&EPA Methods for the Determination
of Nonconventionai Pesticides in
Municipal
Volume If
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Acknowledgments
This methods compendium was prepared under the direction of Thomas E. Fielding, Ph.D., and
William A. Telliard of the Engineering and Analysis Division within EPA's Office of Water. This
document was prepared under EPA Contract No. 68-C9-0019 by the Environmental Services Division
of DynCorp Viar, Inc. The methods contained in this compendium were developed by the U.S. EPA
Environmental Monitoring Systems Laboratory in Cincinnati, Ohio.
Disclaimer
This methods compendium has been reviewed by the Engineering and Analysis Division,
U.S. Environmental Protection Agency, and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation for use.
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Introduction
The Environmental Protection Agency (EPA) is promulgating effluent limitations guidelines and stan-
dards for the Pesticide Chemicals Industry at 40 CFR Part 455 to control the discharge of pollutants,
including certain pesticide active ingredients, into the waters of the United States. This compendium of
test procedures (methods) supports this rulemaking. The purpose of publishing these methods in a
compendium is to create a single reference for analysts seeking to measure infrequently determined active
ingredients.
The proposed rule for the Pesticide Chemicals Manufacturing Subcategory (57 FR 12560) referenced
the original version of Volume I (EPA 821 RR-92-002) of this compendium of methods. That volume has
since been revised and is referenced in the rule as EPA-821-R-93-010-A. Volume II contains methods that
are not included in Volume I but that were incorporated by reference in the proposed rule.
Many of the methods in this two-volume compendium were listed in Appendix E of EPA's original
promulgation of rules for the Pesticides Category (50 FR 40708). These methods were withdrawn as a
part of the remand of the pesticides rules in 1986 (51 FR 44911). Some of the methods that appeared in
the original promulgation have been updated to include more analytes and/or include additional perfor-
mance data.
Many of the methods in the original promulgation were also published by EPA's Effluent Guidelines
Division in 1983 as publication EPA 440/1-83/079-C. This publication is now out of print. The publica-
tion included industry methods and EPA-developed methods.
'The test procedures in this two-volume compendium are methods developed by EPA's Environmental
Monitoring Systems Laboratory in Cincinnati, Ohio (EMSL-Ci), methods developed by EPA's Engineer-
ing and Analysis Division (BAD; formerly the Industrial Technology Division and the Effluent Guidelines
Division) within EPA's Office of Science and Technology (formerly the Office of Water Regulations and
Standards), and an industry method for organotin compounds.
Volume I contains the 600-series and 1600-series methods and one industry method. The 600-series
methods, written by EMSL-Ci, were developed in the late 1970's and early 1980's. Some have been
updated in the interim. The 1600-series methods, written by EAD, were developed to measure active
ingredients in support of the pesticides rulemaking, and have therefore been applied to the specific
wastewater for which they were intended. The industry method for total tin/triorganotin is Method EV-
024/EV-025. Volume II contains 13 of the 500-series methods and one 200-series method developed by
EMSL-Ci since the mid-1980's for the determination of pesticide active ingredients. A summary of the
analytes that may be detected using the methods in this compendium is shown in the cross-reference list.
Some analytes can be detected by more than one method.
Questions about the content of this document or Volume I should be directed to:
W. A. Telliard
U.S. EPA (WH-552)
Office of Science and Technology
Engineering and Analysis Division
401 M Street, SW
Washington, DC 20460
(202)260-5131
-------
Questions about the methods in Volume II—Method 200.9 and the 500-series methods—should be
directed to:
U.S. EPA
Office of Research and Development
Environmental Monitoring Systems Laboratory
Chemistry Research Division
26 W. Dr. Martin Luther King Jr. Drive
Cincinnati, OH 45268
Questions, as well as requests for Volume I, may also be directed to the following address:
Sample Control Center
Operated by DynCorp Viar, Inc.
P.O. Box 1407
Alexandria, VA 22313
(703) 557-5040
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Contents
Method
No. Title Page
200.9 Determination of Trace Elements by Stabilized Temperature
Graphite Furnace Atomic Absorption Spectrometry 1
505. Analysis of Organohalide Pesticides and Commercial
Polychlorinated Biphenyl (PCB) Products in Water
by Microextraction and Gas Chromatography 29
506. Determination of Phthalate and Adipate Esters in Drinking Water
by Liquid-Liquid Extraction or Liquid-Solid Extraction
and Gas Chromatography with Photoionization Detection 61
507. The Determination of Nitrogen- and Phosphorus-Containing
Pesticides in Water by Gas Chromatography
with a Nitrogen-Phosphorus Detector 83
508. Determination of Chlorinated Pesticides in Water
by Gas Chromatography with an Electron Capture Detector 109
515.1 Determination of Chlorinated Acids in Water by Gas Chromatography
with an Electron Capture Detector 135
515.2 Determination of Chlorinated Acids in Water
Using Liquid-Solid Extraction and Gas Chromatography
with an Electron Capture Detector 165
525.1 Determination of Organic Compounds in Drinking Water
by Liquids-Solid Extraction and Capillary Column
Gas Chromatography/Mass Spectrometry 195
Appendix: Detection Limits for Precision and Accuracy
for the Analysis of Pesticide Compounds by EPA Method 525.1 230
531.1 Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates
in Water by Direct Aqueous Injection HPLC
with Post-Column Derivatization 235
547 Determination of Glyphosate In Drinking Water By Direct-Aqueous-Injection
HPLC, Post-Column Derivatization, and Fluorescence Detection 255
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Contents
Method
No. Title Page
548 Determination of Endothall in Drinking Water by Aqueous
Derivatization, Liquid-Solid Extraction, and Gas Chromatography
with Electron-Capture Detection 271
Appendix: Preparation of Endothall-Pentafluorophenylhydrazine 286
548.1 Determination of Endothall in Drinking Water by Ion-Exchange Extraction,Acidic
Methanol Methylation and Gas Chromatography/Mass Spectrometry 291
553. Determination of Benzidines and Nitrogen-Containing Pesticides in Water
by Liquid-Liquid Extraction or Liquid-Solid Extraction and Reverse Phase
High Performance Liquid Chromatography/Particle Beam/Mass Spectrometry ... 319
555. Determination of Chlorinated Acids in Water by High Performance Liquid
Chromatography with a Photodiode Array Ultraviolet Detector 355
IV
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Cross-Reference
Analytes shown in bold italic type are regulated under the Pesticide Chemicals Manufacturing
Rule found at 40 CFR Part 455.
Applicable Method(s)
Analytes CAS No. Volume I Volume II
Acenaphthylene 208-96-8 525.1
Acephote 30560-19-1 1656, 1657 -
Adfluorfen 50594-66-6 - 575.7, 575.2, 555
Alachlor 15972-60-8 645, 1656 505, 507, 525.7
Aldicarb (Temik) 116-06-3 - 537.7
Aldicarb sulfoxide 1646-87-3 531.1
Aldicarb sulfone 1646-88-4 531.1
Aldrin 309-00-2 617, 1656 505, 508, 525.1
Allethrin (Pynamin) 584-79-2 1660 -
Aluminum 7429-90-5 200.9
Ametryn 834-12-8 619 507, 525.7
Aminocarb 2032-59-9 632 -
Amobam 3566-10-7 630, 630.1 -
Anthracene 120-12-7 525.1
Antimony 7440-36-0 200.9
AOP -- 630 --
Aroclor 1232 11141-16-5 505, 508
Aroclor 1221 11104-28-2 505, 508
Aroclor 1260 11096-82-5 505, 508
Aroclor 1242 53469-21-9 505, 508
Aroclor 1254 11097-69-1 505, 508
Aroclor 1248 12672-29-6 505, 508
Aroclor 1016 12674-11-2 505, 508
Arsenic 7440-38-2 200.9
Aspon 3244-90-4 622.1 --
Atraton 1610-17-9 619 507, 525.1
Atrazine 1912-24-9 619, 1656 505, 507, 525.7
Azinphos ethyl 2642-71-9 1657 -
Azinphos methyl (Guthion) 86-50-0 614, 622, 1657 --
Barban 101-27-9 632 -
Basalin (Fluchloralin) 33245-39-5 646 --
Baygon 114-26-1 531.1
Bayleton (Triadimefon) 43121-43-3 633, 1656 507, 525.7
Baythroid (Cyfluthrin) 68359-37-5 1660 -
Bendiocarb 22781-23-3 639 -
Benefin (Benfluralin) 1861-40-1 627, 1656 -
Benfluralin (Benefin) 1861-40-1 627, 1656 -
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Cross-Reference
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Benomyl 17804-35-2 631 -
Bensulide 741-58-2 636 -
Bentazon (Basagran) 25057-89-0 643 515.1, 515.2, 555
Benzidine 92-87-5 553
Benzoylprop ethyl 33878-50-1 553
Benzo[a]pyrene 50-32-8 525.1
Benzo[b]fluoranthene 205-99-2 525.1
Benzo[?,/u]perylene 191-24-2 525.1
Benzo[Jt]fluoranthene 207-08-9 525.1
Benz[a]anthracene 56-55-3 525.1
Beryllium 7440-41-7 200.9
a-BHC 319-84-6 617, 1656 525.1
(8-BHC 319-85-7 617, 1656 525.1
Y-BHC 58-89-9 617, 1656 525.1
6-BHC 319-86-8 617, 1656 525.1
Biphenyl 92-52-4 642 -
Bis(2-ethylhexyl)adipate 103-23-1 506, 525.1
Bis(2-ethylhexyl)phthalate .... 117-81-7 506, 525.1
Bolstur (Sulprofos) 35400-43-2 622, 1657 -
Bromacil Salts & Esters 314-40-9 633, 1656 507, 525.1
Bromoxynil 1689-84-5 1661 -
Bromoxynil octanoate 1689-99-2 1656 -
Busan 40 51026-28-9 630, 630.1 -
Busan 85 128-03-0 630, 630.1 -
Butachlor 23184-66-9 645, 1656 507, 525.1
Butylate 2008-41-5 634 507, 525.1
Butylbenzyl phthalate 85-68-7 506, 525.1
Cadmium 7440-43-9 200.9
Caffeine 58-08-2 553
Captafol 2425-06-1 1656 -
Captan 133-06-2 617, 1656 ~
Carbam-S 128-04-1 630, 630.1' ~
Carbaryl 63-25-2 632 531.1, 553
Carbendazim 10605-21-7 631 -
Carbofuran 1563-66-2 632 531.1
1 Carbam-S was not explicitly listed in these methods, but these methods are applicable to dithiocarbamate
pesticides such as Carbam-S.
VI
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Cross-Reference
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Carbophenothion 786-19-6 617, 1656 -
Carboxin 5234-68-4 507, 525.1
CDN 97-00-7 646 -
Chloramben 133-90-4 515.1, 555
Chlordane 57-74-9 617, 1656 505, 508
a-Chlordane 5103-71-9 1656 505, 508, 525.1
7-Chlordane 5103-74-2 1656 505, 508, 525.1
Chlorfevinphos 470-90-6 1657 -
Chlorobenzilate 510-15-6 608.1, 1656 508, 525.1
2-Chlorobiphenyl 2051-60-7 525.1
Chloroneb 2675-77-6 608.1, 1656 508, 525.1
o-Chlorophenyl thiourea 5344-82-1 553
Chloropicrin 76-06-2 618 -
Chloropropylate 5836-10-2 608.1, 1656 -
Chlorothalonil 1897-45-6 608.2, 1656 508, 525.1
Chlorpropham 101-21-3 632 507, 525.1
Chlorpyrifos methyl 5598-13-0 622, 1657 -
Chlorpyrifos 2921-88-2 622, 1657 508
Chromium 7440-47-3 200.9
Chrysene 218-01-9 525.1
Cobalt 7440-48-4 200.9
Copper 7440-50-8 200.9
Coumaphos 56-72-4 622, 1657 ~
Crotoxyphos 7700-17-6 1657 --
Cyanatine 21725-46-2 629 SOT2
Cycloate 1134-23-2 634 507, 525.1
Cycloprate 54460-46-7 616 -
Cyfluthrin (Baythroid) 68359-37-5 1660 -
2,4-D Salts & Esters 94-75-7 615, 1658 515.1, 515.2, 555
Dacthal (DCPA) 1861-32-1 608.2, 1656 . . 508, 515.1, 515.2, 525.1
Dalapon 75-99-0 615, 1658 515.1
Dazomet 533-74-4 .... 630, 630.1,31659 --
2,4-DB 94-82-6 615, 1658 515.1, 515.2, 555
2 Cyanazine was not specifically listed in this method, but data from EMSL-Ci and from industry indicate that
this method is applicable.
3 Dazomet was not explicitly listed in these methods, but these methods are applicable to dithiocarbamate
pesticides such as Dazomet.
vii
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Cross-Reference (Cont.>
Applicable Method(s)
Analyte CAS No. Volume I Volume II
DBCP 96-12-8 1656 -
DCPA (Dacthal) 1861-32-1 608.2, 1656 . . 508, 515.1, 515.2, 525.1
4,4'-DDD 72-54-8 617, 1656 508, 525.1
4,4'-DDE 72-55-9 617, 1656 508, 525.1
4,4'-DDT 50-29-3 617, 1656 508, 525.1
Deet 134-62-3 633 -
DBF 78-48-8 1657 -
Demeton 8065-48-3 614, 622, 1657 -
Di-fl-butyl phthalate 84-74-2 506, 525.1
Di-«-octyl phthalate 117-84-0 506
Diallate 2303-16-4 1656 -
Diaunon 333-41-5 614, 622, 1657 507, 525.1
Dibenz[a,/z]anthracene 53-70-3 525.1
Dibromochloropropane 96-12-8 608.1 -
Dicamba 1918-00-9 615, 1658 515.1, 515.2, 555
Dichlofenthion 97-17-6 622.1, 1657 --
Dichlone 117-80-6 1656 -
Dichloran 99-30-9 608.2, 617 --
3,3'-Dichlorobenzidine 91-94-1 553
3,5-Dichlorobenzoic acid 51-36-5 515.1, 515.2, 555
2,3-DichIorobiphenyl 16605-91-7 525.1
Dichlorophen 97-23-4 604.1 --
Dichlorprop Salts & Esters 120-36-5 615, 1658 515.1, 515.2, 555
Dichlorvos 62-73-7 622, 1657 507, 525.7
Dicofol 115-32-2 617, 1656 --
Dicrotophos 141-66-2 1657 -
Dieldrin 60-57-1 617, 1656 505, 508, 525.1
Diethyl phthalate 84-66-2 506, 525.1
Dimethoate 60-51-5 1657 -
3,3'-Dimethoxybenzidine 119-90-4 553
Dimethyl phthalate 131-11-3 506, 525.1
3,3'-Dimethylbenzidin 119-93-7 553
Dinocap 39300-45-3 646 --
Dinoseb 88-85-7 615, 1658 515.1, 515.2, 555
Dioxathion 78-34-2 614.1, 1657 -
Diphenamid 957-51-7 645 507, 525.1
Diphenylamine 122-39-4 620 -
Disulfoton 298-04-4 614, 622, 1657 507, 525.1
VIII
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Disulfoton sulfone 2497-06-5 507, 525.1
Disulfoton sulfoxide 2497-07-6 507, 525.1
Diuron 330-54-1 632 553
Endosulfan sulfate 1031-07-8 617, 1656 508, 525.1
Endosulfan I 959-98-8 617, 1656 508, 525.1
Endosulfan II 33213-65-9 617, 1656 508, 525.1
Endotholl Salts & Esters 145-73-3 - 548, 548.1
Endrin 72-20-5 617, 1656 505, 508, 525.1
Endrin aldehyde 7421-93-4 617, 1656 508, 525.1
Endrin ketone 53494-70-5 1656 ~
EPN 2104-64-5 614.1, 1657 --
EPTC 759-94-4 634 507, 525.1
Ethalfluralin 55283-68-6 627, 1656 -
Ethion 563-12-2 614, 614.1, 1657 -
Ethoproprophos (Ethoprop) . . 13194-48-4
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Cross-Reference (com.
Applicable Method(s)
Analyte CAS No. Volume I Volume II
6-HCH 319-86-8 508
a-HCH 319-84-6 508
Heptachlor 76-44-8 617, 1656 505, 508, 525.1
Heptachlor epoxide 1024-57-3 617, 1656 505, 508, 525.1
2,2',3,3',4,4',6-Heptachloro-
biphenyl 52663-71-5 525.1
Hexachlorobenzene 118-74-1 505, 508, 525.1
2,2',4,4',5,6'-HexachIoro-
biphenyl 6-145-22-4 525.1
Hexachlorocyclopentadiene 77-47-4 505, 525.1
Hexachlorophene 70-30-4 604.1 -
Hexazinone 51235-04-2 633 507, 525.1
3-Hydroxycarbofuran 16655-82-6 - 531.1
5-Hydroxydicamba 7600-50-2 515.1, 515.2, 555
Indeno[l,2,3,c,d]pyrene 193-39-5 525.1
Iron 7439-89-6 200.9
Isodrin 465-73-6 617, 1656 -
Isopropalin (Paarlan) 33820-53-0 627, 1656 ~
Kepone 143-50-0 1656 -
Kinoprene 42588-37-4 616 -
KN Methyl 137-41-7 630, 630.1 -
Lead 7439-92-1 200.9
Leptophos 21609-90-5 1657 -
Lethane 112-56-1 645 -
Lindane (T-HCH) 58-89-9 505, 508, 525.1
Linuron (Lorox) 330-55-2 632 553
Malathion 121-75-5 614, 1657 -
Mancozeb 8018-01-7 630 -
Maneb 12427-38-2 630 -
Manganese 7439-96-5 200.9
MBTS 120-78-5 637 -
MCPA 94-74-6 615, 1658 555
MCPP 7085-19-0 615, 1658 555
Mercaptobenzothiazole 149-30-4 640 -
Merphos 150-50-5 622, 1657 507, 525./
Metham (Vapam) 137-42-8 630, 630.1 ~
Methamidophos 10265-92-6 7557 -
Methiocarb 2032-65-7 632 531.1
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Methomyl 16752-77-5 632 531.1
Methoprene 40596-69-8 616 -
Methoxychlor 72-43-5 608.2, 617, 1656 505, 508, 525.1
Methyl paraoxon 950-35-6 507, 525.1
Metolachlor 51218-45-2 507, 525.1
Metribuzin 21087-64-9 633, 1656 507, 525.1
Mevinphos 7786-34-7 622, 1657 507, 525.7
Mexacarbate 315-18-4 632 -
MGK 264 113-48-4 633.1 507, 525.1
MGK 326 136-45-8 633.1 -
Mirex 2385-85-5 617, 1656 . --
Molinate 2212-67-1 634 507, 525.1
Monocrotophos 6923-22-4 1657 -
Monuron 150-68-5 632 553
Monuron-TCA 140-41-0 632 -
Nabam 142-59-6 630, 630.1 -
Nabonate 138-93-2 630.1 -
Noted 300-76-5
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Cross-Reference (cont.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
PCB-1221 11104-28-2 617, 1656 -
PCB-1232 11141-16-5 617, 1656 -
PCB-1242 53469-21-9 617, 1656 -
PCB-1248 12672-29-6 617, 1656 -
PCB-1254 11097-69-1 617, 1656 -
PCB-1260 11096-82-5 617, 1656 --
PCNB 82-68-8 .... 608.1, 617, 1656 --
Pebulate 1114-71-2 634 507, 525.1
Pendimetholin (Prowl) 40487-42-1 1656 -
2,2,3',4,6-Pentachlorobiphenyl . 60233-25-2 525.1
Pentachlorophenol (PCP) 87-86-5 - . . 515.1, 515.2, 525.1, 555
Permethrin5 52645-53-1 . . . 608.2, 1656, 1660 508, 525.1
cis-Permethrin6 61949-76-6 1656, 1660 508, 525.1
trans-Permethrin6 52645-53-1 1656, 1660 505, 525.1
Perthane 72-56-0 617, 1656 --
Phenanthrene 85-01-8 525.1
Phenothrin (Sumithrin) 26002-80-2 1660 -
o-PhenylphenoI 90-43-7 642 -
Phorate 298-02-2 622, 1657 -
Phosmet 732-11-6 622.1, 1657 --
Phosphamidon 13171-21-6 1657 --
Phosphoramide 680-31-9 1657 -
Picloram 1918-02-1 644 515.1, 515.2, 555
Polyram 9006-42-2 630 ~
Profluralin 26399-36-0 627 -
Prometon (Promitol) 1610-18-0 619 507, 525.7
Prometryn 7287-19-6 619 507, 525.7
Pronamide 23950-58-5 633.1 507, 525.7
Propachlor 1918-16-7 608.1, 1656 508, 525.1
Propanil 709-98-8 632.1, 1656 -
Propazine 139-40-2 679, 1656 507, 525.7
Propham 122-42-9 632 -
Propoxur 114-26-1 632 --
Pydrin (Fenvalerate) 51630-58-1 1660 -
5 Detected as cis-Permethrin and trans-Permethrin.
6 Regulated as Permethrin.
XII
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Pynamin (Allethrin) 584-79-2 1660 --
Pyrene 129-00-0 525.1
Pyrethrin I 121-21-1 1660 -
Pyrethrin II 121-29-9 1660 -
Resmethrin 10453-86-8 616, 1660 -
Ronnel 299-84-3 622, 1657 -
Rotenone 83-79-4 635 553
Rubigan (Fenarimol) 60168-88-9 633.1, 1656 507, 525.1
Secbumeton 26259-45-0 619 -
Selenium 7782-49-2 200.9
Siduron 1982-49-6 632 553
Silver 7440-22-4 200.9
Silvex (2,4,5-TP) 93-72-1 615,1658 515.1, 515.2, 555
Simazine 122-34-9 619, 1656 505, 507, 525.1
Simetryn 1014-70-6 619 507, 525.1
Sodium dimethyldithiocarbamate . 128-04-1 630, 630.1 -
Stirofos (Tetrachlorvinphos) . 2224S-79-97 622, 1657 507, 525.1
Strobane 8001-50-1 617, 1656 -
Sulfotepp 3689-24-5 1657 -
Sulprofos (Bolster) 35400-43-2 622, 1657 -
Sumithrin (Phenothrin) 26002-80-2 1660 -
Swep 1918-18-9 632 -
2,4,5-T 93-76-5 615, 1658 515.1, 515.2, 555
TCMTB 21564-17-0 637 -
Tebuthiuron (Spike) 34014-18-1 - 507, 525.7
Temik (Aldicarb) 116-06-3 - 531.1
2,2',4,4'-Tetrachlorobiphenyl . . 2437-79-8 525.1
Tetrachlorvinphos (Stirofos) . 2224S-79-97 622, 1657 507, 525.1
TEPP 107-49-3 1657 -
Terbacil 5902-51-2 633, 1656 507, 525.7
Terbufos (Counter) 13071-79-9 614.1, 1657 507, 525.1
Terbuthylazine (Gardoprim) . . 5915-41-3 619, 1656 -
Terbutryn 886-50-0 619 507, 525.7
Tetramethrin 7696-12-0 1660 -
Thallium 7440-28-0 200.9
Thiabendazole 148-79-8 641 -
1 CAS number in Table 7 of proposed rule is incorrect.
XIII
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Cross-Reference (com.)
Applicable Method (s)
Ana/yte CAS No. Volume I Volume II
Thionazin 297-97-2 622.1 -
Thiram 137-26-8 630, 630.1 -
Tin 7440-31-5 200.9
TOK (Nitrofen) 1836-75-5 1656 -
Tokuthion 34643-46-4 622, 1657 -
Toxaphene 8001-35-2 617, 1656 505, 508, 525.1
2,4,5-TP (Silvex) 93-72-1 615, 1658 515.1, 515.2, 555
Triadimefon (Bayleton) 43121-43-3 633, 1656 507, 525.1
2,4,5-Trichlorobiphenyl 15862-07-4 525.1
Trichlorofon 52-68-6 1657 -
Trichloronate 327-98-0 622, 1657 -
Tricresylphosphate 78-30-8 1657 -
Tricyclazole 41814-78-2 633 507, 525.1
Trifluralin 1582-09-8 617, 627, 1656 508, 525.1
Trimethylphosphate 512-56-1 1657 -
Trithion methyl 953-17-3 1657 -
Vacor 53558-25-1 632.1 -
Vapam (Metham) 137-42-8 630, 630.1 -
Vernolate 1929-77-7 634 507, 525.1
ZAC - 630 -
Zinc 7440-66-6 200.9
Zineb 12122-67-7 630, 630.1 -
Ziram 137-30-4 630, 630.1 -
XIV
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Method 200.9
Determination of Trace Elements by
Stabilized Temperature Graphite Furnace
Atomic Absorption Spectrometry
Revision 1.2 - EPA EMSL-Ci
April 1991
John T. Creed, Theodore D. Martin, Larry B. Lobring and James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
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Method 200.9
Determination of Trace Elements by Stabilized Temperature Graphite
Furnace Atomic Absorption Spectrometry
1. SCOPE AND A PPLICA TION
1.1 This method provides procedures for the determination of dissolved and total recoverable
elements in ground water, surface water, drinking water and wastewater. This method is also
applicable to total recoverable elements in sediment, sludges, biological tissues, and solid
waste samples.
1.2 Dissolved elements are determined after suitable filtration and acid preservation. Acid diges-
tion procedures are required prior to the determination of total recoverable elements. Appro-
priate digestion procedures for biological tissues should be utilized prior to sample analysis.
1.3 This method is applicable to the determination of the following elements by stabilized tempera-
ture graphite furnace atomic absorption spectrometry (STGFAA).
Element
Aluminum
Antimony
Arsenic
Beryllium
Cadmium
Chromium
Cobalt
Copper
Iron
Lead
Manganese
Nickel
Selenium
Silver
Thallium
Tin
Zinc
(Al)
(Sb)
(As)
(Be)
(Cd)
(Cr)
(Co)
(Cu)
(Fe)
(Pb)
(Mn)
(Ni)
(Se)
(Ag)
(Tl)
(Sn)
(Zn)
CAS No.
7429-90-5
7440-36-0
7440-38-2
7440-41-7
7440-43-9
7440-47-3
7440-48-4
7440-50-8
7439-89-6
7439-92-1
7439-96-5
7440-02-0
7782-49-2
7440-22-4
7440-28-0
7440-31-5
7440-66-6
NOTE: Method detection limit and instrumental operating conditions for the applicable
elements are listed in Table 2. These are intended as a guide to instrumental detection
limits typical of a system optimized for the element employing commercial instrumen-
tation. However, actual method detection limits and linear working ranges will be
dependent on the sample matrix, instrumentation and selected operating conditions.
1.4 The sensitivity and limited linear dynamic range (LDR) of GFAA often implies the need to
dilute a sample prior to the analysis. The actual magnitude of the dilution as well as the
cleanliness of the labware used to perform the dilution can dramatically influence the quality of
the analytical results. Therefore, samples types requiring large dilutions should be analyzed by
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Method 200.9
an alternative analytical method which has a larger LDR or which is inherently less sensitive
than GFAA.
1.5 This method should be used by analysts experienced in the use of GFAA.
2. SUMMARY OF METHOD
2.1 This method describes the determination of applicable elements by stabilized temperature
platform graphite furnace atomic absorption (STPGFAA). In STPGFAA the sample (and the
matrix modifier, if required) is first pipetted onto the platform or a device which provides
delayed atomization. The sample is then dried at a relatively low temperature (~ 120°C) to
avoid spattering. Once dried, the sample is normally pretreated in a char or ashing step which
is designed to minimize the interference effects caused by the concomitant sample matrix.
After the char step the furnace is allowed to cool prior to atomization. The atomization cycle
is characterized by rapid heating of the furnace to a temperature where the metal (analyte) is
atomized from the pyrolytic graphite surface. The resulting atomic cloud absorbs the element
specific atomic emission produced by a hollow cathode lamp (HCL) or a electrodeless dis-
charge lamp (EDL). Because the resulting absorbance usually has a nonspecific component
associated with the actual analyte absorbance, an instrumental background correction device is
necessary to subtract from the total signal the component which is nonspecific to the analyte.
In the absence of interferences, the background corrected absorbance is directly related to the
concentration of the analyte. Interferences relating to STPGFAA (Sect. 4) must be recognized
and corrected. Instrumental drift as well as suppressions or enhancements of instrument
response caused by the sample matrix must be corrected for by the method of standard ad-
dition (Sect. 11.5).
3. DEFINITIONS
3.1 Dissolved: Material that will pass through a 0.45-/xm membrane filter assembly, prior to
sample acidification.
3.2 Total Recoverable: The concentration of analyte determined on an unfiltered sample following
treatment with hot dilute mineral acid.
3.3 Instrument Detection Limit (IDL): The concentration equivalent of an analyte signal equal to
three times the standard deviation of the calibration blank signal at the selected absorbance
line.
3.4 Method Detection Limit (MDL): The minimum concentration of an analyte that can be
identified, measured and reported with 99% confidence that the analyte concentration is greater
than zero.
3.5 Linear Dynamic Range (LDR): The concentration range over which the analytical working
curve remains linear.
3.6 Laboratory Reagent Blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, and reagents that are used with sam-
ples. The LRB is used to determine if method analytes or other interferences are present in
the laboratory environment, reagents or apparatus.
-------
Method 200.9
3.7 Calibration Blank: A volume of ASTM type I water acidified such that the acid(s) concentra-
tion is identical to the acid(s) concentration associated with the calibration standards.
3.8 Stock Standard Solution: A concentrated solution containing one analyte prepared in the
laboratory using a assayed reference compound or purchased from a reputable commercial
source.
3.9 Calibration Standard (CAL): A solution prepared from the stock standard solution which is
used to calibrate the instrument response with respect to analyte concentration.
3.10 Laboratory Fortified Blank (LFB): An aliquot of reagent water to which a known quantity of
each method analyte is added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the method is within accepted control limits.
3.11 Laboratory Fortified Sample Matrix (LFM): An aliquot of an environmental sample to which
a known quantity of each method analyte is added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results.
3.12 Quality Control Sample (QCS): A solution containing a known concentration of each method
analyte derived from externally prepared test materials. The QCS is obtained from a source
external to the laboratory and is used to check laboratory performance.
3.13 Matrix Modifier: A substance added to the graphite furnace along with the sample in order to
minimize the interference effects by selective volatilization of either analyte or matrix compo-
nents.
3.14 Standard Addition: The addition of a known amount of analyte to the sample in order to
determine the relative response of the detector to an analyte within the sample matrix. The
relative response is then used to assess the sample analyte concentration.
4. INTERFERENCES
4.1 Several interference sources may cause inaccuracies in the determination of trace elements by
GFAA. These interferences can be classified into three major subdivisions, namely spectral,
non-spectral and memory.
4.1.1 Spectral: Interferences resulting from the absorbance of light by a molecule and/or an
atom which is not the analyte of interest. Spectral interferences caused by an element
only occur if there is a spectral overlap between the wavelength of the interfering
element and the analyte of interest. Fortunately, this type of interference is relatively
uncommon in STPGFAA because of the narrow atomic line widths associated with
STPGFAA. In addition, the use of appropriate furnace temperature programs and
high spectral purity lamps as light sources can minimize the possibility of this type of
interference. However, molecular absorbances can span over several hundred nano-
meters producing broadband spectral interferences. This type of interference is far
more common in STPGFAA. The use of matrix modifiers, selective volatilization and
background correctors are all attempts to eliminate unwanted non-specific absorbance.
The non-specific component of the total absorbance can vary considerably from
sample type to sample type. Therefore, the effectiveness of a particular background
-------
Method 200.9
correction device may vary depending on the actual analyte wavelength used as well
as the nature and magnitude of the interference.
Spectral interferences are also caused by the emission from black body radiation
produced during the atomization furnace cycle. This black body emission reaches the
photomultiplier tube producing erroneous results. The magnitude of this interference
can be minimized by proper furnace tube alignment and monochromator design. In
addition, atomization temperatures which adequately volatilize the analyte of interest
without producing unnecessary black body radiation can help reduce unwanted back-
ground emission produced during atomization.
NOTE: A spectral interference may be manifested by extremely high backgrounds (1.0
abs*) which may exceed the capability of the background corrector and/or it may be
manifested as a non-analyte element which may cause a direct spectral overlap with the
analyte of interest. If a spectral interference is suspected, the analyst is advised to:
1. Dilute the sample if the analyte absorbance is large enough to sacrifice
some of the sensitivity. This dilution may dramatically reduce a molecular
background or reduce it to the point where the background correction
device is capable of adequately removing the remaining nonspecific com-
ponent. If the non-specific component is produced by a spectral overlap
with an interfering element, the change in absorbance caused by dilution of
the sample should decrease in a linear fashion, provided the undiluted and
diluted sample are both within the linear range of the interfering element.
2. If dilution is not acceptable because of the relatively low analyte absor-
bance readings or the dilution produces a linear decrease in the non-
specific absorbance, the analyst is advised to investigate another analyte
wavelength which may eliminate the suspected spectral interference(s).
3. If dilution and alternative spectral lines are not acceptable, the analyst is
advised to attempt to selectively volatilize the analyte or the non-specific
component thereby eliminating the unwanted interference (s) by atomizing
the analyte in an interference-free environment.
4. If none of the above advice is applicable and the spectral interference
persists, an alternative analytical method which is not based on the same
type of physical/chemical principle may be necessary to evaluate the actual
analyte concentration.
This background level is given as a guide and is not intended to serve as an absolute value which
may be applied in all situations.
-------
Method 200.9
4.1.2 Non-spectral: Interferences caused by sample components which inhibit the formation
of free atomic analyte atoms during the atomization cycle. The use of a delayed
atomization device which provides stabilized temperatures is required, because these
devices provide an environment which is more conducive to the formation of free
analyte atoms and thereby minimize this type of interference. This type of interfer-
ence can be detected by analyzing a sample plus a laboratory fortified sample matrix
early within any analysis set. From this data, immediately calculate the percent
recovery (Sect. 10.4.2). If the percent recovery is outside the laboratory determined
control limits (Sect. 10.3.3) a potential problem should be suspected. If the result
indicates a potential matrix effect, the analyst is advised to:
1. Perform the method of standard addition* (see Sect. 11.5); if the "percent
recovery" from the method of standard addition is drastically different
from the percent recovery from LFM, then lab contamination or another
lab related problem should be suspected and corrected.
NOTE: If contamination is suspected, analyze the LFB and calculate a percent recov-
ery.
2. If the two recoveries are approximately equal* and the response from the
standard addition is dramatically different than that which would be cal-
culated from the calibration curve, the sample should be suspected of a
matrix induced interference and analyzed by the method of standard ad-
dition (Sect. 11.5).
4.1.3 Memory interferences resulting from analyzing a sample containing a high concentra-
tion of an element (typically a high atomization temperature element) which cannot be
removed quantitatively in one complete set of furnace steps. The analyte which re-
mains in the furnace can produce false positive signals on subsequent sample(s).
Therefore, the analyst should establish the analyte concentration which can be injected
into the furnace and adequately removed in one complete set of furnace cycles. This
concentration represents the maximum concentration of analyte within a sample which
will not cause a memory interference on the subsequent sample(s). If this con-
centration is exceeded, the sample should be diluted and a blank should be analyzed
(to assure the memory affect has been eliminated) before reanalyzing the diluted
sample.
NOTE: Multiple clean out furnace cycles may be necessary in order to fully utilize the
LDRfor certain elements.
* The limitations listed in Sect. 11.5 must be met in order to apply these recommendations.
-------
Method 200.9
4.1.4 Specific Element Interferences
Antimony: Antimony suffers from an interference produced by K2SO4.5 In the ab-
sence of hydrogen in the char cycle (1300°C*), K2SO4 produces a relatively high
(1.2 abs) background absorbance which can produce a false signal even with Zee-
man background correction. However, this background level can be dramatically
reduced (0.1 abs) by the use of a hydrogen/argon gas mixture in the char step.
This reduction in background is strongly influenced by the temperature of the char
step.
Aluminum: The Pd may have elevated levels of Al which will cause elevated blank
absorbances.
Arsenic: The HC1 present from the digestion procedure can influence the sensitivity
for As. A 1 % HC1 solution with Pd used as a modifier results in a 40% loss in
sensitivity relative to the analyte in a 1 % HNO3 solution. The use of Pd/Mg/H2 as
a modifier reduces this suppression to about 10%.
Cadmium: The HC' present from the digestion procedure can influence the sen-
sitivity for Cd. A 1 % HC1 solution with Pd used as a modifier results in a 70%
loss in sensitivity relative to the analyte in a 1 % HNO3 solution. The use of
Pd/Mg/H2 as a modifier reduces this suppression to less than 10%.
Copper: Pd lines at 324.27 nm and 325.16 nm may produce an interference on the
Cu line at 324.8 nm.5
Lead: The HC1 present from the digestion procedure can influence the sensitivity
for Pb. A 1 % HC1 solution with Pd used as a modifier results in a 70% loss in
sensitivity relative to the analyte response in a 1 % HNO3 solution. The use of
Pd/Mg/H2 as a modifier reduces this suppression to less than 10%.
Selenium: Iron has been shown to suppress Se response with continuum source
background correction.5 In addition, the use of hydrogen as a purge gas during the
dry and char steps can cause a suppression in Se response if not purged from the
furnace prior to atomization.
Silver: The Pd used in the modifier preparation may have elevated levels of Ag
which will cause elevated blank absorbances.
5. SAFETY
5.1 The toxicity or carcinogenicity of reagents used in this method has not been fully established.
Each chemical should be regarded as a potential health hazard, and exposure to these com-
pounds should be as low as reasonably achievable. Each laboratory is responsible for main-
taining a current awareness file of OSHA regulations regarding the safe handling of the
*The actual furnace temperature may vary from instrument to instrument. Therefore, the actual
furnace temperature should be determined on an individual bases.
8
-------
Method 200.9
chemicals specified in this method.''2 A reference file of material data handling sheets should
also be available to all personnel involved in the chemical analysis.
5.2 The graphite tube during atomization emits intense UV radiation. Suitable precautions should
be taken to protect personnel from such a hazard.
5.3 The use of argon/hydrogen gas mixture during the dry and char steps may evolve a consider-
able amount of HC1 gas. Therefore, adequate ventilation is required.
6. APPARATUS AND EQUIPMENT
6.1 Graphite Furnace Atomic Absorbance Spectrophotometer
6.1.1 The GFAA spectrometer must be capable of programmed heating of the graphite tube
and the associated delayed atomization device. The instrument should be equipped
with an adequate background correction device capable of removing undesirable non-
specific absorbance over the spectral region of interest. The capability to record
relatively fast (< 1 sec) transient signals and evaluate data on a peak area basis is
preferred. In addition, a recirculating refrigeration bath is recommended for im-
proved reproduc-ibility of furnace temperatures. The data shown in the tables were
obtained using the stabilized temperature platform and Zeeman background correction.
6.1.2 Single element hollow cathode lamps or single element electrodeless discharge lamps
along with the associated power supplies.
6.1.3 Argon gas supply (high-purity grade, 99.99%).
6.1.4 A 5% hydrogen in argon gas mix and the necessary hardware to use this gas mixture
during specific furnace cycles.
6.1.5 Autosampler: Although not specifically required, the use of an autosampler is highly
recommended.
6.2 Graphite Furnace Operating Conditions: A guide to experimental conditions for the applicable
elements are shown in Table 2.
6.3 Sample Processing Equipment
6.3.1 Balance: Analytical, capable of accurately weighing to 0.1 mg.
6.3.2 Hot Plate: Corning PC100 or equivalent.
6.3.3 Centrifuge: Steel cabinet with guard bowl, electric timer and brake.
6.3.4 Drying Oven capable of ± 3°C temperature control.
6.4 Labware: The determination of trace level elements requires a consideration of potential
sources of contamination and analyte losses. Potential contamination sources include improp-
erly cleaned laboratory apparatus and general contamination within the laboratory environment
from dust, etc. A clean laboratory work area designated for trace element sample handling
must be used. Sample containers can introduce positive and negative errors in the determina-
tion of trace elements by contributing contaminants through surface desorption or leaching
and/or depleting element concentrations through adsorption processes. All reusable labware
(glass, quartz, polyethylene, Teflon etc.), including the sample container, should be cleaned
prior to use. Labware should be soaked overnight and thoroughly washed with laboratory-
grade detergent and water, rinsed with water, and soaked for four hours in a mixture of dilute
-------
Method 200.9
nitric and hydrochloric acid (1 +2 + 9), followed by rinsing with ASTM type I water and oven
drying.
NOTE: Chromic acid must not be used for cleaning glassware.
6.4.1 Glassware: Volumetric flasks and graduated cylinders.
6.4.2 Assorted calibrated pipettes.
6.4.3 Conical Phillips beakers, 250-mL with 50-mm watch glasses. Griffin beakers, 250-
mL with 75-mm watch glasses.
6.4.4 Storage bottles: Narrow mouth bottles, Teflon FEP (fluorinated ethylene propylene)
with Tefzel ETFE (ethylene tetrafluorethylene) screw closure, 125-mL and 250-mL
capacities.
6.4.5 Wash bottle: One piece stem, Teflon FEP bottle with Tefzel ETFE screw closure,
125-mL capacity.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagents: Reagents may contain elemental impurities which might affect analytical data.
Because of the high sensitivity of GFAA, high-purity reagents should be used whenever
possible. All acids used for this method must be ultra high-purity grade. Suitable acids are
available from a number of manufacturers or may be prepared by sub-boiling distillation.
7.1.1 Nitric acid, concentrated (sp.gr. 1.41) (CASRN 7697-37-2).
7.1.2 Nitric acid (1 + 1): Add 500 mL cone, nitric acid to 400 rnL of ASTM type I water
and dilute to 1 L.
7.1.3 Nitric acid (1 +9): Add 100 mL cone, to 400 mL of ASTM type I water and dilute to
1 L.
7.1.4 Hydrochloric acid, concentrated (sp.gr. 1.19) (CASRN 7647-01-0).
7.1.5 Hydrochloric acid (I +4): Add 200 mL cone, hydrochloric acid to 400 mL ASTM
type I water and dilute to 1000 mL.
7.1.6 Tartaric acid. ACS reagent grade (CASRN 87-69-4).
7.1.7 Matrix Modifier, dissolve 300 mg Palladium (Pd) powder in concentrated HNO3 (1 ml
of HNO3, adding 10 mL of concentrated HC1 if necessary). Dissolve 200 mg of
Mg(NO,)2 in ASTM type 1 water. Pour the two solution together and dilute to 100
mL with ASTM type 1 water.
NOTE: It is recommended that the matrix modifier be analyzed separately in order to
assess the contribution of the modifier to the overall laboratory blank.
7.1.8 Ammonium hydroxide, concentrated (sp.gr. 0.902) (CAS no. 1336-21-6).
7.2 Water: For all sample preparation and dilutions, ASTM type I water (ASTM D1193) is
required. Suitable water may be prepared by passing distilled water through a mixed bed of
anion and cation exchange resins.
10
-------
Method 200.9
7.3 Standard Stock Solution: May be purchased from a reputable commercial source or prepared
from ultra high-purity grade chemicals or metal (99.99-99.999% pure). All salts should be
dried for 1 h at 105°C, unless otherwise specified. (CAUTION: Many metal salts are
extremely toxic if inhaled or swallowed. Wash hands thoroughly after handling). The stock
solution should be stored in Teflon bottles. The following procedures may be used for pre-
paring standard stock solutions:
NOTE: Some metals, particularly those which form surface oxides, require cleaning
prior to being weighed. This may be achieved by pickling the surface of the metal in
acid. An amount in excess of the desired weight should be pickled repeatedly, rinsed
with water, dried and weighed until the desired weight is achieved.
7.3.1 Aluminum solution, stock, 1 mL = 1000 /*g Al: Pickle aluminum metal in warm
(1 +1) HC1 to an exact weight of 0.100 g. Dissolve in 10 mL cone. HC1 and 2 mL
cone, nitric acid, heating to effect solution. Continue heating until volume is reduced
to 4 mL. Cool and add 4 mL ASTM type I water. Heat until the volume is reduced
to 2 mL. Cool and dilute to 100 mL with ASTM type I water.
7.3.2 Antimony solution, stock, 1 mL = 1000 pig Sb: Dissolve 0.100 g antimony powder
in 2 mL (1 + 1) nitric acid and 0.5 mL cone, hydrochloric acid, heating to effect
solution. Cool, add 20 ASTM type I water and 0.15g tartaric acid. Warm the solu-
tion to dissolve the white precipitate. Cool and dilute to 100 mL with ATSM type I
water.
7.3.3 Arsenic solution, stock, 1 mL = 1000 ^g As: Dissolve 0.1320 g As2O3 in a mixture
of 50 mL ASTM type I water and 1 mL cone, ammonium hydroxide. Heat gently to
dissolve. Cool and acidify the solution with 2 mL cone, nitric acid. Dilute to 100
mL with ASTM type I water.
7.3.4 Beryllium solution, stock 1 mL = 500 fig Be: Dissolve 1.965 g BeSO4.4H2o (DO
NOT DRY) in 50 mL ASTM Type I Water. Add 2 mL cone, nitric acid. Dilute to
200 mL with ASTM type I water.
7.3.5 Cadmium solution, stock, 1 mL = 1000 fig Cd: Pickle Cd metal in (1+9) nitric acid
to an exact weight of 0.100 g. Dissolve in 5 mL (1 + 1) nitric acid, heating to effect
solution. Cool and dilute to 100 mL with ASTM type I water.
7.3.6 Chromium solution, stock, 1 mL = 1000 fig Cr: Dissolve 0.1923g CrO3 in a solu-
tion mixture of 10 mL ASTM type I water and 1 mL cone, nitric acid. Dilute to 100
mL with ASTM type I water.
7.3.7 Cobalt solution, stock 1 mL = 1000 /ig Co: Pickle Co metal in (1+9) nitric acid to
an exact weight of 0.100 g. Dissolve in 5 mL (1 + 1) nitric acid, heating to effect
solution. Cool and dilute to 100 mL with ASTM type I water.
7.3.8 Copper solution, stock, 1 mL = 1000 /xg Cu: Pickle Cu metal in (1 +9) nitric acid to
an exact weight of 0.100 g. Dissolve in 5 mL (1 +1) nitric acid, heating to effect
solution. Cool and dilute to 100 mL with ASTM type I water.
-------
Method 200.9
7.3.9 Iron solution, stock, 1 mL = 1000 fig Fe: Pickle Fe metal in (1+9) hydrochloric
acid to an exact weight of 0.100 g. Dissolve in 10 mL (1 +1) hydrochloric acid,
heating to effect solution. Cool and dilute to 100 mL with ASTM type I water.
7.3.10 Lead solution, stock, 1 mL = lOOOAigPb: Dissolve 0.1599 g PbNO3 in 5 mL(l + l)
nitric acid. Dilute to 100 mL with ASTM type I water.
7.3.11 Manganese solution, stock, 1 mL = 1000 pig Mn: Pickle manganese flake in (1 +9)
nitric acid to an exact weight of 0.100 g. Dissolve in 5 mL (1 +1) nitric acid, heating
to effect solution. Cool and dilute to 100 mL with ASTM type I water.
7.3.12 Nickel solution, stock, 1 mL = 1000 /xg Ni: Dissolve 0.100 g nickel power in 5 mL
cone, nitric acid, heating to effect solution. Cool and dilute to 100 mL with ASTM
type I water.
7.3.13 Selenium solution, stock, 1 mL = 1000 itg Se: Dissolve 0.1405 g SeO2 in 20 mL
ASTM type I water. Dilute to 100 mL with ASTM type I water.
7.3.14 Silver solution, stock, 1 mL = 1000 /*g Ag: Dissolve 0.100 g silver metal in 5 mL
(1 + 1) nitric acid, heating to effect solution. Cool and dilute to 100 mL with ASTM
type I water. Store in amber container.
7.3.15 Thallium solution, stock 1 mL = 500 /xg Tl: Dissolve 0.1303 g T1NO3 in a solution
mixture of 10 mL ASTM type I water and 2 cone, nitric acid. Dilute to 200 mL with
ASTM type I water.
7.3.16 Tin solution, stock, 1 mL = 1000 ng Sn: Dissolve 0.100 g Sn shot in 20 mL (1 + 1)
hydrochloric acid, heating to effect solution. Cool and dilute to 100 mL with (1 +1)
hydrochloric acid.
7.3.17 Zinc solution, stock, 1 mL = 1000 ng Zn: Pickle zinc metal in (1+9) nitric acid to
an exact weight of 0.100 g. Dissolve in 5 mL (1 +1) nitric acid, heating to effect
solution. Cool and dilute to 100 mL with ASTM type I water.
7.4 Preparation of Calibration Standards: Fresh calibration standards (CAL Solution) should be
prepared every two weeks or as needed. Dilute each of the stock standard solutions to levels
appropriate to the operating range of the instrument using the appropriate acid diluent (see
note). The element concentrations in each CAL solution should be sufficiently high to produce
good measurement precision and to accurately define the slope of the response curve. The
instrument calibration should be initially verified using a quality control sample (Sect. 7.6).
NOTE: The appropriate acid diluent for dissolved elements in water samples is 1 %
HNO,. For total recoverable elements in waters the appropriate acid diluent is 2%
HNO, and I % HCI. Finally, the appropriate add diluent for total recoverable ele-
ments in solid samples is 2% HNO3 and 2% HCI. The reason for these different
diluents is to match the types of acids and the acid concentrations of the samples with
the acid present in the standards and blanks.
7.5 Blanks: Two types of blanks are required for this method. A calibration blank is used to
establish the analytical calibration curve and the laboratory reagent blank (LRB) is used to
12
-------
Method 200.9
assess possible contamination from the sample preparation procedure and to assess spectral
background. All diluent acids should be made from concentrated acids (Sects. 7.1.1, 7.1.4)
and ASTM type I water.
7.5.1 Calibration blank: Consists of the appropriate acid diluent (Sect. 7.4 note)
(HCl/HNO3) in ASTM type I water.
7.5.2 Laboratory reagent blank (preparation blank) must contain all the reagents in the same
volumes as used in processing the samples. The preparation blank must be carried
through the entire sample digestion and preparation scheme.
7.6 Quality Control Sample: Quality control samples are available from various sources. Dilute
(with the appropriate acid (HC1/HNO3) blank solution) an appropriate aliquot of analyte such
that the resulting solution will result in an absorbance of approximately 0.1.
