ana-lytical methods are included in References (17)4and TestMethod D 4657. Other analytical methods can be substitutedby an experienced analyst depe
19、nding on the intended dataquality objectives. Peak maxima intensities of appropriatefluorescence 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
20、PAHs and petroleum oils canbe found in References (818).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, andheterocycle
21、s giving similar fluorescence responses 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 mo
22、st commonly expected PAH mixtures, such 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
23、 other fluorescent chemicals that areexcited 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 2Cauti
24、on: Storage of samples in improper containers (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 ma
25、y increase with storage time evenfor unopened 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. Comp
26、ounds that could be used are dyes thatfluoresce 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 calibrati
27、on standard so the fluorescent fraction 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 Ann
28、ex A1 would be unaffected.7. Apparatus7.1 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 betwee
29、n them for synchronousscanning. The instrument 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(
30、given in Annex A1) is similar to Test Method D 3650 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 approp
31、riate calibration standard for quantification. 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
32、xenon lamphas been proven acceptable). Other 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
33、of at least 45 mm. Stoppered cells may be preferred toprevent sample evaporation and contamination.7.4 Data Recording SystemPreferably the instrumentshould be interfaced to a suitable computer system compatiblewith the instrument and with suitable software for spectral datamanipulation. Use of a str
34、ip chart or X-Y recorder with aresponse time of less than 1 s for full-scale deflection isacceptable.4The boldface numbers in parentheses refer to the list of references at the end ofthis standard.TABLE 1 Specifications for Fluorescence SpectrometersWavelength ReproducibilityExcitation monochromator
35、 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 equivalentSpectral ResolutionsExcitation monoch
36、romator spectral bandpass of 2.5 nm or lessEmission monochromator spectral bandpass 2.5 nm or lessMaximum bandpasses for both monochromators at least 10 nmD 5412 93 (2005)27.5 Micropipet, glass, 10 to 50-L capacity.7.6 Weighing Pans, 5 to 7-mm diameter, 18-mm thick, madeof aluminum or equivalent. Ch
37、eck pans for contamination.8. Reagents 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
38、frequently.8.2 Purity of Water References to water mean Type IVwater conforming to Specification D 1193. 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 bl
39、ank.8.3 Acetone, spectroquality, (CH3COCH3).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
40、from either a TFE-fluorocarbon or glasswash 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 sa
41、mpling neat PAH films onwater as described in Practice D 4489.9. Sampling and Sample Preparation9.1 Collect a representative sample (see Practice D 4489 forwater samples).9.2 Preserve samples in containers as specified in PracticeD 3325. Do not cool samples below 5C to avoid dewaxing ofoil or creoso
42、te samples.9.3 Neat PAH samples (including surface 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 sign
43、al into thelinear range and to avoid self-absorption 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, testth
44、e mixture with appropriate dilutions or preconcentrations 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
45、with thetest method even at high (g/L) levels). This usually becomesa problem only at PAH levels in the low g/L range. Extractionmethods (or separation by column chromatography) are listedin Practice D 3326.9.4.1 An extraction method that proved satisfactory for thecollaborative test is as follows:9
46、.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 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 cyclohex-ane to t
47、he aqueous layer and perform a second extraction.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
48、 min and collect most of the top layer with a 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 th
49、etwo 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 the method. At the mg/L level or above, thePAH mixture might not be totally in solution. If the PAHmixture is emulsified in water, is sparingly soluble in water, orif the concentration of
