1、Designation: D 5659 95 (Reapproved 2006)Standard Test Method forChlorophenoxy Acid Herbicides in Waste Using HPLC1This standard is issued under the fixed designation D 5659; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、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 the analysis of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid
3、(2,4,5-T), and 2,4,5-trichlorophenoxypropionic acid (silvex) in liquids and solids,using high performance liquid chromatography with an ultra-violet detector (HPLC/UV). This test method is applicable fora concentration range from approximately 50 to 1000 ppm.This range takes into consideration the s
4、ample preparation anddilutions outlined in Section 10. Lower detection levels can beobtained by using larger sample sizes, smaller total finalvolumes, or with the use of in-line or solid phase extraction,concentration, and/or cleanup.1.2 The chlorophenoxy herbicides may be present as avariety of sal
5、ts or esters, which are converted to, analyzed, andreported as their respective acids.1.3 This test method is applicable to liquid and solid wasteand waste extract matrices including aqueous, oil, spentsolvent, soil, ash, leachates, etc.1.4 This test method may be applicable to other phenoxyacid her
6、bicides.1.5 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. See Se
7、ction 7 and10.3.1 for specific precautionary statements.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 3478 Test Method for Chlorinated Phenoxy Acid Herbi-cides in Water32.2 EPA Documents:Method 8150 Chlorinated Herbicides, Test Methods forEvaluating Solid Waste P
8、hysical/Chemical Methods SW-846 Third Edition4Method 8000A Gas Chromatography, Test Methods forEvaluating Solid Waste Physical/Chemical Methods, SW-846, 3rd Edition, Final Update 14Method 555 Determination of Chlorinated Acids in Waterby High Performance Liquid Chromatography with aPhotodiode Array
9、Ultraviolet Detector EPA/600/R-92/129, Methods for the Determination of Organic Com-pounds in Drinking Water, Supplement No. 243. Summary of Test Method3.1 The chlorophenoxy acids and esters are hydrolyzed totheir respective salts by heating and stirring the sample withaqueous alkali. The salts are
10、then converted to their respectiveacids by the addition of HCl. The aqueous solutions of the freeacids are then analyzed using High Performance Liquid Chro-matography (HPLC) using ultraviolet detection.4. Significance of Use4.1 Phenoxy acid herbicides are used extensively for weedcontrol. Esters and
11、 salts of 2,4-D, 2,4,5-T, and Silvex havebeen used for agricultural crop and lawn care.5. Interferences5.1 Organic liquids that have high percent levels of chlori-nated organics and are denser than water may interfere with theextraction of Silvex. It is necessary to mix these samples withhexadecane,
12、 typically in a 1:1 ratio, before hydrolysis.5.2 Phenols, especially chlorophenols interfere with theprocedure, by coeluting with the analytes of interest.5.3 Interferences may be encountered from other organiccompounds that absorb UV at the specified wavelengths. Also,closely eluting compounds may
13、complicate identificationbased solely on retention time. When these types of interfer-ences are encountered, the analyst must rely on other sourcesof information for positive identification, such as the follow-ing:5.3.1 Secondary confirmation wavelengths such as 227 nmor 235 nm.1This test method is
14、under the jurisdiction of ASTM Committee D34 on WasteManagement and is the direct responsibility of Subcommittee D34.01.06 onAnalytical Methods.Current edition approved Feb. 1, 2006. Published March 2006. Originallyapproved in 1995. Last previous edition approved in 2001 as D 5659 95(2001).2For refe
15、renced 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 Document Summary page onthe ASTM website.3Withdrawn.4Available from the Superintendent of Documents, U.S. Gover
16、nment PrintingOffice, Washington, DC 20402.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.3.2 Use of a confirmation column.5.3.3 Use of a confirmatory chromatography program suchas changing the mobile phase composition or gradient
17、.6. Apparatus6.1 Analytical High-Pressure Liquid Chromatograph, ca-pable of achieving pressures of 4000 psi and flow rates of 3mL/min.6.2 Variable Wavelength Ultraviolet Detector, capable ofmonitoring at 207, 227, and 235 nm, either simultaneously orindividually.6.3 Chromatographic Column, C18 radia
18、l compression 8 by100 mm, 4-m particle size. Equivalent stainless steel or radialcompression columns may be used.6.4 Guard Column, C18, 4-m particle size.6.5 Injector, manual injection valve, instrument auto-sampler, equipped with a 500-L sample loop, or equivalent.6.6 Data Systems, data systems cap
19、able of controlling theHPLC system and for acquiring data may be used.6.7 Glass Vials, 16-mL capacity with TFE-fluorocarbon-lined screw caps.6.8 Microsyringes, 10, 100, and 500-L capacity.6.9 Balance, analytical, capable of accurately weighing tothe nearest 0.0001 g.6.10 Pipets, Pasteur, disposable
20、glass.6.11 Pipets, disposable glass, 1-mL and 10-mL, calibrated.6.12 pH paper, wide range from 1 to 11.6.13 Hot Plate, with multiple stirring positions.6.14 Water Filtration Apparatus, used for the purification ofwater for HPLC use in 7.4.6.15 Water Filtration Filters, 0.22-m used in 7.4.6.16 Flasks
21、, 100-mL volumetric glass.6.17 Centrifuge.6.18 Stir Bars.6.19 Funnels, glass.6.20 Filter Papers, 15-cm hardened/ashless, fast.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the s
22、pecifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.5Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.