7.7 Laboratory Fortified Blank: To an aliquot of laboratory reagent blank, add an aliquot of the
stock standard to provide a final concentration which will produce an absorbance of approxi-
mately 0.1 for the analyte. The fortified blank must be carried through the entire sample
digestion and preparation scheme.
8. SAMPLE COLLECTION, PRESERVATION AND STORAGE
8.1 Prior to sample collection, consideration should be given to the type of data required so that
appropriate preservation and pretreatment steps can be taken. Filtration, acid preservation etc.
should be performed at the time of sample collection or as soon thereafter as practically
possible.
8.2 For the determination of dissolved elements, the sample should be filtered through a 0.45-/mi
membrane filter. Use a portion of the sample to rinse the filter assembly, discard and then
collect the required volume of filtrate. Acidify the filtrate with (1 +1) nitric acid immediately
following filtration to a pH of less than two.
8.3 For the determination of total recoverable elements in aqueous samples, acidify with (1 + 1)
nitric acid at the time of collection to a pH of less than two. The sample should not be filtered
prior to analysis.
NOTE: Samples that cannot be add preserved at the time of collection because of
sampling limitations or transport restrictions, should be acidified with nitric acid to pH
< 2 upon receipt in the laboratory (normally, 3 mL of (1 + 1) nitric acid per liter of
sample is sufficient for most ambient and drinking water samples). Following acidifica-
tion, the sample should be held for a minimum of 16 h before withdrawing an aliquot
for sample processing.
8.4 Solid samples usually require preservation prior to analysis other than storage at 4°C.
9. CALIBRATION AND STANDARDIZATION
9.1 Calibration: Demonstration and documentation of acceptable initial calibration is required
before any samples are analyzed and is required periodically throughout sample analysis as
13
-------
Method 200.9
dictated by results of continuing calibration checks. After initial calibration is successful, a
calibration check is required at the beginning and end of each period during which analyses are
performed
9.1.1 Initiate proper operating configuration of instrument and data system. Allow a period
of not less than 30 min for the instrument to warm up if an EDL is to be used.
9.1.2 Instrument stability must be demonstrated by analyzing a standard solution of a con-
centration 20 times the IDL a minimum of five times with the resulting relative stan-
dard deviation of absorbance signals less than 5%.
9.1.3 Initial calibration. The instrument must be calibrated for the analyte to be determined
using the calibration blank (Sect. 7.5.1) and calibration standards prepared at three or
more concentration levels within the linear dynamic range of the analyte.
9.2 Instrument Performance: Check the performance of the instrument and verify the calibration
using data gathered from analyses of calibration blanks, calibration standards and the quality
control sample.
9.2.1 After the calibration has been established, it must be initially verified for the analyte
by analyzing the QCS (Sect. 7.6). If measurements exceed ± 10% of the established
QCS value, the analysis should be terminated, the source of the problem identified
and corrected, the instrument recalibrated, and the new calibration must be verified
before continuing analyses.
9.2.2 To verify that the instrument is properly calibrated on a continuing basis, analyze the
calibration blank and an intermediate concentration calibration standard as surrogate
samples after every ten analyses. The results of the analyses of the standard will
indicate whether the calibration remains valid. If the indicated concentration of any
analyte deviates from the true concentration by more than 10%, the instrument must
be recalibrated and the response of the QCS checked as in Sect. 9.2.1. After the QCS
sample has met specifications, the previous ten samples must be reanalyzed in groups
of five with an intermediate concentration calibration standard analyzed after every
fifth sample. If the intermediate concentration calibration standard is found to deviate
by more than 10%, the analyst is instructed to identify the source of instrumental
drift.
NOTE: If the sample matrix is responsible for the calibration drift and/or the sample
matrix is affecting analyte response, it may be necessary to perform standard additions
in order to assess an analyte concentration (Sect. 11.5).
10. QUALITY CONTROL (QC)
10.1 Formal Quality Control: The minimum requirements of this QC program consist of an initial
demonstration of laboratory capability, and the analysis of laboratory reagent blanks and
fortified blanks and samples as a continuing check on performance. The laboratory is required
to maintain performance records that define the quality of the data thus generated.
14
-------
Method 200.9
10.2 Initial Demonstration of Performance
10.2.1 The initial demonstration of performance is used to characterize instrument perfor-
mance (MDLs and linear calibration ranges) for analyses conducted by this method.
10.2.2 Method detection limits (MDL): The method detection limit should be established for
the analyte, using reagent water (blank) fortified at a concentration of two to five
times the estimated detection limit3. To determine MDL values, take seven replicate
aliquots of the fortified reagent water and process through the entire analytical meth-
od. Perform all calculations defined in the method and report the concentration values
in the appropriate units. Calculate the MDL as follows:
MDL = (0 x (S)
where:
t = Student's t value for a 99 % confidence level and a
standard deviation estimate with n-l degrees of freedom
[t = 3.14/or seven replicates],
S = standard deviation of the replicate analyses.
Method detection limits should be determined every six months or whenever a
significant change in background or instrument response is expected.
10.2.3 Linear calibration ranges: Linear calibration ranges are metal dependent. The upper
limit of the linear calibration range should be established by determining the signal
responses from a minimum of four different concentration standards, one of which is
close to the upper limit of the linear range. The linear calibration range which may
be used for the analysis of samples should be judged by the analyst from the resulting
data. Linear calibration ranges should be determined every six months or whenever a
significant change in instrument response maybe expected.
10.3 Assessing Laboratory Performance: Reagent and Fortified Blanks
10.3.1 Laboratory reagent blank (LRB): The laboratory must analyze at least one LRB
(Sect. 7.5.2) with each set of samples. Reagent blank data are used to assess contami-
nation from the laboratory environment and to characterize spectral background from
the reagents used in sample processing. If an analyte value in the reagent blank
exceeds its determined MDL, then laboratory or reagent contamination should be
suspected. Any determined source of contamination should be corrected and the
samples reanalyzed.
10.3.2 Laboratory fortified blank (LFB): The laboratory must analyze at least one LFB
(Sect. 7.7) with each set of samples. Calculate accuracy as percent recovery (Sect.
10.4.2). If the recovery of any analyte falls outside the control limits (Sect. 10.3.3),
that analyte is judged out of control, and the source of the problem should be identi-
fied and resolved before continuing analyses.
10.3.3 Until sufficient data (usually a minimum of 20 to 30 analyses) become available, a
laboratory should assess laboratory performance against recovery limits of 80-120%.
When sufficient internal performance data become available, develop control limits
15
-------
Method 200.9
from the percent mean recovery (x) and the standard deviation (S) of the mean recov-
ery. These data are used to establish upper and lower control limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x — 3S
After each 5-10 new recovery measurements, new control limits should be calculated
using only the most recent 20 to 30 data points.
NOTE: Antimony and Aluminum do manifest relatively low percent recoveries (see
Table I A, NBS River Sediment 1645).
10.4 Assessing Analyte Recovery: Laboratory Fortified Sample Matrix
10.4.1 The laboratory must fortify a minimum of 10% of the samples or one fortified sample
per set, whichever is greater. Ideally for solid samples, the concentration added
should be approximately equal to 0.1 abs units after the solution has been diluted. In
other words if the sample (after dilution) results in an absorbance of 0.05, ideally the
laboratory fortified sample will result in an absorbance of 0.150 (after dilution). Over
time, samples from all routine sample sources should be fortified.
10.4.2 Calculate the percent recovery for the analyte, corrected for background concentra-
tions measured in the unfortified sample, and compare these values to the control
limits established in Sect. 10.3.3 for the analyses of LFBs. Fortified recovery calcu-
lations are not required if the fortified concentration is less than 10% of the sample
background concentration. Percent recovery may be calculated in units appropriate to
the matrix, using the following equation:
R = _ ^1x100
S
where:
R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
S = concentration equivalent of the fortified sample.
10.4.3 If the recovery of the analyte on the fortified sample falls outside the designated
range, and the laboratory performance on the LFB for the analyte is shown to be in
control (Sect. 10.3) the recovery problem encountered with the fortified sample is
judged to be matrix related (Sect. 4), not system related. The data obtained for that
analyte should be verified with the methods of standard additions (Sect. 11.5).
10.5 Quality Control Samples (QCS): Each quarter, the laboratory should analyze one or more
QCS (if available). If criteria provided with the QCS are not met, corrective action should be
taken and documented.
-------
Method 200.9
11. PROCEDURE
11.1 Sample Preparation: Dissolved Elements
11.1.1 For the determination of dissolved elements in drinking water, wastewater, ground
and surface waters, take a 100-mL (± 1 mL) aliquot of the filtered acid preserved
sample, and add 1 mL of concentrated nitric acid. The sample is now ready for
analysis. Allowance should be made in the calculations for the appropriate dilution
factors.
NOTE: If a precipitate is formed during acidification, transport or storage, the sample
aliquot must be treated using the procedure in Sect. 11.2.1 prior to analysis.
11.2 Sample Preparation: Total Recoverable Elements.
11.2.1 For the determination of total recoverable elements in water or waste water, take a
100-mL (±1 mL) aliquot from a well mixed, acid preserved sample and transfer it to
a 250-mL Griffin beaker. Add 1 mL of concentrated HNO3 .and 0.5 mL of con-
centrated HC1. Heat the sample on a hot plate at 85 °C until the volume has been
reduced to approximately 20 mL, ensuring that the sample does not boil. (A spare
beaker containing 20 mL of water can be used as a gauge.)
NOTE: For proper heating adjust the temperature control of the hotplate such that an
uncovered beaker containing 50 mL of water located in the center of the hot plate can
be maintained at approximately but no higher than 85 °C. Evaporation time for 100 mL
of sample at 85 °C is approximately 2 h with the rate of evaporation rapidly increasing
as the sample volume approaches 20 mL.
Cover the beaker with a watch glass and reflux for 30 min. Slight boiling may occur
but vigorous boiling should be avoided. Allow to cool and quantitatively transfer to
either a 50-mL volumetric or a 50-mL class A stoppered graduated cylinder. Dilute
to volume with ASTM type I water and mix. Centrifuge the sample or allow to stand
overnight to separate insoluble material. The sample is now ready for analysis. Prior
to the analysis of samples the calibration standards must be analyzed and the calibra-
tion verified using a QC sample (Sect. 9). Once the calibration has been verified, the
instrument is ready for sample analysis. Because the effects of various matrices on
the stability of diluted samples cannot be characterized, samples should be analyzed as
soon as possible after preparation.
11.2.2 For the determination of total recoverable elements in solid samples (sludge, soils,
and sediments), mix the sample thoroughly to achieve homogeneity and weigh accu-
rately a 1.0 ± 0.01 g portion of the sample. Transfer to a 250-mL Phillips beaker.
Add 4 mL (1 +1) nitric acid and 10 mL (1 +4) HC1. Cover with a watch glass. Heat
the sample on a hot plate and gently reflux for 30 min. Very slight boiling may
occur, however, vigorous boiling must be avoided to prevent the loss of the HC1
azeotrope.
77
-------
Method 200.9
NOTE: For proper heating adjust the temperature control of the hot plate such that an
uncovered Griffin beaker containing 50 mL of water located in the center of the hot
plate can be maintained at a temperature approximately but no higher than 85 °C.
Allow the sample to cool and quantitatively transfer to either 100-mL (± 1 mL)
volumetric flask or a 100-mL class A stoppered graduate cylinder. Dilute to volume
with ASTM type I water and mix. Centrifuge the sample or allow to stand overnight
to separate insoluble material. The sample is now ready for analysis. Prior to the
analysis of samples the calibration standards must be analyzed and the calibration
verified using a QC sample (Sect. 9). Once the calibration has been verified, the
instrument is ready for sample analysis. Because the effects of various matrices on
the stability of diluted samples cannot be characterized, samples should be analyzed as
soon as possible after preparation.
NOTE: Determine the percent solids in the sample for use in calculations and for
reporting data on a dry weight basis.
11.2.3 Appropriate digestion procedures for biological tissues should be utilized prior to
sample analysis.
11.3 For every new or unusual matrix, it is highly recommended that an inductively coupled plasma
atomic emission spectrometer be used to screen for high element concentrations. Information
gained from this may be used to prevent potential damage of the instrument and better estimate
which elements may require analysis by graphite furnace.
11.4 Samples having concentrations higher than the established linear dynamic range should be
diluted into range and reanalyzed. If methods of standard additions are required, follow the
instructions in Sect. 11.5.
11.5 Standard Additions: If methods of standard addition are required, the following procedure is
recommended.
11.5.1 The standard addition technique4 involves preparing new standards in the sample
matrix by adding known amounts of standard to one or more aliquots of the processed
sample solution. This technique compensates for a sample constituent that enhances
or depresses the analyte signal thus producing a different slope from that of the
calibration standards. It will not correct for additive interference which causes a
baseline shift. The simplest version of this technique is the single-addition method.
The procedure is as follows. Two identical aliquots of the sample solution, each of
volume Vx, are taken. To the first (labeled A) is added a small volume Vs, of a
standard analyte solution of concentration Cs. To the second (labeled B) is added the
same volume V,. of the solvent. The analytical signals of A and B are measured and
corrected for nonanalyte signals. The unknown sample concentration cx is calculated:
18
-------
Method 200.9
where SA and SB are the analytical signals (corrected for the blank) of solutions A and
B, respectively. Vs and cs should be chosen so that SA is roughly twice SB on the
average. It is best if Vs is made much less than Vs, and thus cs is much greater than
cx, to avoid excess dilution of the sample matrix. If a separation or concentration step
is used, the additions are best made first and carried through the entire procedure.
For the results from this technique to be valid, the following limitations must be taken
into consideration:
1. The analytical curve must be linear.
2. The chemical form of the analyte added must respond the same as the
analyte in the sample.
3. The interference effect must be constant over the working range of con-
cern.
4. The signal must be corrected for any additive interference.
12. CALCULATIONS
12.1 Do not report element concentrations below the determined MDL.
12.2 For aqueous samples prepared by total recoverable procedure (Sect. 11.2.1), multiply solution
concentrations by the appropriate dilution factor. Round the data to the tenths place and report
the data in /^g/L with up to three significant figures.
12.3 For solid samples prepared by total recoverable procedure (Sect. 11.2.2) round the solution
concentration (/xg/L in the analysis solution) to the tenths place and multiply by the dilution
factor. Data should be reported to a tenth mg/kg up to three significant figures taking into
account the percent solids if the data are reported on a dry weight* basis.
12.4 If additional dilutions were performed, the appropriate dilution factor must be applied to
sample values.
12.5 The QC data obtained during the analyses provide an indication of the quality of the sample
data and should be provided with the sample results.
13. PRECISION AND ACCURACY
13.1 Instrument operating conditions used for single laboratory testing of the method and MDLs are
listed in Table 2.
13.2 Data obtained from single laboratory testing of the method are summarized in Table 1A-C for
three solid samples consisting of SRM 1645 River Sediment, EPA Hazardous Soil and EPA
Electroplating Sludge. Samples were prepared using the procedure described in Sect. 11.2.2.
For each matrix, five replicates were analyzed and an average of the replicates used for
determining the sample background concentration. Two further pairs of duplicates were
fortified at different concentration levels. The sample background concentration, mean spike
The dry weight should be determined on a separate sample aliquot if the sample is available. The
dry weight can be determined by transferring a uniform 1-g aliquot to an evaporating dish and drying the
sample to a constant weight at 103-105 °C.
19
-------
Method 200.9
percent recovery, the standard deviation of the average percent recovery and the relative
percent difference between the duplicate fortified determinations are listed in Table 1A-C. In
addition, Table 1D-F contains a single laboratory testing of the method in aqueous media
including drinking water, pond water and well water. Samples were prepared using the
procedure described in Sect. 11.2.1. For each aqueous matrix, five replicates were analyzed
and an average of the replicates used for determining the sample background concentration.
Four samples were fortified at the levels reported in Table ID-IF. A percent relative standard
deviation is reported in Table ID-IF for the fortified samples. An average percent recovery
is also reported in Tables 1D-F.
20
-------
Method 200.9
References
1. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, revised January, 1976.
2. "Proposed OSHA Safety and Health Standards, Laboratories," Occupational Safety and Health
Administration, Federal Register, July 24, 1986.
3. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
4. Winefordner, J.D., "Trace Analysis: Spectroscopic Methods for Elements, "Chemical Analy-
sis, Vol. 46, pp. 41-42.
5. Waltz, B,, G. Schlemmar and J.R. Mudakavi, JAAS. 1988, 3, 695.
-------
Nj
No
Table 1A. Precision and Recovery Data For NBS River Sediment 1645
Certified
Value +
22600
(51)
(66)
10.2
29600
109
785
1.5
-
—
A verage
Sed. Cone.
(mg/kgj
6810
25.8
69.2
10.8
32800
132
893
0.7
1.7
439
% RSD
4.6
8.2
3.4
3.7
1.6
4.8
5.1
20.4
3.1
4.4
Average
Percent
Recovery
(20 mg/kgr S(r)
*
74.9
69.8
115.3
*
99.1
*
96.0
101.8
-
-
8.3
19.0
2.6
-
14.2
-
15.9
3.8
-
Average
Percent
Recovery
RPD (100 mg/kgr S(r)
-
9.5
12.0
4.0
-
0
-
45.2
9.7
-
*
99.0
89.2
1 10.7
#
111.5
103.2
105.4
93.5
_
.
1.5
4.3
0.7
-
3.6
26.4
4.0
1.9
_
RPD
_
2.7
7.3
1.7
-
2.6
4.7
10.7
5.6
_
Solid Sample
Aluminum
Antimony
Arsenic
Cadmium
Chromium
Copper
Manganese
Selenium
Silver
Tin
% RSD Percent relative standard deviation (n = 5)
S(r) Standard deviation of average percent recovery
RPD Relative percent difference between duplicate recovery determinations
* Fortified concentration < 10% of sample concentration
Not determined
* Values in parenthesis are noncertified
* Fortified concentration
o
Q.
No
O
O
CO
-------
Table 1B. Precision and Recovery Data For EPA Hazardous Soil
Average
Average Percent
Sed. Cone. Recovery
Solid Sample (mg/kg) % RSD (20 mg/kgf
Aluminum 6410 3.3 *
Antimony 4.6 14.7 61.4
Arsenic 8.7 4.6 109.8
Cadmium 1.8 10.3 115.4
Chromium 84.0 4.2 95.5
Copper 127 4.3 108.0
Manganese 453 6.0 *
Selenium 0.6 7.5 95.0
Silver 0.9 18.5 100.1
Tin 18.4 3.7
% RSD Percent relative standard deviation (n = 5)
S(r) Standard deviation of average percent recovery
S(r)
-
2.7
2.1
0.8
33.8
15.2
-
8.4
3.8
-
Average
Percent
Recovery
RPD (100 mg/kgy S(r)
-
7.4
3.5
1.4
17.9
2.6
-
24.1
10.2
-
*
60.9
103.7
99.0
120.8
117.7
99.2
96.9
93.5
-
-
1.7
1.5
4.3
6.6
5.4
13.9
3.3
1.3
-
RPD
-
7.1
3.6
12.1
8.9
5.7
1.6
9.7
3.8
-
RPD Relative percent difference between duplicate recovery determinations
* Fortified concentration < 1 0% of sample concentration
Not determined
" Fortified concentration
I
KJ
o
p
io
-------
Table 1C. Precision and Recovery Data For EPA Electroplating Sludge 286
Average
Sed. Cone.
Solid Sample (mg/kg)
Aluminum 6590
Antimony 7.7
Arsenic 33.7
Cadmium 119
Chromium 8070
Copper 887
Manganese 320
Selenium 0.8
Silver 6.5
Tin 21.8
% BSD Percent relative standard deviation (n = 5)
S(r) Standard deviation of average percent recovery
RPD Relative percent difference between duplicate recovery determinations
* Fortified concentration <10% of sample concentration
Not determined
" Fortified concentration
%RSD
2.7
3.9
2.7
1.3
4.5
1.6
1.6
6.7
2.3
3.2
Average
Percent
Recovery
(20 mg/kgr
*
68.6
87.6
81.9
*
|*
*
99.4
102.8
-
S(r)
-
2.3
2.6
7.9
-
-
-
0.8
2.5
-
RPD
-
5.7
1.7
3.0
-
-
-
2.3
5.3
-
Average
Percent
Recovery
(100 mg/kgr
#
60.7
100.2
112.5
*
99.5
101.0
96.8
92.3
-
S(r)
-
3.1
1.5
3.9
-
21.9
6.4
0.7
1.9
-
RPD
-
12.8
3.1
4.7
-
6.0
4.0
1.9
5.4
-
o
a
§
to
-------
Method 200.9
Table 1 D. Precision and
Average
Cone.
Element (vg/U
Ag
Al
As3
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb
Sb3
Se3
Sn3
Tl
<0.5
550
3.2
0.05
<0.05
<0.7
0.75
2.98
773
751
2.11
1.24
<0.8
<0.6
<1.7
<0.7
Recovery Data for Pond Water
Fortified Cone.
% RSD (fjg/L)1
1.25
T.2
4.1 10
36.4 2.5
0.5
10
8.7 2.5
11.2 10
5.7
2.2
6.8 20
20.5 25
* 25
25
50
75.0 50
< Sample concentration less than the established method
*
1
2
3
Not determined on sample concentrations less than the
% RSD at
Fortified
Cone.2
3.7
-
0.8
14.0
4.5
2.8
1.8
2.9
-
-
1.6
1.8
0.4
1.6
3.3
5.2
detection limit.
method.
Average
Percent
Recovery
107.5
.
100.5
90.0
99.1
97.3
98.5
101.9
-
-
105.6
101.6
115.2
97.8
117.5
101.0
Fortified sample concentration based on 100 ml sample volumes.
RSD are reported on 50
Electrodeless discharge
ml sample volumes.
lamps were used.
25
-------
Method 200.9
Table 1E.
Element
Ag
Al
As
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb
Sb3
Se3
Sn3
Tl
< Sample
Precision and Recovery Data For Drinking Water
%RSD at
Average Fortified Fortified Average Percent
Cone. (pg/L) % RSD Cone. (fjg/U1 Cone.2
<0.5
163.6
0.5
<0.02
<0.05
<0.7
<0.1
2.6
9.1
0.9
0.8
<0.7
<0.8
<0.6
<1.7
<0.7
concentration less
* Not determined on sample
1 Fortified
2 RSD are
*
2.5
10.5
#
*
*
*
7.3
17.6
1.3
32.7
#
#
*
#
#
1.25
150
10
2.5
0.5
10
2.5
10
150
2.5
20
10
15
25
50
20
than the established method detection
5.6
6.4
0.6
9.4
6.3
3.9
3.1
1.2
5.9
0.7
4.3
4.0
14.7
1.5
0.4
2.8
limit.
Recovery
94.6
111.7
88.4
106.0
105.2
88.5
105.7
111.5
107.8
96.7
103.8
101.8
101.4
88.9
100.7
95.4
concentrations less than the method.
sample concentration based on 100
mL sample volumes
reported on 50 mL sample volumes.
3 Electrodeless discharge lamps were used.
26
-------
Method 200.9
Table 1F. Precision and Recovery Data For Well Water
Average
Cone. (vg/U
<0.5
14.4
0.9
<0.02
1.8
4.0
<0.1
35.9
441
3580
11.8
%RSD
*
26.7
14.2
#
11.9
2.9
#
1.2
6.6
2.7
3.2
Fortified Cone.
(tig/U1
1.25 '
150
10
2.5
0.5
10
2.5
10
-
-
20
% RSD at
Fortified
Cone.2
3.6
1.5
2.1
3.4
4.6
1.0
4.0
0.6
-
-
4.0
Average Percent
Recovery
108.3
97.1
101.6
103.7
109.3
95.8
102.6
90.2
-
-
105.7
Element
Ag
Al
As3
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb <0.7 * 25 0.7 102.2
Sb3 <0.8 * 25 1.2 114.3
Se3 <0.6 * 25 1.2 95.9
Sn3 <1.7 * 50 3.0 106.1
Tl <0.7 * 50 1.4 98.0
< Sample concentration less than the established method detection limit.
* Not determined on sample concentration less than the method detection limit.
1 Fortified sample concentration based on 100 mL sample volume.
2 RSD reported on 50 mL sample volume.
3 Electrodeless discharge lamps were used.
27
-------
Method 200.9
Table 2. Recommended Graphite Furnace Operating Conditions and Recommended
Matrix Modifier1^3
Element
Ag
Al
As7
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb
Sb7
Se7
Sn7
Tl
Zn
Temperature
Wave-length
328.1
309.3
193.7
234.9
228.8
242.5
357.9
324.8
248.3
279.5
232.0
283.3
217.6
196.0
286.3
276.8
213.9
Slit
0.7
0.7
0.7
0.7
0.7
0.2
0.7
0.7
0.2
0.2
0.2
0.7
0.7
2.0
0.7
0.7
0.7
Char
1000
1700
1300
1200
800
1400
1650
1300
1400
1400
1400
1250
1100
1000
1400s
1000
700
(Cf Atom.
1800
2600
2200
2500
1600
2500
26006
26006
2400
2200
2500
2000
2000
2000
2300
1600
1800
MDL4 fug/L)
0.59
7.89
0.5
0.02
0.05
0.7
0.1
0.7
0.3
0.6
0.7
0.8
0.6
1.7
0.7
0.3
Matrix Modifier = 0.015 mg Pd + 0.01 mg Mg(NO3)2.
A 5% H2 in Ar gas mix is used during the dry and char steps at 300 mL/min for all elements.
A cool down step between the char and atomization is recommended.
Obtained using a 20 /A. sample size and stop flow atomization.
Actual char and atomization temperatures may vary from instrument to instrument and are best
determined on an individual basis. The actual drying temperature may vary depending on the
temperature of the water used to cool the furnace.
A 7 second atomization is necessary to quantitatively remove the analyte from the graphite
furnace.
An electrodeless discharge lamp was used for this element.
An additional low temperature (approximately 200°C) prechar is recommended.
Pd modifier was determined to have trace level contamination of this element.
28
-------
Method 505
Analysis of Organohalide Pesticides and
Commercial Polychlorinated Biphenyl (PCB)
Products in Water by Microextraction
and Gas Chromatography
Revision 2.0 - EPA EMSL-Ci
T.W. Winfield - Method 505, Revision 1.0 (1986)
T.W. Winfield - Method 505, Revision 2.0 (1989)
-------
-------
Method 505
Analysis of Organohalide Pesticides and Commercial Polychlorinated
Biphenyl (PCB) Products in Water by Microextraction
and Gas Chromatography
1. SCOPE AND APPLICA TION
1.1 This method is applicable to the determination of the following analytes in finished drinking
water, drinking water during intermediate stages of treatment, and the raw source water:'"3
1.2
1.3
Analyte CAS No.
Alachlor 15972-60-8
Aldrin 309-00-2
Atrazine 1912-24-9
Chlordane 57-74-9
a-Chlordane 5103-71-9
7-Chlordane 5103-74-2
Dieldrin 60-57-1
Endrin 72-20-8
Heptachlor 76-44-8
Heptachlor Epoxide 1024-57-3
Hexachlorobenzene 118-74-1
Hexachlorocyclopentadiene 77-74-4
Lindane 58-89-9
Methoxychlor 72-43-5
cis-Nonachlor 5103-73-1
trans-Nonachlor 39765-80-5
Simazine 122-34-9
Toxaphene 8001-35-2
Aroclor1016 12674-11-2
Aroclor1221 11104-28-2
Aroclor1232 11141-16-5
Aroclor1242 53469-21-9
Aroclor1248 12672-29-6
Aroclor1254 11097-69-1
Aroclor1260 11096-82-5
For compounds other than the above mentioned analytes or for other sample sources, the
analyst must demonstrate the applicability of the method by collecting precision and accuracy
data on fortified samples (i.e., groundwater, tap water) and provide qualitative confirmation of
results by Gas Chromatography/Mass Spectrometry (GC/MS), or by GC analysis using
dissimilar columns.4^
Method detection limits (MDL)6 for the above organohalides and Aroclors have been ex-
perimentally determined (Sect. 13.1). Actual detection limits are highly dependent upon the
characteristics of the gas chromatographic system used (e.g. column type, age, and proper
conditioning; detector condition; and injector mode and condition).
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Method 505
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use
of GC and in the interpretation of gas chromatograms. Each analyst must demonstrate the
ability to generate acceptable results with this method using the procedure described in Sect.
II.
1.5 Analytes that are not separated chromatographically, i.e., analytes which have very similar
retention times, cannot be individually identified and measured in the same calibration mixture
or water sample unless an alternative technique for identification and quantitation is used (Sect.
11.4).
1.6 When this method is used to analyze unfamiliar samples for any or all of the analytes above,
analyte identifications should be confirmed by at least one additional qualitative technique.
1.7 Degradation of Endrin, caused by active sites in the injection port and GC columns, may
occur. This is not as much a problem with new capillary columns as with packed columns.
However, high boiling sample residue in capillary columns will create the same problem after
injection of sample extracts.
2. SUMMARY OF METHOD
2.1 Thirty-five mL of sample are extracted with 2 mL of hexane. Two /iL of the extract are then
injected into a gas chromatograph equipped with a linearized electron capture detector for
separation and analysis. Aqueous calibration standards are extracted and analyzed in an
identical manner in order to compensate for possible extraction losses.
2.2 The extraction and analysis time is 30 to 50 min per sample depending upon the analytes and
the analytical conditions chosen. (See Sect. 6.9.)
3. DEFINITIONS
3.1 Laboratory duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.2 Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.3 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.4 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
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Method 505
3.5 Laboratory performance check solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.6 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the
laboratory is capable of making accurate and precise measurements at the required method
detection limit.
3.7 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.8 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.9 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.10 Calibration standard (CAL): a solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.11 Quality control sample (QCS): a sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or
environmental samples. The QCS is obtained from a source external to the laboratory, and is
used to check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.2 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing wih tap and reagent water. Drain
dry, and heat in an oven or muffle furnace at 400°C for 1 hr. Do not heat volumetric
ware. Thermally stable materials might not be eliminated by this treatment. Thor-
ough rinsing with acetone may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any accumulation
of dust or other contaminants. Store inverted or capped with aluminum foil.
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Method 505
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required. WARN-
ING: When a solvent is purified, stabilizers put into the solvent by the manufacturer
are removed thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives put into the solvent by the manufacturer are removed thus
potentially reducing the shelf-life.
4.2 Interfering contamination may occur when a sample containing low concentrations of analytes
is analyzed immediately following a sample containing relatively high concentrations of
analytes. Between-sample rinsing of the sample syringe and associated equipment with hexane
can minimize sample cross contamination. After analysis of a sample containing high
concentrations of analytes, one or more injections of hexane should be made to ensure that
accurate values are obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are coextracted from the sample.
Also, note that all the analytes listed in the scope and application section are not resolved from
each other on any one column, i.e., one analyte of interest may be an interferent for another
analyte of interest. The extent of matrix interferences will vary considerably from source to
source, depending upon the water sampled. Cleanup of sample extracts may be necessary.
Positive identifications should be confirmed (Sect. 11.4).
4.4 It is important that samples and working standards be contained in the same solvent. The
solvent for working standards must be the same as the final solvent used in sample
preparation. If this is not the case, chromatographic comparability of standards to sample may
be affected.
4.5 Caution must be taken in the determination of endrin since it has been reported that the
splitless injector may cause endrin degradation.7 The analyst should be alerted to this possible
interference resulting in an erratic response for endrin.
4.6 Variable amounts of pesticides and commercial PCB products from aqueous solutions adhere to
glass surfaces. It is recommended that sample transfers and glass surface contacts be
minimized.
4.7 Aldrin, hexachlorocyclopentadiene and methoxychlor are rapidly oxidized by chlorine.
Dechlorination with sodium thiosulfate at time of collection will retard further oxidation of
these compounds.
4.8 WARNING: An interfering, erratic peak has been observed within the retention window of
heptachlor during many analyses of reagent, tap, and groundwater. It appears to be related to
dibutyl phthalate; however, the specific source has not yet been definitively determined. The
observed magnitude and character of this peak randomly varies in numerical value from
successive injections made from the same vial.
5. SAFETY
5.1 The toxicity and carcinogenicity of chemicals used in this method have not been precisely
defined; each chemical should be treated as a potential health hazard, and exposure to these
chemicals should be minimized. Each laboratory is responsible for maintaining awareness of
OSHA regulations regarding safe handling of chemicals used in this method. Additional
references to laboratory safety are available for the information of the analyst.8'"
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Method 505
5.2 The following organohalides have been tentatively classified as known or suspected human or
mammalian carcinogens: aldrin, commercial PCB products, chlordane, dieldrin, heptachlor,
hexachlorobenzene, and toxaphene. Pure standard materials and stock standard solutions of
these compounds should be handled in a hood or glovebox.
5.3 WARNING: When a solvent is purified, stabilizers put into the solvent by the manufacturer
are removed thus potentially making the solvent hazardous.
6. APPARATUS AND EQUIPMENT
6.1 Sample Containers: 40-mL screw cap vials (Pierce #13075 or equivalent) each equipped with
a size 24 cap with a flat, disc-like TFE facing backed with a polyethylene film/foam extrusion
(Fisher #02-883-3F or equivalent). Prior to use, wash vials and septa with detergent and rinse
with tap and distilled water. Allow the vials and septa to air dry at room temperature, place
the vials in a 400°C oven for one hour, then remove and allow to cool in an area known to be
free of organics.
6.2 Vials: auto sampler, screw cap with septa, 1.8 mL, Varian #96-000099-00 or equivalent or
any other autosampler vials not requiring more than 1.8 mL sample volumes.
6.3 Auto Sampler: Hewlett-Packard 7671 A, or equivalent.
6.4 Micro Syringes: 10 and 100 /nL.
6.5 Micro Syringe: 25 pL with a 2-inch by 0.006-inch needle; Hamilton 702N or equivalent.
6.6 Pipettes: 2.0 and 5.0 mL transfer.
6.7 Volumetric Flasks: 10 and 100 mL, glass stoppered.
6.8 Standard Solution Storage Containers: 15-mL bottles with PTFE-lined screw caps.
6.9 Gas Chromatograph: Analytical system complete with temperature programmable GC suitable
and split/splitless injector for use with capillary columns and all required accessories including
syringes, analytical columns, gases, a linearized electron capture detector and stripchart
recorder. A data system is recommended for measuring peak areas. Table 1 lists retention
times observed for method analytes using the columns and analytical conditions described
below.
6.9.1 Three gas chromatographic columns are recommended. Column 1 (Sect. 6.9.2)
should be used as the primary analytical column unless routinely occurring analytes
are not adequately resolved. Validation data presented in this method were obtained
using this column. Columns 2 and 3 are recommended for use as confirmatory
columns when GC/MS confirmation is not available. Alternative columns may be
used in accordance with the provisions described in Sect. 10.3.
6.9.2 Column 1 (Primary Column): 0.32 mm ID x 30 M long fused silica capillary with
chemically bonded methyl polysiloxane phase (DB-1, 1.0 jtm film, or equivalent).
Helium carrier gas flow is about 25 cm/sec linear velocity, measured at 180° with
9 psi column head pressure. The oven temperature is programmed from 180°C to
260°C at 4°C/min and held at 260°C until all expected compounds have eluted.
Injector temperature: 200°C. Splitless Mode: 0.5 min. Detector temperature:
290°C. Sample chromatograms for selected pesticides are presented in Figures 1
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Method 505
and 2. Chromatograms of the Aroclors, toxaphene, and technical chlordane are
presented in Figures 3 through 11.
6.9.3 Column 2 (alternative column 1): 0.32mm ID x 30 M long fused silica capillary
with a 1:1 mixed phase of dimethyl silicone and polyethylene glycol (Durawax-DX3,
0.25/*m film, or equivalent). Helium carrier gas flow is about 25 cm/sec linear
velocity and oven temperature is programmed from 100°C to 210°C at 8°C/min, and
held at 210°C until all expected compounds have eluted. Then the post temperature is
programmed to 240°C at 8°C/min for 5 min.
6.9.4 Column 3 (alternative column 2): 0.32mm ID x 25 M long fused silica capillary
with chemically bonded 50:50 Methyl-Phenyl silicone (OV-17, 1.5/mi film thickness,
or equivalent). Helium carrier gas flow is about 40 cm/sec linear velocity and oven
temperature is programmed from 100°C to 260°C at 4°C/min and held at 260°C until
all expected compounds have eluted.
7. REAGENTS AND CONSUMABLE MATERIALS
WARNING: When a solvent is purified stabilizers put into the solvent by the
manufacturer are removed thus potentially making the solvent hazardous. Also, when a
solvent is purified, preservatives put into the solvent by the manufacturer are removed
thus potentially making the shelf-life short.
7.1 Reagents
7.1.1 Hexane extraction solvent: UV Grade, Burdick and Jackson #216 or equivalent.
7.1.2 Methyl alcohol: ACS Reagent Grade, demonstrated to be free of analytes.
7.1.3 Sodium chloride, NaCl, ACS Reagent Grade: For pretreatment before use, pulverize
a batch of NaCl and place in a muffle furnace at room temperature. Increase the
temperature to 400°C and hold for 30 min. Place in a bottle and cap.
7.1.4 Sodium thiosulfate, Na2S2O3, ACS Reagent Grade: For preparation of solution
(0.04 g/mL), mix 1 g of Na2S2O3 with reagent water and bring to 25-mL volume in a
volumetric flask.
7.2 Reagent Water: Reagent water is defined as water free of interference when employed in the
procedure described herein.
7.2.1 A Millipore Super-Q Water System or its equivalent may be used to generate
deionized reagent water.
7.2.2 Test reagent water each day it is used by analyzing it according to Sect. 11.
7.3 Stock Standard Solutions: These solutions may be obtained as certified solutions or prepared
from pure standard materials using the following procedures:
7.3.1 Prepare stock standard solutions (5000 /xg/mL) by accurately weighing about 0.0500 g
of pure material. Dissolve the material in methanol and dilute to volume in a 10-mL
volumetric flask. Larger volumes can be used at the convenience of the analyst.
When compound purity is assayed to be 96% or greater, the weight can be used
without correction to calculate the concentration of the stock standard. Commercially
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Method 505
prepared stock standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.3.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at
4°C and protect from light. Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior to preparing calibration
standards from them.
7.3.3 Stock standard solutions must be replaced after six months, or sooner if comparison
with check standards indicates a problem.
7.4 Primary Dilution Standard Solutions: Use stock standard solutions to prepare primary dilution
standard solutions that contain the analytes in methanol. The primary dilution standards should
be prepared at concentrations that can be easily diluted to prepare aqueous calibration
standards (Sect. 9.1.1) that will bracket the working concentration range. Store the primary
dilution standard solutions with minimal headspace and check frequently for signs of
deterioration or evaporation, especially just before preparing calibration standards. The
storage time described for stock standard solutions in Sect. 7.3.3 also applies to primary
dilution standard solutions.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Sample Collection
8.1.1 Collect all samples in 40-mL bottles into which 3 mg of sodium thiosulfate crystals
have been added to the empty bottles just prior to shipping to the sampling site.
Alternately, 75 /xL of freshly prepared sodium thiosulfate solution (0.04 g/mL) may
be added to empty 40-mL bottles just prior to sample collection.
8.1.2 When sampling from a water tap, open the tap and allow the system to flush until the
water temperature has stabilized (usually about 10 min). Adjust the flow to about 500
mL/min and collect samples from the flowing stream.
8.1.3 When sampling from a well, fill a wide-mouth bottle or beaker with sample, and
carefully fill 40-mL sample bottles.
8.2 Sample Preservation
8.2.1 The samples must be chilled to 4°C at the time of collection and maintained at that
temperature until the analyst is prepared for the extraction process. Field samples that
will not be received at the laboratory on the day of collection must be packaged for
shipment with sufficient ice to insure that they will be maintained at 4°C until arrival
at the laboratory.
8.3 Sample Storage
8.3.1 Store samples and extracts at 4°C until extraction and analysis.
8.3.2 Extract all samples as soon as possible after collection. Results of holding time studies
suggest that all analytes with the possible exception of heptachlor were adequately
stable for 14 days when stored under these conditions. In general, heptachlor showed
inconsistent results. If heptachlor is to be determined, samples should be extracted
within 7 days of collection. Analyte stability may be affected by the matrix;
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Method SOS
therefore, the analyst should verify that the preservation technique is applicable to the
samples under study.
9. CALIBRA TION AND STANDARDIZA TION
9.1 Establish GC operating parameters equivalent to those indicated in Sect. 6.9.
WARNING: Endrin is easily degraded in the injection port if the injection port
or front of the column is dirty. This is the result of buildup of high boiling residue
from sample injection. Check for degradation problems by injecting a mid-level
standard containing only endrin. Look for the degradation products ofendrin (endrin
ketone and endrin aldehyde). If degradation ofendrin exceeds 20%, take corrective
action before proceeding with calibration. Calculate percent breakdown as follows:
Total endrin degradation peak area (endrin aldehyde + endrin ketone)
Total endrin peak area (endrin + endrin aldehyde + endrine ketone)
9.2 At least three calibration standards are needed; five are recommended. One should contain
analytes at a concentration near but greater than the method detection limit for each compound;
the other two should be at concentrations that bracket the range expected in samples. For
example, if the MDL is 0.01 /*g/L, and a sample expected to contain approximately 0.10 ng/L
is to be analyzed, aqueous standards should be prepared at concentrations of 0.02 /ig/L, 0.10
Mg/L, and 0.20 /zg/L.
9.2.1 To prepare a calibration standard (CAL), add an appropriate volume of a secondary
dilution standard to a 35-mL aliquot of reagent water in a 40-mL bottle. Do not add
less than 20 fj.L of an alcoholic standard to the reagent water. Use a 25-/iL micro
syringe and rapidly inject the alcoholic standard into the middle point of the water
volume. Remove the needle as quickly as possible after injection. Mix by inverting
and shaking the capped bottle several times. Aqueous standards must be prepared
fresh daily.
9.2.2 Starting with the standard of lowest concentration, prepare, extract, and analyze each
calibration standard beginning with Sect. 11.2 and tabulate peak height or area
response versus the concentration in the standard. The results are to be used to
prepare a calibration curve for each compound by plotting the peak height or area
response versus the concentration. Alternatively, if the ratio of concentration to
response (calibration factor) is a constant over the working range (20% RSD or less),
linearity to the origin can be assumed and the average ratio or calibration factor can
be used in place of a calibration curve.
9.2.3 The working calibration curve or calibration factor must be verified on each working
day by the measurement of one or more calibration standards. If the response for an
analyte varies from the predicted response by more than ±20%, the test must be
repeated using a fresh calibration standard. If the results still do not agree, generate a
new calibration curve or use a single point calibration standard as described in Sect.
9.2.4.
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Method 505
9.2.4 Single point calibration is an acceptable alternative to a calibration curve. Prepare
single point standards from the secondary dilution standard solutions. The single
point calibration standard should be prepared at a concentration that produces a
response close (±20% or less) to that of the unknowns. Do not use less than 20 jtL
of the secondary dilution standard solution to produce a single point calibration
standard in reagent water.
9.3 Instrument Performance: Check the performance of the entire analytical system daily using
data gathered from analyses of laboratory reagent blanks (LRB), (CAL), laboratory duplicate
samples (LD1 and LD2), and the laboratory performance check solution (LPC) (Sect. 10.6).
9.3.1 Significant peak tailing in excess of that shown for the target compounds in the
method chromatograms (Figures 1-11) must be corrected. Tailing problems are
generally traceable to active sites on the GC column, improper column installation, or
operation of the detector.
9.3.2 Check the precision between replicate analyses. Poor precision is generally traceable
to pneumatic leaks, especially at the injection port. If the GC system is apparently
performing acceptably but with decreased sensitivity, it may be necessary to generate
a new curve or set of calibration factors to verify the decreased responses before
searching for the source of the problem.
9.3.3 Observed relative area responses of endrin (Sect. 4.5) must meet the following general
criteria:
9.3.3.1 The breakdown of endrin into its aldo and keto forms must be adequately
consistent during a period in which a series of analyses is made.
Equivalent relative amounts of breakdown should be demonstrated in the
LRB, LPC, LFB, CAL and QCS. Consistent breakdown resulting in these
analyses would suggest that the breakdown occurred in the instrument
system and that the methodology is in control.
9.3.3.2 Analyses of laboratory fortified matrix (LFM) samples must also be
adequately consistent after corrections for potential background
concentrations are made.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
analysis of laboratory reagent blanks (LRB), laboratory fortified blanks (LFB), laboratory
fortified sample matrix (LFM), and quality control samples (QCS).
10.2 Laboratory Reagent Blanks. Before processing any samples, the analyst must demonstrate that
all glassware and reagent interferences are under control. Each time a set of samples is
extracted or reagents are changed, an LRB must be analyzed. If within the retention time
window of any analyte the LRB produces a peak that would prevent the determination of that
analyte, determine the source of contamination and eliminate the interference before processing
samples.
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Method 505
10.3 Initial Demonstration of Capability
10.3.1 Select a representative concentration (about 10 times MDL or at the regulatory
Maximum Contaminant Level, whichever is lower) for each analyte. Prepare a
primary dilution standard solution (in methanol) containing each analyte at 1000 times
selected concentration. With a syringe, add 35 /zL of the concentrate to each of at
least four 35-mL aliquots of reagent water, and analyze each aliquot according to
procedures beginning in Sect. 11.
10.3.2 For each analyte the recovery value should for at least three out of four consecutively
analyzed samples fall in the range of R±30% (or within R±3SR if broader) using the
values for R and SR for reagent water in Table 2. For those compounds that meet the
acceptance criteria, performance is considered acceptable and sample analysis may
begin. For those compounds that fail these criteria, initial demonstration procedures
should be repeated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. It is expected that as laboratory personnel gain experience with this
method the quality of data will improve beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC conditions, or detectors to improve
separations or lower analytical costs. Each time such method modifications are made, the
analyst must repeat the procedures in Sect. 10.3.
10.5 Assessing Laboratory Performance: Laboratory Fortified Blank (LFB)
10.5.1 The laboratory must analyze at least one laboratory fortified blank (LFB) per sample
set (all samples extracted within a 24-h period). If the sample set contains more than
20 samples, analyze one LFB for every 20 samples. The fortifying concentration of
each analyte in the LFB sample should be 10 times MDL or the MCL, whichever is
less. Calculate accuracy as percent recovery (X,). If the recovery of any analyte falls
outside the control limits (see Sect. 10.5.2), that analyte is judged out of control, and
the source of the problem should be identified and resolved before continuing
analyses.
10.5.2 Until sufficient data become available from within their own laboratory, usually a
minimum of results from 20 to 30 analyses, the laboratory may assess laboratory
performance against the control limits in Sect. 10.3.2 that are derived from the data in
Table 2. When sufficient internal performance data becomes available, develop
control limits from the mean percent recovery (X) and standard deviation (S) of the
percent recovery. These data are used to establish upper and lower control limits as
follows:
Upper Control Limit = X + 3S
Lower Control Limit = X - 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points.
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Method 505
10.5.3 It is recommended that the laboratory periodically determine and document its
detection limit capabilities for analytes of interest.
CAUTION: No attempts to establish low detection limits should be made before
instrument optimization and adequate conditioning of both the column and the GC
system. Conditioning includes the processing ofLFB and LFM samples containing
moderate concentration levels of these analytes.
10.5.4 At least each quarter the laboratory should analyze quality control samples (QCS) (if
available). If criteria provided with the QCS are not met, corrective action should be
taken and documented.
10.6 Assessing Analyte Recovery: Laboratory Fortified Sample Matrix (LFM)
10.6.1 The laboratory must add a known concentration to a minimum of 10% of the routine
samples or one LFM per set, whichever is greater. The fortified concentration should
not be less than the background concentration of the sample selected for fortification.
Ideally the LFM concentration should be the same as that used for the LFB (Sect.
10.5). Periodically, samples from all routine sample sources should be fortified.
10.6.2 Calculate the percent recovery (R;) for each analyte, corrected for background
concentrations measured in the unfortified sample, and compare these values to the
control limits established in Sect. 10.5.2 from the analyses of LFBs.
10.6.3 If the recovery of any such analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control (Sect. 10.5), the
recovery problem encountered with the dosed sample is judged to be matrix related,
not system related. The result for that analyte in the unfortified sample is labeled
suspect/matrix to inform the data user that the results are suspect due to matrix
effects.
10.7 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements or field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 Sample Preparation
11.1.1 Remove samples from storage and allow them to equilibrate to room temperature.
11.1.2 Remove the container caps. Withdraw and discard a 5-mL volume using a 10-mL
graduated cylinder. Replace the container caps and weigh the containers with contents
to the nearest 0.1 g and record these weights for subsequent sample volume
determinations (Sect. 11.3).
11.2 Extraction and Analysis
11.2.1 Remove the container cap of each sample, and add 6 g NaCl (Sect. 7.1.3) to the
sample bottle. Using a transfer or automatic dispensing pipet, add 2.0 mL of hexane.
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Method 505
Recap and shake vigorously by hand for 1 min. Invert the bottle and allow the water
and hexane phases to separate.
11.2.2 Remove the cap and carefully transfer approximately 0.5 mL of hexane layer into an
autosampler vial using a disposable glass pipet.
11.2.3 Transfer the remaining hexane phase, being careful not to include any of the water
phase, into a second autosampler vial. Reserve this second vial at 4°C for an
immediate reanalysis if necessary.
11.2.4 Transfer the first sample vial to an autosampler set up to inject 1-2 /*L portions into
the gas chromatograph for analysis (See Sect. 6.9 for GC conditions). Alternately,
1-2 mL portions of samples, blanks, and standards may be manually injected,
although an autosampler is strongly recommended.
11.3 Determination of Sample Volume in Bottles Not Calibrated
11.3.1 Discard the remaining sample/hexane mixture from the sample bottle. Shake off the
remaining few drops using short, brisk wrist movements.
11.3.2 Reweigh the empty container with original cap and calculate the net weight of sample
by difference to the nearest 0.1 g (Sect. 11.1.2 minus Sect. 11.3.2). This net weight
(in grams) is equivalent to the volume (in mL) of water extracted (Sect. 12.3). By
alternately using 40-mL bottles precalibrated at 35-mL levels, the gravimetric steps
can be omitted, thus increasing the speed and ease of this extraction process.