23、7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type II of Specification D 1193.7.3 Acetone, pesticide quality or equivalent, used for pre-paring standards. Be advised that if methanol is used forpreparing the acid standards, o
24、ver time the acid form of theherbicides will convert to their methyl ester form.7.4 Filtered Water with 0.5 % Phosphoric Acid, (FWPA).Add 5 mL high-purity phosphoric acid to 995-mL reagentwater in a volumetric flask. Filter through a 0.22-m filter.7.5 Herbicide Reference StandardsThe following refer
25、-ence calibration compounds are required. Reference solutionscan be prepared from the pure standard materials or purchasedas certified solutions.7.5.1 2,4-D, 2,4,5-T, Silvex, 2,4-D methyl ester.7.5.2 2,4,5-T methyl ester, Silvex methyl ester.7.6 Hexadecane,99%.7.7 Hydrochloric Acid (density 1.195 g/
26、mL), concentratedhydrochloric acid (HCl).7.8 Methanol, pesticide quality or equivalent.7.9 Acetonitrile, pesticide quality or equivalent.Acetonitrilemay be used as the mobile phase instead of methanol.7.10 Phosphoric Acid, H3PO4, 85 % o-phosphoric acid,HPLC Grade.7.11 Potassium Hydroxide 37 % Soluti
27、oncarefully add 37g of ACS grade potassium hydroxide (KOH) pellets to 50 mLof reagent water in a 100-mL volumetric flask. Mix by swirlingthe flask. Bring to volume with additional reagent water.NOTE 1Caution: Observe caution when adding KOH to water. Thiswill cause an exothermic reaction. Cooling th
28、e flask while mixing issuggested.8. Standard Preparation8.1 Prepare stock standard solutions as follows. For herbi-cide acids and methyl esters, accurately weigh 0.100 g into a10-mL glass volumetric flask. Bring to volume in pesticide-grade acetone. Larger volumes can be used at the convenienceof th
29、e analyst. If compound purity is 96 % or greater, theweight can be used without correction to calculate the concen-tration of the stock standard.8.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials. Store at 4C and protectfrom light. Stock standard solutions should
30、be checked fre-quently for signs of degradation or evaporation especially priorto being used to prepare calibration standards. Marking themeniscus level on the standard vial is recommended whenmonitoring for evaporation.8.3 Stock standard solutions must be replaced after one yearor sooner if compari
31、son with check standard indicates aproblem.8.4 Prepare calibration standard solutions from stock stan-dard solutions at a minimum of 5 concentration levels for eachparameter. Transfer the appropriate volume of stock solution toa volumetric flask and dilute with FWPA. The concentrationlevels should c
32、orrespond to the expected range of concentra-tions found in real samples or should define the working rangeof the HPLC/UV. A working range from 0.5 to 5 ppm has beenfound to be useful. Calibration solutions must be replaced aftersix months, or sooner if comparison with check standardsindicates a pro
33、blem. A 20 % deviation in response between thedaily check and the initial calibration would indicate a potentialproblem.5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemica
34、l Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K. and the United States Pharmacopeia andNational Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.D 5659 95 (2006)29. Sample Collection, Preservation, and Handling9.1 Sample collection should be
35、in accordance with appro-priate sampling protocols.9.2 Sample extracts should be stored in glass containers.Long-term storage should be at 4C. For an aqueous matrix,100 mL of sample are needed and5gofsample are needed forsolids or organic liquids.9.3 Unused sample material, laboratory dilutions, and
36、 wastefrom the samples may be regulated. Consult your specialist andthe regulations, or both, for guidance in the proper handlingand disposal of laboratory waste.10. Procedure10.1 Sample Extraction and Hydrolysis (for Aqueous Ma-trices):10.1.1 Add 50 mLor 50 g of sample to a 100-mLvolumetricor Erlen