11.4 Identification of Analytes
11.4.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound
corresponds, within limits, to the retention time of a standard compound, then
identifiction is considered positive.
11.4.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.4.3 Identification requires expert judgement when sample components are not resolved
chromatographically. When peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of a peak on a
chromatogram, appropriate alternative techniques to help confirm peak identification
need be employed. For example, more positive identification may be made by the use
of an alternative detector which operates on a chemical/physical principle different
from that originally used, e.g., mass spectrometry, or the use of a second
chromatography column. Suggested alternative columns are described in Sect. 6.9.
12. CALCULATIONS
12.1 Identify the organohalides in the sample chromatogram by comparing the retention time of the
suspect peak to retention times generated by the calibration standards and the laboratory
42
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Method 505
fortified blanks. Identify the multicomponent compounds using all peaks that are characteristic
of the specific compound from chromatograms generated with individual standards. Select the
most sensitive and reproducible peaks to obtain a sum for calculation purposes (See Table 1).
12.2 Use the single point calibration (Sect. 9.2.4) or use the calibration curve or calibration factor
(Sect. 9.2.3) to directly calculate the uncorrected concentration (Ci) of each analyte in the
sample (e.g., calibration factor x response).
12.3 Calculate the sample volume (Vs) as equal to the net sample weight:
Vs = Gross weight (Sect. 11.1.2) - bottle tare (Sect. 11.3.2)
12.4 Calculate the corrected sample concentration as:
35(c)
Concentration, uglL = -
12.5 Results should be reported with an appropriate number of significant figures. Experience
indicates that three significant figures may be used for concentrations above 99 fig/L, two
significant figures for concentrations between 1-99 /*g/L, and 1 significant figure for lower
concentrations.
13. ACCURACY AND PRECISION
13.1 Single laboratory (EMSL-Cincinnati) accuracy and precision at several concentrations in
reagent, ground, and tap water matrices are presented in Table 2." These results were
obtained from data generated with a DB-1 column.
13.2 This method has been tested by 10 laboratories using reagent water and groundwater fortified
at three concentration levels. Single operator precision, overall precision, and method accuracy
were found to be directly related to the concentration of the analyte and virtually independent
of the sample matrix. Linear equations to describe the relationships are presented in Table 3.12
43
-------
Method 505
References
\. Glaze, W.W., Lin, C.C., Optimization of Liquid-Liquid Extraction Methods for Analysis of
Organics in Water, EPA-600/S4-83-052, January 1984.
2. Henderson, J.E., Peyton, G.R. and Glaze, W.H. (1976). In "Identification and Analysis of
Organic Pollutants in Water" (L.H. Keith ed.), pp. 105-111. Ann Arbor Sci. Publ., Ann
Arbor, Michigan.
3. Richard, J.J., Junk, G.A., "Liquid Extraction for Rapid Determination of Halomethanes in
Water," Journal AWWA, 69, 62, January 1977.
4. "Handbook for Analytical Quality Control in Water and Wastewater Laboratories,"
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, 45268, March 1979.
5. Budde, W.L., Eichelberger, J.W., "Organic Analyses Using Gas Chromatography-Mass
Spectrometry," Ann Arbor Science, Ann Arbor, Michigan 1979.
6. Glaser, J.A. et al., "Trace Analyses for Wastewaters," Environmental Science and
Technology, 15, 1426(1981).
7. Bellar, T.A., Stemmer, P., Lichtenberg, J.J., "Evaluation of Capillary Systems for the
Analysis of Environmental Extracts," EPA-600/S4-84-004, March 1984.
8. "Carcinogens-Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute of Occupational Safety
and Health, Publication No. 77-206, August, 1977.
9. "OSHA Safety and Health Standards, General Industry," (29CF/?1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
10. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
11. Winfield, T., et al. "Analysis of Organohalide Pesticides and Commercial PCB Products in
Drinking Water by Microextraction and Gas Chromatography." In preparation.
12. Multilaboratory Method Validation Study #40, conducted by the Quality Assurance Branch,
EMSL-Ci. Report in progress.
44
-------
Method SOS
Table 1. Retention Times for Method Analytes
Analyte
Hexachlorocyclopentadiene
Simazine
Atrazine
Hexachlorobenzene
Lindane
Alachlor
Heptachlor
Aldrin
Heptachlor Epoxide
7-Chlordane
a-Chlordane
trans-Nonachlor
Dieldrin
Endrin
cis-Nonachlor
Methoxychlor
Retention Time' (min)
Primary
5.5
10.9
11.2
11.9
12.3
15.1
15.9
17.6
19.0
19.9
20.9
21.3
22.1
23.2
24.3
30.0
I Confirm 1
6.8
25.7
22.6
13.4
18.4
19.7
17.5
18.4
24.6
25.9
26.6
24.8
45.1
33.3
39.0
58.5
| Confirm 2
5.2
19.9
19.6
15.6
18.7
21.1
20.0
21.4
24.6
26.0
26.6
26.3
27.8
29.2
30.4
36.4
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Chlordane
Toxaphene
Primary*
13.6, 14.8, 15.2, 16.2, 17.7
7.7, 9.0, 15.9, 19.1, 24.7
11.2, 14.7, 13.6, 15.2, 17.7
11.2, 13.6, 14.7, 15.2, 17.7, 19.8
14.8, 16.2, 17.1, 17.7, 19.8, 22.0
19.1, 21.9, 23.4, 24.9, 26.7
23.4, 24.9, 26.7, 28.2, 29.9, 32.6
15.1, 15.9, 20.1, 20.9, 21.3
21.7, 22.5, 26.7, 27.2
Columns and analytical conditions are described in Sect. 6.9.2, 6.9.3, and 6.9.4.
Column and conditions described in Sect. 6.9.2. More than one peak listed does not implicate
the total number of peaks characteristic of the multi-component analyte. Listed peaks indicate
only the ones chosen for summation in the quantification.
45
-------
Method 505
Table 2. Single Laboratory Ace
(MDLs) for Analytes from Reage
Concen-
MDLb tration*
Analyte (ug/L) (ug/U
Aldrin 0.075 0.15
Alachlor 0.225 0.50
Aldrm 0.007 0.05
Atrazme 2.4 5.0
20.0
a-Chlordane 0.006 0.06
0.35
7-Chlordane 0.01 2 0.06
0.35
Chlordane 0.14 0.17
3.4
Dieldrin 0.012 0.10
3.6
Endrin 0.063 0.10
3.6
Heptachlor 0.003 0.032
1.2
Heptachlor 0.004 0.04
Epoxide 1.4
Hexachloro- 0.002 0.003
benzene 0.09
Hexachlorocyclo- 0.13 0.15
pentadiene 0.35
Lindane 0.003 0.03
1.2
Methoxychlor 0.96 2.10
7.03
cis-Nonachlor 0.027 0.06
0.45
trans-Nonachlor 0.011 0.06
0.35
Simazine 6.8 25
60
Toxaphene 1.0 10
80
Aroclor1016 0.08 1.0
Aroclor1221 15.0 180
Aroclor 1232 0.48 3.9
Aroclor 1242 0.31 4.7
uracy, Precision and Method Detection Limits
nt Water, Groundwater, and Tap Water3
Accuracy and Standard Deviation Data
Reagent Water
Ff
86
102
106
85
95
95
86
95
86
NA
NA
87
114
119
99
77
80
100
115
104
103
73
73
91
111
100
98
110
82
95
86
99
65
NA
NA
NA
NA
NA
NA
SFf
9.5
13.4
20.0
16.2
5.2
3.5
17.0
0.4
18.5
8.0
3.6
17.1
9.1
29.8
6.5
10.2
7.4
15.6
6.6
13.5
4.4
5.1
11.7
6.5
5.0
21.0
10.9
15.2
21.3
9.6
21.8
8.3
3.6
12.6
15.3
6.6
8.3
13.5
6.0
Ground Water
R
100
-
86
95
86
83
94
86
95
-
-
67
94
94
100
37
71
90
103
91
101
87
69
88
109
-
-
101
93
83
94
97
59
-
-
-
-
-
-
sa
11.0
-
16.3
7.3
9.1
4.4
10.2
5.3
14.5
-
-
10.1
8.6
20.2
11.3
6.8
9.8
14.2
6.9
12.0
4.4
5.1
4.8
7.7
3.4
-
--
7.2
18.3
7.1
17.2
9.2
18.0
-
-
-
-
-
-
Tap Water
R
69
-
-
108
91
85
91
83
91
105
95
92
81
106
85
200
106
112
81
100
88
191
109
103
93
-
-
93
87
73
86
102
67
110
114
97
92
86
96
S*
9.0
-
-
10.9
3.1
7.1
2.4
14.7
6.0
12.4
9.6
15.7
14.0
14.0
12.4
22.6
16.8
7.5
5.9
15.6
15.2
18.5
14.3
8.1
18.4
-
-
14.3
5.4
4.1
5.1
13.4
6.2
9.5
13.5
7.5
9.6
7.3
7.4
46
-------
Method 505
Table 2. Single Laboratory Accuracy, Precision and Method Detection Limits
(MDLs) for Analytes from Reagent Water, Groundwater, and Tap Water (cont.)
Analyte
Aroclor
Aroclor
Aroclor
1248
1254
1260
MDL"
(ug/L)
0.102
0.102
0.189
tration*
(ug/U
3.6
3.4
1.8
1.7
2.0
1.8
Rea,
IT
NA
-
NA
-
NA
NA
Accuracy and Standard Deviation Data
Reagent
* \
NA
Water
SRd
11.5
Ground Water
R
S*
Tap Water
R
s*
10.4
20.7
84
85
88
9.9
11.8
19.8
NA = Not applicable. A separate set of aqueous standards was not analyzed, and the
response factor for reagent water was used to calculate a recovery for the tap water
matrix.
Data corrected for amount detected in blank and represent the mean of 5-8 samples.
MDL= method detection limit in sample in fjg/L; calculated by multiplying standard deviation
(S) times the students' t value appropriate for a 99% confidence level and a standard
deviation estimate with n-1 degrees of freedom.
R = average percent recovery.
SR = Standard deviation about percent recovery.
Refers to concentration levels used to generate R and SR data for the three types of water
Matrices, not for MDL determinations.
No analyses conducted.
47
-------
Method 505
Table 3. Method Accuracy and Precision as Functions of Concentration
Reagent Water
Parameter
Atrazine
Simazine
Hexachlorobenzene
Lindane
Alachlor
Heptachlor
Aldrin
Heptachlor epoxide
Dieldrin
Endrin
Methoxychlor
Chlordane
Toxaphene
PCB-1016
PCS-1254
Applicable
Cone. Range
(pg/U
(3.06-45.90)
(12.55-50.20)
(0.01-0.37)
(0.04-1.39)
(0.50-37.50)
(0.04-1.41)
(0.04-1.42)
(0.04-1.42)
(0.10-7.53)
(0.10-7.50)
(0.20-15.00)
(0.51-50.90)
(5.63-70.40)
(0.50-49.80)
(0.50-50.40)
Accuracy as
Recovery X
1.122C + 0.97
0.892C + 1.446
1.028C-0.00
1.009C-0.00
1.004C-0.08
1.002C + 0.02
1.066C + 0.00
0.952C + 0.00
1.027C+0.00
0.958C + 0.01
0.950C + 0.15
1.037C + 0.06
1.087C + 0.24
0.856C + 0.31
0.872C-0.01
Single Analyst
Precision Sf
(ng/U
O.OOOX + 1.21
-0.049X + 3.52
0.108X + 0.00
0.057X+0.01
0.077X+0.10
0.107X+0.01
0.031 X+0.02
0.032X+0.02
0.091 X+0.01
0.116X+0.01
0.115X+0.12
0.084X+0.06
0.131X-0.31
0.106X+0.31
0.122X+0.11
Overall
Precision S
(ug/U
0.045X + 2.23
0.209X + 1.23
0.227X+0.00
0.142X + 0.00
0.105X+0.16
0.211X+0.02
0.264X-0.00
0.129X+0.02
0.198X+0.02
0.136X+0.02
0.125X+ 0.20
0.125X+0.19
0.269X+0.69
0.147X+0.45
0.281 X+0.05
* The concentration range applicable to the multi-laboratory study from which the data was
generated.
48
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: 1.0^m
Column Dimensions: 0.32mm ID, 30 M Long
S 5
i 1 i r si s 1
§ C * 0 -D g- £
S |j 0 ,_ .C 5 ® Q
5 o> .2 O x
fl
O
.-A-
ID
§
N
1 I
<0 o
o. •=
S 0
0. J8
*
_JJ
c 1
1 1
c
£
s
1
8
i i i
2 4 6 8 10 12 14 '
JUu
o
S
to
0)
^*
CD
TJ
-T3
^c
i
D
c
UJ
•V
I
16 18 20 22
«
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: t.O^m
Column Dimensions: 0.32mm ID,
30 M Long
10 15 20 25
Time (Minutes)
30
35
52-015-17
Figure 2. Extract of Reagent Water Spiked at 20 [ig/L with Atrazine,
60 |ig/Lwith Simazine, 0.45 (ig/Lwith cis-Nonachlor, and
0.35 ng/L with Hexachlorocyclopentadiene, Heptachlor,
y-Chlordane, a-Chlordane, and trans-Nonachlor
50
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: 1.0(im
Column Dimensions: 0.32mm ID, 30 M Long
i
4
T I
6 8
\ i i I I l I I I I I I I I I i I l
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
52-015-18
Figure 3. Hexane Spiked at 11.4 (ig/L with Aroclor 1016
51
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: 1.0|im
Column Dimensions: 0.32mm ID, 30 M Long
~1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I~
4 68 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
Figure 4. Hexane Spiked at 171.4 H.Q/L with Aroclor 1221
52
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: LO^m
Column Dimensions: 0.32mm ID, 30 M Long
I
2
i
4
I i
6 8
l i i
10 12 14
i
16
i i
18 20
i i
22 24
i \
26 28
i 1
30 32
i
34
i r
36 38
i 1 I
40 42 44
Time (Minutes)
52-015-20
Figure 5. Hexane Spiked at 57.1 |j.g/L with Aroclor 1232
53
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: LO^m
Column Dimensions: 0.32mm ID, 30 M Long
i—r
6 8
n—i—i—i—i—i—i—i—i—i—i—i—i—\—i—i—i r
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
Figure 6. Hexane Spiked at 57.1 |j,g/L with Aroclor 1242
54
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: 1.0^m
Column Dimensions: 0.32mm ID, 30 M Long
I
4
\
8
i i rrrri i i i i i IIITTT
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
52-015-22
Figure 7. Hexane Spiked at 57.1 \ig/L with Aroclor 1248
55
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: LO^m
Column Dimensions: 0.32mm ID, 30 M Long
i i i i i I i i i i i i i i i i i i i i i i
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
Figure 8. Hexane Spiked at 42.9 ^L with Aroclor 1254
56
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: 1.0|im
Column Dimensions: 0.32mm ID, 30 M Long
Jl
i
4
I
8
i r r
10 12 14
I I I I I i i i i i i i i i i
16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
52-015-24
Figure 9. Hexane Spiked at 34.3 ng/L with Aroclor 1260
57
-------
Method 505
Column: Fused Silica Capillary
Liquid Phase: DB-1
Film Thickness: 1 .O^m
Column Dimensions: 0.32mm ID, 30 M Long
T
4
T"
8
n—i r~
10 12 14
16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
52-015-25
Figure 10. Hexane Spiked at 28.6 (ig/L with Chlordane
58
-------
Method 505
Column: Fused Silica Capillary
Ljquid Phase: DB-1
Film Thickness: LOfxm
Column Dimensions: 0.32mm ID, 30 M Long
~
4
T
8
iirriiiiiiiiiiiiii
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Time (Minutes)
52-015-26
Figure 11. Hexane Spiked at 57.1 |ig/L with Toxaphene
59
-------
-------
Method 506
Determination of Phthalate and
Adipate Esters in Drinking Water
by Liquid-Liquid Extraction
or Liquid-Solid Extraction
and Gas Chromatography
with Photoionization Detection
EPA EMSL-Ci
July 1990
F.K. Kawahara, J.W. Hodgeson, J.W. Eichelberger
-------
-------
Method 506
Determination of Phthalate and Adipate Esters in Drinking Water
by Liquid-Liquid Extraction or Liquid-Solid Extraction and
Gas Chromatography with Photoionization Detection
1. SCOPE AND APPLICA TION
1.1 This method describes a procedure for the determination of certain phthalate and adipate esters
in drinking water by liquid/liquid or liquid/solid extraction. The following analytes can be
determined by this method:
Parameter CAS No.
Bis (2-ethylhexyl) phthalate 117-81-7
Butylbenzyl phthalate 85-68-7
Di-n-butyl phthalate 84-74-2
Diethyl phthalate 84-66-2
Dimethyl phthalate 131-11-3
Bis(2-ethylhexyl) adipate 103-23-1
Di-n-octyl phthalate 117-84-0
1.2 This is a capillary column gas chromatographic (GC) method applicable to the determination of
the compounds listed above in ground water and finished drinking water. When this method is
used to analyze unfamiliar samples for any or all of the compounds listed above, compound
identifications should be supported by at least one additional qualitative technique. Method
525 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the
qualitative and quantitative confirmation of results for all the analytes listed above, using the
extract produced by this method.
1.3 This method has been validated in a single laboratory and method detection limits (MDLs)
have been determined for the analytes above (Table 2).' Observed detection limits may vary
among waters, depending upon the nature of interferences in the sample matrix and the
specific instrumentation used.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use
of GC and in the interpretation of gas chromatograms obtained by a computerized system.
Each analyst must demonstrate the ability to generate acceptable results with this method using
the procedure described in Sect. 10.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1-L, is extracted with a ternary solvent consist-
ing of methylene chloride, hexane and ethyl acetate using a glass separatory runnel. The
solvent extract is isolated, dried and concentrated to a volume of 5 mL or less. The extract is
further concentrated by gentle use of nitrogen gas blowing to a volume of 1 mL or less. The
analytes in the extract are separated by means of capillary column gas Chromatography using
temperature programming and the phthalate and adipate esters are then measured with a
photoionization detector.2^* Alternatively, a measured volume of sample is extracted with a
63
-------
Method 506
liquid-solid extraction (LSE) cartridge or disk. The LSE cartridge or disk is eluted with
methylene chloride. The eluant is then concentrated by use of a gentle nitrogen purge to a
volume of 1 mL or less.
3. DEFINITIONS
3.1 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.2 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.3 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the labora-
tory is capable of making accurate and precise measurements at the required method detection
limit.
3.4 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.5 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.6 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.7 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.8 Quality control sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
64
-------
Method 506
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in water, solvents, reagents, glassware,
and sampling processing hardware. These lead to discrete artifacts and/or elevated baselines in
gas chromatograms. All of these materials must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks (Sect.
10.2).
4.1.1 Phthalate esters are contaminants in many products found in the laboratory. It is
particularly important to avoid the use of plastics because phthalates are commonly
used as plasticizers and are easily extracted from plastic materials. Great care must
be exercised to prevent contamination. Exhaustive clean up of reagents and glassware
must be required to eliminate background phthalate that is not derived from the
sample.
4.1.2 Glassware must be scrupulously cleaned. Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry and
heat in an oven or muffle furnace at 400°C for 1 hour. Do not heat volumetric
glassware. Thorough rinsing with acetone may be substituted for the heating. After
cooling, the glassware should be sealed with aluminum foil and stored in a clean envi-
ronment to prevent accumulation of dust and other contaminants.
4.1.3 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in an all glass system may be required.
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed thus potentially reducing
the shelf-life.
4.2 Matrix interferences may be caused by contaminants that are co-extracted from the sample.
The extent of matrix interferences will vary from source to source, dependent upon the nature
and diversity of the industrial complex or municipality being sampled. Clean up procedures
can be used to overcome many of these interferences.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
ing the safe handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available and have been identified for
the information of the analyst.5"7 (All specifications are suggested, catalog numbers are
included for illustration only.)
65
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Method 506
6. APPARA rus AND MA TERIALS
6.1 Sampling Equipment
6.1.1 Grab Sample Bottle: 1-L or 1-qt amber glass, fitted with a screw cap lined with
Teflon. Foil may be substituted for Teflon if the sample is not corrosive. Protect
samples from light if amber bottles are not available. The bottle and cap liner must
be washed, rinsed with acetone or methylene chloride and dried before use in order to
minimize contamination. (Sect. 4.1.1.)
6.2 Glassware
6.2.1 Separatory Funnel: 2-L with Teflon stopcock.
6.2.2 Drying Column: Chromatographic column-300 mm long x 10 mm ID, with Teflon
stopcock and coarse frit filter disc at bottom (Kontes K - 420540-0213 or equivalent).
6.2.3 Concentrator Tube: Kuderna-Danish, 10 mL, graduated (Kontes K-570050-1025) or
equivalent), calibration must be checked at the volumes employed in the test. Tight
ground glass stopper is used to prevent evaporation of extracts.
6.2.4 Evaporative Flask: Kuderna-Danish, 500 mL (Kontes K-57000-0500 or equivalent).
Attach to concentrator tube with springs.
6.2.5 Snyder Column: Kuderna-Danish, three-ball macro size (Kontes K-503000-0121 or
equivalent).
6.2.6 Snyder Column: Kuderna-Danish, 2 or 3 ball micro size (Kontes 590025-0125 or
equivalent).
6.2.7 Vials: 10 to 15 mL, amber glass with Teflon-lined screw cap.
6.2.8 Boiling Chips: Approximately 10/40 mesh. Heat to 400°C for 30 min. or extract
with methylene chloride in a Soxhlet apparatus.
6.2.9 Flask, Erlenmeyer: 250 mL. (A.H. Thomas Co. #1590-033 or equivalent.)
6.2.10 Chromatography column similar to 6.2.2.
6.2.11 Pasteur Pipets (and Bulb): VWR Scientific 14672-200 or equivalent.
6.2.12 Autosampler Vials: Equipped with Teflon-lined septum and threaded or crimp top
caps.
6.3 Water Bath: Heated (with concentric ring covers) capable of temperature control (± 2°C).
The water bath should be used in a ventilating hood.
6.4 Balance: Analytical, capable of weighing accurately to nearest 0.0001 gm.
6.5 Gas Chromatograph: An analytical system complete with temperature programmable GC fitted
with split-splitless injection mode system, suitable for use with capillary columns and all
required accessory syringes, analytical columns, gases, detector and stripchart recorder. A
data system for processing chromatographic data is recommended. The gas chromatograph
was interfaced with the Nelson Analytical "760" System, IBM personal computer display,
personal computer AT, IBM Model M Keyboard and Epson FX-85 select type recorder.
6.5.1 Column, Fused Silica Capillary: DB-5 or equivalent, 30 m long x 0.32 mm ID with
a film thickness of 0.25 micron.
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Method 506
6.5.2 The alternate column, Fused Silica Capillary: 30 m long x 0.32 mm ID with a film
thickness of 0.25 micron, DB-1 or equivalent.
6.5.3 Detector: A high temperature photoio.nization detector equipped for 10.0 electron
volts and capable of operating from 250°C to 350°C is required. (Tracor Instru-
ments, Inc., Model 703 or equivalent.)
6.5.4 An automatic injector system is suggested, but was not used for the development of
this method.
6.6 Vacuum pump, 110 VAC, capable of maintaining a vacuum of 8-10 mm Hg.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagent Water: Reagent water is defined as water in which an interfering substance is not
observed at the MDL of the parameters of interest. Reagent water used to generate data in
this method was distilled water obtained from the Millipore L/A-7044 system comprised of
prefiltration, organic adsorption, deionization and Millipore filtration columnar units.
7.2 Acetone, hexane, methylene chloride, ethyl acetate, ethyl ether and iso-octane: Pesticide
quality or equivalent to distillation in glass quality.
7.3 Sodium Sulfate: (ACS) Granular, anhydrous. Several levels of purification may be required
in order to reduce background phthalate levels towards acceptance: 1) Heat 4 h at 400°C in a
shallow tray, 2) Soxhlet extract with methylene chloride for 48 h.
7.4 Florisil: PR grade (60/100 mesh). To prepare for use, place 100 g of Florisil into a 500-mL
beaker and heat for approximately 16 h at 40°C. After heating transfer to a 500-mL reagent
bottle. Tightly seal and cool to room temperature. When cool, add 3 mL of reagent water.
Mix thoroughly by shaking or rolling for 10 min. and let it stand for at least 2 h. Store in the
dark in glass containers with ground glass stoppers or foil-lined screw caps.
7.5 Sodium Chloride: (ACS) Granular. Heat 4 h at 400°C in a shallow tray. When cool, keep in
tightly sealed bottle.
7.6 Ethyl Ether: (ACS) reagent grade.
7.7 Sodium Thiosulfate (Na2S2O3): (ACS) reagent grade.
7.8 Alumina: Neutral activity Super I, W200 series (ICN Life Sciences Group, No. 404583): To
prepare for use, place 100 g of alumina into a 500-mL beaker and heat for approximately 16 h
at 400°C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room
temperature. When cool, add 3 mL of reagent water. Mix thoroughly by shaking or rolling
for 10 min. and let it stand for at least 2 h. Keep the bottle sealed tightly.
7.9 Liquid-solid extraction (LSE) cartridges: Cartridges are inert non-leaching plastic, for exam-
ple polypropylene, or glass, and must not contain plasticizers, such as phthalate esters or
adipates, that leach into methylene chloride. The cartridges are packed with about 1 gram of
silica, or other inert inorganic support, whose surface is modified by chemically bonded
octadecyl (C,8) groups. The packing must have a narrow size distribution and must not leach
organic compounds into methylene chloride. One liter of water should pass through the
cartridge in about 2 hrs with the assistance of a slight vacuum of about 13 cm (5 in.) of
mercury. The extraction time should not vary unreasonably among LSE cartridges.
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Method 506
7.10 Liquid-solid extraction disks, C-18, 47 mm: Disks are manufactured with Teflon and should
contain very little contamination.
7.11 Helium carrier gas, as contaminant free as possible.
7.12 Stock standard solutions (1.00 ng/fiL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
7.12.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure
material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumet-
ric flask. Larger volumes can be used at the convenience of the analyst. When
compound purity is assayed to be 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially pre-
pared stock standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.12.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at
4°C and protect from light. Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior to preparing calibration
standards from them.
7.12.3 Stock standard solutions must be replaced after six months, or sooner if comparison
with check standards indicates a problem. Butylbenzyl phthalate is especially vul-
nerable to autoxidation.
7.2 Laboratory control sample concentrate: See Sect. 10.3.1.
8. SAMPLE COLLECTION, PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional sampling practices should
be followed; however, the bottle must not be prerinsed with sample before collection.8"9
8.2 Sample Preservation and Storage
8.2.1 For sample dechlorination, add 60 mg sodium thiosulfate to the sample bottle at the
sampling site or in the laboratory before shipping to the sampling site.
8.2.2 After the sample is collected in a bottle containing preservative(s), seal the bottle and
shake vigorously for 1 min.
8.2.3 The samples must be iced or refrigerated at 4°C free from light from the time of
collection until extraction. Limited holding studies have indicated that the analytes
thus stored are stable up to 14 days or longer. Analyte stability may be affected by
the matrix; therefore, the analyst should verify that the preservation technique is ap-
plicable to the particular samples under study.
8.3 Extract Storage: Extracts should be stored at 4°C in absence of light. A 14-day maximum
extract storage time is recommended. The analyst should verify appropriate extract holding
times applicable to the samples under study.
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Method 506
9. CALIBRATION
9.1 Establish gas chromatograph operating conditions equivalent to those given in Table 1. The
gas chromatographic system can be calibrated using the external standard technique (Sect.
9.2).
9.1.1 Performance of the detector should be checked daily by a specified procedure given in
the gas chromatograph operator's manual. If the response is weak, the ultraviolet
lamp is removed carefully following disconnection of the power supply. It is cleaned
and then placed into its original position with the aid of a leak detector.
9.2 External standard calibration procedure:
9.2.1 Prepare calibration standards at a minimum of three concentration levels for each
analyte of interest by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with n-hexane. One of the external standards should be
at a concentration near, but above, the MDL (Table 2) and the other concentrations
should correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
9.2.2 Using injection of 1 to 2 /xL, analyze each calibration standard according to Sect. 11.5
and tabulate peak height or area responses against the mass injected. The results can
be used to prepare a calibration curve for each compound. Alternatively, if the ratio
of response to amount injected (calibration factor) is a constant over the working
range (< 10% relative standard deviation, RSD), linearity through the origin can be
assumed and the average ratio or calibration factor can be used in place of a cali-
bration curve.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
analysis of laboratory reagent blanks, laboratory fortified samples, laboratory fortified blanks,
and QC samples. Additional quality control practices are recommended.
10.2 Laboratory reagent blanks: Before processing any samples, the analyst must demonstrate that
all glassware and reagent interferences are under control. Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If within the retention time
window of any analyte of interest the LRB produces a peak that would prevent the determina-
tion of that analyte using a known standard, determine the source of contamination and elimi-
nate the interference before processing samples.
10.3 Initial Demonstration of Capability.
10.3.1 Select a representative spike concentration, about 10 times EDL or at the regulatory
Maximum Contaminant Level (MCL), (whichever is lower) for each analyte. Prepare
a laboratory control sample concentrate (in methanol) containing each analyte at 1000
times selected concentration. With a syringe, add 1 mL of the concentrate to each of
seven 1-L aliquots of reagent water, and analyze each aliquot according to procedures
in Sect. 11.1 or 11.2, and 11.3 and 11.4.
10.3.2 For each analyte, the mean recovery value should fall in the range of R ± 30% (or
within R + 3Sr if broader) using the values for R and Sr for reagent water in Table 3
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Method 506
or Table 4. For those compounds that meet the acceptance criteria, performance is
considered acceptable and sample analysis may begin. For those compounds that fail
these criteria, initial demonstration procedures should be repeated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. It is expected that, as laboratory personnel gain experience with
this method, the quality of data will improve beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC detectors, GC conditions, concentration
techniques, internal standards or surrogate compounds. Each time such method modifications
are made, the analyst must repeat the procedures in Sect. 10.3.
10.5 Assessing laboratory performance: Laboratory Fortified Blank
10.5.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample per
sample set (all samples extracted within a 24-hr period). The spiking concentration
of each analyte in the LFB should be 10 times MDL or the MCL, whichever is less.
Calculate accuracy as percent recovery, R. If the recovery of any analyte falls out-
side the control limits (see Sect. 10.5.2), that analyte is judged out of control, and the
source of the problem should be identified and resolved before continuing analyses.
10.5.2 Until sufficient internal data become available, usually a minimum of results from 20
to 30 analyses, the laboratory should assess laboratory performance against the control
limits in Sect. 10.3.2 that are derived from the control limits developed during the
initial demonstration of capability (10.3). When sufficient internal performance data
becomes available, develop control limits from the mean percent recovery, R, and
standard deviation, Sr, of the percent recovery. These data are used to establish
upper and lower control limits as follows:
Upper Control Limit = R + 3Sr
Lower Control Limit = R — 3Sr
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points.
10.6 Assessing Analyte Recovery: Laboratory Fortified Sample Matrix
10.6.1 The laboratory must fortify each analyte to a minimum of 10% of the routine samples
or one fortified sample per set, whichever is greater. The fortified concentration
should not be less than the background concentration of the sample selected for
fortifying. Ideally, this concentration should be the same as that used for the labo-
ratory fortified blank (Sect. 10.5). Over time, samples from all routine sample
sources should be fortified.
10.6.2 Calculate the accuracy as percent recovery, R, for each analyte, corrected for back-
ground concentrations measured in the unfortified sample, and compare these values
to the control limits established in Sect. 10.5.2 from the analyses of LFBs.
10.6.3 If the recovery of any such analyte falls outside the designated range, and the labora-
tory performance for that analyte is shown to be in control (Sect. 10.5), the recovery
problem encountered with the dosed sample is judged to be matrix related, not system
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Method 506
related. The result for that analyte in the unspiked sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix effects.
10.7 Quality Control Samples (QCS): Each quarter, the laboratory should analyze one or more
QCS (if available). If criteria provided with the QCS are not met, corrective action should be
taken and documented.
10.8 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements.
11. PROCEDURE
11.1 Liquid-Liquid Extraction
11.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel containing 50 g
of NaCl.
11.1.2 Add a mixture of 40 ml CH2Cl2 5 mL hexane, and 5 ml ethyl acetate to the sample
bottle. Seal, and shake gently 5 seconds to rinse the inner walls of the bottle. Trans-
fer the solvent to the separatory funnel. Extract the sample by shaking the funnel for
2 min with initial and periodic venting to release excess pressure. Allow the organic
layer to separate for a minimum of 10 min from the water phase. If the emulsion
interface between layers is more than one-third the volume of the solvent layer, the
analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include stirring, filtration of
the emulsion through glass wool, centrifugation, or other physical methods. Collect
the solvent extract in a 250-mL Erlenmeyer flask.
11.1.3 Add a second 50-mL volume of above mixture to the sample bottle and repeat the
extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
Perform a third extraction in the same manner. Then extract with 40-mL of hexane,
which extract (top phase) is added to the total.
11.1.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator
tube to a 500-mL evaporative flask. Other concentration devices or techniques may
be used in place of the K-D concentrator, provided the concentration factor attained in
Sect. 11.1.6-11.1.8 is achieved without loss of analytes.
11.1.5 Pour the combined extract through a drying column (Sect. 6.2.2) containing about 10
cm of prerinsed anhydrous sodium sulfate, and collect the extract in the K-D concen-
trator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chlo-
ride to complete the quantitative transfer.
11.1.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about I mL of methylene
chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65°C) so that
the concentrator tube is partially immersed in the hot water, and the entire lower
rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of
the apparatus and the water temperature as required to complete the concentration in
77
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Method 506
40 min. At the proper rate of distillation the balls of the column will actively chatter
but the chambers will not flood with condensed solvent. When the apparent volume
of liquid reaches approximately 7 mL, remove the K-D apparatus and allow it to drain
and cool for at least 10 min.
11.1.7 Increase the temperature of the hot water bath to about 85°C. Remove the Snyder
column, rinse the column and the 500-mL evaporative flask with 1-2 mL of methy-
lene chloride. Replace with a micro column and evaporative flask. Concentrate the
extract as in Sect. 11.1.6 to 0.5-1 mL. The elapsed time of concentration should be
approximately 15 min.
11.1.8 Remove the micro Snyder column and rinse the column by flushing with hexane using
a 5-mL syringe. Concentrate to a volume of 1 ml by purging the liquid surface with
a gentle flow of nitrogen. Transfer the extract to an autosampler vial with a Pasteur
pipet. Seal the vial with a threaded or crimp top cap. Store in refrigerator if further
processing will not be performed. If the sample extract requires no further cleanup,
proceed with gas chromatographic analysis (Sect. 11.5). If the sample requires
further cleanup, proceed to Sect. 11.4.
11.1.9 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume
to the nearest 5 mL.
11.2 Liquid-Solid Extraction
11.2.1 This method is applicable to a wide range of organic compounds that are efficiently
partitioned from the water sample onto a C)8 organic phase chemically bonded to a
solid inorganic matrix, and are sufficiently volatile and thermally stable for gas chro-
matography.10 Particulate bound organic matter will not be partitioned, and more than
trace levels of particulates in the water may disrupt the partitioning process. Single
laboratory accuracy and precision data have been determined at a single concentration
for the analytes listed in 1.1 fortified into reagent water and raw source water.
11.2.2 Set up the extraction apparatus shown in Figure 1A. The reservoir is not required,
but recommended for convenient operation. Water drains from the reservoir through
the LSE cartridge and into a syringe needle which is inserted through a rubber stopper
into the suction flask. A slight vacuum of 13 cm (5 in.) of mercury is used during all
operations with the apparatus. With this extraction apparatus, sample elution requires
approximately 2 hours. Acceptable new cartridge and extraction disk technology have
recently become available, which allow significantly faster elution rates.
11.2.3 Mark the water meniscus on the side of the sample bottle (approximately 1 liter) for
later determination of sample volume. Pour the water sample into the 2-L separatory
funnel with the stopcock closed.
11.2.4 Flush each cartridge with two 10 mL aliquots of methylene chloride, followed by two
10 mL aliquots of methanol, letting the cartridge drain dry after each flush. These
solvent flushes may be accomplished by adding the solvents directly to the solvent
reservoir in Figure 1 A. Add 10-mL of reagent water to the solvent reservoir, but
before the reagent water level drops below the top edge of the packing in the LSE
cartridge, open the stopcock of the separatory funnel and begin adding sample water
72
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Method 506
to the solvent reservoir. Close the stopcock when an adequate amount of sample is in
the reservoir.
11.2.5 Periodically open the stopcock and drain a portion of the sample water into the solvent
reservoir. The water sample will drain into the cartridge, and from the exit into the
suction flask. Maintain the packing material in the cartridge immersed in water at all
times. After all of the sample has passed through the LSE cartridge, wash the separa-
tory funnel and cartridge with 10 mL of reagent water, and draw air through the
cartridge for 10 min.
11.2.6 Transfer the 125-mL solvent reservoir and LSE cartridge (from Figure 1A) to the
elution apparatus (Figure IB). The same 125 mL solvent reservoir is used for both
apparatus. Wash the 2-liter separatory funnel with 5 mL of methylene chloride and
collect the washings. Close the stopcock on the 100-mL separatory funnel of the
elution apparatus, add the washings to the reservoir and enough additional methylene
chloride to bring the volume back up to 5 mL and elute the LSE cartridge. Elute the
LSE cartridge with an additional 5 mL of methylene chloride (10-mL total). A small
amount of nitrogen positive pressure may be used to elute the cartridge. Small
amounts of residual water from the LSE cartridge will form an immiscible layer with
the methylene chloride in the 100-mL separatory funnel. Open the stopcock and
allow the methylene chloride to pass through the drying column packed with anhy-
drous sodium sulfate (1-in) and into the collection vial. Do not allow the water layer
to enter the drying column. Remove the 100 mL separatory funnel and wash the
drying column with 2 mL of methylene chloride. Add this to the extract. Concen-
trate the extract to 1 mL under a gentle stream of nitrogen. The extract is now ready
for gas chromatography (Sect. 11.4) or additional cleanup (Sect. 11.3).
11.3 Sample Extraction
11.3.1 Preparation of disks.
11.3.1.1 Insert the disk into the 47 mm filter apparatus. Wash the disk with 5 mL
methylene chloride (MeQ2) by adding the MeCl2 to the disk, pulling about
half through the disk and allowing it to soak the disk for about a minute,
then pulling the remaining MeCl2 through the disk. With the vacuum on,
pull air through the disk for a minute.
11.3.1.2 Pre-wet the disk with 5 mL methanol (MeOH) by adding the MeOH to the
disk, pulling about half through the disk and allowing it to soak for about a
minute, then pulling most of the remaining MeOH through. A layer of
MeOH must be left on the surface of the disk, which shouldn't be allowed
to go dry from this point until the end of the sample extraction. THIS IS
A CRITICAL STEP FOR A UNIFORM FLOW AND GOOD RECOV-
ERY.
11.3.1.3 Rinse the disk with 5 mL reagent water by adding the water to the disk
and pulling most through, again leaving a layer on the surface of the disk.
11.3.2 Add 5 mL MeOH per liter of water sample. Mix well.
11.3.3 Add the water sample to the reservoir and turn on the vacuum to begin the filtration.
Full aspirator vacuum may be used. Particulate-free water may filter in as little as 10
73
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Method 506
minutes or less. Filter the entire sample, draining as much water from the sample
container as possible.
11.3.4 Remove the filtration top from the vacuum flask, but don't disassemble the reservoir
and fritted base. Empty the water from the flask and insert a suitable sample tube to
contain the eluant. The only constraint on the sample tube is that it fit around the
drip tip of the fritted base. Reassemble the apparatus.
Add 5 mL of acetonitrile (CH,CN) to rinse the sample bottle. Allow the CH3CN to
settle to the bottom of the bottle and transfer to the disk with a dispo-pipet, rinsing the
sides of the glass filtration reservoir in the process. Pull about half of the CH3CN
through the disk, release the vacuum, and allow the disk to soak for a minute. Pull
the remaining CH3CN through the disk.
Repeat the above step twice, using MeCl2 instead of CH3CN. Pour the combined
eluates thru a small funnel with filter paper containing 3 grams of anhydrous sodium
sulfate. Rinse the test tube and sodium sulfate with two 5 mL portions of MeCl2.
Collect the filtrate in a concentrator tube.
11.3.5 With the concentrator tube in a 28°C heating block, evaporate the eluate with a
stream of N: to 0.5 mL.
11.4 Extract Cleanup: Cleanup procedures may not be necessary for a relatively clean sample
matrix, such as most drinking waters. If particular circumstances demand the use of a
cleanup procedure, the analyst may use either procedure below or any other appropriate
procedure. However, the analyst first must demonstrate that the requirements of Sect. 10.3
and 10.5 can be met using the method as revised to incorporate the cleanup procedure.
11.4.1 Florisil column cleanup for phthalate esters:
11.4.1.1 Place 10 g of Florisil (Sect. 7.4) into a chromatographic column. Tap the
column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to
the top.
11.4.1.2 Preelute the column with 40 mL of hexane. Discard the eluate and just
prior to exposure of the sodium sulfate layer to the air, quantitatively
transfer the sample extract (Sect. 11.1.8 or 11.2.6) onto the column, using
an additional 2 mL of hexane to complete the transfer. Just prior to ex-
posure of the sodium sulfate layer to the air, add 40 mL of hexane and
continue the elution of the column. Discard this hexane eluate.
11.4.1.3 Next, elute the column with 100 mL of 20% ethyl ether in hexane (V/V)
into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Elute
the column at a rate of about 2 mL/min for all fractions. Concentrate the
collected fraction as in Sect. 11.1. No solvent exchange is necessary. .
Adjust the volume of the cleaned extract to 1 mL in the concentrator tube
and analyze by gas chromatography.
11.4.2 Alumina column cleanup for phthalate esters:
11.4.2.1 Place 10 g of alumina into a chromatographic column. Tap the column to
settle the alumina and add I cm of anhydrous sodium sulfate to the top.
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Method 506
11.4.2.2 Preelute the column with 40 mL of hexane. The rate for all elutions
should be about 2 mL/min. Discard the eluate and just prior to exposure
of the sodium sulfate layer to the air, quantitatively transfer the sample
extract (Sect. 11.1.8 or 11.2.6) onto the column, using an additional 2 mL
of hexane to complete the transfer. Just prior to exposure of the sodium
sulfate layer to the air, add 35 mL of hexane and continue the elution of
the column. Discard this hexane eluate.
11.4.2.3 Next, elute the column with 140 mL of 20% ethyl ether in hexane (V/V)
into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Con-
centrate the collected fraction as in Sect. 11.1. No solvent exchange is
necessary. Adjust the volume of the cleaned extract to 1 mL in the con-
centrator tube and analyze by gas chromatography.
11.5 Gas Chromatography
11.5.1 Table 1 summarizes the recommended operating conditions for the gas chromato-
graph. Included are retention data for the primary and confirmation columns. Other
capillary (open-tubular) columns, chromatographic conditions, or detectors may be
used if the requirements of Sect. 10 are met.
11.5.2 Calibrate the system daily as described in Sect. 9.
11.5.3 Inject 1 to 2 pL of the sample extract or standard into the gas chromatograph. Small-
er (1.0 jiL) volumes may be injected if automatic devices are employed. For opti-
mum reproducibility, an auto injector is recommended.
11.5.4 Identify the analytes in the sample by comparing the retention times of the peaks in
the sample chromatogram with those of the peaks in standard chromatograms. The
width of the retention time window used to make identifications should be based upon
measurements of actual retention time variations of standards over the course of a
day. Three times the standard deviation of a retention time for a compound can be
used to calculate a suggested window size; however, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.5.5 If the response for a peak exceeds the working range of the system, dilute the extract
and reanalyze
11.5.6 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11.5.7 The calibration curves should be linear over the range of concentrations in Tables
2-5.
12. CALCULATIONS
12.1 Calculate the amount of material injected from the peak response using the calibration curve or
calibration factor determined in Sect. 9.2.2. The concentration in the sample can be calculated
from Equation 1.
75
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Method 506
Equation 1
Concentration (ug/L) =
where:
A = Amount of material injected (ng).
V = Volume of extract injected
Vt = Volume of total extract
V = Volume of water extracted (mL).
12.2 Report results in ^g/L without correction for recovery data. All QC data obtained should be
reported with the sample results.
13. METHOD PERFORMANCE
Single laboratory accuracy and precision data were obtained by replicate liquid-liquid extrac-
tion analyses of reagent water fortified at two sets of concentrations of method analytes. The
data are given in Tables 2 and 3. Accuracy and precision data by liquid-solid extraction of
reagent water fortified at a single concentration are given in Table 4. Finally, method valida-
tion data obtained by the analyses of fortified tap water and raw source water are given in
Tables 5-7.
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Method 506
References
1. Glaser, J.V., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analysis for
Waste Waters." Environ. Sci. Technol. 15. 1426, 1981,
2. "Determination of Phthalates in Industrial and Municipal Wastewaters," EPA-600/4-81-063,
U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, October 1981.
3. Giam, C.S., Chan, H.S. and Nef, G.S. "Sensitive Method for Determination of Phthalate
Ester Plasticizers in Open-Ocean Biota Samples," Anal. Chem.. 47, 2225 (1975),
4. Giam, C.S., and Chan, H.S. "Control of Blanks in the Analysis of Phthalates in Air and
Ocean Biota Samples," U.S. National Bureau of Standards, Special Publication 442, pp.
701-708, 1976.
5. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
6. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, D3694-78. "Standard Practices for Preparation of
Sample Containers and for Preservation of Organic Constituents," American Society for
Testing and Materials, Philadelphia.
9. ASTM Annual Book of Standards, Part 31, D3370. "Standard Practices for Sampling Water,"
American Society for Testing and Materials, Philadelphia.
10. J.W. Eichelberger, T.D. Behymer and W.L. Budde, "Determination of Organic Compounds in
Drinking Water By Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass
Spectrometry", EPA Method 525, in Methods for the Determination of Organic Compounds in
Drinking Water. EPA-600/4-88/039, Environmental Monitoring Systems Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio 45268, December 1988, pp. 325-356.
77
-------
Method 506
Table 1. Retention Data and Chromatographic Conditions
Retention Time (min)
Parameter
Dimethyl phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butylbenzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
Column
17.23
20.29
27.57
34.19
34.85
37.51
41.77
Column 2
17.89
21.13
28.67
35.34
36.76
39.58
44.44
Column 1: DB-5, fused silica capillary, 30 m x 0.32 mm I.D., 0.25 micron film thickness, Helium
linear velocity = 30 cm/s.
Column 2: DB-1, fused silica capillary, 30 m x 0.32 mm I.D., 0.25 micron film thickness, Helium
linear velocity = 30 cm/s.
Chromatographic Conditions:
Injector temperature = 295 °C
Detector temperature = 295°C
Program - 1 min hold at 60°C,
6°C/min to 260°C, 10 min hold.
Splitless injection with 45 s delay
Table 2. Accuracy, Precision, and Method Detection Limit Data from Six Liquid-
Liquid Extraction Analyses of Fortified Reagent Water
Analyte
Dimethyl phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
True Cone.
(ug/U
2.02
1.51
2.62
6.00
6.03
5.62
17.18
Mean
Meas.
Cone.
(ug/U
1.42
1.16
1.78
3.27
3.94
2.92
7.96
Std. Dev.
(ug/L)
0.38
0.28
0.41
0.89
1.44
0.75
2.14
Mean
Accuracy
(% of True
Cone.)