37、meyer flask.10.1.2 Add 10 mL of 37 % KOH solution to the flask.Check the pH. If the pH is not 10 or greater, add additionalKOH solution. Record the KOH solution used, in millilitres,and the final pH.10.1.3 Add a stir bar to the flask. Place the flask on a stirringhot plate and heat and mix at 70 to
38、90C for 2 h.10.1.4 Continue to 10.3.10.2 Sample Extraction and Hydrolysis (All Other Matri-ces):10.2.1 Weigh5gofsample into a 100-mL volumetric orErlenmeyer flask.NOTE 2Be cognizant of sample matrix type, especially for solidmaterials such as soils and ash. It may be necessary to crush or powder the
39、sample to ensure complete extraction.10.2.1.1 If the sample is a dense organic liquid for example,high PCB oils, chlorinated solvents, and so forth, weigh 2.5 gof sample into a 100-mL volumetric flask and add 2.5 ghexadecane. Mix by swirling the flask.NOTE 3It was determined through recovery studies
40、 that the chlo-rophenoxy herbicides remained in highly chlorinated organic liquids onthe bottom of the volumetric flask after hydrolysis. Mixing the samplewith hexadecane brought the sample to the top of the volumetric flask andthe herbicides were extracted into and remained in the aqueous phase aft
41、erhydrolysis.10.2.1.2 Add 50 mL reagent water to the flask. Mix byswirling the flask.10.2.1.3 Add 10 mL of 37 % KOH solution to the flask.Check the pH. If the pH is not 10 or greater add additionalKOH solution. Record millilitres of KOH solution used and thefinal pH.10.2.1.4 Add a stir bar to the fl
42、ask. Place the flask on astirring hot plate and heat and mix at 70 to 90C for 2 h.10.2.1.5 For oil samples, after stirring is complete, removestir bar and bring volumetric flask to volume with reagentwater. The water meniscus should be at the mark. Let standuntil the layers separate.10.2.1.6 Draw of
43、f and discard any oil layer that may bepresent on top of the water.10.2.1.7 For solid samples, filter extract through filter paper(see 6.20) into a separate 100-mL volumetric flask. Rinse thefilter paper being careful not to exceed volume. Bring tovolume with reagent water.10.2.1.8 Continue to 10.4.
44、NOTE 4This is a good stopping point in the method if needed. Storesample extracts overnight in the refrigerator.10.3 Preparation for Analysis (Aqueous Matrices):10.3.1 Add 5 mL of concentrated HCl to the volumetricflask containing the solution prepared in 10.1. Mix well.Caution: always wear personal
45、 protective gear when add-ing concentrated acid to water because of the potential forheat generation.10.3.2 Check the pH using wide-range pH paper. The pHmust be less than two before HPLC analysis.10.3.3 If the pH is not less than two, add additional HCl tothe flask. Check and record the final pH.10
46、.3.4 Bring the volumetric flask to volume with reagentwater.10.4 Preparation for Analysis (All Other Matrices):10.4.1 Add 8 mL of FWPA prepared in 7.4, 1 mL concen-trated HCl, and 1 mL of the sample extract prepared in 10.2 toa 16-mL glass vial. Shake.10.4.2 Check the pH using wide-range pH paper. T
47、he pHmust be less than two before HPLC analysis.10.4.3 If the pH is not less than two, re-prepare the sampleas in 10.4.1 using 7 mL FWPA and 2 mL concentrated HCl.Check and record the final pH.10.4.4 If visible suspended solids are present in the sample,centrifuge the sample before injecting into th
48、e HPLC. It is veryimportant to limit the amount of suspended solids injected ontothe column and into the HPLC system because of the nature ofHPLC and the potential for problems.10.5 HPLC Calibration:10.5.1 Using the calibration standards prepared in 8.4,generate a calibration curve that defines the
49、working range orrange of interest of the HPLC system. A correlation coefficientof 0.995 with a percent RSD of less than 20 is recommendedfor the acceptance of a valid calibration curve.10.5.2 Check the initial calibration curve daily by injectinga mid-level standard. If the response varies from the initialcalibration by more than 20 %, recalibration is recommended.If the daily standard and initial calibration is acceptable, usethe response of the daily standard for Hstdvalue in Section 11.10.6 HPLC AnalysisInject the solution prepared in 10.3or 10.4 into the HPLC.