70.3
76.8
67.9
54.5
65.3
52.0
46.3
MDL
(ug/L)
1.14
0.84
1.23
2.67
11.82
2.25
6.42
78
-------
Method 506
Table 3. Accuracy and Precision Data from Seven Liquid-liquid Extraction Analyses
of Fortified Reagent Water
Mean Accuracy Relative Standard
True Concentration <% of True Concen- Deviation
Analyte (ug/U tration) (%)
Dimethyl phthalate 15 73 16
Diethyl phthalate 15 71 16
Di-n-butyl phthalate 15 68 15
Butyl benzyl phthalate 15 71 15
Bis(2-ethylhexyl) adipate 30 69 18
Bis(2-ethylhexyl) phthalate 30 67 21
Di-n-octyl phthalate 30 62 23
Table 4. Accuracy And Precision Data from Six Liquid-Solid Extraction Analyses of
Fortified Reagent Water
Mean Accuracy Relative Standard
True Concentration (% of True Concen- Deviation
Analyte (ug/U tration) (%)
Dimethyl phthalate 15 74 11
Diethyl phthalate 15 85 10
Di-n-butyl phthalate 15 74 11
Butyl benzyl phthalate 15 72 14
Bis(2-ethylhexyl) adipate 30 84 11
Bis(2-ethylhexyl) phthalate 30 101 13
Di-n-octyl phthalate 30 85 13
Table 5. Accuracy and Precision Data from Six Liquid-liquid Extraction Analyses of
Fortified Tap Water
Mean Accuracy Relative Standard
True Concentration (% of True Concen- Deviation
Analyte (ug/U tration) (%)
Dimethyl phthalate 5 103 10.0
Diethyl phthalate 5 106 10.0
Di-n-butyl phthalate 5 94 6.8
Butyl benzyl phthalate 5 93 9.1
Bis(2-ethylhexyl) adipate 5 87 12.0
Bis(2-ethylhexyl) phthalate 5 93 4.9
Di-n-octyl phthalate 5 72 26.0
79
-------
Method 506
Table 6. Accuracy and Precision Data from Six Liquid-Liquid Extraction Analyses
of Fortified Raw Source Water
Mean Accuracy Relative Standard
True Concentration (% of True Concen- Deviation
Analyte (ug/L) tration) (%)
Dimethyl phthalate 5 59 51
Diethyl phthalate 5 78 45
Di-n-butyl phthalate 5 99 29
Butyl benzyl phthalate 5 72 23
Bis(2-ethylhexyl) adipate 5 115 32
Bis(2-ethylhexyl) phthalate 5 91 35
Di-n-octyl phthalate 5 54 24
Table 7. Accuracy and Precision Data From Six Liquid-Solid Extraction Analyses of
Fortified Raw Source Water
Mean Accuracy Relative Standard
True Concentration (% of True Concen- Deviation
Analyte (ug/L) tration) (%)
Dimethyl phthalate 5 110 20
Diethyl phthalate 5 111 32
Di-n-butyl phthalate 5 95 30
Butyl benzyl phthalate 5 82 20
Bis(2-ethylhexyl) adipate 5 65 24
Bis(2-ethylhexyl) phthalate 5 60 21
Di-n-octyl phthalate 5 53 15
80
-------
Method 506
2 Liter
Separately Funnel
H
\7
125mL
Solvent
Reservoir
Ground Glass
Stopper 14/35
LSE Cartridge
125mL
Solvent
Reservoir
Ground Glass
Stopper 14/35
LSE Cartridge
Rubber Stopper
No. 18-20 Luer-lok
Syringe Needle
H
100mL
Separately
Funnel
Drying Column
(Na2SO4)
1.2 cm x 40 cm
10 mL
Graduated
Vial
52-015-31
A. Extraction Apparatus
B. Elution Apparatus
Figure 1
81
-------
Method 506
o>
w
a
w
a>
cc
Time (Minutes)
52-015-32
Peaks obtained by injecting 5 ng for the 1st, 2nd, 4th and 5th
compounds, 10 ng for the 6th, 7th and 8th compounds, and 2.5 ng for
the 3rd compound. (Table 1)
Figure 2
82
-------
Method 507
The Determination of Nitrogen- and
Phosphorus-Containing Pesticides in Water
by Gas Chromatography with
a Nitrogen-Phosphorus Detector
Revision 2.0 - EPA EMSL-Ci
T. Engels (Battelle Columbus Laboratories) — National Pesticide Survey
Method 1, Revision 1.0 (1987)
R.L. Graves - Method 507, Revision 2.0 (1989)
-------
-------
Method 507
Determination of Nitrogen- and Phosphorus-Containing Pesticides in
Water by Gas Chromatography with a Nitrogen-Phosphorus Detector
1. SCOPE AND APPLICA TION
1.1
This is a gas chromatographic (GC) method applicable to the determination of certain
nitrogen- and phosphorus-containing pesticides in ground water and finished drinking
water." The following compounds can be determined using this method:
Analyte
Alachlor
Ametryn
Atraton
Atrazine
Bromacil
Butachlor
Butylate
Carboxin
Chlorpropham
Cycloate
Diazinon'*
Dichlorvos
Diphenarnid
Disulfoton"
Disulfoton sulfone*
Disulfoton sulfoxide'*
EPTC
Ethoprop
Fenamiphos
Fenarimol
Fluridone
Hexazinone
Merphos*
Methyl paraoxon
Metolachlor
Metribuzin
Mevinphos
MGK 264
Molinate
Napropamide
Norflurazon
Pebulate
Prometon
Prometryn
Pronamide3*
Propazine
Simazine
Simetryn
Stirofos
CAS No.
15972-60-8
834-12-8
1610-17-9
1912-24-9
314-40-9
23184-66-9
2008-41-5
5234-68-4
101-21-3
1134-23-2
333-41-5
62-73-7
957-51-7
298-04-4
2497-06-5
2497-07-6
759-94-4
13194-48-4
22224-92-6
60168-88-9
59756-60-4
51235-04-2
150-50-5
950-35-6
51218-45-2
21087-64-9
7786-34-7
113-48-4
2212-67-1
15299-99-7
27314-13-2
1114-71-2
1610-18-0
7287-19-6
23950-58-5
139-40-2
122-34-9
1014-70-6
22248-79-9
85
-------
Method 507
Analyte CAS No.
Tebuthiuron 34014-18-1
Terbacil 5902-51-2
Terbufos3* 13071-79-9
Terbutryn 886-50-0
Triadimefon 43121-43-3
Tricyclazole 41814-78-2
Vernolate 1929-77-7
a Compound exhibits aqueous instability. Samples for which this com-
pound is an analyte of interest must be extracted immediately (Sec-
tions 11.1 through 11.3).
* These compounds are only qualitatively identified in the National
Pesticides Survey (NPS) Program. These compounds are not quanti-
tated because control over precision has not been accomplished.
1.2 This method has been validated in a single laboratory and estimated detection limits (EDLs)
have been determined for the analytes above (Sect. 13). Observed detection limits may vary
among waters, depending upon the nature of interferences in the sample matrix and the
specific instrumentation used.
1.3 This method is restricted to use by or under the supervision of analysts experienced in the use
of GC and in the interpretation of gas chromatograms. Each analyst must demonstrate the
ability to generate acceptable results with this method using the procedure described in Sect.
10.3.
1.4 Analytes that are not separated chromatographically, i.e., analytes which have very similar
retention times, cannot be individually identified and measured in the same calibration mixture
or water sample unless an alternative technique for identification and quantitation exist (Section
11.5).
1.5 When this method is used to analyze unfamiliar samples for any or all of the analytes above,
analyte identifications should be confirmed by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample of approximately 1 L is extracted with methylene chloride by
shaking in a separatory funnel or mechanical tumbling in a bottle. The methylene chloride
extract is isolated, dried and concentrated to a volume of 5 mL during a solvent exchange to
methyl tertbutyl ether (MTBE). Chromatographic conditions are described which permit the
separation and measurement of the analytes in the extract by Capillary Column GC with a
nitrogen-phosphorus detector (NPD).
3. DEFINITIONS
3.1 Internal standard: A pure analyte(s) added to a solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of
the same solution. The internal standard must be an analyte that is not a sample component.
86
-------
Method 507
3.2 Surrogate analyte: A pure analyte(s), which is extremely unlikely to be found in any sample,
and which is added to a sample aliquot in known amount(s) before extraction and is measured
with the same procedures used to measure other sample components. The purpose of a surro-
gate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.4 Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.7 Laboratory performance check solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the labora-
tory is capable of making accurate and precise measurements at the required method detection
limit.
3.9 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.11 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
87
-------
Method 5O7
3.12 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality control sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.2 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with tap and reagent water. Drain
dry, and heat in an oven or muffle furnace at 400°C for 1 hour. Do not heat volu-
metric ware. Thermally stable materials might not be eliminated by this treatment.
Thorough rinsing with acetone may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any accumulation
of dust or other contaminants. Store inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
WARNING: When a solvent is purified, stabilizers added by the manufacturer
may be removed thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed thus potentially reducing
the shelf-life,
4.2 Interfering contamination may occur when a sample containing low concentrations of analytes
is analyzed immediately following a sample containing relatively high concentrations of
analytes. Between-sample rinsing of the sample syringe and associated equipment with MTBE
can minimize sample cross contamination. After analysis of a sample containing high concen-
trations of analytes, one or more injections of MTBE should be made to ensure that accurate
values are obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are coextracted from the sample.
Also, note that all the analytes listed in the scope and application section are not resolved from
each other on any one column, i.e., one analyte of interest may be an interferant for another
analyte of interest. The extent of matrix interferences will vary considerably from source to
source, depending upon the water sampled. Further processing of sample extracts may be
necessary. Positive identifications should be confirmed (Sect. 11.5).
88
-------
Method 507
4.4 It is important that samples and working standards be contained in the same solvent. The
solvent for working standards must be the same as the final solvent used in sample prepara-
tion. If this is not the case, chromatographic comparability of standards to sample may be
affected.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
ing the safe handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available and have been identified3 5
for the information of the analyst.
5.2 WARNING: When a solvent is purified, stabilizers added by the manufacturer may be
removed thus potentially making the solvent hazardous.
6. APPARATUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1 Sample bottle: Borosilicate, 1-L volume with graduations (Wheaton Media/Lab bottle 219820
or equivalent), fitted with screw caps lined with TFE-fluorocarbon. Protect samples from
light.The container must be washed and dried as described in Sect. 4.1.1 before use to mini-
mize contamination. Cap liners are cut to fit from sheets (Pierce Catalog No. 012736 or
equivalent) and extracted with methanol overnight prior to use.
6.2 Glassware
6.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground glass or
TFE-fluorocarbon stopper.
6.2.2 Tumbler bottle: 1.7-L (Wheaton Roller Culture Vessel or equivalent), with TFE-
fluorocarbon lined screw cap. Cap liners are cut to fit from sheets (Pierce Catalog
No. 012736) and extracted with methanol overnight prior to use.
6.2.3 Flask, Erlenmeyer: 500-mL.
6.2.4 Concentrator tube, Kuderna-Danish (K-D): 10- or 25-mL, graduated (Kontes
K-570050-2525 or K-570050-1025 or equivalent). Calibration must be checked at the
volumes employed in the test. Ground glass stoppers are used to prevent evaporation
of extracts.
6.2.5 Evaporative flask, K-D: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
6.2.6 Snyder column, K-D: Three-ball macro (Kontes K-503000-0121 or equivalent).
6.2.7 Snyder column, K-D: Two-ball micro (Kontes K-569001-0219 or equivalent).
6.2.8 Vials: Glass, 5- to 10-mL capacity with TFE-fluorocarbon lined screw cap.
89
-------
Method 507
6.3 Separator}' funnel shaker (Optional): Capable of holding 2-L separatory funnels and shaking
them with rocking motion to achieve thorough mixing of separatory funnel contents (available
from Eberbach Co. in Ann Arbor, MI or other suppliers).
6.4 Tumbler: Capable of holding tumbler bottles and tumbling them end-over-end at 30 turns/min
(Associated Design and Mfg. Co., Alexandria, VA. or other suppliers).
' *rf
6.5 Boiling stones: Carborundum, #12 granules (Arthur H. Thomas Co. #1590-033 or equiva- '
lent). Heat at 400°C for 30 min prior to use. Cool and store in desiccator.
6.6 Water bath: Heated, capable of temperature control (± 2°C). The bath should be used in a
hood.
6.7 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.8 Gas Chromatograph: Analytical system complete with temperature programmable GC suitable
for use with capillary columns and all required accessories including syringes, analytical
columns, gases, detector and stripchart recorder. A data system is recommended for measuring
peak areas. Table 1 lists retention times observed for method analytes using the columns and
analytical conditions described below.
6.8.1 Column 1 (Primary column): 30 m long x 0.25 mm I.D. DB-5 bonded fused silica
column, 0.25 jim film thickness (J&W Scientific) or equivalent. Helium carrier gas
flow is established at 30 cm/sec linear velocity and oven temperature is programmed
from 60°C to 300°C at 4°C/min. Data presented in this method were obtained using
this column. The injection volume was 2 uL in splitless mode with a 45 s delay. The
injector temperature was 250°C and the detector temperature was 300°C. Alternative
columns may be used in accordance with the provisions described in Sect. 10.4.
6.8.2 Column 2 (Confirmation column): 30 m long x 0.25 mm I.D.DB-1701 bonded fused
silica column, 0.25 /xm film thickness (J&W Scientific) or equivalent. Helium carrier
gas flow is established at 30 cm/sec linear velocity and oven temperature is pro-
grammed from 60C to 300°C at 4°C/min.
6.8.3 Detector: Nitrogen-phosphorus (NPD). A NPD was used to generate the validation
data presented in this method. Alternative detectors, including a mass spectrometer,
may be used in accordance with the provisions described in Sect. 10.4.
7. REAGENTS AND CONSUMABLE MATERIALS
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed thus potentially reducing
the shelf-life.
7.1 Acetone, methylene chloride, methyl tert.-butyl ether (MTBE): Distilled-in-glass quality or
equivalent.
7.2 Phosphate buffer, pH 7: Prepare by mixing 29.6 mL 0.1 N Hcl and 50 mL 0.1 M dipotassi-
um phosphate.
90
-------
Method 507
7.3 Sodium chloride (NaCl), crystal, ACS grade: Heat treat in a shallow tray at 450°C for a
minimum of 4 hours to remove interfering organic substances.
7.4 Sodium sulfate, granular, anhydrous, ACS grade: Heat treat in a shallow tray at 450°C for a
minimum of 4 hours to remove interfering organic substances.
7.5 Sodium thiosulfate, granular, anhydrous, ACS grade.
7.6 Triphenylphosphate (TPP): 98% purity, for use as internal standard (available from Aldrich
Chemical Co.).
7.7 l,3-Dimethyl-2-nitrobenzene: 98% purity, for use as surrogate standard (available from
Aldrich Chemical Co.).
7.8 Mercuric Chloride: ACS grade (Aldrich Chemical Co.) - for use as a bactericide. If any
other bactericide can be shown to work as well as mercuric chloride, it may be used instead.
7.9 Reagent water: Reagent water is defined as a water that is reasonably free of contamination
that would prevent the determination of any analyte of interest. Reagent water used to gener-
ate the validation data in this method was distilled water obtained from the Magnetic Springs
Water Co., Columbus, Ohio.
7.10 Stock Standard Solutions (1.00 /ig/^L): Stock standard solutions may be purchased as certi-
fied solutions or prepared from pure standard materials using the following procedure:
7.10.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
pure material. Dissolve the material in MTBE and dilute to volume in a 10-mL
volumetric flask. The stock solution for simazine should be prepared in methanol.
Larger volumes may be used at the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight may be used without correction to calculate the
concentration of the stock standard. Commercially prepared stock standards may be
used at any concentration if they are certified by the manufacturer or by an indepen-
dent source.
7.10.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw cap amber
vials. Store at room temperature and protect from light.
7.10.3 Stock standard solutions should be replaced after two months or sooner if comparison
with laboratory fortified blanks, or QC samples indicate a problem.
7.11 Internal Standard Solution: Prepare the internal standard solution by accurately weighing
approximately 0.0500 g of pure TPP. Dissolve the TPP in MTBE and dilute to volume in a
100-mL volumetric flask. Transfer the internal standard solution to a TFE-fluorocarbon-sealed
screw cap bottle and store at room temperature. Addition of 50 ^L of the internal standard
solution to 5 mL of sample extract results in a final TPP concentration of 5.0 /ng/mL. Solu-
tion should be replaced when ongoing QC (Sect. 10) indicates a problem. Note that TPP has
been shown to be an effective internal standard for the method analytes,' but other compounds
may be used if the quality control requirements in Sect. 10 are met.
7.12 Surrogate Standard Solution: Prepare the surrogate standard solution by accurately weighing
approximately 0.0250 g of pure l,3-dimethyl-2-nitrobenzene. Dissolve the 1,3-dimethyl-
2-nitrobenzene in MTBE and dilute to volume in a 100-mL volumetric flask. Transfer the
surrogate standard solution to a TFE-fluorocarbon-sealed screw cap bottle and store at room
temperature. Addition of 50 p,L of the surrogate standard solution to a 1-L sample prior to
91
-------
Method 507
extraction results in a 1,3-dimethyl-2-nitrobenzene concentration in the sample of 12.5
Solution should be replaced when ongoing QC (Sect. 10) indicates a problem. Note that
1,3-dimethyl-2-nitrobenzene has been shown to be an effective surrogate standard for the
method analytes,1 but other compounds may be used if the quality control requirements in
Sect. 10 are met.
7.13 Laboratory Performance Check Solution: Prepare the laboratory performance check solution
by adding 5 /*L of the vernolate stock solution, 0.5 mL of the bromacil stock solution, 30 fiL
of the prometon stock solution, 15 pL of the atrazine stock solution, 1.0 mL of the surrogate
solution, and 500 pL of the internal standard solution to a 100-mL volumetric flask. Dilute to
volume with MTBE and thoroughly mix the solution. Transfer to a TFE-fluorocarbon-sealed
screw cap bottle and store at room temperature. Solution should be replaced when ongoing
QC (Section 10) indicates a problem.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional sampling practices6 should
be followed; however, the bottle must not be prerinsed with sample before collection.
8.2 Sample Preservation and Storage
8.2.1 Add mercuric chloride (See 7.8) to the sample bottle in amounts to produce a concen-
tration of 10 mg/L. Add 1 mL of a solution containing 10 mg/mL of mercuric
chloride in reagent water to the sample bottle at the sampling site or in the laboratory
before shipping to the sampling site. A major disadvantage of mercuric chloride is
that it is a highly toxic chemical; mercuric chloride must be handled with caution, and
samples containing mercuric chloride must be disposed of properly.
8.2.2 If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
the sample bottle prior to collecting the sample.
8.2.3 After the sample is collected in a bottle containing preservative(s), seal the bottle and
shake vigorously for 1 min.
8.2.4 The samples must be iced or refrigerated at 4°C away from light from the time of
collection until extraction. Preservation study results indicated that most method
analytes present in samples were stable for 14 days when stored under these condi-
tions.' The analytes disulfoton sulfoxide, diazinon, pronamide, and terbufos exhibited
significant aqueous instability, and samples to be analyzed for these compounds must
be extracted immediately. The analytes carboxin, EPTC, fluridone, metolachlor,
napropamide, tebuthiuron, and terbacil exhibited recoveries of less than 60% after 14
days. Analyte stability may be affected by the matrix; therefore, the analyst should
verify that the preservation technique is applicable to the samples under study.
8.3 Extract Storage: Extracts should be stored at 4°C away from light. Preservation study results
indicate that most analytes are stable for 28 days; however, a 14-day maximum extract storage
time is recommended. The analyst should verify appropriate extract holding times applicable
to the samples under study.
92
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Method 507
9. CALIBRATION
9.1 Establish GC operating parameters equivalent to those indicated in Sect. 6.8. The GC system
may be calibrated using either the internal standard technique (Sect. 9.2) or the external
standard technique (Sect. 9.3). Be aware that NPDs may exhibit instability (i.e., fail to hold
calibration curves over time). The analyst may, when analyzing samples for target analytes
which are very rarely found, prefer to analyze on a daily basis a low level (e.g. 5 to 10 times
detection limit or 1/2 times the regulatory limit, whichever is less), sample (containing all
analytes of interest) and require some minimum sensitivity (e.g. 1/2 full scale deflection) to
show that if the analyte were present it would be detected. The analyst may then quantitate
using single point calibration (Sect. 9.2.5 or 9.3.4).
NOTE: Calibration standard solutions must be prepared such that no unresolved
analytes are mixed together.
9.2 Internal Standard Calibration Procedure: To use this approach, the analyst must select one or
more internal standards compatible in analytical behavior to the compounds of interest. The
analyst must further demonstrate that the measurement of the internal standard is not affected
by method or matrix interferences. TPP has been identified as a suitable internal standard.
9.2.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest by adding volumes of one or more stock standards
to a volumetric flask. If Merphos is to be determined, calibrate with DBF (S,S,S-
tributylphosphoro-trithioate). To each calibration standard, add a known constant
amount of one or more of the internal standards, and dilute to volume with MTBE.
The lowest standard should represent analyte concentrations near, but above, their
respective EDLs. The remaining standards should bracket the analyte concentrations
expected in the sample extracts, or should define the working range of the detector.
9.2.2 Analyze each calibration standard according to the procedure described in Sect. 11.4.
Tabulate response (peak height or area) against concentration for each compound and
internal standard. Calculate the response factor (RF) for each analyte and surrogate
using Equation 1.
Equation 1
RF
Where:
As = Response for the analyte.
Ais = Response for the internal standard.
Ca = Concentration of the internal standard \i.glL.
Cs = Concentration of the analyte to be measured
93
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Method 507
9.2.3 If the RF value over the working range is constant (20% RSD or less) the average RF
can be used for calculations. Alternatively, the results can be used to plot a calibration
curve of response ratios (AS/A,5) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than +20%, the test must be repeated
using a fresh calibration standard. If the repetition also fails, a new calibration curve
must be generated for that analyte using freshly prepared standards.
9.2.5 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards in MTBE. The single point
standard should be prepared at a concentration that produces a response that deviates
from the sample extract response by no more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check calibra-
tion.
9.3 External Standard Calibration Procedure
9.3.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest and surrogate compound by adding volumes of one
or more stock standards to a volumetric flask. If Merphos is to be determined,
calibrate with DEF (S,S,S-tributylphosphoro-trithioate). Dilute to volume with
MTBE. The lowest standard should represent analyte concentrations near, but above,
their respective EDLs. The remaining standards should bracket the analyte concentra-
tions expected in the sample extracts, or should define the working range of the
detector.
9.3.2 Starting with the standard of lowest concentration, analyze each calibration standard
according to Sect. 11.4 and tabulate response (peak height or area) versus the concen-
tration in the standard. The results can be used to prepare a calibration curve for
each compound. Alternatively, if the ratio of response to concentration (calibration
factor) is a constant over the working range (20% RSD or less), linearity through the
origin can be assumed and the average ratio or calibration factor can be used in place
of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be verified on each working
day by the measurement of a minimum of two calibration check standards, one at the
beginning and one at the end of the analysis day. These check standards should be at
two different concentration levels to verify the calibration curve. For extended periods
of analysis (greater than 8 hrs.), it is strongly recommended that check standards be
interspersed with samples at regular intervals during the course of the analyses. If the
response for any analyte varies from the predicted response by more than ±20%, the
test must be repeated using a fresh calibration standard. If the results still do not
agree, generate a new calibration curve.
9.3.4 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards in MTBE. The single point
94
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Method 507
standard should be prepared at a concentration that produces a response that deviates
from the sample extract response by no more than 20%.
9.3.5 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check calibra-
tion.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
determination of surrogate compound recoveries in each sample and blank, monitoring internal
standard peak area or height in each sample and blank (when internal standard calibration
procedures are being employed), analysis of laboratory reagent blanks, laboratory fortified
samples, laboratory fortified blanks, and QC samples.
10.2 Laboratory Reagent Blanks. Before processing any samples, the analyst must demonstrate that
all glassware and reagent interferences are under control. Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If within the retention time
window of any analyte of interest the LRB produces a peak that would prevent the determina-
tion of that analyte, determine the source of contamination and eliminate the interference
before processing samples.
10.3 Initial Demonstration of Capability.
10.3.1 Select a representative fortified concentration (about 10 times EDL or at the regulato-
ry Maximum Contaminant Level, whichever is lower) for each analyte. Prepare a
sample concentrate (in methanol) containing each analyte at 1000 times selected
concentration. With a syringe, add 1 mL of the concentrate to each of at least four
1-L aliquots of reagent water, and analyze each aliquot according to procedures
beginning in Sect. 11.
10.3.2 For each analyte the recovery value for all four of these samples must fall in the
range of R ± 30% (or within R ± 3SR if broader) using the values for R and SR for
reagent water in Table 2. For those compounds that meet the acceptance criteria,
performance is considered acceptable and sample analysis may begin. For those
compounds that fail these criteria, this procedure must be repeated using four fresh
samples until satisfactory performance has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. It is expected that as laboratory personnel gain experience with this
method the quality of data will improve beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC detectors, GC conditions, continuous
extraction techniques, concentration techniques (i.e. evaporation techniques), internal standards
or surrogate compounds. Each time such method modifications are made, the analyst must
repeat the procedures in Sect. 10.3.
95
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Method 507
10.5 Assessing Surrogate Recovery
10.5.1 When surrogate recovery from a sample or method blank is <70% or > 130%, check
(1) calculations to locate possible errors, (2) fortifying solutions for degradation, (3)
contamination, and (4) instrument performance. If those steps do not reveal the cause
of the problem, reanalyze the extract.
10.5.2 If a blank extract reanalysis fails the 70-130% recovery criterion, the problem must
be identified and corrected before continuing.
10.5.3 If sample extract reanalysis meets the surrogate recovery criterion, report only data
for the reanalyzed extract. If sample extract reanalysis continues to fail the recovery
criterion, report all data for that sample as suspect.
10.6 Assessing the Internal Standard
10.6.1 When using the internal standard calibration procedure, the analyst is expected to
monitor the IS response (peak area or peak height) of all samples during each analysis
day. The IS response for any sample chromatogram should not deviate from the daily
calibration check standard's IS response by more than 30%.
10.6.2 If >30% deviation occurs with an individual extract, optimize instrument perfor-
mance and inject a second aliquot of that extract.
10.6.2.1 If the reinjected aliquot produces an acceptable internal standard response
report results for that aliquot.
10.6.2.2 If a deviation of greater than 30% is obtained for the reinjected extract,
analysis of the sample should be repeated beginning with Sect. 11, provid-
ed the sample is still available. Otherwise, report results obtained from the
re-injected extract, but annotate as suspect.
10.6.3 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
calibration check standard.
10.6.3.1 If the check standard provides a response factor (RF) within 20% of the
predicted value, then follow procedures itemized in Sect. 10.6.2 for each
sample failing the IS response criterion.
10.6.3.2 If the check standard provides a response factor which deviates more than
20% of the predicted value, then the analyst must recalibrate, as specified
in Sect. 9.
10.7 Assessing Laboratory Performance: Laboratory Fortified Blank
10.7.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
every twenty samples or one per sample set (all samples extracted within a 24-h
period) whichever is greater. The fortified concentration of each analyte in the LFB
should be 10 times EDL or the MCL, whichever is less. Calculate accuracy as
percent recovery (X,). If the recovery of any analyte falls outside the control limits
(see Sect. 10.7.2), that analyte is judged out of control, and the source of the problem
should be identified and resolved before continuing analyses.
10.7.2 Until sufficient data become available from within their own laboratory, usually a
minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
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Method 507
performance against the control limits in Sect. 10.3.2 that are derived from the data in
Table 2. When sufficient internal performance data becomes available, develop
control limits from the mean percent recovery (X) and standard deviation (S) of the
percent recovery. These data are used to establish upper and lower control limits as
follows:
Upper Control Limit = X + 3S
Lower Control Limit = X — 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points. These calculated control
limits should never exceed those established in Sect. 10.3.2.
10.7.3 It is recommended that the laboratory periodically determine and document its detec-
tion limit capabilities for analytes of interest.
10.7.4 At least quarterly, analyze a QC sample from an outside source.
10.7.5 Laboratories are encouraged to participate in external performance evaluation studies
such as the laboratory certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as independent checks
on the analyst's performance.
10.8 Assessing Analyte Recovery: Laboratory Fortified Sample Matrix
10.8.1 The laboratory must add a known concentration to a minimum of 5% of the routine
samples or one sample concentration per set, whichever is greater. The fortified
concentration should not be less then the background concentration of the sample
selected for fortification. Ideally, the concentration should be the same as that used
for the laboratory fortified blank (Sect. 10.7). Over time, samples from all routine
sample sources should be fortified.
10.8.2 Calculate the percent recovery, P of the concentration for each analyte, after correct-
ing the analytical result, X, from the fortified sample for the background concentra-
tion, b, measured in the unfortified sample, i.e.,:
p = 100 (X - b)
fortifying concentration
and compare these values to control limits appropriate for reagent water data collected
in the same fashion. If the analyzed unfortified sample is found to contain NO back-
ground concentrations, and the added concentrations are those specified in Sect. 10.7,
then the appropriate control limits would be the acceptance limits in Sect. 10.7. If,
on the other hand, the analyzed unfortified sample is found to contain background
concentration, b, estimate the standard deviation at the background concentration, sb,
using regressions or comparable background data and, similarly, estimate the mean,
Xa, and standard deviation, sa, of analytical results at the total concentration after
fortifying. Then the appropriate percentage control limits would be P ± 3sP , where:
97
-------
Method 507
o 100 X
r -
(b + fortifying concentration)
and
sp = 100
fortifying concentration
For example, if the background concentration for Analyte A was found to be 1 /ug/L
and the added amount was also 1 jug/L, and upon analysis the laboratory fortified
sample measured 1.6 /u./L, then the calculated P for this sample would be (1.6 /ig/L
minus 1.0 /xg/L)/l /zg/L or 60%. This calculated P is compared to control limits
derived from prior reagent water data. Assume it is known that analysis of an inter-
ference free sample at 1 ^g/L yields an s of 0.12 /xg/L and similar analysis at 2.0
Hg/L yields X and s of 2.01 /xg/L and 0.20 //g/L, respectively. The appropriate limits
to judge the reasonableness of the percent recovery, 60%, obtained on the fortified
matrix sample is computed as follows:
100 (2.01
2.0 . _
+ (0.20
l.Opg/L
100.5% ± 300 (0.233) =
100.5% + 70% or 30% to 170% recovery of the added analyte.
10.9 Assessing Instrument System—Laboratory Performance Check (LPC): Instrument performance
should be monitored on a daily basis by analysis of the LPC sample. The LPC sample
contains compounds designed to indicate appropriate instrument sensitivity, column perfor-
mance (primary column) and chromatographic performance. LPC sample components and
performance criteria are listed in Table 3. Inability to demonstrate acceptable instrument
performance indicates the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method. If laboratory EDLs differ
from those listed in this method, concentrations of the instrument QC standard compounds
must be adjusted to be compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements or field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
98
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Method 507
11. PROCEDURE
11.1 Extraction (Manual Method)
11.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume (Sect. 11.1.6). Add preservative to blanks and QC check standards.
Fortify the sample with 50 pL of the surrogate standard solution. Pour the entire
sample into a 2-L separatory funnel.
11.1.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.
11.1.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
11.1.4 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by
vigorously shaking the funnel for 2 min with periodic venting to release excess pres-
sure. Allow the organic layer to separate from the water phase for a minimum of 10
min. If the emulsion interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical techniques to complete the
phase separation. The optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, centrifugation, or other physical
methods. Collect the methylene chloride extract in a 500-mL Erlenmeyer flask.
11.1.5 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
Perform a third extraction in the same manner.
11.1.6 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11.2 Extraction (Automated Method): Data presented in this method were generated using the
automated extraction procedure with the mechanical tumbler.
11.2.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume (Sect. 11.2.6). Add preservative to blanks and QC check standards.
Fortify the sample with 50 /tL of the surrogate standard solution. If the mechanical
separatory funnel shaker is used, pour the entire sample into a 2-L separatory funnel.
If the mechanical tumbler is used, pour the entire sample into a tumbler bottle.
11.2.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.
11.2.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
11.2.4 Add 300 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the
inner walls. Transfer the solvent to the sample contained in the separatory funnel or
tumbler bottle, seal, and shake for 10 s, venting periodically. Repeat shaking and
venting until pressure release is not observed. Reseal and place sample container in
appropriate mechanical mixing device (separatory funnel shaker or tumbler). Shake
or tumble the sample for 1 hour. Complete mixing of the organic and aqueous phases
should be observed within about 2 min after starting the mixing device.
11.2.5 Remove the sample container from the mixing device. If the tumbler is used, pour
contents of tumbler bottle into a 2-L separatory funnel. Allow the organic layer to
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Method 507
separate from the water phase for a minimum of 10 min. If the emulsion interface
between layers is more than one third the volume of the solvent layer, the analyst
must employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring, filtration through glass
wool, centrifugation, or other physical methods. Collect the methylene chloride
extract in a 500-mL Erlenmeyer flask.
11.2.6 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11.3 Extract Concentration
11.3.1 Assemble a K-D concentrator by attaching a 25-mL concentrator tube to a 500-mL
evaporative flask. Other concentration devices or techniques may be used in place of
the K-D if the requirements of Sect. 10.3 are met.
11.3.2 Dry the extract by pouring it through a solvent-rinsed drying column containing about
10 cm of anhydrous sodium sulfate. Collect the extract in the K-D concentrator, and
rinse the column with 20-30 mL methylene chloride. Alternatively, add about 5 g
anhydrous sodium sulfate to the extract in the Erlenmeyer flask; swirl flask to dry
extract and allow to sit for 15 min. Decant the methylene chloride extract into the
K-D concentrator. Rinse the remaining sodium sulfate with two 25-mL portions of
methylene chloride and decant the rinses into the K-D concentrator.
11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and attach a macro Snyder
column. Prewet the Snyder column by adding about 1 mL methylene chloride to the
top. Place the K-D apparatus on a hot water bath, 65 to 70°C, so that the concentra-
tor tube is partially immersed in the hot water, and the entire lower rounded surface
of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and
the water temperature as required to complete the concentration in 15 to 20 min. At
the proper rate of distillation the balls of the column will actively chatter, but the
chambers will not flood. When the apparent volume of liquid reaches 2 mL, remove
the K-D apparatus and allow it to drain and cool for at least 10 min.
11.3.4 Remove the Snyder column and rinse the flask and its lower joint into the concentra-
tor tube with 1 to 2 mL of MTBE. Add 5-10 mL of MTBE and a fresh boiling stone.
Attach a micro-Snyder column to the concentrator tube and prewet the column by
adding about 0.5 mL of MTBE to the top. Place the micro K-D apparatus on the
water bath so that the concentrator tube is partially immersed in the hot water. Adjust
the vertical position of the apparatus and the water temperature as required to com-
plete concentration in 5 to 10 min. When the apparent volume of liquid reaches 2
mL, remove the micro K-D from the bath and allow it to drain and cool. Add 5-10
mL MTBE to the micro K-D and reconcentrate to 2 mL. Remove the micro K-D
from the bath and allow it to drain and cool. Remove the micro Snyder column, and
rinse the walls of the concentrator tube while adjusting the volume to 5.0 mL with
MTBE. NOTE: If methylene chloride is not completely removed from the final
extract, it may cause detector problems.
11.3.5 Transfer extract to an appropriate- sized TFE-fluorocarbon- sealed screw-cap vial and
store, refrigerated at 4°C, until analysis by GC-NPD.
100
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Method 507
11.4 Gas Chromatography
11.4.1 Sect. 6.8 summarizes the recommended operating conditions for the gas chromato-
graph. Included in Table 1 are retention times observed using this method. Other GC
columns, chromatographic conditions, or detectors may be used if the requirements of
Sect. 10.3 are met.
11.4.2 Calibrate the system daily as described in Sect. 9. The standards and extracts must be
in MTBE.
11.4.3 If the internal standard calibration procedure is used, add 50 ftL of the internal stan-
dard solution to the sample extract, seal, and shake to distribute the internal standard.
11.4.4 Inject 2 ^L of the sample extract. Record the resulting peak size in area units.
11.4.5 If the response for the peak exceeds the working range of the system, dilute the
extract and reanalyze.
11.5 Identification of Analytes
11.5.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound corre-
sponds, within limits, to the retention time of a standard compound, then identification
is considered positive.
11.5.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.5.3 Identification requires expert judgement when sample components are not resolved
chromatographically. When peaks obviously represent more than one sample compo-
nent (i.e., broadened peak with shoulder(s) or valley between two or more maxima),
or any time doubt exists over the identification of a peak on a chromatogram, appro-
priate alternative techniques to help confirm peak identification, need be employed.
For example, more positive identification may be made by the use of an alternative
detector which operates on a chemical/physical principle different from that originally
used, e.g., mass spectrometry, or the use of a second chromatography column. A
suggested alternative column is described in Sect. 6.8.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for the analyte using the
calibration procedure described in Sect. 9.
12.2 If the internal standard calibration procedure is used, calculate the concentration (C) in the
sample using the response factor (RF) determined in Sect. 9.2 and Equation 2, or determine
sample concentration from the calibration curve.
707
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Method 507
Equation 2
(A
C (pglL) =
(X,,) (RF) (Vo)
Where:
A s = Response for the parameter to be measured.
An = Response for the internal standard.
/ = Amount of internal standard added to each extract
Vo = Volume of water extracted (L).
12.3 If the external standard calibration procedure is used, calculate the amount of material injected
from the peak response using the calibration curve or calibration factor determined in Sect.
9.3.2. The concentration (C) in the sample can be calculated from Equation 3.
Equation 3
Concentration (us/L) =
Where:
A = Amount of material injected (ng).
V( = Volume of extract injected (fj.L) .
Vt = Volume of total extract (/iL).
Vt = Volume of water extracted (mL).
13. PRECISION AND A CCURA c Y
13.1 In a single laboratory, analyte recoveries from reagent water were determined at five concen-
tration levels. Results were used to determine analyte EDLs and demonstrate method range.1
Analytes were divided into five groups for recovery studies. Analyte EDLs and analyte
recoveries and standard deviation about the percent recoveries at one concentration are given
in Table 2.
13.2 In a single laboratory, analyte recoveries from two standard synthetic ground waters were
determined at one concentration level. Results were used to demonstrate applicability of the
method to different ground water matrices.' Analyte recoveries from the two synthetic matri-
ces are given in Table 2.
102
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Method 5O7
References
1.National Pesticide Survey Method No. 1: Determination of Nitrogen- and Phosphorus-Containing
Pesticides in Groundwater by Gas Chromatography with a Nitrogen-Phosphorus Detector.
2.ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation", American Society for Testing and Materials,
Philadelphia, PA, 1986.
3. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Halth Standards, General Industry," (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82," Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
103
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Method 507
Table 1 . Retention Times for Method Analytes
Analyte
1 ,3-Dimethyl-2-nitrobenzene (surrogate)
Dichlorvos
Disulfoton sulfoxide
EPIC
Butylate
Mevinphos
Vernolate
Pebulate
Tebuthiuron
Molinate
Ethoprop
Cycloate
Chlorpropham
Atraton
Simazine
Prometon
Atrazine
Propazine
Terbufos
Pronamide
Diazinon
Disulfoton
Terbacil
Metribuzin
Methyl paraoxon
Simetryn
Alachlor
Ametryn
Prometryn
Terbutryn
Bromacil
Metolachlor
Triadimefon
MGK 264C
Diphenamid
Stirofos
Disulfoton sulfone
Butachlor
Fenamiphos
Napropamide
Tricyclazole
Merphos"
Carboxin
Norflurazon
Triphenyl phosphate (int. std.)
Hexazinone
Fenanmol
Flundone
Retention
Col. 1 |
14.48
16.54
19.08
20.07
22.47
22.51
22.94
23.41
25.15
25.66
28.58
28.58
29.09
31.26
31.49
31.58
31.77
32.01
32.57
32.76
33.23
33.42
33.79
35.20
35.58
35.72
35.96
36.00
36.14
36.80
37.22
37.74
38.12
38.73
38.87
41.27
41.31
41.45
41.78
41.83
42.25
42.35
42.77
45.92
47
46.58
51.32
56.68
Time'
Col. 2
b
15.35
_ b
16.57
18.47
21.92
19.25
19.73
42.77
22.47
26.42
29.67
_ b
29.97
31.32
30
31.23
31.13
b
32.63
b
30.9
b
34.73
34.1
34.55
34.1
34.52
34.23
34.8
40
35.7
37
36.73
37.97
39.65
42.42
39
41
b
44.33
39.28
42.05
47.58
45.4
47.8
50.02
59.07
704
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Method 507
Table 1. Retention Times for Method Analytes (com.)
3 Columns and analytical conditions are described in Sect, 6.8.1 and 6.8.2.
b Data not available
0 MGK 264 gives two peaks; peak identified in this table used for quantification.
d Merphos is converted to S,S,S-tributylphosphoro-trithioate (DEF) in the hot GC injection port;
DEF is actually detected using these analyses conditions.
Table 2. Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters3
Analyte
Alachlor
Ametryn
Ametraton
Atrazine
Bromacil
Butachlor
Butylate
Carboxin
Chlorpropham
Cycloate
Diazinon
Dichlorvos
Diphenamid
Disulfoton
Disulfoton
sulfone
Disulfoton
sulfoxide
EPIC
Ethoprop
Fenamiphos
Fenarimol
Fluridone
Hexazinone
Merphos
Methyl
paraoxon
Metolachlor
Metribuzin
Mevinphos
MGK 264
Molinate
Napropamide
Norflurazon
Pebulate
Prometon
Prometryn
Pronamide
Propazine
EDL"
frg/L)
0.38
2
0.6
0.13
2.5
0.38
0.15
0.6
0.5
0.25
0.25
2.5
0.6
0.3
3.8
0.38
0.25
0.19
1
0.38
3.8
0.76
0.25
2.5
0.75
0.15
5
0.5
0.15
0.25
0.5
0.13
0.3
0.19
0.76
0.13
Cone.
3.8
20
6
1.3
25
3.8
1.5
6
5
2.5
2.5
25
6
3
7.5
3.8
2.5
1.9
10
3.8
38
7.6
2.5
25
7.5
1.5
50
5
1.5
2.5
5
1.3
3
1.9
7.6
1.3
Reagent
f¥ \
95
91
91
92
91
96
97
102
93
89
115
97
93
89
98
87
85
103
90
99
87
90
96
98
93
101
95
100
98
101
94
94
78
93
91
92
Water
s/
11
10
11
8
9
4
21
4
11
9
7
6
8
10
10
11
9
5
8
5
9
7
8
10
4
5
11
4
18
6
5
9
9
8
10
8
Synthetic
R
82
102
84
89
81
93
36
98
82
97
83
86
88
107
92
88
83
91
87
89
91
86
90
97
92
99
93
91
83
89
101
80
89
91
84
89
Water 1
SR
6
11
7
6
5
15
8
13
7
14
8
6
4
12
5
22
5
7
5
6
11
6
4
8
10
10
6
11
8
5
15
6
5
8
7
6
Synthetic
R
90
96
91
92
88
84
83
87
93
93
84
106
93
95
96
54
86
79
89
89
86
95
92
94
84
86
92
83
89
104
87
98
63
93
92
92
Water 2
SR
8
4
8
5
8
5
8
5
8
3
3
16
5
5
3
19
4
3
2
6
10
9
4
4
4
4
6
9
18
4
15
2
4
8
5
105
-------
Method 507
Table 2. Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters3 (cont.)
Analyte
Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
Triadimefon
Tricyclazole
Vernolate
EDL"
0.075
0.25
0.76
1.3
4.5
0.5
0.25
0.65
1
0.13
Cone.
(fjg/U
0.75
2.5
7.6
13
45
5
2.5
6.5
10
1.3
Reagent
Ff |
100
99
98
84
97
97
94
93
86
93
Water
S"
•>R
7
5
6
9
6
4
9
8
7
6
Synthetic
*
86
88
84
85
86
80
91
94
90
79
Water 1
SR
5
4
6
10
5
6
8
5
6
9
Synthetic
"
103
103
95
98
102
77
92
95
90
81
Water 2
s,
14
14
10
13
12
7
4
5
11
2
Data corrected for blank and represent the analysis of 7-8 samples using mechanical tumbling
and internal standard calibration.
EDL = estimated detection limit; defined as either MDL (Appendix B to 40 CFR Part 136 —
Definition and Procedure for the Determination of the Method Detection Limit—Revision 1.11)
or a level of compound in a sample yielding a peak in the final extract with signal-to-noise ratio
of approximately 5, whichever value is higher. The concentration used in determining the EDL
is not the same as the concentration presented in this table.
R = average percent recovery.
S = standard deviation of the percent recovery.
Corrected for amount found in blank; Absopure Nature Artesian Spring Water obtained from
the Absopure Water Company in Plymouth, Michigan.
Corrected for amount found in blank; reagent water fortified with fulvic acid at the 1 mg/L
concentration level. A well-characterized fulvic acid, available from the International Humic
Substances Society (associated with the United States Geological Survey in Denver, Colorado),
was used.
106
-------
Method 507
Table 3. Laboratory Performance Check Solution
Cone.
Test Analyte (fjg/mU Requirements
Sensitivity Vernolate 0.05 Detection of analyte; S/N> 3
Chromatographic performance Bromacil 5.0 0.80 < PGF < 1.20a
Column performance Prometon 0.30 Resolution > 0.7b
Atrazine 0.15
a PGF = peak Gaussian factor. Calculated using the equation:
1.83xW -1
PGF = ' 2
(•» f ~\
.1 is the peak width at half height and W Ur^r is the peak width at tenth height
b Resolution between the two peaks as defined by the equation:
w
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
707
-------
-------
Method 508
Determination of Chlorinated Pesticides in
Water by Gas Chromatography with an
Electron Capture Detector
Revision 3.0 — EPA EMSL-Ci
J.J. Lichtenberg, J.E. Longbottom, T.A. Bellar, J.W. Eichelberger,
and R.C. Dressman - EPA 600/4-81-053, Revision 1.0 (1981)
T. Engels (Battelle Columbus Laboratories) — National Pesticide Survey Method 2,
Revision 2.0 (1987)
R. L. Graves - Method 508, Revision 3.0 (1989)
-------
-------
Method SOS
Determination of Chlorinated Pesticides in Water by Gas
Chromatography with an Electron Capture Detector
1. SCOPE AND APPLICA TION
1.1 This is a gas chromatographic (GC) method applicable to the determination of certain chlori-
nated pesticides in groundwater and finished drinking water.' The following compounds can
be determined using this method:
Compound
Aldrin
Chlordane-a
Chlordane-7
Chloroneb
Chlorobenzilate*
Chlorothalonil
Chlorpyrifos
DCPA
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Etridiazole
HCH-a
HCH-/3
HCH-5a
HCH-7 (Lindane)
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Methoxychlor
cis-Permethrin
trans-Permethrin
Propachlor
Trifluralin
Aroclor 1016*
Aroclor 1221*
Aroclor 1232*
Aroclor 1242*
Aroclor 1248*
Aroclor 1254*
Aroclor 1260*
Toxaphene*
Chlordane*
CAS No.
309-00-2
5103-71-9
5103-74-2
2675-77-6
510-15-6
1897-45-6
2921-88-2
1861-32-1
72-54-8
72-55-9
50-29-3
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
2593-15-9
319-84-6
319-85-7
319-86-8
58-89-9
76-44-8
1024-57-3
118-74-1
72-43-5
61949-76-6
61949-77-7
1918-16-7
1582-09-8
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
8001-35-2
57-74-9
The extraction conditions of this method are comparable to USEPA Method
608, which does measure the multicomponent constituents: commercial
777
-------
Method 508
polychlorinated biphenyl (PCB) mixtures (Aroclors), toxaphene, and chlordane.
The extract derived from this procedure may be analyzed for these
constituents by using the GC conditions prescribed in either Method 608
(packed column) or Method 505 (capillary column). The columns used in this
method may well be adequate, however, no data were collected for these
constituents during methods development.
d These compounds are only qualitatively identified in the National Pesticides
Survey (NFS) Program. These compounds are not quantitated because control
over precision has not been accomplished.
1.2 This method has been validated in a single laboratory and estimated detection limits (EDLs)
have been determined for the analytes above (Sect. 13). Observed detection limits may vary
between waters, depending upon the nature of interferences in the sample matrix and the
specific instrumentation used.
1.3 This method is restricted to use by or under the supervision of analysts experienced in the use
of GC and in the interpretation of gas chromatograms. Each analyst must demonstrate the
ability to generate acceptable results with this method using the procedure described in Sect.
10.3.
1.4 Degradation of DDT and Endrin caused by active sites in the injection port and GC columns
may occur. This is not as much a problem with new capillary columns as with packed
columns. However, high boiling sample residue in capillary columns will create the same
problem after injection of sample extracts.
1.5 Analytes that are not separated chromatographically, i.e., analytes which have very similar
retention times cannot be individually identified and measured in the same calibration mixture
or water sample unless an alternative technique for identification and quantitation exist (Sect.
ll.5).
1.6 When this method is used to analyze unfamiliar samples for any or all of the analytes above,
analyte identifications must be confirmed by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample of approximately 1 L is solvent extracted with methylene
chloride by shaking in a separatory funnel or mechanical tumbling in a bottle. The methylene
chloride extract is isolated, dried and concentrated to a volume of 5 mL after solvent substitu-
tion with methyl tert-butyl ether (MTBE). Chromatographic conditions are described which
permit the separation and measurement of the analytes in the extract by capillary column/GC
with an electron capture detector (ECD).
3. DEFINITIONS
3.1 Internal standard: A pure analyte(s) added to a solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of
the same solution. The internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte: A pure analyte(s), which is extremely unlikely to be found in any sample,
and which is added to a sample aliquot in known amount(s) before extraction and is measured
772
-------
Method 508
with the same procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.7 Laboratory performance check solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of the
method analytes are added in the laboratory. The LFB is analyzed exactly like a sample, and
its purpose is to determine whether the methodology is in control, and whether the laboratory
is capable of making accurate and precise measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.11 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
113
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Method 508
3.13 Quality control sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or
environmental samples. The QCS is obtained from a source external to the laboratory, and is
used to check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.2 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with tap and reagent water. Drain
dry, and heat in an oven or muffle furnace at 400°C for 1 hour. Do not heat
volumetric ware. Thermally stable materials such as PCBs might not be eliminated by
this treatment. Thorough rinsing with acetone may be substituted for the heating.
After drying and cooling, seal and store glassware in a clean environment to prevent
any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed thus potentially reducing
the shelf-life.
4.2 Interferences by phthalate esters can pose a major problem in pesticide analysis when using the
electron capture detector. These compounds generally appear in the chromatogram as large
peaks. Common flexible plastics contain varying amounts of phthalates that are easily
extracted or leached during laboratory operations. Cross contamination of clean glassware
routinely occurs when plastics are handled during extraction steps, especially when
solvent-wetted surfaces are handled. Interferences from phthalates can best be minimized by
avoiding the use of plastics in the laboratory. Exhaustive cleanup of reagents and glassware
may be required to eliminate background phthalate contamination.3'4
4.3 Interfering contamination may occur when a sample containing low concentrations of analytes
is analyzed immediately following a sample containing relatively high concentrations of
analytes. Between-sample rinsing of the sample syringe and associated equipment with MTBE
can minimize sample cross contamination. After analysis of a sample containing high
concentrations of analytes, one or more injections of MTBE should be made to ensure that
accurate values are obtained for the next sample.
114
-------
Method 508
4.4 Matrix interferences may be caused by contaminants that are coextracted from the sample.
Also, note that all the analytes listed in the Scope and Application Section are not resolved
from each other on any one column, i.e., one analyte of interest may be an interferant for
another analyte of interest. The extent of matrix interferences will vary considerably from
source to source, depending upon the water sampled. Cleanup of sample extracts may be
necessary. Positive identifications should be confirmed (Sect. 11.5).
4.5 It is important that samples and standards be contained in the same solvent, i.e., the solvent
for final working standards must be the same as the final solvent used in sample preparation. If
this is not the case chromatographic comparability of standards to sample may be affected.
4.6 WARNING: A dirty injector insert will cause the late eluting compounds to drop off.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method. A reference file of
material safety data sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified5"7 for the information of the analyst.
5.2 WARNING: When a solvent is purified stabilizers added by the manufacturer are removed
thus potentially making the solvent hazardous.
6. APPARA TUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1 Sample Bottle: Borosilicate, 1-L volume with graduations (Wheaton Media/Lab bottle 219820
or equivalent), fitted with screw caps lined with TFE-fluorocarbon. Protect samples from
light. The container must be washed and dried as described in Sect. 4.1.1 before use to
minimize contamination. Cap liners are cut to fit from sheets (Pierce Catalog No. 012736)
and extracted with methanol overnight prior to use.
6.2 Glassware
6.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground glass or
TFE-fluorocarbon stopper.
6.2.2 Tumbler bottle 1.7-L (Wheaton Roller Culture Vessel or equivalent), with
TFE-fluorocarbon lined screw cap. Cap liners are cut to fit from sheets (Pierce
Catalog No. 012736) and extracted with methanol overnight prior to use.
6.2.3 Flask, Erlenmeyer: 500-mL.
6.2.4 Concentrator tube, Kuderna-Danish (K-D) 10- or 25-mL, graduated (Kontes
K-570050-1025 or K-570050-2525 or equivalent). Calibration must be checked at the
volumes employed in the test. Ground glass stoppers are used to prevent evaporation
of extracts.
115
-------
Method 508
6.2.5 Evaporative flask, K-D 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
6.2.6 Snyder column, K-D three-ball macro (Kontes K-503000-0121 or equivalent).
6.2.7 Snyder column, K-D two-ball micro (Kontes K-569001-0219 or equivalent).
6.2.8 Vials: Glass, 5- to 10-mL capacity with TFE-fluorocarbon lined screw cap.
6.3 Separatory Funnel Shaker: Capable of holding 2-L separatory funnels and shaking them with
rocking motion to achieve thorough mixing of separatory funnel contents (available from
Eberbach Co. in Ann Arbor, MI or other suppliers).
6.4 Tumbler: Capable of holding tumbler bottles and tumbling them end-over-end at 30 turns/min
(Associated Design and Mfg. Co., Alexandria, VA or other suppliers.).
6.5 Boiling Stones Carborundum, #12 granules (Arthur H. Thomas Co. #1590-033 or equivalent).
Heat at 400°C for 30 min prior to use. Cool and store in a desiccator.
6.6 Water Bath: Heated, capable of temperature control (± 2°C). The bath should be used in a
hood.
6.7 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.8 Gas Chromatograph: Analytical system complete with temperature programmable GC suitable
for use with capillary columns and all required accessories including syringes, analytical
columns, gases, detector and stripchart recorder. A data system is recommended for measuring
peak areas. Table 1 lists retention times observed for method analytes using the columns and
analytical conditions described below.
6.8.1 Column 1 (Primary column): 30 m long x 0.25 mm I.D. DB-5 bonded fused silica
column, 0.25 ^m film thickness (J&W Scientific). Helium carrier gas flow is
established at 30 cm/sec linear velocity and oven temperature is programmed from
60°C to 300°C at 4°C/min. Data presented in this method were obtained using this
column. The injection volume was 2 /nL splitless mode with a 45 sec. delay. The
injector temperature was 250°C and the detector temperature was 320°C. Column
performance criteria are presented in Table 3 (See Section 10.9). Alternative columns
may be used in accordance with the provisions described in Sect. 10.4.
6.8.2 Column 2 (Alternative column): 30 m long X 0.25 mm I.D.DB-1701 bonded fused
silica column, 0.25 /*m film thickness (J&W Scientific). Helium carrier gas flow is
established at 30 cm/sec linear velocity and oven temperature is programmed from
60°C to 300°C at 4°C/min.
6.8.3 Detector: Electron capture. This detector has proven effective in the analysis of
spiked reagent and artificial ground waters. An ECD was used to generate the
validation data presented in this method. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Sect. 10.4.
7. REAGENTS AND CONSUMABLE MATERIALS
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed thus potentially making the solvent hazardous. Also, when a solvent is
116
-------
Method 508
purified, preservatives added by the manufacturer are removed thus potentially reducing
the shelf-life.
7.1 Acetone, methylene chloride, MTBE: Distilled-in-glass quality or equivalent.
7.2 Phosphate Buffer, pH7: Prepare by mixing 29.6 mL 0.1 N Hcl and 50 mL 0.1 M dipotassium
phosphate.
7.3 Sodium chloride, crystal, ACS grade. Heat treat in a shallow tray at 450°C for a minimum of
4 hours to remove interfering organic substances.
7.4 Sodium sulfate, granular, anhydrous, ACS grade. Heat treat in a shallow tray at 450°C for a
minimum of 4 hours to remove interfering organic substances.
7.5 Sodium thiosulfate, granular, anhydrous, ACS grade.
7.6 Pentachloronitrobenzene (PCNB) 98% purity, for use as internal standard.
7.7 4,4'-Dichlorobiphenyl (DCB) 96% purity, for use as surrogate standard (available from
Chemicals Procurement Inc.).
7.8 Mercuric Chloride—ACS grade: For use as a bactericide. If any other bactericide can be
shown to work as well as mercuric chloride, it may be used instead.
7.9 Reagent Water: Reagent water is defined as water that is reasonably free of contamination that
would prevent the determination of any analyte of interest. Reagent water used to generate the
validation data in this method was distilled water obtained from the Magnetic Springs Water
Co., Columbus, Ohio.
7.10 Stock Standard Solutions (1.00 ^g//*L): Stock standard solutions may be purchased as
certified solutions or prepared from pure standard materials using the following procedure:
7.10.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
pure material. Dissolve the material in MTBE and dilute to volume in a 10-mL volu-
metric flask. Larger volumes may be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.10.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw cap amber
vials. Store at room temperature and protect from light.
7.10.3 Stock standard solutions should be replaced after two months or sooner if comparison
with laboratory fortified blanks, or QC samples indicate a problem.
7.11 Internal Standard Solution: Prepare an internal standard fortifying solution by accurately
weighing approximately 0.0010 g of pure PCNB. Dissolve the PCNB in MTBE and dilute to
volume in a 10-mL volumetric flask. Transfer the internal standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature. Addition of 5 jiL of the
internal standard fortifying solution to 5 mL of sample extract results in a final internal
standard concentration of 0.1 jug/mL. Solution should be replaced when ongoing QC (Sect.
10) indicates a problem. Note that PCNB has been shown to be an effective internal standard
for the method analytes,' but other compounds may be used if the quality control requirements
in Section 10 are met.
777
-------
Method 508
7.12 Surrogate Standard Solution: Prepare a surrogate standard fortifying solution by accurately
weighing approximately 0.0050 g of pure DCB. Dissolve the DCS in MTBE and dilute to
volume in a 10-inL volumetric flask. Transfer the surrogate standard fortifying solution to a
TFE-fluorocarbon-sealed screw cap bottle and store at room temperature. Addition of 50 /xL
of the surrogate standard fortifying solution to a 1-L sample prior to extraction results in a
surrogate standard concentration in the sample of 25 ^ig/L and, assuming quantitative recovery
of DCB, a surrogate standard concentration in the final extract of 5.0 jug/mL. Solution should
be replaced when ongoing QC (Sect. 10) indicates a problem. Note DCB has been shown to
be an effective surrogate standard for the method analytes,1 but other compounds may be used
if the quality control requirements in Section 10 are met.
7.13 Laboratory Performance Check Solution: Prepare by accurately weighing 0.0010 g each of
chlorothalonil, chlorpyrifos, DCPA, and HCH-6. Dissolve each analyte in MTBE and dilute
to volume in individual 10-mL volumetric flasks. Combine 2 fj.L of the chloropyrifos stock
solution, 50 ^L of the DCPA stock solution, 50 /*L of the chlorothalonil stock solution, and
40 /xL of the HCH-5 stock solution to a 100-mL volumetric flask and dilute to volume with
MTBE. Transfer to a TFE-fluorcarbon-sealed screw cap bottle and store at room temperature.
Solution should be replaced when ongoing QC (Section 10) indicates a problem.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional sampling practices (8)
should be followed; however, the bottle must not be prerinsed with sample before collection.
8.2 Sample Preservation
8.2.1 Add mercuric chloride (See 7.8) to the sample bottle in amounts to produce a
concentration of 10 mg/L. Add 1 mL of a 10 mg/mL solution of mercuric chloride in
reagent water to the sample bottle at the sampling site or in the laboratory before
shipping to the sampling site. A major disadvantage of mercuric chloride is that it is
a highly toxic chemical; mercuric chloride must be handled with caution, and samples
containing mercuric chloride must be disposed of properly.
8.2.2 If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
the sample bottle prior to collecting the sample.
8.2.3 After adding the sample to the bottle containing preservative(s), seal the sample bottle
and shake vigorously for 1 min.
8.2.4 Samples must be iced or refrigerated at 4°C from the time of collection until
extraction. Preservation study results indicate that most of the target analytes present
in the samples are stable for 7 days when stored under these conditions.' Preservation
data for the analytes chlorthalonil, a-HCH, 6-HCH, 7-HCH, cis-permethrin, trans-
permethrin, and trifluralin are nondefinitive, and therefore if these are analytes of
interest, it is recommended that the samples be analyzed immediately. Analyte
stability may be affected by the matrix; therefore, the analyst should verify that the
preservation technique is applicable to the samples under study.
775
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Method 508
8.3 Extract Storage
8.3.1 Sample extracts should be stored at 4°C away from light. A 14-day maximum extract
storage time is recommended. However, analyte stability may be affected by the
matrix; therefore, the analyst should verify appropriate extract holding times
applicable to the samples under study.
9. CALIBRATION
9.1 Establish GC operating parameters equivalent to those indicated in Sect. 6.8. The GC system
must be calibrated using the internal standard technique (Sect. 9.2) or the external standard
technique (Sect. 9.3). WARNING: DDT and endrin are easily degraded in the injection port
if the injection port or front of the column is dirty. This is the result of buildup of high
boiling residue from sample injection. Check for degradation problems by injecting a mid-
level standard containing only 4,4'-DDT and endrin. Look for the degradation products of
4,4'-DDT (4,4'-DDE and 4,4'-DDD) and endrin (endrin ketone and endrin aldehyde). If
degradation of either DDT or endrin exceeds 20%, take corrective action before proceeding
with calibration. Calculate percent breakdown as follows:
% breakdown for 4,4' -DDT =
Total DDT degradation peak area (DDT + DDD)
Total DDT peak area (DDT + DDE + DDD)
x
% breakdown for Endrin =
Total endrin degradation peak area (endrin aldehyde + endrin ketone) , ,,„
- X 1UU
Total endrin peak area (endrin + endrin aldehyde + endrin ketone)
NOTE: Calibration standard solutions must be prepared such that no unresolved
analytes are mixed together.
9.2 Internal Standard Calibration Procedure: To use this approach, the analyst must select one or
more internal standards compatible in analytical behavior to the compounds of interest. The
analyst must further demonstrate that the measurement of the internal standard is not affected
by method or matrix interferences. PCNB has been identified as a suitable internal standard.
Data presented in this method were generated using the internal standard calibration procedure.
9.2.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest and surrogate compound by adding volumes of one
or more stock standards to a volumetric flask. To each calibration standard, add a
known constant amount of one or more of the internal standards, and dilute to volume
with MTBE. The lowest standard should represent analyte concentrations near, but
above, their respective EDLs. The remaining standards should correspond to the
range of concentrations expected in the sample concentrates, or should define the
779
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Method 508
working range of the detector. The calibration standards must bracket the analyte
concentrations found in the sample extracts.
9.2.2 Analyze each calibration standard according to the procedure (Sect. 11.4). Tabulate
response ( peak height or area) against concentration for each compound and internal
standard. Calculate the response factor (RF) for each analyte and surrogate using
Equation 1.
Equation 1
Where:
A s = Response for the analyte.
An = Response for the internal standard.
Cn = Concentration of the internal standard
Ct = Concentration of the analyte to be measured
9.2.3 If the RF value over the working range is constant (20% RSD or less) the average RF
can be used for calculations. Alternatively, the results can be used to plot a
calibration curve of response ratios (As/Ais) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ±20%, the test must be repeated
using a fresh calibration standard. Alternatively, a new calibration curve must be
prepared for that analyte.
9.2.5 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces a response that deviates
from the sample extract response by no more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check
calibration.
9.3 External Standard Calibration Procedure
9.3.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest and surrogate compound by adding volumes of one
or more stock standards to a volumetric flask. Dilute to volume with MTBE. The
lowest standard should represent analyte concentrations near, but above, their
respective EDLs. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or should define the working
range of the detector. The calibration standards must bracket the analyte
concentrations found in the sample extracts.
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Method 508
9.3.2 Starting with the standard of lowest concentration, analyze each calibration standard
according to Sect. 11.4 and tabulate response (peak height or area) versus the
concentration in the standard. The results can be used to prepare a calibration curve
for each compound. Alternatively, if the ratio of response to concentration
(calibration factor) is a constant over the working range (20% RSD or less), linearity
through the origin can be assumed and the average ratio or calibration factor can be
used in place of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be verified on each working
day by the measurement of a minimum of two calibration check standards, one at the
beginning and one at the end of the analysis day. These check standards should be at
two different concentration levels to verify the calibration curve. For extended periods
of analysis (greater than 8 hrs.), it is strongly recommended that check standards be
interspersed with samples at regular intervals during the course of the analyses. If the
response for any analyte varies from the predicted response by more than ±20%, the
test must be repeated using a fresh calibration standard. If the results still do not
agree, generate a new calibration curve.
9.3.4 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces a response that deviates
from the sample extract response by no more than 20%.
9.3.5 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check
calibration.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
determination of monitoring internal standard peak area or height in each sample and blank
(when internal standard calibration procedures are being employed), analysis of laboratory
reagent blanks, laboratory fortified samples, laboratory fortified blanks, and QC samples.
10.2 Laboratory Reagent Blanks: Before processing any samples, the analyst must demonstrate that
all glassware and reagent interferences are under control. Each time a set of samples is
extracted or reagents are changed, a laboratory reagent blank (LRB) must be analyzed. If
within the retention time window of any analyte of interest the LRB produces a peak that
would prevent the determination of that analyte, determine the source of contamination and
eliminate the interference before processing samples.
10.3 Initial Demonstration of Capability
10.3.1 Select a representative fortified concentration (about 10 times EDL or at the
regulatory Maximum Contaminant Level, whichever is lower) for each analyte.
Prepare a sample concentrate (in methanol) containing each analyte at 1000 times
selected concentration. With a syringe, add 1 mL of the concentrate to each of at
least four 1-L aliquots of reagent water, and analyze each aliquot according to
procedures beginning in Section 11.
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Method 508
10.3.2 For each analyte the recovery value for all four of these samples must fall in the
range of R + 30% (or within R ± 3SR if broader) using the values for R and SR for
reagent water in Table 2. For those compounds that meet the acceptance criteria,
performance is considered acceptable and sample analysis may begin. For those
compounds that fail these criteria, this procedure must be repeated using four fresh
samples until satisfactory performance has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. It is expected that as laboratory personnel gain experience with
this method the quality of data will improve beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC conditions, GC detectors, continuous
extraction techniques, concentration techniques (i.e. evaporation techniques), internal standards
or surrogate compounds. Each time such method modifications are made, the analyst must
repeat the procedures in Section 10.3.
10.5 Assessing Surrogate Recovery
10.5.1 When surrogate recovery from a sample or method blank is <70% or > 130%, check
(1) calculations to locate possible errors, (2) fortifying solutions for degradation, (3)
contamination or other obvious abnormalities, and (4) instrument performance. If
those steps do not reveal the cause of the problem, reanalyze the extract.
10.5.2 If a blank extract reanalysis fails the 70-130% recovery criterion, the problem must
be identified and corrected before continuing.
10.5.3 If sample extract reanalysis meets the surrogate recovery criterion, report only data
for the reanalyzed extract. If sample extract reanalysis continues to fail the surrogate
recovery criterion, report all data for that sample as suspect.
10.6 Assessing the Internal Standard
10.6.1 When using the internal standard calibration procedure, the analyst is expected to
monitor the IS response (peak area or peak height) of all samples during each analysis
day. The IS response for any sample chromatogram should not deviate from the daily
calibration check standards IS response by more than 30%.
10.6.2 If >30% deviation occurs with an individual extract, optimize instrument
performance and inject a second aliquot of that extract.
10.6.2.1 If the reinjected aliquot produces an acceptable internal standard response
report results for that aliquot.
10.6.2.2 If a deviation of greater than 30% is obtained for the re-injected extract,
analysis of the sample should be repeated beginning with Section 11,
provided the sample is still available. Otherwise, report results obtained
from the re-injected extract, but annotate as suspect.
10.6.3 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
calibration check standard.
10.6.3.1 If the check standard provides a response factor (RF) within 20% of the
predicted value, then follow procedures itemized in Section 10.6.2 for each
sample failing the IS response criterion.
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Method 508
10.6.3.2 If the check standard provides a response factor which deviates more than
20% of the predicted value, then the analyst must recalibrate, as specified
in Section 9.
10.7 Assessing Laboratory Performance—Laboratory Fortified Blank
10.7.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
every twenty samples or one per sample set (all samples extracted within a 24-h
period) whichever is greater. The fortified concentration of each analyte in the LFB
should be 10 times EDL or the MCL, whichever is less. Calculate accuracy as
percent recovery (X,). If the recovery of any analyte falls outside the control limits
(see Sect. 10.7.2), that analyte is judged out of control, and the source of the problem
should be identified and resolved before continuing analyses.
10.7.2 Until sufficient data become available from within their own laboratory, usually a
minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
performance against the control limits in Sect. 10.3.2 that are derived from the data in
Table 2. When sufficient internal performance data becomes available, develop
control limits from the mean percent recovery (x) and standard deviation (S) of the
percent recovery. These data are used to establish upper and lower control limits as
follows:
Upper Control Limit = X + 3S
Lower Control Limit = X — 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points. These calculated control
limits should never exceed those established in Sect. 10.3.2.
10.7.3 It is recommended that the laboratory periodically document and determine its
detection limit capabilities for the analytes of interest.
10.7.4 At least quarterly, analyze a QC sample from an outside source.
10.7.5 Laboratories are encouraged to participate in external performance evaluation studies
such as the laboratory certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as independent checks
on the analyst's performance.
10.8 Assessing Method Performance—Laboratory Fortified Sample Matrix
10.8.1 The laboratory must add a known concentration to a minimum of 10% of the routine
samples or one sample concentration per set, whichever is greater. The added
concentration should not be less then the background concentration of the sample
selected for fortification. Ideally, the fortified analyte concentrations should be the
same as that used for the LFB (Section 10.7). Over time, samples from all routine
sample sources should be fortified.
10.8.2 Calculate the percent recovery, P of the concentration for each analyte, after
correcting the analytical result, X, from the fortified sample for the background
concentration, b, measured in the unfortified sample, i.e.,:
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Method 508
p = 100 (X - b)
fortifying concentration
and compare these values to control limits appropriate for reagent water data collected
in the same fashion. If the analyzed unfortified sample is found to contain NO
background concentrations, and the added concentrations are those specified in Sect.
10.7, then the appropriate control limits would be the acceptance limits in Sect. 10.7.
If, on the other hand, the analyzed unfortified sample is found to contain background
concentration, b, estimate the standard deviation at the background concentration, sb,
using regressions or comparable background data and, similarly, estimate the mean,
Xa and standard deviation, sa, of analytical results at the total concentration after
fortifying. Then the appropriate percentage control limits would be P ± 3sp , where:
p = 100 X
(b + fortifying concentration)
and
fortifying concentration
For example, if the background concentration for Analyte A was found to be 1 jtg/L
and the added amount was also 1 /ig/L, and upon analysis the laboratory fortified
sample measured 1.6 /x/L, then the calculated P for this sample would be (1.6 fj,g/L
minus 1.0 /xg/L)/l /xg/L or 60%. This calculated P is compared to control limits
derived from prior reagent water data. Assume it is known that analysis of an
interference free sample at 1 /xg/L yields an s of 0.12 jig/L and similar analysis at 2.0
jtg/L yields X and s of 2.01 /xg/L and 0.20 fig/L, respectively. The appropriate
limits to judge the reasonableness of the percent recovery, 60%, obtained on the
fortified matrix sample is computed as follows:
100 (2.01
2.0 fig/L
+ 3 no0) - + (0.20
100.5% ± 300 (0.233) =
100.5% ± 70% or30% to 170% recovery of the added analyte.
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Method 508
10.8.3 If the recovery of any such analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control (Sect. 10.7), the
recovery problem encountered with the dosed sample is judged to be matrix related,
not system related. The result for that analyte in the unfortified sample is labeled
suspect/matrix to inform the data user that the results are suspect due to matrix
effects.
10.9 Assessing Instrument System—Laboratory Performance Check Sample: Instrument
performance should be monitored on a daily basis by analysis of the LPC sample. The LPC
sample contains compounds designed to indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. LPC sample components
and performance criteria are listed in Table 3. Inability to demonstrate acceptable instrument
performance indicates the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method. If laboratory EDLs differ
from those listed in this method, concentrations of the instrument QC standard compounds
must be adjusted to be compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to asses
the precision of the environmental measurements or filed reagent blanks may be used to asses
contamination of samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 Extraction (Manual Method)
11.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume (Sect. 11.1.6). Add preservative to blanks and QC check standards.
Fortify the sample with 50 jiL of the surrogate standard fortifying solution. Pour the
entire sample into a 2-L separatory funnel.
11.1.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer. Check pH: add
H2SO4 or NaOH if necessary.
11.1.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
11.1.4 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by
vigorously shaking the funnel for 2 min with periodic venting to release excess
pressure. Allow the organic layer to separate from the water phase for a minimum of
10 min. If the emulsion interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical techniques to complete the
phase separation. The optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, centrifugation, or other physical
methods. Collect the methylene chloride extract in a 500-mL Erlenmeyer flask.
11.1.5 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
Perform a third extraction in the same manner.
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Method 508
11.1.6 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11.2 Automated Extraction Method: Data presented in this method were generated using the
automated extraction procedure with the mechanical tumbler.
11.2.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume (Sect. 11.2.6). Add preservative to blanks and QC check standards.
Fortify the sample with 50 /tL of the surrogate standard fortifying solution. If the
mechanical separately funnel shaker is used, pour the entire sample into a 2-L
separatory funnel. If the mechanical tumbler is used, pour the entire sample into a
tumbler bottle.
11.2.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer. Check pH: add
H2SO4 or NaOH if necessary.
11.2.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
11.2.4 Add 300 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the
inner walls. Transfer the solvent to the sample contained in the separately funnel or
tumbler bottle, seal, and shake for 10 s, venting periodically. Repeat shaking and
venting until pressure release is not observed during venting. Reseal and place sample
container in appropriate mechanical mixing device (separately funnel shaker or
tumbler). Shake or tumble the sample for 1 hour. Complete mixing of the organic
and aqueous phases should be observed within about 2 min after starting the mixing
device.
11.2.5 Remove the sample container from the mixing device. If the tumbler is used, pour
contents of tumbler bottle into a 2-L separatory funnel. Allow the organic layer to
separate from the water phase for a minimum of 10 min. If the emulsion interface
between layers is more than one third the volume of the solvent layer, the analyst
must employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring, filtration through glass
wool, centrifugation, or other physical methods. Collect the methylene chloride
extract in a 500-mL Erlenmeyer flask.
11.2.6 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11.3 Extract Concentration
11.3.1 Assemble a K-D concentrator by attaching a 25-mL concentrator tube to a 500-mL
evaporative flask. Other concentration devices or techniques may be used in place of
the K-D if the requirements of Sect. 10.3 are met.
11.3.2 Dry the extract by pouring it through a solvent-rinsed drying column containing about
10 cm of anhydrous sodium sulfate. Collect the extract in the K-D concentrator, and
rinse the column with 20-30 mL methylene chloride. Alternatively, add about 5 g
anhydrous sodium sulfate to the extract in the Erlenmeyer flask; swirl flask to dry
extract and allow to sit for 15 min. Decant the methylene chloride extract into the
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Method 508
K-D concentrator. Rinse the remaining sodium sulfate with two 25-mL portions of
methylene chloride and decant the rinses into the K-D concentrator.
11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and attach a macro Snyder
column. Prewet the Snyder column by adding about 1 mL methylene chloride to the
top. Place the K-D apparatus on a hot water bath, 65 to 70°C, so that the
concentrator tube is partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the concentration in 15 to
20 min. At the proper rate of distillation the balls of the column will actively chatter,
but the chambers will not flood. When the apparent volume of liquid reaches 2 mL,
remove the K-D apparatus and allow it to drain and cool for at least 10 min.
11.3.4 Remove the Snyder column and rinse the flask and its lower joint into the
concentrator tube with 1 to 2 mL of MTBE. Add 5-10 mL of MTBE and a fresh
boiling stone. Attach a micro-Snyder column to the concentrator tube and prewet the
column by adding about 0.5 mL of MTBE to the top. Place the micro K-D apparatus
on the water bath so that the concentrator tube is partially immersed in the hot water.
Adjust the vertical position of the apparatus and the water temperature as required to
complete concentration in 5 to 10 min. When the apparent volume of liquid reaches 2
mL, remove the micro K-D from the bath and allow it to drain and cool. Add 5-10
mL MTBE to the micro K-D and reconcentrate to 2 mL. Remove the micro K-D
from the bath and allow it to drain and cool. Remove the micro Snyder column, and
rinse the walls of the concentrator tube while adjusting the volume to 5.0 mL with
MTBE.
11.3.5 Transfer extract to an appropriate-sized TFE-fluorocarbon-sealed screw-cap vial and
store, refrigerated at 4°C, until analysis by GC-NPD.
11.4 Gas Chromatography
11.4.1 Sect. 6.8 summarizes the recommended operating conditions for the gas chromato-
graph. Included in Table 1 are retention times observed using this method. Other
GC columns, chromatographic conditions, or detectors may be used if the
requirements of Sect. 10.3 are met.
11.4.2 Calibrate the system daily as described in Sect. 9. The standards and extracts must be
in MTBE.
11.4.3 If the internal standard calibration procedure is used, add 5 /xL of the internal standard
fortifying solution to the sample extract, seal, and shake to distribute the internal
standard.
11.4.4 Inject 2 /xL of the sample extract. Record the resulting peak size in area units.
11.4.5 If the response for the peak exceeds the working range of the system, dilute the
extract and reanalyze.
11.5 Identification of Analytes
11.5.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound
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Method 508
corresponds, within limits, to the retention time of a standard compound, then
identification is considered positive.
11.5.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.5.3 Identification requires expert judgment when sample components are not resolved
chromatographically. When GC peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of a peak on a
chromatogram, appropriate alternate techniques, to help confirm peak identification,
need to be employed. For example, more positive identification may be made by the
use of an alternative detector which operates on a chemical/physical principle different
from that originally used; e.g., mass spectrometry, or the use of a second
chromatography column. A suggested alternative column is described in Sect. 6.8.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for the analyte using the
calibration procedure described in Sect. 9.
12.2 If the internal standard calibration procedure is used, calculate the concentration (C) in the
sample using the calibration curve or response factor (RF) determined in Sect. 9.2 and
Equation 2.
Equation 2
C (jiglL) =
(A,) (RF) (V)
Where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each extract
V = Volume of water extracted (L).
12.3 If the external standard calibration procedure is used, calculate the amount of material injected
from the peak response using the calibration curve or calibration factor determined in Section
9.3. The concentration (C) in the sample can be calculated from Equation 3.
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Method 508
Equation 3
Concentration (us/L) =
Where:
A = Amount of material injected (ng).
Vt = Volume of extract injected
Vt = Volume of total extract
V = Volume of water extracted (mL).
13, PRECISION AND A CCURA c Y
13.1 In a single laboratory, analyte recoveries from reagent water were determined at five
concentration levels. Results were used to determine analyte EDLs and demonstrate method
range.1 Analytes were divided into two fortified groups for recovery studies. Analyte EDLs
and analyte recoveries and standard deviation about the percent recoveries at one concentration
are given in Table 2.
13.2 In a single laboratory, analyte recoveries from two standard synthetic ground waters were
determined at one concentration level. Results were used to demonstrate applicability of the
method to different ground water matrices.' Analyte recoveries from the two synthetic
matrices are given in Table 2.
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Method 508
References
I. National Pesticide Survey Method No. 2: Determination of Chlorinated Pesticides in
Groundwater by Gas Chromatography with a Electron Capture Detector.
2. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation", American Society for Testing and
Materials, Philadelphia, PA, 1986.
3. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
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Method 508
Table 1 . Retention Times
Analyte
Etridiazole
Chloroneb
Propachlor
Trifluralin
HCH-a
Hexachlorobenzene
HCH-/3
HCH-i
PCNB (internal std.)
HCH-6
Chlorthalonil
Heptachlor
Aldrin
Chlorpyrifos
DCPA
Heptachlor epoxide
Chlordane-7
Endosulfan I
Chlordane-a
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
Chlorobenzilate
4,4'-DDD
Endrin aldehyde
Endosulfan sulfate
4,4'-DDT
Methoxychlor
cis-Permethrin
trans-Permethrin
DCB
for Method Analytes
Primary
23.46
25.50
28.90
31.62
31.62
31.96
33.32
33.66
34
35.02
35.36
37.74
40.12
40.6
41.14
42.16
43.52
44.20
44.54
45.90
45.90
46.92
47.60
47.94
48.28
48.62
49.98
50.32
53.38
58.48
58.82
64.1
Retention Time*
(minutes)
| Alternative
22.78
26.18
30.94
b
32.98
b
40.12
35.36
34
41.48
39.78
36.72
38.08
b
41.14
42.16
43.86
43.52
44.54
44.88
45.90
b
51.68
48.28
46.92
46.92
49.30
50.32
53.72
b
b
b
Columns and analytical conditions are described in Sect. 6.8.1 and 6.8.2.
Data not available.
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Method 508
Table 2. Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters3
EDL Cone. Reagent Water Synthetic Water T Synthetic Water 2'
Analyte
Aldrin
Chlordane-r*
Chlordane-y
Chloroneb
Chlorobenzilate
Chlorthalonil
DCPA
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrm
Endosulfan 1
Endosulfan
sulfate
Endrin
Endrin
aldehyde
Endosulfan II
Etridiazole
HCH-a
HCH-j3
HCH-5
HCH-y
Heptachlor
Heptachlor
epoxide
Hexachloro-
benzene
Methoxychlor
cis-Permethrin
trans-Permethrin
Propachlor
Trifluralin
(V9/U
0.075
0.0015
0.0015
0.5
5
0.025
0.025
0.0025
0.01
0.06
0.02
0.015
0.015
0.015
0.025
0.024
0.025
0.025
0.01
0.01
0.015
0.01
0.015
0.0077
0.05
0.5
0.5
0.5
0.025
(iig/U
0.15
0.15
0.15
5
10
0.25
0.25
0.25
0.1
0.6
0.2
0.15
0.15
0.15
0.25
0.15
0.25
0.05
0.1
0.1
0.15
0.1
0.15
0.05
0.5
5
5
5
0.25
RC
86
99
99
97
108
91
103
107
99
112
87
87
102
88
88
92
103
92
95
102
89
98
87
99
105
91
111
103
103
ss\
9.5
11.9
11.9
11.6
5.4
8.2
12.4
6.4
11.9
16.8
8.7
8.7
15.3
8.8
7.9
10.1
6.2
10.1
6.7
11.2
9.8
11.8
8.7
21.8
13.7
9.1
6.7
9.3
5.2
R
100
96
96
95
98
103
100
96
96
98
103
102
94
98
103
98
91
106
92
99
115
85
103
82
101
96
97
116
86
I *
11.0
12.5
12.5
6.7
10.8
10.3
13.0
8.6
12.5
11.8
9.3
8.2
1.3
9.8
11.3
10.8
6.4
7.4
5.5
11.9
6.9
11.1
7.2
9.8
10.1
11.5
9.7
4.6
10.3
/?
69
99
99
75
102
71
101
101
99
84
82
84
72
104
84
76
98
86
100
103
85
85
82
68
104
86
102
95
87
sa
9.0
7.9
6.9
8.3
9.2
9.2
6.1
7.1
6.9
8.4
7.4
8.4
12.2
9.4
9.2
6.8
3.9
7.7
6.0
6.2
7.7
7.7
9.8
4.8
6.2
9.5
7.1
7.6
9.6
Data corrected for amount detected in blank and represent the mean of 7-8 samples.
EDL= estimated detection limit; defined as either MDL (Appendix B to 40 CFR Part 136 —
Definition and Procedure for the Determination of the Method Detection Limit —Revision 1.11)
or a level of compound in a sample yielding a peak in the final extract with signal-to-noise ratio
of approximately 5, whichever value is higher. The concentration level used in determining the
EDL is not the same as the concentration level presented in this table.
R = average percent recovery.
SR = standard deviation of the percent recovery.
Corrected for amount found in blank; Absopure Nature Artesian Spring Water Obtained from
the Absopure Water Company in Plymouth, Michigan.
Corrected for amount found in blank; reagent water fortified with fulvic acid at the 1 mg/L
concentration level. A well-characterized fulvic acid, available from the International Humic
Substances Society (associated with the United States Geological Survey in Denver, Colorado),
was used.
732
-------
Method 508
Table 3. Laboratory Performance Check Solution
Cone.
Test Analyte f/jg/U Requirements
Sensitivity Chlorpyrifos 0.0020 Detection of analyte; S/N > 3
Chromatographic performance DCPA 0.0500 PSF between 0.80 and 1.15a
Column performance Chlorothalonil 0.0500 Resolution > 0.50b
HCH-6 0.0400
3 PGF - peak Gaussian factor. Calculated using the equation:
1.83xW fl
PGF - I 2
where W I _ is the peak width at half height and W _ is the peak width at tenth height
b Resolution between the two peaks as defined by the equation:
/?-JL
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
133
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-------
Method 515.1
Determination of Chlorinated Acids in Water
by Gas Chromatography with
an Electron Capture Detector
Revision 4.0 - EPA EMSL-Ci
B.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
J.W. Hodgeson - Method 515, Revision 2.0 (1986)
T. Engels (Battelle Columbus Laboratories) — National Pesticide Survey
Method 3, Revision 3.0 (1987)
R.L. Graves - Method 515.1, Revision 4.0 (1989)
-------
-------
Method 515.1
Determination of Chlorinated Acids in Water by Gas
Chromatography with an Electron Capture Detector
1. SCOPE AND APPLICA TION
1.1 This is a gas chromatographic (GC) method applicable to the determination of certain chlori-
nated acids in ground water and finished drinking water.1 The following compounds can be
determined by this method:
Analyte CAS No.
Acifluorfen* 50594-66-6
Bentazon 25057-89-0
Chloramben* 133-90-4
2,4-D 94-75-7
Dalapon* 75-99-0
2,4-DB 94-8.2-6
DCPA acid metabolites*
Dicamba 1918-00-9
3,5-Dichlorobenzoic acid 51-36-5
Dichlorprop 120-36-5
Dinoseb 88-85-7
5-Hydroxydicamba 7600-50-2
4-Nitrophenol* 100-02-7
Pentachlorophenol (PCP) 87-86-5
Picloram 1918-02-1
2,4,5-T 93-76-5
2,4,5-TP 93-72-1
a DCPA monoacid and diacid metabolites included in method scope; DCPA
diacid metabolite used for validation studies.
* These compounds are only qualitatively identified in the National Pesticides
Survey (NPS) Program. These compounds are not quantitated because control
over precision has not been accomplished.
1.2 This method may be applicable to the determination of salts and esters of analyte acids. The
form of each acid is not distinguished by this method. Results are calculated and reported for
each listed analyte as the total free acid.
1.3 This method has been validated in a single laboratory and estimated detection limits (EDLs)
have been determined for the analytes above (Sect. 13). Observed detection limits may vary
between ground waters, depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use
of GC and in the interpretation of gas chromatograms. Each analyst must demonstrate the
ability to generate acceptable results with this method using the procedure described in Sect.
10.3.
137
-------
Method 515.1
1.5 Analytes that are not separated chromatographically i.e., which have very similar retention
times, cannot be individually identified and measured in the same calibration mixture or water
sample unless an alternate technique for identification and quantitation exist (Sect. 11.8).
1.6 When this method is used to analyze unfamiliar samples for any or all of the analytes above,
analyte identifications must be confirmed by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample of approximately 1 L is adjusted to pH 12 with 6 N sodium
hydroxide and shaken for 1 hr to hydrolyze derivatives. Extraneous organic material is
removed by a solvent wash. The sample is acidified, and the chlorinated acids are extracted
with ethyl ether by shaking in a separatory funnel or mechanical tumbling in a bottle. The
acids are converted to their methyl esters using diazomethane as the derivatizing agent. Excess
derivatizing reagent is removed, and the esters are determined by capillary column/GC using
an electron capture detector (ECD).
2.2 The method provides a Florisil cleanup procedure to aid in the elimination of interferences that
may be encountered.
3. DEFINITIONS
3.1 Internal standard: A pure analyte(s) added to a solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of
the same solution. The internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte: A pure analyte(s), which is extremely unlikely to be found in any sample,
and which is added to a sample aliquot in known amount(s) before extraction and is measured
with the same procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.4 Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
138
-------
Method 515.1
3.7 Laboratory performance check solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the labora-
tory is capable of making accurate and precise measurements at the required method detection
limit.
3.9 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.11 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality control sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.2 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with dilute acid, tap and reagent
water. Drain dry, and heat in an oven or muffle furnace at 400°C for 1 hr. Do not
heat volumetric ware. Thermally stable materials such as PCBs might not be elimi-
nated by this treatment. Thorough rinsing with acetone may be substituted for the
heating. After drying and cooling, seal and store glassware in a clean environment to
753
-------
Method 515.1
prevent any accumulation of dust or other contaminants. Store inverted or capped
with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
WARNING: When a solvent is purified, stabilizers added by the manufacturer are
removed, thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed, thus potentially
reducing the shelf-life.
4.2 The acid forms of the analytes are strong organic acids which react readily with alkaline
substances and can be lost during sample preparation. Glassware and glass wool must be
acid-rinsed with IN hydrochloric acid and the sodium sulfate must be acidified with sulfuric
acid prior to use to avoid analyte losses due to adsorption.
4.3 Organic acids and phenols, especially chlorinated compounds, cause the most direct interfer-
ence with the determination. Alkaline hydrolysis and subsequent extraction of the basic
sample removes many chlorinated hydrocarbons and phthalate esters that might otherwise
interfere with the electron capture analysis.
4.4 Interferences by phthalate esters can pose a major problem in pesticide analysis when using the
ECD. These compounds generally appear in the chromatogram as large peaks. Common
flexible plastics contain varying amounts of phthalates, that are easily extracted or leached
during laboratory operations. Cross contamination of clean glassware routinely occurs when
plastics are handled during extraction steps, especially when solvent-wetted surfaces are
handled. Interferences from phthalates can best be minimized by avoiding the use of plastics
in the laboratory. Exhaustive purification of reagents and glassware may be required to
eliminate background phthalate contamination.3'4
4.5 Interfering contamination may occur when a sample containing low concentrations of analytes
is analyzed immediately following a sample containing relatively high concentrations of
analytes. Between-sample rinsing of the sample syringe and associated equipment with methyl-
t-butyl-ether (MTBE) can minimize sample cross contamination. After analysis of a sample
containing high concentrations of analytes, one or more injections of MTBE should be made to
ensure that accurate values are obtained for the next sample.
4.6 Matrix interferences may be caused by contaminants that are coextracted from the sample.
Also, note that all analytes listed in the Scope and Application Section are not resolved from
each other on any one column, i.e., one analyte of interest may be an interferant for another
analyte of interest. The extent of matrix interferences will vary considerably from source to
source, depending upon the water sampled. The procedures in Sect. 11 can be used to over-
come many of these interferences. Positive identifications should be confirmed (Sect. 11.8).
4.7 It is important that samples and working standards be contained in the same solvent. The
solvent for working standards must be the same as the final solvent used in sample prepara-
tion. If this is not the case, chromatographic comparability of standards to sample may be
affected.
140
-------
Method 515.1
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
ing the safe handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available and have been identified for
the information of the analyst.6"8
5.2 Diazomethane: A toxic carcinogen which can explode under certain conditions. The follow-
ing precautions must be followed:
5.2.1 Use only a well ventilated hood: do not breath vapors.
5.2.2 Use a safety screen.
5.2.3 Use mechanical pipetting aides.
5.2.4 Do not heat above 90°C: EXPLOSION may result.
5.2.5 Avoid grinding surfaces, ground glass joints, sleeve bearings, glass stirrers: EX-
PLOSION may result.
5.2.6 Store away from alkali metals: EXPLOSION may result.
5.2.7 Solutions of diazomethane decompose rapidly in the presence of solid materials such
as copper powder, calcium chloride, and boiling chips.
5.2.8 The diazomethane generation apparatus used in the esterification procedures (Sect.
11.4 and 11.5) produces micromolar amounts of diazomethane to minimize safety
hazards.
5.3 Ethyl Ether: Nanograde, redistilled in glass, if necessary.
5.3.1 Ethyl ether is an extremely flammable solvent. If a mechanical device is used for
sample extraction, the device should be equipped with an explosion-proof motor and
placed in a hood to avoid possible damage and injury due to an explosion.
5.3.2 Must be free of peroxides as indicated by EM Quant test strips (available from Scien-
tific Products Co., Cat. No. P1126-8, and other suppliers).
5.4 WARNING: When a solvent is purified, stabilizers added by the manufacturer are removed,
thus potentially making the solvent hazardous.
6. APPARA TUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1 Sample Bottle: Borosilicate, 1-L volume with graduations (Wheaton Media/Lab bottle 219820
or equivalent), fitted with screw caps lined with TFE-fluorocarbon. Protect samples from
light. The container must be washed and dried as described in Sect. 4.1.1 before use to
minimize contamination. Capliners are cut to fit from sheets (Pierce Catalog No. 012736) and
extracted with methanol overnight prior to use.
141
-------
Method 515 1
6.2 Glassware
6.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcocks, ground glass or
TFE-fluorocarbon stoppers.
6.2.2 Tumbler bottle: 1.7-L (Wheaton Roller Culture Vessel or equivalent), with
TFE-fluorocarbon lined screw cap. Cap liners are cut to fit from sheets (Pierce
Catalog No. 012736) and extracted with methanol overnight prior to use.
6.2.3 Concentrator tube, Kuderna-Danish (K-D): 10- or 25-mL, graduated (Kontes
K-570050-2525 or Kontes K-570050-1025 or equivalent). Calibration must be
checked at the volumes employed in the test. Ground glass stoppers are used to
prevent evaporation of extracts.
6.2.4 Evaporative flask, K-D: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
6.2.5 Snyder column, K-D: three-ball macro (Kontes K-503000-0121 or equivalent).
6.2.6 Snyder column, K-D: two-ball micro (Kontes K-569001-0219 or equivalent).
6.2.7 Flask, round-bottom: 500-mL with 24/40 ground glass joint.
6.2.8 Vials: glass, 5- to 10-mL capacity with TFE-fluorocarbon lined screw cap.
6.2.9 Disposable pipets: sterile plugged borosilicate glass, 5-mL capacity (Corning
7078-5N or equivalent).
6.3 Separatory Funnel Shaker: Capable of holding 2-L separatory funnels and shaking them with
rocking motion to achieve thorough mixing of separatory funnel contents (available from
Eberbach Co. in Ann Arbor, MI or other suppliers).
6.4 Tumbler: Capable of holding tumbler bottles and tumbling them end-over-end at 30 turns/min
(Associated Design and Mfg. Co., Alexandria, VA and other suppliers).
6.5 Boiling Stones: Teflon, Chemware (Norton Performance Plastics No. 015021 and other
suppliers).
6.6 Water Bath: Heated, capable of temperature control (+ 2°C). The bath should be used in a
hood.
6.7 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.8 Diazomethane Generator: Assemble from two 20 x 150 mm test tubes, two Neoprene rubber
stoppers, and a source of nitrogen as shown in Figure 1 (available from Aldrich Chemical
Co.). When esterification is performed using diazomethane solution, the diazomethane collec-
tor is cooled in an approximately 2-L thermos for ice bath or a cryogenically cooled vessel
(Thermoelectrics Unlimited Model SK-12 or equivalent).
6.9 Glass Wool: Acid washed (Supelco 2-0383 or equivalent) and heated at 450°C for 4 hr.
6.10 Gas Chromatograph: Analytical system complete with temperature programmable GC suitable
for use with capillary columns and all required accessories including syringes, analytical
columns, gases, detector and stripchart recorder. A data system is recommended for measur-
ing peak areas. Table I lists retention times observed for method analytes using the columns
and analytical conditions described below.
742
-------
Method 515.1
6.10.1 Column 1 (Primary column): 30 m long x 0.25 mm I.D. DB-5 bonded fused silica
column, 0.25 /^m film thickness (J&W Scientific). Helium carrier gas flow is estab-
lished at 30 cm/sec linear velocity and oven temperature is programmed from 60°C to
300°C at 4°C/min. Data presented in this method were obtained using this column.
The injection volume was 2 pL splitless mode with 45 second delay. The injector
temperature was 250°C and the detector was 320°C. Alternative columns may be
used in accordance with the provisions described in Sect. 10.2.
6.10.2 Column 2 (Confirmation column): 30 m long x 0.25 mm I.D. DB-1701 bonded
fused silica column, 0.25 um film thickness (J&W Scientific). Helium carrier gas
flow is established at 30 cm/sec linear velocity and oven temperature is programmed
from 60°C to 300°C at 4°C/min.
6.10.3 Detector: Electron capture. This detector has proven effective in the analysis of
fortified reagent and artificial ground waters. An BCD was used to generate the
validation data presented in this method. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Sect. 10.3.
7. REAGENTS AND CONSUMABLE MATERIALS
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed, thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed, thus potentially reduc-
ing the shelf-life.
7.1 Acetone, Methanol, Methylene Chloride, MTBE: Pesticide quality or equivalent.
7.2 Ethyl Ether, Unpreserved: Nanograde, redistilled in glass if necessary. Must be free of
peroxides as indicated by EM Quant test strips (available from Scientific Products Co., Cat.
No. PI126-8, and other suppliers). Procedures recommended for removal of peroxides are
provided with the test strips.
7.3 Sodium Sulfate, Granular, Anhydrous, ACS Grade: Heat treat in a shallow tray at 450°C for
a minimum of 4 hr to remove interfering organic substances. Acidify by slurry ing 100 g
sodium sulfate with enough ethyl ether to just cover the solid. Add 0.1 mL concentrated
sulfuric acid and mix thoroughly. Remove the ether under vacuum. Mix 1 g of the resulting
solid with 5 mL of reagent water and measure the pH of the mixture. The pH must be below
pH 4. Store at 130°C.
7.4 Sodium Thiosulfate, Granular, Anhydrous: ACS grade.
7.5 Sodium Hydroxide (NAOH), Pellets: ACS grade.
7.5.1 NaOH, 6 N: Dissolve 216 g NaOH in 900 mL reagent water.
7.6 Sulfuric Acid, Concentrated: ACS grade,sp. gr. 1.84.
7.6.1 Sulfuric acid, 12 N: Slowly add 335 mL concentrated sulfuric acid to 665 mL of
reagent water.
143
-------
Method 515.1
7.7 Potassium Hydroxide (KOH), Pellets: ACS grade.
7.7.1 KOH, 37% (w/v): Dissolve 37 g KOH pellets in reagent water and dilute to 100 mL.
7.8 Carbitol (Diethylene Glycol Monoethyl Ether): ACS grade. Available from Aldrich Chemical
Co.
7.9 Diazald, ACS grade: Available from Aldrich Chemical Co.
7.10 Diazald Solution: Prepare a solution containing 10 g Diazald in 100 mL of a 50:50 by volume
mixture of ethyl ether and carbitol. This solution is stable for one month or longer when
stored at 4°C in an amber bottle with a Teflon-lined screw cap.
7.11 Sodium Chloride (NACL), Crystal, ACS Grade: Heat treat in a shallow tray at 450°C for a
minimum of 4 hr to remove interfering organic substances.
7.12 4,4-Dibromooctafluorobiphenyl (DBOB): 99% purity, for use as internal standard (available
from Aldrich Chemical Co).
7.13 2,4-Dichlorophenylacetic ACID (DCAA): 99% purity, for use as surrogate standard (available
from Aldrich Chemical Co).
7.14 Mercuric Chloride: ACS grade (Aldrich Chemical Co.) - for use as a bacteriocide. If any
other bactericide can be shown to work as well as mercuric chloride, it may be used instead.
7.15 Reagent Water: Reagent water is defined as water that is reasonably free of contamination that
would prevent the determination of any analyte of interest. Reagent water used to generate the
validation data in this method was distilled water obtained from the Magnetic Springs Water
Co., Columbus, Ohio.
7.16 Silicic Acid, ACS Grade.
7.17 Florisil: 60-100/PR mesh (Sigma No. F-9127). Activate by heating in a shallow container at
150°C for at least 24 and not more than 48 hr.
7.18 Stock Standard Solutions (1.00 /*g//LtL): Stock standard solutions may be purchased as certi-
fied solutions or prepared from pure standard materials using the following procedure:
7.18.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
pure material. Dissolve the material in MTBE and dilute to volume in a 10-mL volu-
metric flask. Larger volumes may be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially pre-
pared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.18.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw cap amber
vials. Store at room temperature and protect from light.
7.18.3 Stock standard solutions should be replaced after two months or sooner if comparison
with laboratory fortified blanks, or QC samples indicate a problem.
7.19 Internal Standard Solution: Prepare an internal standard solution by accurately weighing
approximately 0.0010 g of pure DBOB. Dissolve the DBOB in MTBE and dilute to volume in
a 10-mL volumetric flask. Transfer the internal standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature. Addition of 25 pL of the
internal standard solution to 10 mL of sample extract results in a final internal standard
144
-------
Method 515.1
concentration of 0.25 /xg/mL. Solution should be replaced when ongoing QC (Sect. 10)
indicates a problem. Note that DBOB has been shown to be an effective internal standard for
the method analytes, but other compounds may be used if the quality control requirements in
Sect. 10 are met.1
7.20 Surrogate Standard Solution: Prepare a surrogate standard solution by accurately weighing
approximately 0.0010 g of pure DCAA. Dissolve the DCAA in MTBE and dilute to volume
in a 10-mL volumetric flask. Transfer the surrogate standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature. Addition of 50 pL of the
surrogate standard solution to a 1-L sample prior to extraction results in a surrogate standard
concentration in the sample of 5 jug/L and, assuming quantitative recovery of DCAA, a
surrogate standard concentration in the final extract of 0.5 jtg/mL. Solution should be re-
placed when ongoing QC (Sect. 10) indicates a problem. Note DCAA has been shown to be
an effective surrogate standard for the method analytes(l), but other compounds may be used
if the quality control requirements in Sect. 10.4 are met.
7.21 Laboratory Performance Check Solutions: Prepare a diluted dinoseb solution by adding 10 pL
of the 1.0 /ig/jttL dinoseb stock solution to the MTBE and diluting to volume in a 10-mL
volumetric flask. To prepare the check solution, add 40 jtL of the diluted dinoseb solution, 16
nL of the 4-nitrophenol stock solution, 6 /iL of the 3,5-dichlorobenzoic acid stock solution, 50
/iL of the surrogate standard solution, 25 /xL of the internal standard solution, and 250 (j,L of
methanol to a 5-mL volumetric flask and dilute to volume with MTBE. Methylate sample as
described in Sects. 11.4 or 11.5. Dilute the sample to 10 mL in MTBE. Transfer to a TFE-
fluorocarbon-sealed screw cap bottle and store at room temperature. Solution should be
replaced when ongoing QC (Sect. 10) indicates a problem.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional sampling practices should
be followed; however, the bottle must not be prerinsed with sample before collection.8
8.2 Sample Preservation and Storage
8.2.1 Add mercuric chloride (See 7.14) to the sample bottle in amounts to produce a con-
centration of 10 mg/L. Add 1 mL of a 10 mg/mL solution of mercuric chloride in
water to the sample bottle at the sampling site or in the laboratory before shipping to
the sampling site. A major disadvantage of mercuric chloride is that it is a highly
toxic chemical; mercuric chloride must be handled with caution, and samples contain-
ing mercuric chloride must be disposed of properly.
8.2.2 If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
the sample bottle prior to collecting the sample.
8.2.3 After the sample is collected in the bottle containing preservative(s), seal the bottle
and shake vigorously for 1 min.
8.2.4 The samples must be iced or refrigerated at 4°C away from light from the time of
collection until extraction. Preservation study results indicate that the analytes (mea-
sured as total acid) present in samples are stable for 14 days when stored under these
conditions.1 However, analyte stability may be affected by the matrix; therefore, the
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analyst should verify that the preservation technique is applicable to the samples under
study.
8.3 Extract Storage
8.3.1 Extracts should be stored at 4°C away from light. Preservation study results indicate
that most analytes are stable for 28 days; however, the analyst should verify appro-
priate extract holding times applicable to the samples under study.'
9. CALIBRA TION
9.1 Establish GC operating parameters equivalent to those indicated in Sect. 6.10. The GC system
may be calibrated using either the internal standard technique (Sect. 9.2) or the external
standard technique (Sect. 9.3).
NOTE: Calibration standard solutions must be prepared such that no unresolved
analytes are mixed together.
9.2 Internal standard calibration procedure: To use this approach, the analyst must select one or
more internal standards compatible in analytical behavior to the compounds of interest. The
analyst must further demonstrate that the measurement of the internal standard is not affected
by method or matrix interferences. DBOB has been identified as a suitable internal standard.
9.2.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest by adding volumes of one or more stock standards
to a volumetric flask. To each calibration standard, add a known constant amount of
one or more of the internal standards and 250 /*L methanol, and dilute to volume with
MTBE. Esterify acids with diazomethane as described in Sect. 11.4 or 11.5. The
lowest standard should represent analyte concentrations near, but above, the respective
EDLs. The remaining standards should bracket the analyte concentrations expected in
the sample extracts, or should define the working range of the detector.
9.2.2 Analyze each calibration standard according to the procedure (Sect. 11.7). Tabulate
response (peak height or area) against concentration for each compound and internal
standard. Calculate the response factor (RF) for each analyte and surrogate using
Equation 1.
Equation 1
where:
A^ = Response for the analyte to be measured.
/4(1 = Response for the internal standard.
CM = Concentration of the internal standard
C( = Contentration of the analyte to be measured
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Method 515.1
9.2.3 If the RF value over the working range is constant (20% RSD or less) the average RF
can be used for calculations. Alternatively, the results can be used to plot a calibra-
tion curve of response ratios (AS/A1S) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than +20%, the test must be repeated
using a fresh calibration standard. If the repetition also fails, a new calibration curve
must be generated for that analyte using freshly prepared standards.
9.2.5 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces a response that deviates
from the sample extract response by no more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source. Re-
sults from these analyses must be within the limits used to routinely check calibration.
9.3 External Standard Calibration Procedure
9.3.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest and surrogate compound by adding volumes of one
or more stock standards and 250 pL methanol to a volumetric flask. Dilute to volume
with MTBE. Esterify acids with diazomethane as described in Sect. 11.4 or 11.5.
The best standard should represent analyte concentrations near, but above, the respec-
tive EDL. The remaining standards should bracket the analyte concentrations expect-
ed in the sample extracts, or should define the working range of the detector.
9.3.2 Starting with the standard of lowest concentration, analyze each calibration standard
according to Sect. 11.7 and tabulate response (peak height or area) versus the concen-
tration in the standard. The results can be used to prepare a calibration curve for
each compound. Alternatively, if the ratio of response to concentration (calibration
factor) is a constant over the working range (20% RSD or less), linearity through the
origin can be assumed and the average ratio or calibration factor can be used in place
of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be verified on each working
day by the measurement of a minimum of two calibration check standards, one at the
beginning and one at the end of the analysis day. These check standards should be at
two different concentration levels to verify the calibration curve. For extended
periods of analysis (greater than 8 hr), it is strongly recommended that check stan-
dards be interspersed with samples at regular intervals during the course of the analy-
ses. If the response for any analyte varies from the predicted response by more than
±20%, the test must be repeated using a fresh calibration standard. If the results still
do not agree, generate a new calibration curve or use a single point calibration
standard as described in Sect. 9.3.3.
9.3.4 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards in MTBE. The single point
147
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Method 515.1
standards should be prepared at a concentration that produces a response that deviates
from the sample extract response by no more than 20%.
9.3.5 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check
calibration.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
determination of surrogate compound recoveries in each sample and blank, monitoring internal
standard peak area or height in each sample and blank (when internal standard calibration
procedures are being employed), analysis of laboratory reagent blanks, laboratory fortified
samples, laboratory fortified blanks, and QC samples.
10.2 Laboratory reagent blanks (LRB). Before processing any samples, the analyst must
demonstrate that all glassware and reagent interferences are under control. Each time a set of
samples is extracted or reagents are changed, a LRB must be analyzed. If within the retention
time window of any analyte the LRB produces a peak that would prevent the determination of
that analyte, determine the source of contamination and eliminate the interference before
processing samples.
10.3 Initial demonstration of capability
10.3.1 Select a representative fortified concentration (about 10 times EDL) for each analyte.
Prepare a sample concentrate (in methanol) containing each analyte at 1000 times
selected concentration. With a syringe, add 1 mL of the concentrate to each of at
least four 1-L aliquots of reagent water, and analyze each aliquot according to
procedures beginning in Sect. 11.
10.3.2 For each analyte the recovery value for all four of these samples must fall in the
range of R ± 30% (or within R ± 3SR if broader) using the values for R and SR for
reagent water in Table 2. For those compounds that meet the acceptable criteria,
performance is considered acceptable and sample analysis may begin. For those
compounds that fail these criteria, this procedure must be reported using five fresh
samples until satisfactory performance has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. It is expected that as laboratory personnel gain experience with
this method the quality of data will improve beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC conditions, detectors, continuous
extraction techniques, concentration techniques (i.e., evaporation techniques), internal standard
or surrogate compounds. Each time such method modifications are made, the analyst must
repeat the procedures in Sect. 10.3
10.5 Assessing Surrogate Recovery.
10.5.1 When surrogate recovery from a sample or method blank is <70% or > 130%, check
(1) calculations to locate possible errors, (2) spiking solutions for degradation, (3)
748
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Method 515.1
contamination, and (4) instrument performance. If those steps do not reveal the cause
of the problem, reanalyze the extract.
10.5.2 If a blank extract reanalysis fails the 70-130% recovery criterion, the problem must
be identified and corrected before continuing.
10.5.3 If sample extract reanalysis meets the surrogate recovery criterion, report only data
for the analyzed extract. If sample extract continues to fail the recovery criterion,
report all data for that sample as suspect.
10.6 Assessing the internal standard
10.6.1 When using the internal standard calibration procedure, the analyst is expected to
monitor the IS response (peak area or peak height) of all samples during each analysis
day. The IS response for any sample chromatogram should not deviate from the daily
calibration check standard's IS response by more than 30%.
10.6.2 If >30% deviation occurs with an individual extract, optimize instrument
performance and inject a second aliquot of that extract.
10.6.2.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
10.6.2.2 If a deviation of greater than 30% is obtained for the reinjected extract,
analysis of the samples should be repeated beginning with Sect. 11,
provided the sample is still available. Otherwise, report results obtained
from the reinjected extract, but annotate as suspect.
10.6.3 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
calibration check standard.
10.6.3.1 If the check standard provides a response factor (RF) within 20% of the
predicted value, then follow procedures itemized in Sect. 10.6.2 for each
sample failing the IS response criterion.
10.6.3.2 If the check standard provides a response factor which deviates more than
20% of the predicted value, then the analyst must recalibrate, as specified
in Sect. 9.
10.7 Assessing laboratory performance: Laboratory Fortified Blank
10.7.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
every 20 samples or one per sample set (all samples extracted within a 24-hr period)
whichever is greater. The concentration of each analyte in the LFB should be 10
times EDL or the MCL, whichever is less. Calculate accuracy as percent recovery
(X,). If the recovery of any analyte falls outside the control limits (Sect. 10.7.2), that
analyte is judged out of control, and the source of the problem should be identified
and resolved before continuing analyses.
10.7.2 Until sufficient data become available from within their own laboratory, usually a
minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
performance against the control limits in Sect. 10.3.2 that are derived from the data in
Table 2. When sufficient internal performance data becomes available, develop
control limits from the mean percent recovery (X) and standard deviation (S) of the
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Method 515.1
percent recovery. These data are used to establish upper and lower control limits as
follows:
Upper Control Limit = x + 3S
Lower Control Limit = X - 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points. These calculated control
limits should never exceed those established in Section 10.3.2.
10.7.3 It is recommended that the laboratory periodically determine and document its
detection limit capabilities for the analytes of interest.
10.7.4 At least quarterly, analyze a QC sample from an outside source.
10.7.5 Laboratories are encouraged to participate in external performance evaluation studies
such as the laboratory certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as independent checks
on the analyst's performance.
10.8 Assessing Analyte Recovery—Laboratory Fortified Sample Matrix
10.8.1 The laboratory must add a known concentration to a minimum of 10% of the routine
samples or one sample concentration per set, whichever is greater. The concentration
should not be less then the background concentration of the sample selected for
fortification. Ideally, the concentration should be the same as that used for the
laboratory fortified blank (Sect. 10.7). Over time, samples from all routine sample
sources should be fortified.
10.8.2 Calculate the percent recovery, P of the concentration for each analyte, after
correcting the analytical result, X, from the fortified sample for the background
concentration, b, measured in the unfortified sample:
p = 100 (X - V)
fortifying concentration
and compare these values to control limits appropriate for reagent water data collected
in the same fashion. If the analyzed unfortified sample is found to contain NO
background concentrations, and the added concentrations are those specified in Sect.
10.7, then the appropriate control limits would be the acceptance limits in Sect. 10.7.
If, on the other hand, the analyzed unfortified sample is found to contain background
concentration, b, estimate the standard deviation at the background concentration, sb,
using regressions or comparable background data and, similarly, estimate the mean,
xa and standard deviation, sa, of analytical results at the total concentration after
fortifying. Then the appropriate percentage control limits would be P + 3sP, where:
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Method 515.1
p = 100X
(b + fortifying concentration)
and
,, - 100 _ _
fortifying concentration
For example, if the background concentration for Analyte A was found to be 1 /xg/L
and the added amount was also 1 /*g/L, and upon analysis the laboratory fortified
sample measured 1.6 jig/L, then the calculated P for this sample would be (1.6 /*g/L
minus 1.0 /^g/L)/l pig/L or 60%. This calculated P is compared to control limits
derived from prior reagent water data. Assume it is known that analysis of an
interference free sample at 1 ^g/L yields an s of 0.12 jtg/L and similar analysis at 2.0
/ig/L yields X and s of 2.01 /xg/L and 0.20 /ig/L, respectively. The appropriate limits
to judge the reasonableness of the percent recovery, 60%, obtained on the fortified
matrix sample is computed as follows:
100 (2.01 pg/L)
2.0 v-glL
+ 3 (100) [(0-12 ng/L)2 + (0.20
1.0 /ig/L
100.5% ± 300 (0.233) =
100.5% ± 70% or30% to 170% recovery of the added analyte.
10.8.3 If the recovery of any such analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control (Sect. 10.7), the
recovery problem encountered with the fortified sample is judged to be matrix related,
not system related. The result for that analyte in the unfortified sample is labeled
suspect/matrix to inform the data user that the results are suspect due to matrix
effects.
10.9 Assessing instrument system: Laboratory Performance Check Sample. Instrument
performance should be monitored on a daily basis by analysis of the LPC sample. The LPC
sample contains compounds designed to indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. LPC sample components
and performance criteria are listed in Table 3. Inability to demonstrate acceptable instrument
performance indicates the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method. If laboratory EDLs differ
from those listed in this method, concentrations of the instrument QC standard compounds
must be adjusted to be compatible with the laboratory EDLs.
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Method 515.1
10.10 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements or field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
; 1. PROCEDURE
11.1 Manual Hydrolysis, Preparation, and Extraction
11.1.1 Add preservative to blanks and QC check standards. Mark the water meniscus on the
side of the sample bottle for later determination of sample volume (Sect. 11.1.9).
Pour the entire sample into a 2-L separatory funnel. Fortify sample with 50 /*L of the
surrogate standard solution.
11.1.2 Add 250 g NaCl to the sample, seal, and shake to dissolve salt.
11.1.3 Add 17 mL of 6 N NaOH to the sample, seal, and shake. Check the pH of the
sample with pH paper; if the sample does not have a pH greater than or equal to 12,
adjust the pH by adding more 6 N NaOH. Let the sample sit at room temperature for
1 hr, shaking the separatory funnel and contents periodically.
11.1.4 Add 60 mL methylene chloride to the sample bottle to rinse the bottle, transfer the
methylene chloride to the separatory funnel and extract the sample by vigorously
shaking the funnel for 2 min with periodic venting to release excess pressure. Allow
the organic layer to separate from the water phase for a minimum of 10 min. If the
emulsion interface between layers is more than one-third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample, but may include
stirring, filtration through glass wool, centrifugation, or other physical methods.
Discard the methylene chloride phase.
11.1.5 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
extraction procedure a second time, discarding the methylene chloride layer. Perform
a third extraction in the same manner.
11.1.6 Add 17 mL of 12 N H2SO4 to the sample, seal, and shake to mix. Check the pH of
the sample with pH paper; if the sample does not have a pH less than or equal to 2,
adjust the pH by adding more 12 N H2SO4.
11.1.7 Add 120 mL ethyl ether to the sample, seal, and extract the sample by vigorously
shaking the funnel for 2 min with periodic venting to release excess pressure. Allow
the organic layer to separate from the water phase for a minimum of 10 min. If the
emulsion interface between layers is more than one third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample, but may include
stirring, filtration through glass wool, centrifugation, or other physical methods.
Remove the aqueous phase to a 2-L Erlenmeyer flask and collect the ethyl ether phase
in a 500-mL round-bottom flask containing approximately 10 g of acidified anhydrous
sodium sulfate. Periodically, vigorously shake the sample and drying agent. Allow
the extract to remain in contact with the sodium sulfate for approximately 2 hours.
152
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Method 515.1
11.1.8 Return the aqueous phase to the separatory funnel, add a 60-mL volume of ethyl ether
to the sample, and repeat the extraction procedure a second time, combining the
extracts in the 500-mL erlenmeyer flask. Perform a third extraction with 60 mL of
ethyl ether in the same manner.
11.1.9 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11.2 Automated Hydrolysis, Preparation, and Extraction: Data presented in this method were
generated using the automated extraction procedure with the mechanical separatory funnel
shaker.
11.2.1 Add preservative (Sect. 8.2) to any samples not previously preserved, e.g., blanks and
QC check standards. Mark the water meniscus on the side of the sample bottle for
later determination of sample volume (Sect. 11.2.9). Fortify sample with 50 /*L of
the surrogate standard solution. If the mechanical separatory funnel shaker is used,
pour the entire sample into a 2-L separatory funnel. If the mechanical tumbler is
used, pour the entire sample into a tumbler bottle.
11.2.2 Add 250 g NaCl to the sample, seal, and shake to dissolve salt.
11.2.3 Add 17 mL of 6 N NaOH to the sample, seal, and shake. Check the pH of the
sample with pH paper; if the sample does not have a pH greater than or equal to 12,
adjust the pH by adding more 6 N NaOH. Shake sample for 1 hr using the
appropriate mechanical mixing device.
11.2.4 Add 300 mL methylene chloride to the sample bottle to rinse the bottle, transfer the
methylene chloride to the separatory funnel or tumbler bottle, seal, and shake for 10s,
venting periodically. Repeat shaking and venting until pressure release is not
observed during venting. Reseal and place sample container in appropriate
mechanical mixing device. Shake or tumble the sample for 1 hr. Complete and
thorough mixing of the organic and aqueous phases should be observed at least 2 min
after starting the mixing device.
11.2.5 Remove the sample container from the mixing device. If the tumbler is used, pour
contents of tumbler bottle into a 2-L separatory funnel. Allow the organic layer to
separate from the water phase for a minimum of 10 min. If the emulsion interface
between layers is more than one third the volume of the solvent layer, the analyst
must employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring, filtration through glass
wool, centrifugation, or other physical methods. Drain and discard the organic phase.
If the tumbler is used, return the aqueous phase to the tumbler bottle.
11.2.6 Add 17 mL of 12 N H2SO4 to the sample, seal, and shake to mix. Check the pH of
the sample with pH paper; if the sample does not have a pH less than or equal to 2,
adjust the pH by adding more 12 N H2SO4.
11.2.7 Add 300 mL ethyl ether to the sample, seal, and shake for 10 s, venting periodically.
Repeat shaking and venting until pressure release is not observed during venting.
Reseal and place sample container in appropriate mechanical mixing device. Shake or
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Method 515.1
tumble sample for 1 hr. Complete and thorough mixing of the organic and aqueous
phases should be observed at least 2 min after starting the mixing device.
11.2.8 Remove the sample container from the mixing device. If the tumbler is used, pour
contents of tumbler bottle into a 2-L separatory funnel. Allow the organic layer to
separate from the water phase for a minimum of 10 min. If the emulsion interface
between layers is more than one third the volume of the solvent layer, the analyst
must employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring, filtration through glass
wool, centrifugation, or other physical methods. Drain and discard the aqueous
phase. Collect the extract in a 500-mL round-bottom flask containing about 10 g of
acidified anhydrous sodium sulfate. Periodically vigorously shake the sample and
drying agent. Allow the extract to remain in contact with the sodium sulfate for
approximately 2 hr.
11.2.9 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11.3 Extract Concentration
11.3.1 Assemble a K-D concentrator by attaching a concentrator tube to a 500-mL
evaporative flask.
11.3.2 Pour the dried extract through a funnel plugged with acid washed glass wool, and
collect the extract in the K-D concentrator. Use a glass rod to crush any caked
sodium sulfate during the transfer. Rinse the round-bottom flask and funnel with 20
to 30 mL of ethyl ether to complete the quantitative transfer.
11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and attach a macro Snyder
column. Prewet the Snyder column by adding about 1 mL ethyl ether to the top.
Place the K-D apparatus on a hot water bath, 60 to 65°C, so that the concentrator
tube is partially immersed in the hot water, and the entire lower rounded surface of
the flask is bathed with hot vapor. At the proper rate of distillation the balls of the
column will actively chatter but the chambers will not flood. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 min.
11.3.4 Remove the Snyder column and rinse the flask and its lower joint into the
concentrator tube with 1 to 2 mL of ethyl ether. Add 2 mL of MTBE and a fresh
boiling stone. Attach a micro-Snyder column to the concentrator tube and prewet the
column by adding about 0.5 mL of ethyl ether to the top. Place the micro K-D
apparatus on the water bath so that the concentrator tube is partially immersed in the
hot water. Adjust the vertical position of the apparatus and the water temperature as
required to complete concentration in 5 to 10 min. When the apparent volume of
liquid reaches 0.5 mL, remove the micro K-D from the bath and allow it to drain and
cool. Remove the micro Snyder column and add 250 /zL of methanol. If the gaseous
diazomethane procedure (Sect. 11.4) is used for esterification of pesticides, rinse the
walls of the concentrator tube while adjusting the volume to 5.0 mL with MTBE. If
the pesticides will be esterified using the diazomethane solution (Sect. 11.5), rinse the
walls of the concentrator tube while adjusting the volume to 4.5 mL with MTBE.
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Method 515.1
11.4 Esterification of Acids Using Gaseous Diazomethane: Results presented in this method were
generated using the gaseous diazomethane derivatization procedure. See Section 11.5 for an
alternative procedure.
11.4.1 Assemble the diazomethane generator (Figure 1) in a hood.
11.4.2 Add 5 mL of ethyl ether to Tube 1. Add 1 mL of ethyl ether, 1 mL of carbitol, 1.5
mL of 37% aqueous KOH, and 0.2 grams Diazald to Tube 2. Immediately place the
exit tube into the concentrator tube containing the sample extract. Apply nitrogen
flow (10 mL/min) to bubble diazomethane through the extract for 1 min. Remove
first sample. Rinse the tip of the diazomethane generator with ethyl ether after
methylation of each sample. Bubble diazomethane through the second sample extract
for 1 min. Diazomethane reaction mixture should be used to esterify only two
samples; prepare new reaction mixture in Tube 2 to esterify each two additional
samples. Samples should turn yellow after addition of diazomethane and remain
yellow for at least 2 min. Repeat methylation procedure if necessary.
11.4.3 Seal concentrator tubes with stoppers. Store at room temperature in a hood for 30
min.
11.4.4 Destroy any unreacted diazomethane by adding 0.1 to 0.2 grams silicic acid to the
concentrator tubes. Allow to stand until the evolution of nitrogen gas has stopped (ap-
proximately 20 min). Adjust the sample volume to 5.0 mL with MTBE.
11.5 Esterification of Acids Using Diazomethane Solution: Alternative procedure.
11.5.1 Assemble the diazomethane generator (Figure 2) in a hood. The collection vessel is a
10- or 15-mL vial, equipped with a Teflon-lined screw cap and maintained at 0-5C.
11.5.2 Add a sufficient amount of ethyl ether to tube 1 to cover the first impinger. Add 5
mL of MTBE to the collection vial. Set the nitrogen flow at 5-10 mL/min. Add 2
mL Diazald solution (Sect. 7.10) and 1.5 mL of 37% KOH solution to the second
impinger. Connect the tubing as shown and allow the nitrogen flow to purge the
diazomethane from the reaction vessel into the collection vial for 30 min. Cap the
vial when collection is complete and maintain at 0-5°C. When stored at 0-5°C this
diazomethane solution may be used over a period of 48 hr.
11.5.3 To each concentrator tube containing sample or standard, add 0.5 mL diazomethane
solution. Samples should turn yellow after addition of the diazomethane solution and
remain yellow for at least 2 min. Repeat methylation procedure if necessary.
11.5.4 Seal concentrator tubes with stoppers. Store at room temperature in a hood for 30
min.
11.5.5 Destroy any unreacted diazomethane by adding 0.1 to 0.2 grams silicic acid to the
concentrator tubes. Allow to stand until the evolution of nitrogen gas has stopped (ap-
proximately 20 min). Adjust the sample volume to 5.0 mL with MTBE.
11.6 Florisil Separation
11.6.1 Place a small plug of glass wool into a 5-mL disposable glass pipet. Tare the pipet,
and measure 1 g of activated Florisil into the pipet.
11.6.2 Apply 5 mL of 5 percent methanol in MTBE to the Florisil. Allow the liquid to just
reach the top of the Florisil. In this and subsequent steps, allow the liquid level to
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Method 515.1
just reach the top of the Florisil before applying the next rinse, however, do not allow
the Florisil to go dry. Discard eluate.
11.6.3 Apply 5 mL methylated sample to the Florisil leaving silicic acid in the tube. Collect
eluate in K-D tube.
11.6.4 Add I mL of 5 percent methanol in MTBE to the sample container, rinsing walls.
Transfer the rinse to the Florisil column leaving silicic acid in the tube. Collect eluate
in a K-D tube. Repeat with 1-mL and 3-mL aliquots of 5 percent methanol in MTBE,
collecting eluates in K-D tube.
11.6.5 If necessary, dilute eluate to 10 mL with 5 percent methanol in MTBE.
11.6.6 Seal the vial and store in a refrigerator if further processing will not be performed
immediately. Analyze by GC-ECD.
11.7 Gas Chromatography
11.7,1 Sect. 6.10 summarizes the recommended operating conditions for the GC. Included
in Table 1 are retention times observed using this method. Other GC columns,
chromatographic conditions, or detectors may be used if the requirements of Sect.
10.4 are met.
11.7.2 Calibrate the system daily as described in Sect. 9. The standards and extracts must be
in MTBE.
11.7.3 If the internal standard calibration procedure is used, fortify the extract with 25 /iL of
internal standard solution. Thoroughly mix sample and place aliquot in a GC vial for
subsequent analysis.
11.7.4 Inject 2 ;iL of the sample extract. Record the resulting peak size in area units.
11.7,5 If the response for the peak exceeds the working range of the system, dilute the
extract and reanalyze.
11.8 Identification of Analytes
11.8.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound
corresponds, within limits, to the retention time of a standard compound, then
identification is considered positive.
11.8.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.8.3 Identification requires expert judgement when sample components are not resolved
chromatographically. When GC peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley between two or more
maxima, or any time doubt exists over the identification of a peak on a
chromatogram, appropriate alternative techniques, to help confirm peak identification,
need to be employed. For example, more positive identification may be made by the
use of an alternative detector which operates on a chemical/physical principle different
756
-------
Method 515.1
from that originally used, e.g., mass spectrometry, or the use of a second
chromatography column. A suggested alternative column in described in Sect. 6.10.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for the analyte using the
calibration procedure described in Sect. 9.
12.2 If the internal standard calibration procedure is used, calculate the concentration (C) in the
sample using the response factor (RF) determined in Sect. 9.2 and Equation 2, or determine
sample concentration from the calibration curve.
Equation 2
C (»glL) = -
where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each extract
Vo = Volume of water extracted (L).
12.3 If the external standard calibration procedure is used, calculate the amount of material injected
from the peak response using the calibration curve or calibration factor determined in Sect.
9.3. The concentration (C) in the sample can be calculated from Equation 3.
Equation 3
where:
A = Amount of material injected (ng).
V, = Volume of extract injected (/xL).
V; = Volume of total extract (pL).
Vs = Volume of water extracted (mL).
13. PRECISION AND ACCURACY
13.1 In a single laboratory, analyte recoveries from reagent water were determined at five
concentration levels. Results were used to determine analyte EDLs and demonstrate method
range.' Analyte EDLs and analyte recoveries and standard deviation about the percent
recoveries at one concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from one standard synthetic ground waters were
determined at one concentration level. Results were used to demonstrate applicability of the
757
-------
Method 515.1
method to different ground water matrices.' Analyte recoveries from the one synthetic matrix
are given in Table 2.
758
-------
Method 515.1
References
1. National Pesticide Survey Method No. 3, "Determination of Chlorinated Acids in Water by
Gas Chromatography with an Electron Capture Detector."
2. "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No. 68-03-2697.
Available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268.
3. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society for Testing and
Materials, Philadelphia, PA, p. 86, 1986.
4. Giam, C. S., Chan, H. S., and Nef, G. S. "Sensitive Method for Determination of Phthalate
Ester Plasticizers in Open-Ocean Biota Samples," Analytical Chemistry. 47, 2225 (1975).
5. Giam, C. S., and Chan, H. S. "Control of Blanks in the Analysis of Phthalates in Air and
Ocean Biota Samples," U.S. National Bureau of Standards, Special Publication 442, pp.
701-708, 1976.
6. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, Aug. 1977.
7. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
8. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
9. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, p. 130,
1986.
759
-------
Method 515.1
Table 1. Retention Times for Method Analytes
Retention Time* (minutes)
Analyte
Dalapon
3,5-Dichlorobenzoic acid
4-Nitropheno(
DCAA (surrogate)
Dicamba
Dichlorprop
2,4-D
DBOB (int. std.)
Pentachlorophenol (PCP)
Chloramben
2,4,5-TP
5-Hydroxydicamba
2,4,5-T
2,4-DB
Dinoseb
Bentazon
Picloram
DCPA acid metabolites
Acifluorfen
3 Columns and analytical conditions are described in Sect. 6.10.1 and 6.10.2.
Primary
3.4
18.6
18.6
22.0
22.1
25.0
25.5
27.5
28.3
29.7
29.7
30.0
30.5
32.2
32.4
33.3
34.4
35.8
41.5
Confirmation
4.7
17.7
20.5
14.9
22.6
25.6
27.0
27.6
27.0
32.8
29.5
30.7
30.9
32.2
34.1
34.6
37.5
37.8
42.8
160
-------
Method 515.1
Table 2. Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters8
EDL Concentrate
Analyte fag/if fag/L)
Acifluorfen 0.096 0.2
Bentazon 0.2 1
Chloramben 0.093 0.4
2,4-D 0.2 1
Dalapon 1.3 10
2,4-DB 0.8 4
DCPA acid metabolites 0.02 0.2
Dicamba 0.081 0.4
3,5-Dichlorobenzoic 0.061 0.6
acid
Dichlorprop 0.26 2
Dinoseb 0.19 0.4
5-Hydroxydicamba 0.04 0.2
4-Nitrophenol 0.13 1
Pentachlorophenol 0.076 0.04
(PCP)
Picloram 0.14 0.6
2,4,5-T 0.08 0.4
2,4,5-TP 0.075 0.2
Reagent Water
If
121
120
111
131
100
87
74
135
102
107
42
103
131
130
91
117
134
-------
Method 515.1
Table 3. Laboratory Performance Check Solution
Cone
Test Analyte (pg/mL) Requirements
Sensitivity Dinoseb 0.004 Detection of analyte; S/N > 3
Chromatographic performance 4-Nitrophenol 1.6 0.70 0.40b
4-Nitrophenol 1.6
a PGF = peak Gaussian factor. Calculated using the equation:
1.83xW fl
PGF = LI
where W | _
WIJS
is the peak width at half height and W I _L I is the peak width at tenth height
2 ' "| TO
b Resolution between the two peaks as defined by the equation:
R-l.
w
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
762
-------
.1
Nitrogen
Tubel
Tube 2
r
Sample
Tube
52-015-27
Figure 1. Gaseous Diazorhethane Generator
163
-------
Method 515.1
Glass Tubing
Nitrogen
Rubber Stopper
Tubel
Tube 2
Collection
52-015-28
Thermos or
Cryogenic Cooler
Figure 2. Diazomethane Solution Generator
764
-------
Method 515.2
Determination of Chlorinated Acids in Water
Using Liquid-Solid Extraction
and Gas Chromatography
with an Electron Capture Detector
Revision 1.0 - EPA EMSL-Ci
August 1992
B.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
J.W. Hodgeson - Method 515, Revision 2.0 (1986)
T. Engels (Battelle Columbus Laboratories) - National Pesticide Survey
Method 3, Revision 3.0 (1987)
R.L. Graves - Method 515.1, Revision 4.0 (1989)
J.W. Hodgeson - Method 515.2, Revision 1.0 (1992)
-------
-------
Method 515.2
Determination of Chlorinated Acids in Water Using Liquid-Solid
Extraction and Gas Chromatography with an
Electron Capture Detector
1. SCOPE AND APPLICA TION
1.1 This is a gas chromatographic (GC) method applicable to the determination of certain chlori-
nated acids in ground water and finished drinking water. The following compounds can be
determined by this method:
Analyte CAS No.
Acifluorfen 50594-66-6
Bentazon 25057-89-0
2,4-D 94-75-7
2,4-DB 94-82-6
Dacthal3 1861-32-1
Dicamba 1918-00-9
3,5-Dichlorobenzoic acid 51-36-5
Dichlorprop 120-36-5
Dinoseb 88-85-7
5-Hydroxydicamba 7600-50-2
Pentachlorophenol (PCP) 87-86-5
Picloram 1918-02-1
2,4,5-T 93-76-5
2,4,5-TP (Silvex) 93-72-1
3 Dacthal monoacid and diacid metabolites included in method scope; Dacthal
diacid metabolite used for validation studies.
1.2 This method is applicable to the determination of salts and esters of analyte acids. The form
of each acid is not distinguished by this method. Results are calculated and reported for each
listed analyte as the total free acid.
1.3 Single laboratory accuracy and precision data and method detection limits (MDLs) have been
determined for the analytes above (Sect. 13). Observed detection limits may vary among
water matrices, depending upon the nature of interferences in the sample matrix and the
specific instrumentation used.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use
of GC and in the interpretation of gas chromatograms. Each analyst must demonstrate the
ability to generate acceptable results with this method using the procedure described in Sect.
9.3.
1.5 Analytes that are not separated chromatographically, (i.e., have very similar retention times)
cannot be individually identified and measured in the same calibration mixture or water sample
unless an alternative technique for identification and quantitation exists (Sect. 11.6).
167
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Method 515.2
1.6 When this method is used to analyze unfamiliar samples for any or all of the analytes above,
analyte identifications should be confirmed by analysis on a second gas chromatographic
column or by gas chromatography/mass spectrometry (GC/MS).
2. SUMMARY OF METHOD
2.1 A 250-mL measured volume of sample is adjusted to pH 12 with 6 N sodium hydroxide for 1
hr to hydrolyze derivatives. Extraneous organic material is removed by a solvent wash. The
sample is acidified, and the chlorinated acids are extracted with a 47 mm resin based extrac-
tion disk. The acids are converted to their methyl esters using diazomethane. Excess
derivatizing reagent is removed, and the esters are determined by capillary column GC using
an electron capture detector (ECD).
3. DEFINITIONS
3.1 Internal Standard (IS): A pure analyte(s) added to a sample, extract, or standard solution in
known amount(s), and used to measure the relative responses of other method analytes and
surrogates that are components of the same sample or solution. The IS must be an analyte that
is not a sample component.
3.2 Surrogate Analyte (SA): A pure analyte(s), which is extremely unlikely to be found in any
sample, and which is added to a sample aliquot in known amount(s) before extraction or other
processing, and is measured with the same procedures used to measure other sample compo-
nents. The purpose of the SA is to monitor method performance with each sample.
3.3 Laboratory Duplicates (LD1 and LD2): Two aliquots of the same sample taken in the analyti-
cal laboratory and analyzed separately with identical procedures. Analyses of LD1 and LD2
indicate the precision associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field Duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory Reagent Blank (LRB): An aliquot of reagent water or other blank matrix that is
treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents,
internal standards, and surrogates that are used with other samples. The LRB is used to
determine if method analytes or other interferences are present in the laboratory environment,
the reagents, or the apparatus.
3.6 Field Reagent Blank (FRB): An aliquot of reagent water or other blank matrix that is placed
in a sample container in the laboratory and treated as a sample in all respects, including
shipment to the sampling site, exposure to sampling site conditions, storage, preservation and
all analytical procedures. The purpose of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.7 Instrument Performance Check Solution (IPC): A solution of one or more method analytes,
surrogates, internal standards, or other test substances used to evaluate the performance of the
instrument system with respect to a defined set of criteria.
168
-------
Method 515.2
3.8 Laboratory Fortified Blank (LFB): An aliquot of reagent water or other blank matrix to which
known quantities of the method analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine whether the methodology is in control,
and whether the laboratory is capable of making accurate and precise measurements.
3.9 Laboratory Fortified Sample Matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot, and the measured values in the LFM correct-
ed for background concentrations.
3.10 Stock Standard Solution (SSS): A concentrated solution containing one or more method
analytes prepared in the laboratory using assayed reference materials or purchased from a
reputable commercial source.
3.11 Primary Dilution Standard Solution (PDS): A solution of several analytes prepared in the
laboratory from stock standard solutions, and diluted as needed to prepare calibration solutions
and other needed analyte solutions.
3.12 Calibration Standard (CAL): A solution prepared from the primary dilution standard solution
or stock standard solutions and the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality Control Sample (QCS): A solution of method analytes of known concentrations which
is used to fortify an aliquot of LRB or sample matrix. The QCS is obtained from a source
external to the laboratory and different from the source of calibration standards. It is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under analytical conditions by analyzing laboratory reagent blanks as described in
Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned.1 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with dilute acid, tap and reagent
water. Drain dry, and heat in an oven or muffle furnace at 400°C for 1 hr. Do not
heat volumetric ware. Thermally stable materials such as PCBs might not be elimi-
nated by this treatment. Thorough rinsing with acetone may be substituted for the
heating. After glassware is dry and cool, store it in a clean environment to prevent
any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
169
-------
Method 515.2
WARNING: When a solvent is purified, stabilizers and preservatives added by
the manufacturer are removed, thus potentially making the solvent hazardous and
reducing the shelf life.
4.2 The acid forms of the analytes are strong organic acids which react readily with alkaline
substances and can be lost during sample preparation. Glassware and glass wool must be
acid-rinsed with I N hydrochloric acid and the sodium sulfate must be acidified with sulfuric
acid prior to use to avoid analyte losses due to adsorption.
4.3 Organic acids and phenols, especially chlorinated compounds, cause the most direct interfer-
ence with the determination. Alkaline hydrolysis and subsequent extraction of the basic
sample removes many chlorinated hydrocarbons and phthalate esters that might otherwise
interfere with the electron capture analysis.
4.4 Interferences by phthalate esters can pose a major problem in pesticide analysis when using the
ECD. Phthalates generally appear in the chromatogram as large peaks. Common flexible
plastics contain varying amounts of phthalates, that are easily extracted or leached during
laboratory operations. Cross-contamination of clean glassware routinely occurs when plastics
are handled during extraction steps, especially when solvent-wetted surfaces are handled.
Interferences from phthalates can best be minimized by avoiding the use of plastics in the
laboratory. Exhaustive purification of reagents and glassware may be required to eliminate
background phthalate contamination.2-3
4.5 Interfering contamination may occur when a sample containing low concentrations of analytes
is analyzed immediately following a sample containing relatively high concentrations of
analytes. Between-sample rinsing of the sample syringe and associated equipment with methyl-
tert-butyl-ether (MTBE) can minimize sample cross- contamination. After analysis of a sample
containing high concentrations of analytes, one or more injections of MTBE should be made to
ensure that accurate values are obtained for the next sample.
4.6 Matrix interferences may be caused by contaminants that are coextracted from the sample.
Also, note that all analytes listed in the Scope and Application Section are not resolved from
each other on any one column, i.e., one analyte of interest may interfere with another analyte
of interest. The extent of matrix interferences will vary considerably from source to source,
depending upon the water sampled. The procedures in Sect. 11 can be used to overcome
many of these interferences. Tentative identifications should be confirmed (Sect. 11.6).
4.7 It is important that samples and working standards be contained in the same solvent. The
solvent for working standards must be the same as the final solvent used in sample prepara-
tion. If this is not the case, chromatographic comparability of standards to sample extracts
may be affected.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
770
-------
Method 515.2
ing the safe handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available and have been identified5"7
for the information of the analyst.
5.2 Diazomethane: A toxic carcinogen which can explode under certain conditions. The follow-
ing precautions must be followed:
5.2.1 Use the diazomethane generator behind a safety shield in a well ventilated fume hood.
Under no circumstances can the generator be heated above 90°C, and all grinding
surfaces such as ground glass joints, sleeve bearings, and glass stirrers must be
avoided. Diazomethane solutions must not be stored. Only generate enough for the
immediate needs. The diazomethane generator apparatus used in the esterification
procedure (Sect. 11.4) produces micromolar amounts of diazomethane in solution to
minimize safety hazards. If the procedure is followed exactly, no possibility for
explosion exists.
5.3 Methyltertbutyl Ether: Nanograde, redistilled in glass, if necessary. Must be free of perox-
ides as indicated by EM Quant test strips (available from Scientific Products Co., Cat. No.
PI 126-8, and other suppliers).
5.4 WARNING: When a solvent is purified, stabilizers added by the manufacturer are removed,
thus potentially making the solvent hazardous.
6. EQUIPMENT AND SUPPLIES
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1 Kontes Filter Funnels: Fisher Cat. No. 953755-0000 or equivalent.
6.2 Vacuum Flasks: 1000 mL with glass side arm
6.3 Vacuum Manifold: The manifold should be capable of holding 6-8 filter flasks in series with
house vacuum. Commercial manifolds are available from a number of suppliers, e.g., Baker,
Fisher, and Varian.
6.4 Culture Tubes (25 x 200 mm) With Teflon-lined Screw Caps: Fisher Cat. No. 14-933-1C, or
equivalent.
6.5 Pasteur Pipets: Glass disposable (5 mL)
6.6 Large Volume Pipets: Disposable, Fisher Cat. No. 13-678-8 or equivalent.
6.7 Balance: Analytical, capable of weighing to .0001 g.
6.8 pH Meter: Wide range capable of accurate measurements in the pH = 1-12 range.
6.9 Diazomethane Generator: See Figure 1 for a diagram of an all glass system custom made for
these validation studies. A micromolar generator is also available from Aldrich Chemical.
6.10 Analytical Concentrator: Six or twelve positions, Organomation N-EVAP Model No. 111-
6917 or equivalent.
6.11 Gas Chromatography: Analytical system complete with gas chromatograph equipped with
ECD, split/splitless capillary injector, temperature programming, differential flow control and
all required accessories. A data system is recommended for measuring peak areas. An
autoinjector is recommended to improve precision of analysis.
777
-------
Method 515.2
6.12 GC Columns and Recommended Operating Conditions
6.12.1 Primary: DB-5 or equivalent, 30 m x .32 mm ID, 0.25 /xm film thickness. Injector
Temp. = 200°C, Detector Temp. = 280°C, Helium linear velocity is 30 cm/sec at
200°C and 10 psi, 2 piL splitless injection with purge on 3 min. Program: Hold at
60°C 1 min., increase to 260°C at 5°C/min. and hold 5 min.
6.12.2 Confirmation: DB-1701 or equivalent, 30 m x .32 mm ID, 0.25 itm film thickness.
Injector Temp. = 200 °C, Detector Temp. = 280°C, Helium linear velocity is 30
cm/sec at 200°C and 10 psi, 2 /xL splitless injection with purge on 3 min. Program:
Hold at 60°C 1 min., increase to 260°C at 5°C/min. and hold 5 min.
6.13 Glass Wool: Acid washed with IN HCl and heated at 450°C for 4 hr.
6.14 Short Range pH Paper (pH=0-3).
6.15 Volumetric Flasks: 50 mL, 100 mL, and 250 mL
6.16 Microsyringes: 25 /xL, 50 /xL, 100 pL, 250 /xL, 500 /xL
6.17 Amber Bottles: 15 mL, with Teflon-lined screw caps
6.18 Graduated Cylinder: 250 mL
6.19 Separatory Funnel: 500 mL
6.20 Graduated Centrifuge Tubes: 15 mL or 10 mL Kuderna Danish Concentrator tubes
7. REAGENTS AND STANDARDS
7.1 Extraction Disks, 47 mm: Resin based polystyrenedivinylbenzene
7.2 Reagent Water: Reagent water is defined as a water in which an interference is not observed
at the MDL of each analyte of interest.
7.2.1 A Millipore Super-Q water system or its equivalent may be used to generate deionized
reagent water. Distilled water that has been passed through granular charcoal may
also be suitable.
7.2.2 Test reagent water each day it is used by analyzing according to Sect. 11.
7.3 Methanol: Pesticide quality or equivalent.
7.4 Methyltertbutyl Ether (MTBE): Nanograde, redistilled in glass if necessary. Ether must be
demonstrated to be free of peroxides. One test kit (EM Quant Test Strips), is available from
EM Science, Gibbstown, NJ. Procedures for removing peroxides from the ether are provided
with the test strips. Ethers must be periodically tested (at least monthly) for peroxide forma-
tion during use. Any reliable test kit may be used.
7.5 Sodium Sulfate: (ACS) Granular, Anhydrous: Heat in a shallow tray at 400°C for a mini-
mum of 4 hr to remove phthalates and other interfering organic substances. Alternatively,
extract with methylene chloride in a Soxhlet apparatus for 48 hr.
7.5.1 Sodium sulfate drying tubes: Plug the bottom of a large volume disposable pipet with
a minimum amount of acidified glass wool (Supelco Cat. No. 20383 or equivalent).
Fill the pipet halfway (3 g) with acidified sodium sulfate (See Sect. 7.9).
772
-------
Method 515.2
7.6 Sulfuric Acid: Reagent grade.
7.6.1 Sulfuric acid, 12 N: Slowly add 335 mL concentrated sulfuric acid to 665 mL of
reagent water.
7.7 Sodium Hydroxide: ACS reagent grade or equivalent.
7.7.1 Sodium hydroxide IN: Dissolve 4.0 g reagent grade sodium hydroxide in reagent
water and dilute to 100 mL in volumetric flasks.
7.7.2 Sodium hydroxide 6N
7.8 Ethyl Ether, Unpreserved: Nanograde, redistilled in glass if necessary. Must be free of
peroxides as indicated by EM Quant test strips (available from Scientific Products Co., Cat.
No. PI126-8, and other suppliers). Procedures recommended for removal of peroxides are
provided with the test strips.
7.9 Acidified Sodium Sulfate: Cover 500 g sodium sulfate (Sect. 7.5) with ethyl ether (Sect. 7.8).
While agitating vigorously, add dropwise approximately 0.7 mL concentrated sulfuric acid.
Remove the ethyl ether overnight under vacuum and store the sodium sulfate in a 100°C oven.
7.10 Carbitol, ACS Grade: Available from Aldrich Chemical.
7.11 Diazald, ACS Grade: Available from Aldrich Chemical.
7.12 Diazald Solution: Prepare a solution containing 10 g Diazald in 100 mL of a 50:50 by volume
mixture of ethyl ether and carbitol. This solution is stable for 1-month or longer when stored
at 4°C in an amber bottle with a Teflon-lined screw cap.
7.13 4,4'-Dibromooctafluorobiphenyl (DBOB): 99% purity, for use as internal standard.
7.14 2,4-Dichlorophenylacetic Acid (DCAA): 99% purity, for use as surrogate standard.
7.15 Potassium Hydroxide: ACS reagent grade or equivalent.
7.15.1 Potassium hydroxide solution, 37%: Using extreme caution, dissolve 37 g reagent
grade potassium hydroxide in reagent water and dilute to 100 mL.
7.16 Stock Standard Solutions (1.00-2.00 /xg//xL): Stock standard solutions may be purchased as
certified solutions or prepared from pure standard materials using the following procedure:
7.16.1 Prepare stock standard solutions by accurately weighing approximately 0.0100-0.0200
g of pure material. Dissolve the material in methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes may be used at the convenience of the
analyst. If compound purity is certified at 96% or greater, the weight may be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.16.2 Transfer the stock standard solutions into 15-mL TFE-fluorocarbon-sealed screw cap
amber vials. Store at 4°C or less when not in use.
7.16.3 Stock standard solutions should be replaced after 2 months or sooner if comparison
with laboratory fortified blanks, or QC samples indicate a problem.
7.16.4 Primary Dilution Standards: Prepare two sets of standards according to the sets
labeled A and B in Table 1. For each set, add approximately 25 mL of methanol to a
50 mL volumetric flask. Add aliquots of each stock standard in the range of approxi-
173
-------
Method 515.2
mately 20 to 400 /zL and dilute to volume with methanol. Individual analyte concen-
trations will then be in the range of 0.4 to 8 /xg/mL (for a 1.0 mg/mL stock). The
minimum concentration would be appropriate for an analyte with strong electron
capture detector (ECD) response, e.g. pentachlorophenol. The maximum concentra-
tion is for an analyte with weak response, e.g., 2,4-DB. The concentrations given in
Table 2 reflect the relative volumes of stock standards used for the primary dilution
standards used in generating the method validation data. Use these relative values to
determine the aliquot volumes of individual stock standards above.
7.17 Internal Standard Solution: Prepare a stock internal standard solution by accurately weighing
approximately 0.050 g of pure DBOB. Dissolve the DBOB in methanol and dilute to volume
in a 10-mL volumetric flask. Transfer the DBOB solution to a TFE-fluorocarbonsealed screw
cap bottle and store at room temperature. Prepare a primary dilution standard at approximate-
ly 1.00 /xg/mL by the addition of 20 /xL of the stock standard to 100 mL of methanol. Addi-
tion of 100 jxL of the primary dilution standard solution to the final 5 mL of sample extract
(Sect. 11) results in a final internal standard concentration of 0.020 jig/mL. Solution should
be replaced when ongoing QC (Sect. 9) indicates a problem. Note that DBOB has been shown
to be an effective internal standard for the method analytes, but other compounds may be used
if the QC requirements in Sect. 9 are met.
7.18 Surrogate Analyte Solution: Prepare a surrogate analyte stock standard solution by accurately
weighing approximately 0.050 g of pure DCAA. Dissolve the DCAA in methanol and dilute
to volume in a 10-mL volumetric flask. Transfer the surrogate analyte solution to a
TFE-fluorocarbon-sealed screw cap bottle and store at room temperature. Prepare a primary
dilution standard at approximately 2.0 /xg/mL by addition of 40 /xL at the stock standard to
100 mL of methanol. Addition of 250 /xL of the surrogate analyte solution to a 250-mL
sample prior to extraction results in a surrogate concentration in the sample of 2 uglL and,
assuming quantitative recovery of DCAA, a surrogate analyte concentration in the final 5 mL
extract of 0.1 /xg/mL. The surrogate standard solution should be replaced when ongoing QC
(Sect. 9) indicates a problem. DCAA has been shown to be an effective surrogate standard for
the method analytes, but other compounds may be used if the QC requirements in Sect. 10 are
met.
7.19 Instrument Performance Check Solution: Prepare a diluted dinoseb solution by adding 10 itL
of the 1.0 /xg//zL dinoseb stock solution to the MTBE and diluting to volume in a 10-mL
volumetric flask. To prepare the check solution, add 40 /xL of the diluted dinoseb solution, 16
ixL of the 4-nitrophenol stock solution, 6 /xL of the 3,5-dichlorobenzoic acid stock solution, 50
/xL of the surrogate standard solution, 25 iiL of the internal standard solution, and 250 tiL of
methanol to a 5-mL volumetric flask and dilute to volume with MTBE. Methylate sample as
described in Sect. 11.4. Dilute the sample to 10 mL in MTBE. Transfer to a TFE-fluorocar-
bon-sealed screw cap bottle and store at room temperature. Solution should be replaced when
ongoing QC (Sect. 9) indicates a problem.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Grab samples should be collected in 1-L amber glass containers. Conventional sampling
practices should be followed; however, the bottle must not be prerinsed with sample before
collection.7
774
-------
Method 515.2
8.2 Sample Preservation and Storage
8.2.1 Add hydrochloric acid (diluted 1:1 in water) to the sample at the sampling site in
amounts to produce a sample pH < 2. Short range (0-3) pH paper (Sect. 6.14) may
be used to monitor the pH.
8.2.2 If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
the sample bottle prior to collecting the sample.
8.2.3 After the sample is collected in the bottle containing preservative(s), seal the bottle
and shake vigorously for 1 min.
8.2.4 The samples must be iced or refrigerated at 4°C away from light from the time of
collection until extraction. Preservation study results indicate that the sample analytes
(measured as total acid), except 5-hydroxy-dicamba, are stable in water for 14 days
when stored under these conditions (Tables 8 and 9). The concentration of 5-
hydroxydicamba is seriously degraded over 14 days in a biologically active matrix.
However, analyte stability will very likely be affected by the matrix; therefore, the
analyst should verify that the preservation technique is applicable to the samples under
study.
8.3 Extract Storage
8.3.1 Extracts should be stored at 4°C or less away from light. Preservation study results
indicate that most analytes are stable for 14 days (Tables 8 and 9); however, the
analyst should verify appropriate extract holding times applicable to the samples under
study.
9. QUALITY CONTROL
9.1 Minimum QC requirements are initial demonstration of laboratory capability, determination of
surrogate compound recoveries in each sample and blank, monitoring internal standard peak
area or height in each sample and blank (when internal standard calibration procedures are
being employed), analysis of laboratory reagent blanks, laboratory fortified samples, laborato-
ry fortified blanks, and QC samples.
9.2 Laboratory Reagent Blanks (LRB): Before processing any samples, the analyst must demon-
strate that all glassware and reagent interferences are under control. Each time a set of
samples is extracted or reagents are changed, a LRB must be analyzed. If within the retention
time window of any analyte the LRB produces a peak that would prevent the determination of
that analyte, determine the source of contamination and eliminate the interference before
processing samples.
9.3 Initial Demonstration of Capability
9.3.1 Select a representative fortified concentration (about 10 to 20 times MDL) for each
analyte. Prepare a sample concentrate (in methanol) containing each analyte at 1000
times selected concentration. With a syringe, add 250 pL of the concentrate to each
of at least four 250 mL aliquots of reagent water, and analyze each aliquot according
to procedures beginning in Sect. 11.
9.3.2 For each analyte the recovery value for all four of these samples must fall in the
range of + 40% of the fortified concentration. For those compounds that meet the
175
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Method 515.2
acceptance criteria, performance is considered acceptable and sample analysis may
begin. For compounds failing this criteria, this procedure must be repeated using five
fresh samples until satisfactory performance has been demonstrated for all analytes.
9.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. As laboratory personnel gain experience with this method the
quality of data should improve beyond those required here.
9.4 The analyst is permitted to modify GC columns, GC conditions, detectors, concentration
techniques (i.e., evaporation techniques), internal standard or surrogate compounds. Each
time such method modifications are made, the analyst must repeat the procedures in Sect. 9.3.
9.5 Assessing Surrogate Recovery
9.5.1 When surrogate recovery from a sample or a blank is <60% or > 140%, check (1)
calculations to locate possible errors, (2) fortifying solutions for degradation, (3)
contamination, and (4) instrument performance. If those steps do not reveal the cause
of the problem, reanalyze the extract.
9.5.2 If a blank extract reanalysis fails the 60-140% recovery criteria, the problem must be
identified and corrected before continuing.
9.5.3 If sample extract reanalysis meets the surrogate recovery criteria, report only data for
the reanalyzed extract. If sample extract continues to fail the recovery criteria, report
all data for that sample as suspect.
9.6 Assessing the Internal Standard
9.6.1 When using the internal standard (IS) calibration procedure, the analyst is expected to
monitor the IS response (peak area or peak height) of all samples during each analysis
day. The IS response for any sample chromatogram should not deviate from the daily
calibration check standard's IS response by more than 30%.
9.6.2 If >30% deviation occurs with an individual extract, optimize instrument perfor-
mance and inject a second aliquot of that extract.
9.6.2.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
9.6.2.2 If a deviation of greater than 30% is obtained for the reinjected extract,
analysis of the samples should be repeated beginning with Sect. 11, pro-
vided the sample is still available. Otherwise, report results obtained from
the reinjected extract, but annotate as suspect.
9.6.3 If consecutive samples fail the IS response acceptance criteria, immediately analyze a
medium calibration standard.
9.6.3.1 If the standard provides a response factor (RF) (Sect. 10.2.2) within 20%
of the predicted value, then follow procedures itemized in Sect. 9.6.2 for
each sample failing the IS response criterion.
9.6.3.2 If the check standard provides a response factor which deviates more than
20% of the predicted value, then the analyst must recalibrate as specified
in Sect. 10.
176
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Method 515.2
9.7 Assessing Laboratory Performance: Laboratory Fortified Blank
9.7.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
every 20 samples or one per sample set (all samples extracted within a 24-hr period)
whichever is greater. The concentration of each analyte in the LFB should be 10
times the MDL. Calculate percent recovery (X,). If the recovery of any analyte falls
outside the control limits (See Sect. 9.7.2), that analyte is judged out of control, and
the source of the problem should be identified and resolved before continuing analy-
ses.
9.7.2 Until sufficient data become available, usually a minimum of results from 20 to 30
analyses, each laboratory should assess laboratory performance against the control
limits in Sect. 9.3.2 that are derived from the data in Table 2. When sufficient
internal performance data become available, develop control limits from the mean
percent recovery (X) and standard deviation (S) of the percent recovery. These data
are used to establish upper and lower control limits as follows:
Upper Control Limit = X + 3S
Lower Control Limit = X - 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points. These calculated control
limits should never exceed those established in Sect. 9.3.2.
9.7.3 Method detection limits (MDL) must be determined using the procedure given in
reference 8. The MDLs must be sufficient to detect analytes at the required levels
according to SDWA regulations.
9.7.4 At least quarterly, analyze a QCS (Sect. 3.13) from an outside source.
9.7.5 Laboratories are encouraged to participate in external performance evaluation studies
such as the laboratory certification programs offered by many states or the studies
conducted by USEPA.
9.8 Assessing Analyte Recovery - Laboratory Fortified Sample Matrix
9.8.1 Each laboratory must analyze a LFM for 10% of the samples or one sample con-
centration per set, whichever is greater. The concentration should not be less then the
background concentration of the sample selected for fortification. Ideally, the concen-
tration should be the same as that used for the laboratory fortified blank (Sect. 9.7).
Over time, samples from all routine sample sources should be fortified.
9.8.2 Calculate the percent recovery, P of the concentration for each analyte, after correct-
ing the measured concentration, X, from the fortified sample for the background
concentration, b, measured in the unfortified sample.
p = 100 (X - b)
fortifying concentration
177
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Method 515.2
and compare these values to control limits appropriate for reagent water data
collected in the same fashion. If the analyzed unfortified sample is found to con-
tain NO background concentrations and the added concentrations are those speci-
fied in Sect. 9.7, then the appropriate control limits would be the acceptance limits
in Sect. 9.7. If, on the other hand, the analyzed unfortified sample is found to
contain background concentration, b, estimate the standard deviation at the back-
ground concentration, sh, using regressions or comparable background data and,
similarly, estimate the mean, Xa and standard deviation, sa, of analytical results at
the total concentration after fortifying. Then the appropriate percentage control
limits would be P ± 3sP , where:
100 X
(b + fortifying concentration)
and
,,.1.0 _ _
fortifying concentration
For example, if the background concentration for Analyte A was found to be 1
pg/L and the added amount was also 1 Mg/L, and upon analysis the laboratory
fortified sample measured 1.6 /xg/L, then the calculated P for this sample would be
(1.6 /ig/L minus 1.0 ^g/L) /I pig/L or 60%. This calculated P is compared to
control limits derived from prior reagent water data. Assume that analysis of an
interference free sample at 1 fig/L yields an s of 0.12 jtg/L and similar analysis at
2.0 /xg/L yields X and s of 2.01 jig/L and 0.20 /xg/L, respectively. The appropri-
ate limits to judge the reasonableness of the percent recovery, 60%, obtained on
the fortified matrix sample is computed as follows:
100 (2.01
2.0
3
(0.20
1.0 pg/L
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
9.8.3 If the recovery of any such analyte falls outside the designated range, and the labora-
tory performance for that analyte is shown to be in control (Sect. 9.7), the recovery
problem encountered with the fortified sample is judged to be matrix related, not
775
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Method 515.2
system related. The result for that analyte in the unfortified sample is labeled sus-
pect/matrix to inform the data user that the results are suspect due to matrix effects.
9.9 Assessing Instrument System/Instrument Performance Check (IPC) Sample: Instrument
performance should be monitored on a daily basis by analysis of the IPC sample. The IPC
sample contains compounds designed to indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. IPC sample components
and performance criteria are listed in Table 11. Inability to demonstrate acceptable instrument
performance indicates the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the MDLs published in this method. MDLs will vary from
laboratory to laboratory.
9.10 The laboratory may adopt additional QC practices for use with this method. The specific
practices that are most productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be analyzed to assess the preci-
sion of the environmental measurements or field reagent blanks may be used to assess contami-
nation of samples under site conditions, transportation, and storage.
1 0. CALIBRA TION AND STANDARDIZA TION
10.1 Establish GC operating parameters equivalent to those indicated in Sect. 6.12. This calibration
procedure employs procedural standards, i.e., fortified aqueous standards which are processed
through most of the method (Sect. 11). The GC system is calibrated by means of the internal
standard technique (Sect. 10.2). NOTE: Calibration standard solutions must be prepared such
that no unresolved analytes are mixed together (See Table 1).
10.2 Internal Standard Calibration Procedure: To use this approach, the analyst must select one or
more internal standards compatible in analytical behavior to the compounds of interest. The
analyst must further demonstrate that the measurement of the internal standard is not affected
by method or matrix interferences. DBOB (Sect. 7.13) has been identified as a suitable
internal standard.
10.2.1 Prepare aqueous calibration standards at a minimum of three (five are recommended)
concentration levels for each method analyte as follows: for each concentration, fill a
250-mL volumetric flask with 240 mL of reagent water at pH 1 and containing 20%
by weight of dissolved sodium sulfate. Add an appropriate aliquot of the primary
dilution standard (Sect. 7.16.4) and dilute to 250 mL with the same reagent water.
Process each aqueous calibration sample through the analytical procedure beginning
with Sect. 11.2, i.e., omit the hydrolysis and cleanup step (Sect. 11.1). The lowest
calibration standard should represent analyte concentrations near, but above, the
respective MDLs. The remaining standards should bracket the analyte concentrations
expected in the sample extracts, or should define the working range of the detector.
The internal standard is added to the final 5 mL extract as specified in Sect. 11.
10.2.2 Analyze each calibration standard according to the procedure beginning in Sect. 11.2.
Tabulate response (peak height or area) against concentration for each compound and
internal standard. Calculate the response factor (RF) for each analyte and surrogate
using Equation 1.
179
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Method 515.2
Equation 1
where:
As = Response for the analyte to be measured.
Ais = Response for the internal standard.
Ca = Concentration of the internal standard
Cs = Concentration of the analyte to be measured (/j.g/L).
10.2.3 If the RF value over the working range is constant (30% RSD or less) the average RF
can be used for calculations. Alternatively, the results can be used to plot a calibra-
tion curve of response ratios (As/Ais) vs. Cs. A data station may be used to collect the
chromatographic data, calculate response factors and generate linear or second order
regression curves.
10.2.4 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. A new calibration standard need
not be derivatized each day. The same standard extract can be used up to 14 days. If
the response for any analyte varies from the predicted response by more than ±30%,
the test must be repeated using a fresh calibration standard. If the repetition also
fails, a new calibration curve must be generated for that analyte using freshly pre-
pared standards.
10.2.5 Verify calibration standards periodically, at least quarterly is recommended, by ana-
lyzing a standard prepared from reference material obtained from an independent
source. Results from these analyses must be within the limits used to routinely check
calibration.
1 1. PROCEDURE
11.1 Manual Hydrolysis and Clean-up
11.1.1 Remove the sample bottles from cold storage and allow them to equilibrate to room
temperature. Acidify and add sodium thiosulfate to blanks and QC check standards as
specified in Sect. 8.
1 1 .1 .2 Measure a 250-mL aliquot of each sample with a 250-mL graduated cylinder and pour
into a 500-mL separatory funnel. Add 250 pL of the surrogate primary dilution stan-
dard (Sect. 7.18) to each 250-mL sample. The surrogate will be at a concentration of
2 ng/L. Dissolve 50 g sodium sulfate in the sample.
1 1 .1 .3 Add 4 mL of 6 N NaOH to each sample, seal, and shake. Check the pH of the
sample with pH paper or a pH meter; if the sample does not have a pH greater than
or equal to 12, adjust the pH by adding more 6 N NaOH. Let the sample sit at room
temperature for 1 hr, shaking the separatory funnel and contents periodically.
1 1 .1 .4 Add 15 mL methylene chloride to the graduated cylinder to rinse the walls, transfer
the methylene chloride to the separatory funnel and extract the sample by vigorously
shaking the funnel for 2 min with periodic venting to release excess pressure. Allow
180
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Method 515.2
the organic layer to separate from the water phase for a minimum of 10 min. If the
emulsion interface between layers is more than one-third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete the phase separa-
tion. The optimum technique depends upon the sample, but may include stirring,
filtration through glass wool, centrifugation, or other physical methods. Discard the
methylene chloride phase.
11.1.5 Add a second 15-mL volume of methylene chloride to the separatory funnel and
repeat the extraction procedure a second time, discarding the methylene chloride
layer. Perform a third extraction in the same manner.
11.1.6 Drain the contents of the separatory funnel into a 500-mL beaker. Adjust the pH to
1.0 + 0.1 by the dropwise addition of concentrated sulfuric acid with constant stir-
ring. Monitor the pH with a pH meter (Sect. 6.8) or short range (0-3) pH paper
(Sect. 6.14).
11.2 Sample Extraction
11.2.1 Vacuum Manifold: Assemble a manifold (Sect. 6.3) consisting of 6-8 vacuum flasks
with filter funnels (Sect. 6.1,6.2). Individual vacuum control, on-off and vacuum
release valves and vacuum gauges are desirable. Place the 47 mm extraction disks
(Sect. 7.1) on the filter frits.
11.2.2 Add 20 mL of 10% by volume of methanol in MTBE to the top of each disk without
vacuum and allow the solvent to remain for 2 min. Turn on full vacuum and pull the
solvent through the disks, followed by room air for 5 min.
11.2.3 Adjust the vacuum to approximately 5 in. (mercury) and add the following in series to
the filter funnel (a) 20 mL methanol (b) 20 mL reagent water (c) sample. Do not
allow the disk to dry between steps and maintain the vacuum at 5 in.
11.2.4 After the sample is extracted completely, apply maximum vacuum and draw room air
through the disks for 20 min.
11.2.5 Place the culture tubes (Sect. 6.4) in the vacuum tubes to collect the eluates. Elute
the disks with two each 2-mL aliquots of 10% methanol in MTBE. Allow each
aliquot to remain on the disk for one min before applying vacuum.
11.2.6 Rinse each 500-mL beaker (Sect. 11.1.6) with 4 mL of pure MTBE and elute the disk
with this solvent as in Sect. 11.2.5.
11.2.7 Remove the culture tubes and cap.
11.3 Extract Preparation
11.3.1 Pre-rinse the drying tubes (Sect. 7.5.1) with 2 mL of MTBE.
11.3.2 Remove the entire extract with a 5-mL pipet and drain the lower aqueous layer back
into the culture tube. Add the organic layer to the sodium sulfate drying tube (Sect.
7.5.1). Maintain liquid in the drying tube between this and subsequent steps. Collect
the dried extract in a 15-mL graduated centrifuge tube or a 10-mL Kuderna-Danish
tube.
11.3.3 Rinse the culture tube with an additional 1 mL of MTBE and repeat Sect. 11.3.2.
787
-------
Method 515.2
11.3.4 Repeat step Sect. 11.3.3 and finally add a 1-mL aliquot of MTBE to the drying tube
before it empties. The final volume should be 6-9 mL. In this form the extract is
esterified as described below.
11.4 Extract Esterification
11.4.1 Assemble the diazomethane generator (Figure 1) in a hood.
11.4.2 Add 5 mL of ethyl ether to Tube 1. Add 4 mL of Diazald solution (Sect. 7.12) and
3 mL of 37% KOH solution (Sect. 7.15.1) to the reaction tube 2. Immediately place
the exit tube into the collection tube containing the sample extract. Apply nitrogen
flow (10 mL/min) to bubble diazo-methane through the extract. Each charge of the
generator should be sufficient to esterify four samples. The appearance of a persistent
yellow color is an indication that esterification is complete. The first sample should
require 30 sec to 1 min and each subsequent sample somewhat longer. The final
sample may require 2-3 min.
11.4.3 Cap each collection tube and allow to remain stored at room temperature in a hood
for 30 min. No significant fading of the yellow color should occur during this period.
Fortify each sample with 100 /*L of the internal standard primary dilution solution
(Sect. 7.17) and reduce the volume to 5.0 mL with the analytical concentrator (Sect.
6.10), a stream of dry nitrogen, or an equivalent concentration technique. NOTE:
The excess diazomethane is volatilized from the extract during the concentration
procedure.
11.4.4 Cap the tubes and store in a refrigerator if further processing will not be performed
immediately. Analyze by GC-ECD.
11.5 Gas Chromatography
11.5.1 Sect. 6.12 summarizes the recommended GC operating conditions. Included in Table
1 are retention times observed using this method. Figures 2A and 2B illustrate the
chromatographic performance of the primary column (Sect. 6.12.1) for groups A and
B of the method analytes. Other GC columns, chromatographic conditions, or detec-
tors may be used if the requirements of Sect. 9.3 are met.
11.5.2 Calibrate the system daily as described in Sect. 10.
11.5.3 Inject 2 pL of the sample extract. Record the resulting peak size in area units.
11.5.4 If the response for any sample peak exceeds the working range of the detector, dilute
the extract and reanalyze.
11.6 Identification of Analytes
11.6.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound
corresponds, within limits, to the retention time of a standard compound, then an
analyte is considered to be identified.
11.6.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in interpretation of chromatograms.
752
-------
Method S15.2
11.6.3 Identification requires expert judgment when sample components are not resolved
chromatographically. When GC peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley between two or more
maxima, or any time doubt exists over the identification of a peak in a chromatogram,
appropriate alternative techniques to help confirm peak identification need to be
employed. For example, more positive identification may be made by the use of an
alternative detector which operates on a chemical/physical principle different from that
originally used, e.g., mass spectrometry, or the use of a second chromatography
column. A suggested alternative column is described in Sect. 6.12.2.
72. DA TA ANAL vs/s AND CALCULA TIONS
12.1 Calculate analyte concentrations in the sample from the response for the analyte using the
calibration procedure described in Sect. 10.
12.2 Calculate the concentration (C) in the sample using the response factor (RF) determined in
Sect. 10.2.2 and Equation 2, or determine sample concentration from the calibration curve
(Sect. 10.2.3).
Equation 2
(Ais)(RF)(Vo)
where:
As = Response for the parameter to be measured.
Au = Response for the internal standard.
Is ~ Amount of internal standard added to each extract (/j.g).
V = Volume of water extracted (L).
13. METHOD PERFORMANCE
13.1 In a single laboratory, analyte recoveries from reagent water were determined at three
concentration levels, Tables 2-4. Results were used to determine the analyte MDLs8 listed in
Table 2.
13.2 In a single laboratory, analyte recoveries from dechlorinated tap water were determined at two
concentrations, Tables 5 and 6. In addition, analyte recoveries were determined at two
concentrations from an ozonated surface (river) water, Tables 7 and 8, and at one level from a
high humectant surface (reservoir) water, Table 10. Finally, a holding study was conducted on
the preserved, ozonated surface water and recovery data are presented for day 1 and day 14 of
this study, Tables 8 and 9. The ozonated surface water was chosen as the matrix in which to
study analyte stability during a 14-day holding time because it was very biologically active.
183
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Method 515.2
14. POLLUTION PREVENTION
14.1 This method utilizes the new liquid-solid extraction technology which requires the use of very
small quantities of organic solvents. This feature eliminates the hazards involved with the use
of large volumes of potentially harmful organic solvents needed for conventional liquid-liquid
extractions. Also, mercuric chloride, a highly toxic and environmentally hazardous chemical,
has been replaced with hydrochloric acid as the sample preservative. These features make this
method much safer and a great deal less harmful to the environment. Some of the phenolic
herbicides on the analyte list are very difficult to methylate and diazomethane is still required
to derivatize these compounds.
14.2 For information about pollution prevention that may be applicable to laboratory operations,
consult "Less is Better: Laboratory Chemical Management for Waste Reduction" available
from the American Chemical Society's Department of Government Relations and Science
Policy, H55 I6th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 Due to the nature of this method, there is little need for waste management. No large volumes
of solvents or hazardous chemicals are used. The matrices of concern are finished drinking
water or source water. However, the Agency requires that laboratory waste management
practices be conducted consistent with all applicable rules and regulations, and that laboratories
protect the air, water, and land by minimizing and controlling all releases from fume hoods
and bench operations. Also, compliance is required with any sewage discharge permits and
regulations, particularly the hazardous waste identification rules and land disposal restrictions.
For further information on waste management, consult "The Waste Management Manual for
Laboratory Personnel," also available from the American Chemical Society at the address in
Sect. 14.2.
184
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Method 515.2
References
1. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society for Testing and
Materials, Philadelphia, PA, p. 86, 1986.
2. Giam, C.S., H.S. Chan, and G.S. Nef. "Sensitive Method for Determination of Phthalate
Ester Plasticizers in Open-Ocean Biota Samples," Analytical Chemistry. 47, 2225 (1975).
3. Giam, C.S., and H.S. Chan. "Control of Blanks in the Analysis of Phthalates in Air and
Ocean Biota Samples," U.S. National Bureau of Standards, Special Publication 442, pp.
701-708, 1976.
4. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, Aug. 1977.
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
7. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, p. 130,
1986.
8. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., "Trace Analyses
for Wastewaters," Environ. Sci. Technol. 1981,15, 1426-1435.
9. 40 CFR, Part 136, Appendix B.
185
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Method 515.2
Retention Time (min.f
Table 1. Retention Data
Analyte
3,5-Dichlorobenzoic acid
2,4-Dichlorophenylacetic acid (SA)
Dicamba
Dichlorprop
2,4-D
4,4'-Dibromooctafluorobiphenyl (IS)
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
2,4-DB
Dinoseb
Bentazon
Picloram
Dacthal
Acifluorfen
* Analytes were divided into two groups during method development to avoid chromatographic
overlap.
b Columns and chromatographic conditions are described in Sect. 6.12.
Table 2. Single Laboratory Recovery, Precision Data and Method Detection Limit
With Fortified Reagent Water —Level 1
Group'
A
A,B
B
A
B
A,B
A
B
B
A
B
A
B
B
A
B
Primary
16.72
19.78
20.18
22.53
23.13
24.26
25.03
25.82
26.28
26.57
27.95
28.03
28.70
29.93
31.02
35.62
I Confirmation
18.98
22.83
23.42
25.90
27.01
26.57
27.23
29.08
30.18
30.33
31.47
33.02
33.58
35.90
34.32
40.58
Fortified Cone. Mean* Recovery Relative Std.
Analyte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
(itg/U
0.50
2.50
0.25
2.50
0.25
0.75
1.25
0.25
0.50
0.75
0.25
0.75
0.25
0.25
70
70
96
79
96
109
126
106
87
90
103
95
116
98
Dev. (%)
21
11
38
12
16
11
24
15
22
12
18
15
18
9
MDL
fag/U
0.25
0.63
0.28
0.72
0.13
0.28
1.23
0.13
0.28
0.25
0.16
0.35
0.16
0.06
Based on the analyses of seven replicates.
Measurement includes the mono- and diacid metabolites.
756
-------
Method 515.2
Table 3. Single Laboratory Recovery and Precision Data For Fortified Reagent
Water-Level 2
Analyte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified Cone.
frg/U
0.80
4.0
0.40
4.0
0.40
1.20
2.00
0.40
0.80
1.20
0.40
1.20
0.40
0.40
Mean* Recovery Relative Std. Dev.
61
81
96
90
96
109
126
76
87
90
66
68
116
105
27
8
38
13
16
11
24
21
22
12
26
21
18
7
Based on the analyses of six-seven replicates.
Measurement includes the mono- and diacid metabolites.
Table 4. Single Laboratory Recovery and Precision Data For Fortified Reagent
Water—Level 3
Analyte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified Cone.
(V9/U
2.0
10.0
1.0
10.0
1.0
3.0
5.0
1.0
2.0
3.0
1.0
3.0
1.0
1.0
Mean* Recovery Relative Std. Dev.
59
68
90
74
60
75
62
97
63
77
69
66
64
68
13
8
20
6
10
9
18
17
10
8
11
9
15
8
Based on the analyses of six-seven replicates.
Measurement includes the mono- and diacid metabolites.
757
-------
Method 515.2
Table 5. Single Laboratory Recovery and Precision Data For Fortified,
Dechlorinated Tap Water —Level 1
Fortified Cone. Mean' Recovery Relative Std. Dev.
Analyte (f/g/LJ (%) (o/o}
Acifluorfen 0.50 117 21
Bentazon 2.50 96 12
2,4-D 0.25 59C 55
2,4-DB 2.50 112 15
Dacthal" 0.25 101 10
Dicamba 0.75 91 14
3,5-Dichlorobenzoic acid 1.25 103 15
Dichlorprop 0.25 218d 37
Dinoseb 0.50 134 10
5-Hydroxydicamba 0.75 90 14
Pentachlorophenol 0.25 91 8
Picloram 0.75 76 28
2,4,5-T 0.25 118 16
2,4,5-TP 0.25 99 10
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
' 2,4-D background value was 0.29 /vg/L.
d Probable interference.
Table 6. Single Laboratory Recovery and Precision Data For Fortified,
Dechlorinated Tap Water—Level 2
Fortified Cone. Mean* Recovery Relative Std. Dev.
Analyte fag/L) (%) (%)
Acifluorfen 2.0 150 7
Bentazon 10.0 112 9
2,4-D 1.0 90 16
2,4-DB 10.0 111 10
Dacthal" 1.0 118 8
Dicamba 3.0 86 10
3,5-Dichlorobenzoic acid 5.0 111 5
Dichlorprop 1.0 88 30
Dinoseb 2.0 121 6
5-Hydroxydicamba 3.0 96 6
Pentachlorophenol 1.0 96 6
Picloram 3.0 132 12
2,4,5-T 1.0 108 10
2,4,5-TP 1.0 115 7
2,4-Dichlorophenylacetic acidc 1.0 120 19
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Surrogate analyte.
188
-------
Method 515.2
Table 7. Single Laboratory Recovery and Precision Data For Fortified, Ozonated
Surface Water—Level 1
Fortified Cone. Mean' Recovery Relative Std. Dev.
Analyte frg/U (%) (%)
Acifluorfen .50 172 14
Bentazon 2.50 92 22
2,4-D 0.25 127 13
2,4-DB 2.50 154 19
Dacthal" 0.25 113 17
Dicamba 0.75 107 13
3,5-Dichlorobenzoic acid 1.25 100 17
Dichlorprop 0.25 115 20
Dinoseb 0.50 134 28
5-Hydroxydicamba 0.75 89 13
Pentachlorophenol 0.25 110 22
Picloram 0.75 109 27
2,4,5-T 0.25 102 19
2,4,5-TP 0.25 127 8
2,4-Dichlorophenylacetic acidc 0.25 72 31
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Surrogate analyte.
Table 8. Single Laboratory Recovery and Precision Data For Fortified, Ozonated
Surface Water—Level 2, Stability Study Day 1°
Fortified Cone. Mean* Recovery Relative Std. Dev.
Analyte (f/g/U (%) (%)
Acifluorfen 2.0 173 11
Bentazon 10.0 122 7
2,4-D 1.0 126 10
2,4-DB 10.0 130 7
Dacthal" 1.0 116 11
Dicamba 3.0 109 9
3,5-Dichlorobenzoic acid 5.0 115 11
Dichlorprop 1.0 116 11
Dinoseb 2.0 116 9
5-Hydroxydicamba 3.0 121 9
Pentachlorophenol 1.0 118 10
Picloram 3.0 182 14
2,4,5-T 1.0 112 9
2,4,5-TP 1.0 122 10
2,4-Dichlorophenylacetic acid" 1.0 110 26
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Samples preserved at pH = 2.0.
d Surrogate analyte.
755
-------
Method 515.2
Table 9. Single Laboratory Recovery and Precision Data For Fortified, Ozonated
Surface Water-Level 2, Stability Study Day 14°
Fortified Cone. Mean* Recovery Relative Std. Dev.
Analyte (jjg/U (%) (%)
Acifluorfen 2.0 151 18
Bentazon 10.0 97 9
2,4-D 1.0 84 11
2,4-DB 10.0 128 10
Dacthal6 1.0 116 7
Dicamba 3.0 103 9
3,5-Dichlorobenzoic acid 5.0 81 12
Dichlorprop 1.0 107 11
Dinoseb 2.0 118 7
5-Hydroxydicamba 3.0 20 14
Pentachlorophenol 1.0 94 7
Picloram 3.0 110 32
2,4,5-T 1.0 113 8
2,4,5-TP 1.0 113 11
2,4-Dichlorophenylacetic acid" 1.0 87 6
3 Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
0 Samples preserved at pH = 2.0.
d Surrogate analyte.
Table 10. Single Laboratory Recovery and Precision Data For Fortified, High Humic
Content Surface Water
Fortified Cone. Mean" Recovery Relative Std. Dev.
Analyte (tig/U (%) (%)
Acifluorfen 2.0 120 13
Bentazon 10.0 87 11
2,4-D 1.0 59 7
2,4-DB 10.0 80 14
Dacthal" 1.0 100 6
Dicamba 3.0 76 9
3,5-Dichlorobenzoic acid 5.0 87 4
Dichlorprop 1.0 110 22
Dinoseb 2.0 97 6
5-Hydroxydicamba 3.0 82 9
Pentachlorophenol 1.0 70 5
Picloram 3.0 124 9
2,4,5-T 1.0 101 4
2,4,5-TP 1.0 80 6
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
190
-------
Method 515.2
Table 11. Laboratory Performance Check Solution
Test
Sensitivity
Analyte
Dinoseb
Chromatographic performance 4-Nitrophenol
Column performance 3,5-Dichlorobenzoic acid
4-Nitrophenol
Cone,
(ug/mL) Requirements
0.004 Detection of analyte; S/N >
3
1.6 0.70 0.40b
1.6
PGF = peak Gaussian factor. Calculated using the equation:
1.83xW
PGF =
where W | _ is the peak width at half height and W — | is the peak width at tenth height
" Resolution between the two peaks as defined by the equation:
R-J-
w
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
191
-------
Method 515.2
N2 Flow
Flat Joint with O Ring and Clamp
Diethyl Ether Level
Flat Joint with O Ring and Clamp
Diazald Level
KOH Level
52-015-1
Figure 1. Diazomethane Generator
192
-------
Method 515.2
4.0 -
3.0 -
v>
I
o
X
35DCBA
2.0 -
1.0 -
1.5
PCP
Dacthal
2.0
2.5
x101 Minutes
3.0
3.5
Figure 2A. Chromatogram of Group A Analytes Extracted from
Ozonated Surface Water (bottom Chromatogram is
the laboratory reagent blank)
52-015-2
193
-------
Method 515.2
3.0 —
ID
*-
o
> 2.0-
'o
1.0 —
Dicamba
I
SURR
2.0
Bentazon
Silvex
IS
24D
24DB
5HD
2.5
3.0
x101 Minutes
ACIP
3.5
52-015-3
Figure 2B. Chromatogram of Group B Analytes Extracted from
Ozonated Surface Water (bottom Chromatogram is
the laboratory reagent blank)
194
-------
Method 525.1
Determination of Organic Compounds
in Drinking Water
by Liquid-Solid Extraction
and Capillary Column
Gas Chromatography/Mass Spectrometry
Revision 2.2 - EPA EMSL-Ci
May 1991
J.W. Eichelberger, T.D. Behymer, W.L. Budde — Method 525,
Revision 1.0, 2.0, 2.1 (1988)
-------
-------
Method 525.1
Determination of Organic Compounds in Drinking Water
by Liquid-Solid Extraction and Capillary Column
Gas Chromatography/Mass Spectrometry
1. SCOPE AND APPLICA TION
1.1 This is a general purpose method that provides procedures for determination of organic
compounds in finished drinking water, raw source water, or drinking water in any treatment
stage. The method is applicable to a wide range of organic compounds that are efficiently
partitioned from the water sample onto a C,g organic phase chemically bonded to a solid silica
matrix in a cartridge or disk, and sufficiently volatile and thermally stable for gas chromatog-
raphy. Single-laboratory accuracy and precision data have been determined at two concentra-
tions with two instrument systems for the following compounds:
Compound
Acenaphthylene
Alachlor
Aldrin
Anthracene
Atrazine
Benz[a]anthracene
Benzo[6]fluoranthene
Benzo[Ar]fluoranthene
Benzo[a]pyrene
Benzo[<7,/?,/]perylene
Butylbenzyl phthalate
Chlordane Components
a-Chlordane
7-Chlordane
trans-Nonachlor
2-Chlorobiphenyl
Chrysene
Dibenz[a,/7]anthracene
Di-n-butyl phthalate
2,3-dichlorobiphenyl
Diethyl phthatate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexy) phthalate
Dimethyl phthalate
Endrin
Fluorene
Heptachlor
Heptachlor epoxide
2,2'3,3',4,4',6-Heptachlorobiphenyl
Hexachlorobenzene
2,2',4,4',5,6'-Hexachlorobiphenyl
Hexachlorocyclopentadiene
lndeno[1,2,3,c,£/]pyrene
MW
CAS No.
152
269
362
178
215
228
252
252
252
276
312
406
406
440
188
228
278
278
222
222
222
390
194
378
166
370
386
392
282
358
270
276
208-96-8
15972-60-8
309-00-2
120-12-7
1912-24-9
56-55-3
205-99-2
207-08-9
50-32-8
191-24-2
85-68-7
5103-71-9
5103-74-2
39765-80-5
2051-60-7
218-01-9
53-70-3
84-72-2
16605-91-7
84-66-2
103-23-1
117-81-7
131-11-3
72-20-8
86-73-7
76-44-8
1024-57-3
52663-71-5
118-74-1
6-145-22-4
77-47-4
193-39-5
757
-------
Method 525.1
Compound
Lindane
Methoxychlor
2,2',3,3',4,5',6,6'-Octachlorobiphenyl
2,2,3',4,6-Pentachlorobiphenyl
Pentachlorophenol
Phenanthrene
Pyrene
Simazine
2,2',4,4'-Tetrachlorobipheneyl
Toxaphene mixture
2,4,5-Trichlorobiphenyl
MW
288
344
426
324
264
178
202
201
290
256
CAS No.
58-89-9
72-43-5
40186-71-8
60233-25-2
87-86-5
85-01-8
1 29-00-0
122-34-9
2437-79-8
8001-35-2
15862-07-4
a Monoisotopic molecular weight calculated from the atomic masses of the iso-
topes with the smallest masses.
A laboratory may use this method to identify and measure additional analytes after the labora-
tory obtains acceptable (defined in Sect. 10) accuracy and precision data for each added
analyte.
1.2 Method detection limit (MDL) is defined as the statistically calculated minimum amount that
can be measured with 99% confidence that the reported value is greater than zero.1 The MDL
is compound dependent and is particularly dependent on extraction efficiency and sample
matrix. For the listed analytes, MDLs vary from 0.01 to 15 ng/L. The concentration calibra-
tion range of this method is 0.1 jttg/L to 10 ng/L.
2. SUMMARY OF METHOD
2.1 Organic compound analytes, internal standards, and surrogates are extracted from a water
sample by passing 1 liter of sample water through a cartridge or disk containing a solid inor-
ganic matrix coated with a chemically bonded CI8 organic phase (liquid-solid extraction, LSE).
The organic compounds are eluted from the LSE cartridge or disk with a small quantity of
methylene chloride, and concentrated further by evaporation of some of the solvent. The
sample components are separated, identified, and measured by injecting an aliquot of the
concentrated methylene chloride extract into a high resolution fused silica capillary column of
a gas chromatography/mass spectrometry (GC/MS) system. Compounds eluting from the GC
column are identified by comparing their measured mass spectra and retention times to refer-
ence spectra and retention times in a data base. Reference spectra and retention times for
analytes are obtained by the measurement of calibration standards under the same conditions
used for samples. The concentration of each identified component is measured by relating the
MS response of the quantitation ion produced by that compound to the MS response of the
quantitation ion produced by a compound that is used as an internal standard. Surrogate
analytes, whose concentrations are known in every sample, are measured with the same
internal standard calibration procedure.
198
-------
Method 525.1
3. DEFINITIONS
3.1 Internal standard: A pure analyte(s) added to a solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of
the same solution. The internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte: A pure analyte(s), which is extremely unlikely to be found in any sample,
and which is added to a sample aliquot in known amount(s) before extraction and is measured
with the same procedures used to measure other sample components. The purpose of a surro-
gate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.4 Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation, and storage, as well as with laboratory procedures.
3.5 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.7 Laboratory performance check solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the labora-
tory is capable of making accurate and precise measurements at the required method detection
limit.
3.9 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
199
-------
Method 525. 1
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.11 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL solu-
tions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality control sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 During analysis, major contaminant sources are reagents and liquid-solid extraction columns.
Analyses of field and laboratory reagent blanks provide information about the presence of
contaminants.
4.2 Interfering contamination may occur when a sample containing low concentrations of com-
pounds is analyzed immediately after a sample containing relatively high concentrations of
compounds. Syringes and splitless injection port liners must be cleaned carefully or replaced
as needed. After analysis of a sample containing high concentrations of compounds, a labora-
tory reagent blank should be analyzed to ensure that accurate values are obtained for the next
sample.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method has not been precisely de-
fined; each chemical should be treated as a potential health hazard, and exposure to these
chemicals should be minimized. Each laboratory is responsible for maintaining awareness of
OSHA regulations regarding safe handling of chemicals used in this method. Additional
references to laboratory safety are cited.2~*
5.2 Some method analytes have been tentatively classified as known or suspected human or mam-
malian carcinogens. Pure standard materials and stock standard solutions of these compounds
should be handled with suitable protection to skin, eyes, etc.
6. APPARATUS AND EQUIPMENT
6.1 All glassware must be meticulously cleaned. This may be accomplished by washing with
detergent and water, rinsing with water, distilled water, or solvents, air-drying, and heating
(where appropriate) in an oven. Volumetric glassware is never heated.
6.2 Sample containers. 1-liter or 1-quart amber glass bottles fitted with a Teflon-lined screw cap.
(Bottles in which high purity solvents were received can be used as sample containers without
additional cleaning if they have been handled carefully to avoid contamination during use and
after use of original contents.)
200
-------
Method 525.1
6.3 Separatory funnels. 2-liter and 100-mL with a Teflon stopcock.
6.4 Liquid chromatography column reservoirs. Pear-shaped 100- or 125-mL vessels without a
stopcock but with a ground glass outlet joint sized to fit the liquid-solid extraction column.
(Lab Glass, Inc. part no. ML-700-706S, with a 24/40 top outer joint and a 14/35 bottom inner
joint, or equivalent). A 14/35 outlet joint fits some commercial cartridges.
6.5 Syringe needles. No. 18 or 20 stainless steel.
6.6 Vacuum flasks. 1- or 2-liter with solid rubber stoppers.
6.7 Volumetric flasks, various sizes.
6.8 Laboratory or aspirator vacuum system. Sufficient capacity to maintain a slight vacuum of
13 cm (5 in.) of mercury in the vacuum flask.
6.9 Micro syringes, various sizes.
6.10 Vials. Various sizes of amber vials with Teflon-lined screw caps.
6.11 Drying column. Approximately 1.2 cm x 40 cm with 10 mL graduated collection vial.
6.12 Analytical balance. Capable of weighing 0.0001 g accurately.
6.13 Fused silica capillary gas chromatography column. Any capillary column that provides ade-
quate resolution, capacity, accuracy, and precision (Sect. 10) can be used. A 30 m x
0.25 mm id fused silica capillary column coated with a 0.25 /mi bonded film of polyphenyl-
methylsilicone is recommended (J&W DB-5 or equivalent).
6.14 Gas chromatograph/mass spectrometer/data system (GC/MS/DS)
6.14.1 The GC must be capable of temperature programming and be equipped for
splitless/split or on-column capillary injection. The injection tube liner should be
quartz and about 3 mm in diameter. The injection system must not allow the analytes
to contact hot stainless steel or other metal surfaces that promote decomposition.
6.14.2 The GC/MS interface should allow the capillary column or transfer line exit to be
placed within a few mm of the ion source. Other interfaces, for example the open
split interface, are acceptable as long as the system has adequate sensitivity (see
Sect. 9 for calibration requirements).
6.14.3 The mass spectrometer must be capable of electron ionization at a nominal electron
energy of 70 eV. The spectrometer must be capable of scanning from 45 to 450 amu
with a complete scan cycle time (including scan overhead) of 1.5 sec or less. (Scan
cycle time = Total MS data acquisition time in sec divided by number of scans in the
chromatogram). The spectrometer must produce a mass spectrum that meets all
criteria in Table 1 when 5 ng or less of DFTPP is introduced into the GC. An aver-
age spectrum across the DFTPP GC peak may be used to test instrument
performance.
6.14.4 An interfaced data system is required to acquire, store, reduce, and output mass spec-
tral data. The computer software must have the capability of processing stored
GC/MS data by recognizing a GC peak within any given retention time window,
comparing the mass spectra from the GC peak with spectral data in a user-created data
base, and generating a list of tentatively identified compounds with their retention
times and scan numbers. The software must also allow integration of the ion abun-
207
-------
Method 525.1
dance of any specific ion between specified time or scan number limits, calculation of
response factors as defined in Sect. 9.2.6 (or construction of a second or third order
regression calibration curve), calculation of response factor statistics (mean and
standard deviation), and calculation of concentrations of analytes using either the
calibration curve or the equation in Sect. 12.
6.15 Millipore Standard Filter Apparatus, All Glass. This will be used if the disks are to be used to
carry out the extraction instead of the cartridges.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Helium carrier gas, as contaminant free as possible.
7.2 Liquid-solid extraction (LSE) cartridges. Cartridges are inert non- leaching plastic, for exam-
ple polypropylene, or glass, and must not contain plasticizers, such as phthalate esters or
adipates, that leach into methylene chloride. The cartridges are packed with about 1 gram of
silica, or other inert inorganic support, whose surface is modified by chemically bonded
octadecyl (C,8) groups. The packing must have a narrow size distribution and must not leach
organic compounds into methylene chloride. One liter of water should pass through the
cartridge in about 2 hrs with the assistance of a slight vacuum of about 13 cm (5 in.) of
mercury. Sect. 10 provides criteria for acceptable LSE cartridges which are available from
several commercial suppliers.The extraction disks contain approximately 0.5 grams of 8 um
octadecyl bonded silica uniformly enmeshed in a matrix of inert PFTE fibrils. The size of the
disks is 47mm x 0.5mm. As with cartridges, the disks should not contain any organic com-
pounds, either from the PFTE or the bonded silica, which will leach into the methylene chlo-
ride eluant. One liter of reagent water should pass through the disks in 5-20 minutes using a
vacuum of about 66cm (26 in.) of mercury. Section 10 provides criteria for acceptable LSE
disks which are available commercially.
7.3 Solvents
7.3.1 Methylene chloride, acetone, toluene and methanol. High purity pesticide quality or
equivalent.
7.3.2 Reagent water. Water in which an interferent is not observed at the method detection
limit of the compound of interest. Prepare reagent water by passing tap water through
a filter bed containing about 0.5 kg of activated carbon or by using a water purifica-
tion system. Store in clean, narrow-mouth bottles with Teflon-lined septa and screw
caps.
7.4 Hydrochloric acid. 6N.
7.5 Sodium sulfate, anhydrous. (Soxhlet extracted with methylene chloride for a minimum of 4
hrs.)
7.6 Stock standard solutions. Individual solutions of analytes, surrogates, and internal standards
may be purchased as certified solutions or prepared from pure materials. To prepare, add 10
mg (weighed on an analytical balance to 0.1 mg) of the pure material to 1.9 mL of methanol
or acetone in a 2-mL volumetric flask, dilute to the mark, and transfer the solution to an
amber glass vial. If the analytical standard is available only in quantities smaller than 10 mg,
reduce the volume of solvent accordingly. Some polycyclic aromatic hydrocarbons are not
soluble in methanol or acetone, and their stock standard solutions are prepared in toluene.
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Method 525.1
Methylene chloride should be avoided as a solvent for standards because its high vapor pres-
sure leads to rapid evaporation and concentration changes. Methanol and acetone are not as
volatile as methylene chloride, but their solutions must also be handled with care to avoid
evaporation. Compounds 10, 11, and 35 in Table 2 are soluble in acetone. Compounds 12,
13, and 20 in Table 2 are soluble in toluene. If compound purity is certified by the supplier at
>96%, the weighed amount can be used without correction to calculate the concentration of
the solution (5 ftg//*L). Store the amber vials in a dark cool place.
7.7 Primary dilution standard solution. The stock standard solutions are used to prepare a primary
dilution standard solution that contains multiple analytes. The recommended solvent for this
dilution is acetone. Aliquots of each of the stock standard solutions are combined to produce
the primary dilution in which the concentration of the analytes is at least equal to the concen-
tration of the most concentrated calibration solution, that is, 10 ng//*L. Store the primary
dilution standard solution in an amber vial in a dark cool place, and check frequently for signs
of deterioration or evaporation, especially just before preparing calibration solutions.
7.8 Fortification solution of internal standards and surrogates. Prepare a solution of
acenaphthene-D10, phenanthrene-D,0, chrysene-D,2, and perylene-D,2 in methanol or acetone at
a concentration of 500 /*g/mL of each. This solution is used in the preparation of the calibra-
tion solutions. Dilute a portion of this solution by 10 to 50 /xg/mL and use this solution to
fortify the actual water samples (see Sect. 11.2). Other surrogates, for example, caffeine-'5N2
and pyrene-D10 may be included in this solution as needed (a 100-/*L aliquot of this 50 /ig/mL
solution added to 1 liter of water gives a concentration of 5 /xg/L of each internal standard or
surrogate). Store this solution in an amber vial in a dark cool place.
7.9 MS performance check solution. Prepare a 5 ng//xL solution of DFTPP in methylene chlo-
ride. Store this solution in an amber vial in a dark cool place.
7.10 Calibration solutions (CAL1 through CAL6). Prepare a series of six concentration calibration
solutions in acetone which contain all analytes except pentachlorophenol and toxaphene at
concentrations of 10, 5, 2, 1, 0.5, and 0.1 ng//*L, with a constant concentration of 5 ng//iL of
each internal standard and surrogate in each CAL solution. CAL1 through CAL6 are prepared
by combining appropriate aliquots of the primary dilution standard solution (7.7) and the
fortification solution (500 /^g/mL) of internal standards and surrogates (7.8). Pentachlorophe-
nol is included in this solution at a concentration four times the other analytes. Toxaphene
CAL solutions should be prepared as separate solutions at concentrations of 250, 200, 100, 50,
25, and 10 ng//xL. Store these solutions in amber vials in a dark cool place. Check these
solutions regularly for signs of deterioration, for example, the appearance of anthraquinone
from the oxidation of anthracene.
7.11 Reducing agents. Sodium sulfite or sodium arsenite. Sodium thiosulfate is not recommended
as it may produce a residue of elemental sulfur that can interfere with some analytes.
7.12 Fortification solution for optional recovery standard. Prepare a solution of terphenyl-D,4 in
methylene chloride at a concentration of 500 /xg/mL. An aliquot of this solution may be added
(optional) to the extract of the LSE cartridge to check on the recovery of the internal standards
in the extraction process.
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Method 525.1
8. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
8.1 Sample collection. When sampling from a water tap, open the tap and allow the system to
flush until the water temperature has stabilized (usually about 2-5 min). Adjust the flow to
about 500 mL/min and collect samples from the flowing stream. Keep samples sealed from
collection time until analysis. When sampling from an open body of water, fill the sample
container with water from a representative area. Sampling equipment, including automatic
samplers, must be free of plastic tubing, gaskets, and other parts that may leach analytes into
water. Automatic samplers that composite samples over time must use refrigerated glass
sample containers.
8.2 Sample dechlorination and preservation. All samples should be iced or refrigerated at 4°C
from the time of collection until extraction. Residual chlorine should be reduced at the sam-
pling site by addition of a reducing agent. Add 40-50 mg of sodium sulfite or sodium arsenite
(these may be added as solids with stirring until dissolved) to each liter of water. Hydrochlo-
ric acid should be used at the sampling site to retard the microbiological degradation of some
analytes in unchlorinated water. The sample pH is adjusted to <2 with 6 N hydrochloric
acid. This is the same pH used in the extraction, and is required to support the recovery of
pentachlorophenol.
8.3 Holding time. Samples must be extracted within 7 days and the extracts analyzed within 30
days of sample collection.
8.4 Field blanks.
8.4.1 Processing of a field reagent blank (FRB) is recommended along with each sample
set, which is composed of the samples collected from the same general sample site at
approximately the same time. At the laboratory, fill a sample container with reagent
water, seal, and ship to the sampling site along with the empty sample containers.
Return the FRB to the laboratory with filled sample bottles.
8.4.2 When hydrochloric acid is added to samples, use the same procedures to add the same
amount to the FRB.
9. CALIBRATION
9.1 Demonstration and documentation of acceptable initial calibration is required before any
samples are analyzed and is required intermittently throughout sample analysis as dictated by
results of continuing calibration checks. After initial calibration is successful, a continuing
calibration check is required at the beginning of each 8 hr. period during which analyses are
performed. Additional periodic calibration checks are good laboratory practice.
9.2 Initial Calibration
9.2.1 Calibrate the mass and abundance scales of the MS with calibration compounds and
procedures prescribed by the manufacturer with any modifications necessary to meet
the requirements in Sect. 9.2.2.
9.2.2 Inject into the GC a l-/xL aliquot of the 5 ng//iL DFTPP solution and acquire a mass
spectrum that includes data for m/z 45-450. Use GC conditions that produce a nar-
row (at least five scans per peak) symmetrical peak. If the spectrum does not meet all
criteria (Table 1), the MS must be retuned and adjusted to meet all criteria before
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Method 525.1
proceeding with calibration. An average spectrum across the GC peak may be used to
evaluate the performance of the system.
9.2.3 Inject a 1-pL aliquot of a medium concentration calibration solution, for example
0.5-2 /ig/L, and acquire and store data from m/z 45-450 with a total cycle time
(including scan overhead time) of 1.5 sec or less. Cycle time should be adjusted to
measure at least five or more spectra during the elution of each GC peak.
9.2.3.1 Multi-ramp temperature program GC conditions. Adjust the helium carrier
gas flow rate to about 33 cm/sec. Inject at 45°C and hold in splitless
mode for 1 min. Heat rapidly to 130°C. At 3 min start the temperature
program: 130-180°C at 12°/min; 180-240°C at 7°/min; 240-320°C at
12°/min. Start data acquisition at 5 min.
9.2.3.2 Single ramp linear temperature program. Adjust the helium carrier gas
flow rate to about 33 cm/sec. Inject at 40°C and hold in splitless mode
for 1 min. Heat rapidly to 160°C. At 3 min start the temperature pro-
gram: 160-320°C at 6°/min; hold at 320° for 2 min. Start data acqui-
sition at 3 min.
9.2.4 Performance criteria for the medium calibration. Examine the.stored GC/MS data
with the data system software. Figure 1 shows an acceptable total ion chromatogram.
9.2.4.1 GC performance. Anthracene and phenanthrene should be separated by
baseline. Benz[a]anthracene and chrysene should be separated by a valley
whose height is less than 25% of the average peak height of these two
compounds. If the valley between benz[a]anthracene and chrysene exceeds
25%, the GC column requires maintenance. See Sect. 9.3.6.
9.2.4.2 MS sensitivity. The GC/MS/DS peak identification software should be
able to recognize a GC peak in the appropriate retention time window for
each of the compounds in calibration solution, and make correct tentative
identifications. If fewer than 99% of the compounds are recognized,
system maintenance is required. See Sect. 9.3.6.
9.2.4.3 Lack of degradation of endrin. Examine a plot of the abundance of m/z 67
in the region of 1.05-1.3 of the retention time of endrin. This is the re-
gion of elution of endrin aldehyde, a product of the thermal isomerization
of endrin. Confirm that the abundance of m/z 67 at the retention time of
endrin aldehyde is < 10% of the abundance of m/z 67 produced by endrin.
If more than 10% endrin aldehyde is observed, system maintenance is
required to correct the problem. See Sect. 9.3.6.
9.2.5 If all performance criteria are met, inject a 1-/*L aliquot of each of the other CAL
solutions using the same GC/MS conditions.
9.2.6 Calculate a response factor (RF) for each analyte and surrogate for each CAL solution
using the internal standard whose retention time is nearest the retention time of the
analyte or surrogate. Table 2 contains suggested internal standards for each analyte
and surrogate, and quantitation ions for all compounds. This calculation is supported
in acceptable GC/MS data system software (Sect. 6.14.4), and many other software
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Method 525.1
programs. RF is a unitless number, but units used to express quantities of analyte and
internal standard must be equivalent.
where:
A^ = intergrated abundance of the quantitation ion of the analyte.
An = integrated abundance of the quantitation ion internal standard.
(2t = quantity of analyte injected in ng or concentration units.
Qn = quantity of internal injected in ng or concentration units.
9.2.6.1 For each analyte and surrogate, calculate the mean RF from the analyses
of the six CAL solutions. Calculate the standard deviation (SD) and the
relative standard deviation (RSD) from each mean: RSD = 100 (SD/M).
If the RSD of any analyte or surrogate mean RF exceeds 30%, either ana-
lyze additional aliquots of appropriate CAL solutions to obtain an accep-
table RSD of RFs over the entire concentration range, or take action to
improve GC/MS performance. See Sect. 9.2.7.
9.2.7 As an alternative to calculating mean response factors and applying the RSD test, use
the GC/MS data system software or other available software to generate a linear,
second, or third order regression calibration curve.
9.3 Continuing calibration check. Verify the MS tune and initial calibration at the beginning of
each 8 hr. work shift during which analyses are performed using the following procedure.
9.3.1 Inject a l-/nL aliquot of the 5ng//xL DFTPP solution and acquire a mass spectrum that
includes data for m/z 45-450. If the spectrum does not meet all criteria (Table 1), the
MS must be retuned and adjusted to meet all criteria before proceeding with the
continuing calibration check.
9.3.2 Inject a 1-/*L aliquot of a medium concentration calibration solution and analyze with
the same conditions used during the initial calibration.
9.3.3 Demonstrate acceptable performance for the criteria shown in Sect. 9.2.4.
9.3.4 Determine that the absolute areas of the quantitation ions of the internal standards and
surrogate(s) have not decreased by more than 30% from the areas measured in the
most recent continuing calibration check, or by more than 50% from the areas mea-
sured during initial calibration. If these areas have decreased by more than these
amounts, adjustments must be made to restore system sensitivity. These adjustments
may require cleaning of the MS ion source, or other maintenance as indicated in Sect.
9.3.6, and recalibration. Control charts are useful aids in documenting system sen-
sitivity changes.
9.3.5 Calculate the RF for each analyte and surrogate from the data measured in the con-
tinuing calibration check. The RF for each analyte and surrogate must be within 30%
of the mean value measured in the initial calibration. Alternatively, if a second or
third order regression is used, the point from the continuing calibration check for each
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Method 525.1
analyte and surrogate must fall, within the analyst's judgement, on the curve from the
initial calibration. If these conditions do not exist, remedial action must be taken
which may require reinitial calibration.
9.3.6 Some possible remedial actions. Major maintenance such as cleaning an ion source,
cleaning quadrupole rods, etc. require returning to the initial calibration step.
9.3.6.1 Check and adjust GC and/or MS operating conditions; check the MS
resolution, and calibrate the mass scale.
9.3.6.2 Clean or replace the splitless injection liner; silanize a new injection liner.
9.3.6.3 Flush the GC column with solvent according to manufacturer's instruc-
tions.
9.3.6.4 Break off a short portion (about 1 meter) of the column from the end near
the injector; or replace GC column. This action will cause a change in
retention times.
9.3.6.5 Prepare fresh CAL solutions, and repeat the initial calibration step.
9.3.6.6 Clean the MS ion source and rods (if a quadrupole).
9.3.6.7 Replace any components that allow analytes to come into contact with hot
metal surfaces.
9.3.6.8 Replace the MS electron multiplier, or any other faulty components.
10. QUALITY CONTROL
10.1 Quality control (QC) requirements are the initial demonstration of laboratory capability fol-
lowed by regular analyses of laboratory reagent blanks, laboratory fortified blanks, and
laboratory fortified matrix samples. The laboratory must maintain records to document the
quality of the data generated. Additional quality control practices are recommended.
10.2 Initial demonstration of low system background and acceptable particle size and packing.
Before any samples are analyzed, or any time a new supply of cartridges or disks is received
from a supplier, it must be demonstrated that a laboratory reagent blank (LRB) is reasonably
free of contamination that would prevent the determination of any analyte of concern. In this
same experiment, it must be demonstrated that the particle size and packing of the LSE
cartridge or disk are acceptable. Consistent flow rate is an indication of acceptable particle
size distribution and packing.
10.2.1 A major source of potential contamination is the liquid-solid extraction (LSE) car-
tridge which could contain phthalate esters, silicon compounds, and other contami-
nants that could prevent the determination of method analytes.5 Although disks are
made of a teflon matrix, they may still contain phthalate materials. Generally, phtha-
late esters will be leached from the cartridges into methylene chloride and produce a
variable background that is equivalent to < 2 /xg/L in the water sample. If the back-
ground contamination is sufficient to prevent accurate and precise analyses, the con-
dition must be corrected before proceeding with the initial demonstration. Figure 2
shows unacceptable background contamination from a poor quality commercial LSE
207
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Method 525.1
cartridge. The background contamination is the large broad peak, and the small peaks
are method analytes present at a concentration equivalent to 2 /*g/L. Several sources
of LSE cartridges may be evaluated before an acceptable supply is identified.
10.2.2 Other sources of background contamination are solvents, reagents, and glassware.
Background contamination must be reduced to an acceptable level before proceeding
with the next section. In general, background from method analytes should be below
the method detection limit.
10.2.3 One liter of water should pass through a cartridge in about 2 hrs with a partial vacu-
um of about 13 cm (5 in.) of mercury. The flow rate through a disk should be about
5-20 minutes for a liter of drinking water, using full aspirator or pump vacuum. The
extraction time should not vary unreasonably among a set of LSE cartridges or disks.
10.3 Initial demonstration of laboratory accuracy and precision. Analyze four to seven replicates of
a laboratory fortified blank containing each analyte of concern at a concentration in the range
of 2-5 fig/L (see regulations and maximum contaminant levels for guidance on appropriate
concentrations).
10.3.1 Prepare each replicate by adding an appropriate aliquot of the primary dilution stan-
dard solution, or another certified quality control sample, to reagent water. Analyze
each replicate according to the procedures described in Sect. 11 and on a schedule that
results in the analyses of all replicates over a period of several days.
10.3.2 Calculate the measured concentration of each analyte in each replicate, the mean
concentration of each analyte in all replicates, and mean accuracy (as mean percentage
of true value) for each analyte, and the precision (as relative standard deviation, RSD)
of the measurements for each analyte. Calculate the MDL of each analyte using the
procedures described in Sect. 13.1.2.1
10.3.3 For each analyte and surrogate, the mean accuracy, expressed as a percentage of the
true value, should be 70-130% and the RSD should be <30%. Some analytes, par-
ticularly the polycyclic aromatic hydrocarbons with molecular weights > 250, are
measured at concentrations below 2 ftg/L, with a mean accuracy of 35-130% of true
value. The MDLs should be sufficient to detect analytes at the regulatory levels. If
these criteria are not met for an analyte, take remedial action and repeat the measure-
ments for that analyte to demonstrate acceptable performance before samples are
analyzed.
10.3.4 Develop and maintain a system of control charts to plot the precision and accuracy of
analyte and surrogate measurements as a function of time. Charting of surrogate
recoveries is an especially valuable activity since these are present in every sample
and the analytical results will form a significant record of data quality.
10.4 Monitor the integrated areas of the quantitation ions of the internal standards and surrogates in
continuing calibration checks (see Sect. 9.3.4). In laboratory fortified blanks or samples, the
integrated areas of internal standards and surrogates will not be constant because the volume of
the extract will vary (and is difficult to keep constant). But the ratios of the areas should be
reasonably constant in laboratory fortified blanks and samples. The addition of 10 /*L of the
208
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Method 525.1
recovery standard, terphenyl-D,4 (500 jig/mL), to the extract is optional, and may be used to
monitor the recovery of internal standards and surrogates in laboratory fortified blanks and
samples. Internal standard recovery should be in excess of 70%.
10.5 Laboratory reagent blanks. With each batch of samples processed as a group within a work
shift, analyze a laboratory reagent blank to determine the background system contamination.
Any time a new batch of LSE cartridges or disks is received, or new supplies of other reagents
are used, repeat the demonstration of low background described in 10.2.
10.6 With each batch of samples processed as a group within a work shift, analyze a single labora-
tory fortified blank (LFB) containing each analyte of concern at a concentration as determined
in 10.3. If more than 20 samples are included in a batch, analyze a LFB for every 20 sam-
ples. Use the procedures described in 10.3.3 to evaluate the accuracy of the measurements,
and to estimate whether the method detection limits can be obtained. If acceptable accuracy
and method detection limits cannot be achieved, the problem must be located and corrected
before further samples are analyzed. Add these results to the on-going control charts to
document data quality.
10.7 Determine that the sample matrix does not contain materials that adversely affect method
performance. This is accomplished by analyzing replicates of laboratory fortified matrix
samples and ascertaining that the precision, accuracy, and method detection limits of analytes
are in the same range as obtained with laboratory fortified blanks. If a variety of different
sample matrices are analyzed regularly, for example, drinking water from groundwater and
surface water sources, matrix independence should be established for each. A laboratory
fortified sample matrix should be analyzed for every 20 samples processed in the same batch.
10.8 With each set of field samples a field reagent blank (FRB) should be analyzed. The results of
these analyses will help define contamination resulting from field sampling and transportation
activities.
10.9 At least quarterly, replicates of laboratory fortified blanks should be analyzed to determine the
precision of the laboratory measurements. Add these results to the on-going control charts to
document data quality (as in Sect. 10.3).
10.10 At least quarterly, analyze a quality control sample from an external source. If measured
analyte concentrations are not of acceptable accuracy (Sect. 10.3.3), check the entire analytical
procedure to locate and correct the problem source.
10.11 Numerous other quality control measures are incorporated into other parts of this procedure,
and serve to alert the analyst to potential problems.
7 1. PROCEDURE
11.1 Cartridge Extraction
11.1.1 Setup the extraction apparatus shown in Figure 3A. The reservoir is not required, but
recommended for convenient operation. Water drains from the reservoir through the
LSE cartridge and into a syringe needle which is inserted through a rubber stopper
into the suction flask. A slight vacuum of 13 cm (5 in.) of mercury is used during all
209
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Method 525.1
operations with the apparatus. The pressure used is critical as a vacuum > than 13
cm may result in poor precision. About 2 hrs is required to draw a liter of water
through the system.
11.1.2 Pour the water sample into the 2-L separatory funnel with the stopcock closed, add 5
mL methanol, and mix well. Residual chlorine should not be present as a reducing
agent should have been added at the time of sampling. Also the pH of the sample
should be about 2. If residual chlorine is present and/or the pH is >2, the sample
may be invalid. Add a 100-/iL aliquot of the fortification solution (50 /ig/mL) for
internal standards and surrogates, and mix immediately until homogeneous. The
concentration of these compounds in the water should be 5 /zg/L.
11.1.3 Flush each cartridge with two 10 mL aliquots of methylene chloride, followed by two
10 mL aliquots of methanol, letting the cartridge drain dry after each flush. These
solvent flushes may be accomplished by adding the solvents directly to the solvent
reservoir in Figure 3A. Add 10 mL of reagent water to the solvent reservoir, but
before the reagent water level drops below the top edge of the packing in the LSE
cartridge, open the stopcock of the separatory funnel and begin adding sample water
to the solvent reservoir. Close the stopcock when an adequate amount of sample is in
the reservoir.
11.1.4 Periodically open the stopcock and drain a portion of the sample water into the solvent
reservoir. The water sample will drain into the cartridge, and from the exit into the
suction flask. Maintain the packing material in the cartridge immersed in water at all
times. After all of the sample has passed through the LSE cartridge, wash the separa-
tory funnel and cartridge with 10 mL of reagent water, and draw air through the
cartridge for 10 min.
11.1.5 Transfer the 125-mL solvent reservoir and LSE cartridge (from Figure 3A) to the
elution apparatus (Figure 3B). The same 125-mL solvent reservoir is used for both
apparatus. Wash the 2-liter separatory funnel with 5 mL of methylene chloride and
collect the washings. Close the stopcock on the 100-mL separatory funnel of the
elution apparatus, add the washings to the reservoir and enough additional methylene
chloride to bring the volume back up to 5 mL and elute the LSE cartridge. Elute the
LSE cartridge with an additional 5 mL of methylene chloride (10-mL total). A small
amount of nitrogen positive pressure may be used to elute the cartridge. Small
amounts of residual water from the LSE cartridge will form an immiscible layer with
the methylene chloride in the 100-mL separatory funnel. Open the stopcock and
allow the methylene chloride to pass through the drying column packed with an-
hydrous sodium sulfate (1-in) and into the collection vial. Do not allow the water
layer to enter the drying column. Remove the 100 mL separatory funnel and wash
the drying column with 2 mL of methylene chloride. Add this to the extract. Con-
centrate the extract to 1 mL under a gentle stream of nitrogen. If desired, gently
warm the extract in a water bath to evaporate to between 0.5-1.0 mL (without gas
flow). Do not concentrate the extract to less than 0.5 mL (or dryness) as this will
2/0
-------
Method 525.1
result in losses of analytes. If desired, add an aliquot of the recovery standard to the
concentrated extract to check the recovery of the internal standards (see Sect. 10.4).
11.2 Disk Extraction (This may be manual or automatic)
11.2.1 Preparation of Disks
11.2.1.1 Insert the disk into the 47mm filter apparatus as shown in Figure 4. Wash
the disk with 5mL methylene chloride (MeC12) by adding the MeCl2 to the
disk, drawing about half through the disk, allowing it to soak the disk for
about a minute, then drawing the remaining MeC12 through the disk.
11.2.1.2 Pre-wet the disk with 5 mL methanol (MeOH) by adding the MeOH to the
disk and allowing it to soak for about a minute, then drawing most of the
remaining MeOH through. A layer of MeOH must be left on the surface
of the disk, which should not be allowed to go dry from this point until the
end of the sample extraction. THIS IS A CRITICAL STEP FOR A UNI-
FORM FLOW AND GOOD RECOVERY.
11.2.1.3 Rinse the disk with 5 mL reagent water by adding the water to the disk
and drawing most through, again leaving a layer on the surface of the disk.
11.2.2 Add 5 mL MeOH per liter of water sample. Mix well.
11.2.3 Add the water sample to the reservoir and turn on the vacuum to begin the extraction.
Full aspirator vacuum may be used. Particulate-free water may pass through the disk
in as little as ten minutes or less. Extract the entire sample, draining as much water
from the sample container as possible.
11.2.4 Remove the filtration top from the vacuum flask, but do not disassemble the reservoir
and fritted base. Empty the water from the flask, and insert a suitable sample tube to
contain the eluant. The only constraint on the sample tube is that it fit around the
drip tip of the fritted base. Reassemble the apparatus.
11.2.5 Add 5 mL methylene chloride to the sample bottle, and rinse the inside walls thor-
oughly. Allow the methylene chloride to settle to the bottom of the bottle, and trans-
fer to the disk with a pipet or syringe, rinsing the sides of the glass filtration reservoir
in the process. Draw about half of the methylene chloride through the disk, release
the vacuum, and allow the disk to soak for a minute. Draw the remaining methylene
chloride through the disk.
11.2.6 Repeat the above step twice. Pour the combined eluates through a small funnel with
filter paper containing three grams of anhydrous sulfate. Rinse the test tube and
sodium sulfate with two 5 mL portions of methylene chloride. Collect all the extract
and washings in a concentrator tube.
11.2.7 Concentrate the extract to 1 mL under a gentle stream of nitrogen. If desired, gently
warm the extract in a water bath or heating block to concentrate to between 0.5 and 1
mL. Do not concentrate the extract to less than 0.5 mL, since this will result in
losses of analytes.
11.3 Analyze a 1-2 juL aliquot with the GC/MS system under the same conditions used for the
initial and continuing calibrations (Sect. 9.2.3).
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Method 525.1
11.4 At the conclusion of data acquisition, use the same software that was used in the calibration
procedure to tentatively identify peaks in retention time windows of interest. Use the data
system software to examine the ion abundances of components of the chromatogram. If any
ion abundance exceeds the system working range, dilute the sample aliquot and analyze the
diluted aliquot.
11.5 Identification of analytes. Identify a sample component by comparison of its mass spectrum
(after background subtraction) to a reference spectrum in the user-created data base. The GC
retention time of the sample component should be within 10 sec of the time observed for that
same compound when a calibration solution was analyzed.
11.5.1 In general, all ions that are present above 10% relative abundance in the mass spec-
trum of the standard should be present in the mass spectrum of the sample component
and should agree within absolute 20%. For example, if an ion has a relative abun-
dance of 30% in the standard spectrum, its abundance in the sample spectrum should
be in the range of 10 to 50%. Some ions, particularly the molecular ion, are of
special importance, and should be evaluated even if they are below 10% relative abun-
dance.
11.5.2 Identification is hampered when sample components are not resolved chromatograph-
ically and produce mass spectra containing ions contributed by more than one analyte.
When GC peaks obviously represent more than one sample component (i.e., broad-
ened peak with shoulder(s) or valley between two or more maxima), appropriate
analyte spectra and background spectra can be selected by examining plots of charac-
teristic ions for tentatively identified components. When analytes coelute (i.e., only
one GC peak is apparent), the identification criteria can be met but each analyte
spectrum will contain extraneous ions contributed by the coeluting compound.
11.5.3 Structural isomers that produce very similar mass spectra can be explicitly identified
only if they have sufficiently different GC retention times. See Sect. 9.2.4.1. Accep-
table resolution is achieved if the height of the valley between two isomer peaks is
less than 25% of the average height of the two peak heights. Otherwise, structural
isomers are identified as isomeric pairs. Benzofb] and benzo[k]fluoranthene are
measured as an isomeric pair.
11.5.4 Phthalate esters and other background components appear in variable quantities in
laboratory and field reagent blanks, and generally cannot be accurately measured at
levels below about 2 /xg/L. Subtraction of the concentration in the blank from the
concentration in the sample at or below the 2 ^ig/L level is not recommended because
the concentration of the background in the blank is highly variable.
12. CALCULATIONS
12.1 Complete chromatographic resolution is not necessary for accurate and precise measurements
of analyte concentrations if unique ions with adequate intensities are available for quantitation.
For example, although two listed analytes, dibenz[a,h]anthracene and indeno[l,2,3,c,d]pyrene,
were not resolved with the GC conditions used, and produced mass spectra containing common
ions, concentrations (Tables 3-6) were calculated by measuring appropriate characteristic ions.
12.1.1 Calculate analyte and surrogate concentrations.
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Method 525.1
c =
(AJRF V
where:
Cx = concentration of analyte or surrogate in ng/L in the water sample.
Ax = integrated abundance of the quantitation ion of the analyte in the sample.
Ais = integrated abundance of the quantitation ion of the analyte in the sample.
Qis = total quantity (in micrograms) of internal standard added to the water sample.
V = original water sample volume in liters.
RF = mean response factor of analyte from the initial calibration.
12.1 .2 Alternatively, use the GC/MS system software or other available proven software to
compute the concentrations of the analytes and surrogates from the second or third
order regression curves.
1 2.1 .3 Calculations should utilize all available digits of precision, but final reported concen-
trations should be rounded to an appropriate number of significant figures (one digit
of uncertainity). Experience indicates that three significant figures may be used for
concentrations above 99 /tg/L, two significant figures for concentrations between 1-99
and one significant figure for lower concentrations.
13. METHOD PERFORMANCE
13.1 Single laboratory accuracy and precision data (Tables 3-7) for each listed analyte was obtained
at two concentrations with the same extracts analyzed on more than two different instrument
systems. Seven 1-liter aliquots of reagent water containing 2 /xg/L of each analyte, and five to
seven 1-liter aliquots of reagent water containing 0.2 /xg/L of each analyte were analyzed with
this procedure. Tables 8-10 list data gathered using C-18 disks. These data were results from
different extracts generated by a volunteer laboratory, Environmental Health Laboratories.
13.1.2 With these data, method detection limits (MDL) were calculated using the formula:
MDL = $Vi, .-0 = 0.99,
where:
l
-------
Method 525.1
13.2.2 Some polycyclic aromatic hydrocarbons are rapidly oxidized and/or chlorinated in
water containing residual chlorine. Therefore residual chlorine must be reduced
before analysis.
13.2.3 In water free of residual chlorine, some polycyclic aromatic hydrocarbons (for exam-
ple, compounds 9, 12, 13, 20, and 35) are not accurately measured because of low
recoveries in the extraction process.
13.2.4 Pentachlorophenol No. 40 and hexachlorocyclopentadiene No. 34 may not be accu-
rately measured. Pentachlorophenol is a strong acid and elutes as a broad weak peak.
Hexachlorocyclopentadiene is susceptible to photochemical and thermal decom-
position.
214
-------
Method 525.1
References
1. Glaser, J. A., D. L. Foerst, G. D. McKee, S. A. Quave, and W. L. Budde, "Trace Analyses
for Wastewaters," Environ. Sci. Technol. 198115, 1426-1435.
2. "Carcinogens - Working With Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, Aug. 1977.
3. "OSHA Safety and Health Standards, General Industry," (29CFK1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. Junk, G.A., M. J. Avery, J. J. Richard, "Interferences in Solid-Phase Extraction Using C-18
Bonded Porous Silica Cartridges," Anal. Chem. 1988, 60, 1347.
215
-------
Method 525.1
Table 1. Ion Abundance Criteria for Bis(perfluorophenyl)phenyl Phosphine (Deca-
fluorotriphenylphosphine, DFTPP)
Mass
(M/z) Relative Abundance Criteria
51 10-80% of the base peak
68 <2% of mass 69
70 < 2% of mass 69
127 10-80% of the base peak
197 <2% of mass 198
198 base peak or >50% of 442
199 5-9% of mass 198
275 10-60% of the base peak
365 > 1 % of the base peak
441 Present and < mass 443
442 base peak or >50% of 198
443 15-24% of mass 442
Purpose of Checkpoint1
Low mass sensitivity
Low mass resolution
Low mass resolution
Low-mid mass sensitivity
Mid-mass resolution
Mid-mass resolution and sensitivity
Mid-mass resolution and isotope ratio
Mid-high mass sensitivity
Baseline threshold
High mass resolution
High mass resolution and sensitivity
High mass resolution and isotope ratio
All ions are used primarily to check the mass measuring accuracy of the mass spectrometer
and data system, and this is the most important part of the performance test. The three
resolution checks, which include natural abundance isotope ratios, constitute the next most
important part of the performance test. The correct setting of the baseline threshold, as
indicated by the presence of low intensity ions, is the next most important part of the perfor-
mance test. Finally, the ion abundance ranges are designed to encourage some standardization
to fragmentation patterns.
276
-------
Method 525.1
Table 2. Retention Time Data, Quantitation Ions, and Internal Standard References
for Method Analytes
Retention
Compound
Internal standard
Acenapththene-D10
Phenanthrene-D10
Chrysene-D12
Surrogate
Perylene-D12
Target analytes
Acenphthylene
Aldrin
Anthracene
Atrazine
Benz[a]anthracene
Benzo[6]fluoranthene
Benzo[/r]fluoranthene
Benzofajpyrene
Benzo[5r,/7,/]perylene
Buthylbenzyl phthalate
Chlordane Components
a-Chlordane
•y-Chlordane
trans-Nonachlor
2-Chlorobiphenyl
Chrysene
Dibenz[a,/;]anthracene
Di-/?-butyl phthalate
2,3-Dichlorobiphenyl
Diethyl phthalate
Di(2-ethylhexyl) phthalate
Di(2-ethylhexyl) adipate
Dimethyl phthalate
Endrin
Fluorene
Heptaclor
Heptachlor epoxide
2,2',3,3',4,4',6-Hepta-
chlorobiphenyl
Hexachlorobenzene
2,2',4,4',5,6'-Hexa-
chlorobiphenyl
Hexachloro-
cyclopentadiene
lndeno[1,2,3,c,cflpyrene
Compound
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
lime (i
A'
4:49
8:26
18:14
23:37
4:37
11:21
8:44
7:56
18:06
22:23
22:28
23:28
27:56
16:40
13:44
13:16
13:54
4:56
18:24
27:15
10:58
7:20
5:52
19:19
17:17
4:26
15:52
6:00
10:20
12:33
18:25
7:37
14:34
3:36
27:09
mn:sec/
I *
7:45
11:08
19:20
22:55
7:25
13:36
11:20
10:42
19:14
22:07
22:07
22:47
26:44
18:09
15:42
15:18
15:50
7:55
19:23
25:57
13:20
10:12
8:50
20:01
18:33
7:21
16:53
8:53
12:45
14:40
19:25
10:20
16:30
6:15
25:50
Quantitatk
Ion (m/z)
164
188
240
264
152
66
178
200/215
228
252
252
252
276
149
375
375
409
188
228
278
149
222
149
149
129
163
81
166
100/160
81/353
394/396
284/286
360
237
276
Internal
Standard
Reference
1
2
2
1/2
3
3
3
3
3
2/3
2/3
2/3
2/3
1
3
3
2
1
1
2/3
2/3
1
2/3
1
2
2
3
1/2
1
3
277
-------
Method 525.1
Table 2. Retention Time Data, Quantitation Ions, and Internal Standard References
for Method Analytes (cont.)
Compound
Lindane
Methoxychlor
2,2',3,3',4,5',6,6'-
Octachlorobiphenyl
2,2',3',4,6-Penta-
chlorobiphenyl
Pentachlorophenol
Phenanthrene
Pyrene
Simazine
2,2',4,4'-Tetrachloro-
biphenyl
Toxaphene
2,4,5-Trichlorobiphenyl
Alachlor
Compound_
Number
36
37
38
39
40
41
42
43
Retention
Time (min:sec)
A'
8:17
18:34
18:38
12:50
8:11
8:35
13:30
7:47
44 11:01
45 11:30-23:30
46 9:23
47
10:57
19:30
19:33
15:00
10:51
11:13
15:29
10:35
13:25
13:00-21:30
11:59
13:19
Internal
.Quantitation Standard
Ion (m/zj Reference
181/183 1/2
227 3
430
326
266
178
202
201
292
159
256
160
2
2
2
2/3
1/2
2
2
2
2
Single ramp linear temperature program conditions (Sect. 9.2.3.2).
Multi-ramp linear temperature program conditions (Sect. 9.2.3.1).
218
-------
Method 525.1
Table 3. Accuracy and Precision Data from Seven Determinations of the Method
Analytes at 2 //g/L With Liquid-Solid Extraction and the Ion Trap Mass
Spectrometer
Mean
Compound
Number
(Table 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean"
True Cone.
(ug/L)
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
25
2
2
Mean
Observed
Cone.
(U9/U
5.0
1.9
1.6
1.7
2.2
1.8
not separated
4.2
0.8
0.7
2.0
2.0
2.2
2.7
1.9
2.2
0.3
2.2
2.3
2.0
1.9
1.6
1.9
1.8
2.2
2.2
2.3
1.4
1.7
1.6
1.1
0.4
2.1
1.8
1.8
1.9
8.2
2.4
1.9
2.1
1.5
28.
1.7
1.8
Method
Pel. Std. Accuracy
Std. Dev.
(ug/L)
0.3
0.2
0.2
0.1
0.3
0.2
from No. 1 1;
0.3
0.2
0.1
0.3
0.2
0.3
1.0
0.1
0.1
0.3
0.3
0.1
0.3
0.2
0.3
0.2
0.1
0.2
0.3
0.2
0.2
0.2
0.4
0.1
0.2
0.2
0.2
0.2
0.1
1.2
0.1
0.1
0.2
0.1
4.7
0.1
0.2
Dev.
(%)
6.0
11.
13.
5.9
14.
11.
measured
7.1
25.
14.
15.
10.
14.
37.
5.2
4.5
100.
14.
4.3
15.
11.
19.
11.
5.5
9.1
14.
8.7
14.
12.
25.
9.1
50.
9.5
11.
11.
5.3
15.
4.2
5.3
9.5
6.7
17.
5.9
15.
(% of True
Cone.)
100
95
80
85
110
90
with No. 1 1
105
40
35
100
100
110
135
95
110
15
110
115
100
95
80
95
90
110
110
115
70
85
80
55
20
105
90
90
95
102
120
95
105
75
112
85
91
Method
Detection
Limit (MDL)
(ug/L)
_ a
_ a
_ a
_ a
_ a
a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
__ a
_ a
_ a
_ a
_ "
_ a
_ a
_ a
__ a
_ a
_ a
__ a
_ a
__ a
_ a
_ a
_^ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
15.
_ a
6.6
8 See Table 4.
" Compounds 4, 40, and 45 excluded from the means.
219
-------
Method 525.1
Table 4. Accuracy and Precision Data from Five to Seven Determinations of the
Method Anaytes at 0.2 //g/L with Liquid-Solid Extraction and the Ion Trap Mass
Spectrometer
Compound
Number
(Table 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean3
True Cone.
(ug/L)
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Mean
Observed
Cone. (u/L)
0.45
0.13
0.13
0.13
0.24
0.14
0.25
0.03
0.03
0.32
0.17
0.19
0.17
0.19
0.21
0.03
0.48
0.20
0.45
0.39
0.31
0.21
0.12
0.21
0.22
0.19
0.19
0.16
0.19
0.04
0.04
0.22
0.11
0.19
0.13
0.78
0.20
0.18
0.25
0.14
Std. Dev
(ug/L)
0.6
0.03
0.03
0.01
0.03
0.01
not separated
0.04
0.01
0.02
0.07
0.04
0.03
0.08
0.03
0.01
0.02
0.09
0.03
0.21
0.16
0.16
0.01
0.12
0.05
0.01
0.04
0.03
0.04
0.03
0.01
0.03
0.02
0.01
0.05
0.02
0.08
0.004
0.005
0.04
0.04
not measured at this
0.2
0.2
0.13
0.18
0.02
0.04
RelStd.
Dev.
(%)
13.
23.
23.
7.7
13.
7.1
from No. 1
16.
33.
67.
22.
24.
16.
47.
16.
4.8
67.
19.
15.
47.
41.
52.
4.8
100.
24.
4.5
21.
16.
25.
16.
25.
75.
9.1
9.1
26.
15.
10.
2.0
2.8
16.
29.
level
15.
25.
Mean Method
Accuracy
(% of True Cone.)
90
65
65
65
120
70
Method
Detection Limit
(MDU (ug/L)
0.1
0.1
0.1
0.04
0.1
0.04
1; measured with No. 1 1
62
15
15
160
85
95
85
95
105
150
240
100
225
195
155
105
60
105
110
95
95
80
95
20
20
110
55
95
65
97
100
90
125
70
65
95
0.2
0.04
0.1
0.3
0.2
0.1
0.3
0.1
0.04
0.1
0.3
0.1
0.8
0.6
0.6
0.04
0.5
0.2
0.04
0.2
0.1
0.1
0.1
0.03
0.1
0.1
0.04
0.2
0.1
0.3
0.01
0.02
0.02
0.1
0.06
0.16
"Compound 4, 40, and 45 excluded from the means.
220
-------
Method 525.1
Table 5. Accuracy and Precision data From Five to Seven Determinations of the
Method Anaytes at 0.2 /yg/L with Liquid-Solid Extraction and a Magnetic Sector
Mass Spectrometer
Compound
Number
(Table 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean3
True Cone.
(fjg/U
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
25
2
2
Afea/i
Observed
Cone. (u/L)
5.7
1.9
1.6
2.2
2.4
2.2
Pel. Std. Mean Method
Std. Dev
(ug/L)
0.34
0.22
0.18
0.67
0.46
0.87
not separated from No.
4.0
0.85
0.69
2.0
2.2
2.1
1.9
2.0
2.1
0.75
2.5
2.0
3.5
2.0
1.4
2.9
1.7
2.6
1.2
2.6
1.5
1.5
1.9
0.89
0.83
2.2
2.0
1.5
1.6
12.
2.3
2.0
2.5
1.6
28.
1.9
1.8
0.37
0.15
0.12
0.20
0.41
0.38
0.10
0.29
0.32
0.18
0.32
0.23
1.8
0.28
0.16
0.70
0.45
1.0
0.10
0.42
0.19
0.35
0.17
0.11
0.072
0.10
0.88
0.11
0.14
2.6
0.18
0.26
0.34
0.17
2.7
0.073
0.32
Dev.
<%) (%
6.0
12.
11.
30.
19.
40
1 1 ; measured
9.3
18.
17.
10.
19.
18.
5.2
14.
15.
24.
13.
12.
51.
14.
11.
24.
26.
38.
8.3
16.
13.
23.
8.9
12.
8.7
4.5
44.
7.3
8.8
22.
7.8
13.
14.
11.
10.
3.8
16.
Accuracy
of True Cone.)
114
95
80
110
120
110
with No. 1 1
100
43
35
100
110
105
95
100
105
38
125
100
175
100
70
145
85
130
60
130
75
75
95
45
42
110
100
75
80
150
115
100
125
80
112
95
88
Method
Detection Limit
(MOD (ug/U
a
_ a
_ a
_ a
_ a
a
_ a
_ a
_ a
_ a
a
a
_ a
a
a
a
_ a
_ a
_ a
_ a
_ a
_ a
a
a
a
a
_ a
_ a
a
_ a
a
a
a
_ a
_ a
_ a
_ a
_ a
a
a
9.
a
1.
3 See Table 4.
Compound 4, 40, and 45 exluded from the means.
221
-------
Method 525.7
Table 6. Accuracy and Precision Data from Six or Seven Determinations of the
Method Anaytes at 0.2 jjg/L with Liquid-Solid Extraction and a Magnetic Sector
Spectrometer
Compound
Number
(Tab/e 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean8
True
Cone.
(ug/L)
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.8
0.2
0.2
0.2
0.2
Mean
Observed
Conc.(p/L)
0.67
0.11
0.11
0.14
0.26
0.24
Pel. Std. Mean Method Method Detection
Std.Dev
(ug/L)
0.07
0.03
0.02
0.02
0.08
0.06
not separated from
0.40
0.08
0.07
0.33
0.19
0.17
0.19
0.17
0.27
0.09
1.1
0.18
0.29
0.42
0.32
0.20
0.53
0.18
0.11
0.33
0.17
0.11
0.17
0.05
0.08
0.27
0.24
0.15
0.13
1.8
0.21
0.19
0.27
0.13
0.10
0.02
0.01
0.16
0.02
0.08
0.04
0.02
0.08
0.01
1.2
0.05
0.17
0.23
0.16
0.09
0.30
0.03
0.05
0.08
0.01
0.04
0.03
0.02
0.06
0.03
0.09
0.02
0.02
0.82
0.07
0.04
0.07
0.03
Dev.
(%) (%
9.4
24.
21.
17.
31.
26.
No. 1 1 ; measured
25.
27.
22.
48.
13.
45.
18.
13.
28.
15.
109.
30.
59.
55.
50.
47.
57.
15.
42.
26.
7.1
40.
15.
35.
8.1
11.
39.
12.
13.
46.
33.
23.
27.
22.
Accuracy
of True Cone.)
134
55
56
70
130
120
with No. 1 1
100
38
33
160
95
85
95
85
135
46
550
90
145
210
160
100
265
90
55
165
85
55
85
24
40
135
120
75
65
225
105
95
135
65
Limit (MDL)
(ug/L)
0.2
0.1
0.1
0.1
0.3
0.2
0.3
0.1
0.1
0.5
0.1
0.3
0.1
0.1
0.3
0.1
4.
0.2
0.6
0.8
0.5
0.3
1.
0.1
0.2
0.3
0.04
0.2
0.1
0.1
0.02
0.1
0.3
0.1
0.1
3.
0.2
0.1
0.2
0.1
not measured at this point
0.2
0.2
0.16
0.21
0.04
0.09
23.
28.
80
102
0.12
0.3
Compound 4, 40, and 45 excluded from the means.
222
-------
Method 525.1
Table 7. Accuracy and Precision Data from Seven Determinations at 2 jug/L with
Liquid-Solid Extraction and a Quadrupole Mass Spectrometer
Compound True Mean Rel. Std. Mean Method Method Detection
Number Cone. Observed Std. Dev Dev. Accuracy Limit (MDL)
(Table 2) (ug/U Cone. (u/LJ (ug/L) (%) (% of True Cone.) (ug/L)
47 2 2.4 0.4 16. 122 1.0
223
-------
Method 525.1
Table 8. Accuracy and Precision Data from Seven Replicates at 0.2 /vg/L with
Liquid-solid C-1
Compound
Number
(Table 2)
1
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
8 Disk Extraction and an ion Trap Mass Spectrometer
Target
Concentration
(ug/L)
0.2
5.0
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
2.0
0.2
0.2
0.2
0.2
20.0
0.2
0.2
Standard
Deviation
(ug/L)
0.01
0.37
0.03
0.03
0.04
0.07
0.16
0.03
0.04
0.03
0.07
0.12
0.06
0.18
0.01
0.02
0.05
0.08
0.02
0.02
0.50
0.04
0.02
0.05
0.01
0.05
0.08
0.08
0.04
0.06
0.01
0.05
0.01
0.03
0.04
0.03
0.02
0.02
0.04
0.06
2.47
0.04
0.03
Relative
Deviation
<%)
5.3
7.4
13.2
13.7
22.4
33.2
77.6
13.7
21.7
14.9
32.5
61.1
31.9
91.3
7.2
10.9
22.9
40.3
9.7
11.9
252.0
20.8
7.6
25.4
7.3
22.9
38.9
38.0
17.7
31.9
5.2
27.0
6.5
13.4
21.1
15.1
12.2
10.2
18.8
28.1
12.3
21.4
14.7
Mean
(ng/U
0.22
5.55
0.26
0.22
0.21
0.29
0.40
0.21
0.26
0.23
0.37
0.19
0.19
0.55
0.16
0.27
0.18
0.47
0.17
0.27
1.54
0.36
0.23
0.23
0.20
0.28
0.36
0.28
0.22
0.19
0.34
0.29
0.22
0.20
0.20
0.17
0.20
0.24
0.19
0.21
24.80
0.19
0.11
Accuracy
(% of target)
110
111
130
108
105
147
199
107
128
115
183
95
93
276
78
136
90
233
87
133
771
180
117
117
101
139
181
141
109
96
170
143
110
100
99
84
102
121
94
107
123
95
55
224
-------
Method 525.1
Table 9. Accuracy and Precision Data from
Seven Replicates at 2.
0 //g/L with
Liquid-Solid C-18 Disk Extraction and an ion Trap Mass Spectrometer
Compound
Number
(Table 2)
1
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Target
Concentration
(ug/L)
2
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
20
2
2
2
2
100
2
2
Standard
Deviation
(ug/U
0.18
0.45
0.30
0.17
0.47
0.21
0.62
0.57
0.31
0.28
0.33
0.62
1.02
1.39
0.22
0.23
0.27
0.23
0.38
0.22
0.38
0.26
0.69
0.12
0.19
0.30
0.15
0.64
0.85
0.52
0.22
0.37
0.42
0.34
0.77
0.29
15.16
0.20
0.17
0.27
0.15
3.36
0.58
0.07
Relative
Deviation
(%)
9.2
9.1
14.8
8.6
23.5
10.4
30.9
28.7
15.6
13.9
16.7
31.1
51.2
69.3
11.2
11.6
13.4
11.3
18.9
11.1
19.1
12.8
34.6
6.1
9.7
15.0
7.4
32.2
42.3
25.9
11.0
18.3
21.2
16.8
38.5
14.7
75.8
9.9
8.3
13.3
7.4
3.4
28.8
3.5
Mean
(ug/L)
2.00
5.22
2.14
2.25
2.78
2.21
2.84
2.30
2.61
2.28
2.92
1.21
1.92
3.29
2.52
1.99
2.25
2.45
2.35
2.23
3.25
2.49
1.80
1.97
2.15
2.10
2.41
2.46
1.96
2.05
1.42
2.31
2.69
2.34
0.97
2.11
19.51
2.20
2.34
2.37
2.11
98.33
1.65
1.55
Accuracy
(% of target)
100
104
107
112
139
111
142
115
130
114
146
61
96
164
126
100
113
123
117
111
163
124
90
98
108
105
121
123
98
102
71
115
134
117
49
106
98
110
117
119
106
98
82
77
225
-------
Method 525.1
Table 10. Minimum Detection Limits From Seven Replicates Using Liquid-Solid
Extraction C-18 Disks and an Ion Trap Mass Spectrometer
Chemical Name Minimum Detection Limits
Acenaphytiene 0.033
Alachlor 0.092
Aldrin 0.083
Anthracene 0.086
Atrazine 0.140
Benz[a]anthracene 0.224
Benzo|6]fluoranthene 0.488
Benzo[*]fluoranthene 0.086
Benzo[a]pyrene 0.137
BenzolgA/lperylene 0.094
Butylbenzyl phthalate 0.204
Chlordane-a 0.384
Chlordane-7 0.200
Chlordane (trans-Nonachlor) 0.574
Chrysene 0.068
Dibenz[a,/?]anthracene 0.144
Di-n-butyl phthalate 0.253
Diethyl phthalate 0.075
Di(2-ethylhexyl) phthalate 1.584
Di(2-ethlyhexyl) adipate 0.131
Dimethyl phthalate 0.048
Endrin 0.160
Fluorene 0.046
Heptachlor 0.144
Heptachlorepoxide 0.244
Hexachlorobenzene 0.111
Hexachlorocyclopentadiene 0.039
lndeno[1,2,3,c,rf]pyrene 0.170
Lindane 0.041
Methoxychlor 0.084
PCB-mono-CI-isomer 0.045
PCB-di-CI-isomer 0.061
PCB-tri-CI-isomer 0.135
PCB-tetra-CI-isomer 0.177
PCB-penta-CI-isomer 0.200
PCB-hepta-CI-isomer 0.239
PCB-octa-CI-isomer 0.133
Pentachlorophenol 47.648
Phenanthrene 0.076
Pyrene 0.064
Simazine 0.118
Toxaphene 7.763
226
-------
Method 525.1
TIC
100-
80-
60-
40-
20-
34 26 J
i U
44
A°
8925696
33
27
A
Scan
R.T.
100
4:55
300
9:46
400
12:12
500
14:38
TIC
100-
80-
60-
40-
20-
25
38
24
600
17:04
700
19:29
10 11
900
24:21
2513216
13
35 20 A
-uv^A-J^
1000
26:47
52-015-11
Figure 1. Total Ion Chromatogram of Two Nanograms of Analytes
And Five Nanograms of Surrogates and Internal Standard
227
-------
Method 525.1
ioo%H
TOT-
600
10:01
900
15:01
1200
20:01
52-015-13
Figure 2, Total Ion Chromatogram from a Laboratory Blank
With an Unacceptably High Background
228
-------
Method 525.1
HD
2 Liter
Separatory Funnel
HO
HD
125 ml.
Solvent
Reservoir
Ground Glass
Stopper 14/35
LSE Cartridge
Rubber Stopper
No. 18-20 Luer-lok
Syringe Needle
HD
125ml_
Solvent
Reservoir
Ground Glass
Stopper 14/35
LSE Cartridge
100mL
Separatory
Funnel
Drying Column
(Na2SO4)
1.2 cm x 40 cm
10 mL
Graduated
Vial
52-015-14
A. Extraction Apparatus
B. Elution Apparatus
Figure 3.
229
-------
Method §25.1
Source
Vacuum
1 Liter
Suction Flask
Pinch Clamp
52-015-15
Figure 4. Disk Extraction Apparatus
230
-------
Method 525.1
Appendix
Detection Limits for Precision and Accuracy for the Analysis
of Pesticide Compounds by EPA Method 525.1
Table 7. Method 525.1 Detection Limits —507 & 508 Compounds
Compound
Alachlor
Aldrin
Ametryn
Atraton
Atrazine
BHC, a
BHC, 0
BHC, 6
BHC, y
Bromacil
Butachlor
Butylate
Carboxin
Chlordane, a
Chlordane, 7
Chloroneb
Chlorobenzilate
Chlorothalonil
Chlorpropham
Cycloate
DCPA
ODD, 4,4'-
DDE, 4,4'-
DDT, 4,4-
Diazinon
Dichlorvos
Dieldrin
Diphenamid
Disulfoton
Disulfoton sulfone
Disulfoton sulfoxide
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
EPIC
Ethoprop
Etridiazole
Fenamiphos
Fenarimol
Fluridone
Target
(pg/U
0.2
0.2
0.1
2.0
0.2
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.2
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
2.0
0.1
0.1
0.1
2.0
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
2.0
#
Reps
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
%
Rec
73
33
68
51
78
143
76
78
73
117
98
78
65
61
61
62
315
77
84
67
78
73
143
74
98
75
86
87
75
86
131
72
101
79
85
92
56
152
61
140
136
105
SD
7
10
14
8
7
31
9
9
12
13
8
23
9
11
11
8
25
9
9
14
23
14
16
17
14
28
4
7
16
8
11
10
30
13
16
11
20
2
24
12
9
19
MS
MDL
0.044
0.060
0.043
0.533
0.041
0.970
0.027
0.029
0.072
0.041
0.025
0.071
0.027
0.069
0.066
0.026
0.077
0.028
0.028
0.043
0.071
0.045
0.051
0.052
0.043
0.089
0.265
0.022
0.050
0.025
0.723
0.031
0.096
0.042
0.102
0.036
0.061
0.007
0.076
0.037
0.028
1.166
EC-NPD
MDL
0.380
0.075
2.000
0.600
0.130
0.025
0.010
0.010
0.015
2.500
0.380
0.150
0.600
0.002
0.002
0.500
5.000
0.025
0.500
0.025
0.025
0.003
0.010
0.060
0.250
2.500
0.020
0.600
0.300
3.800
0.380
0.015
0.024
0.015
0.015
0.025
0.250
0.250
0.190
1.000
0.380
3.800
231
-------
Method 525.1
Table 7. Method 525.1 Detection Limits— 507 & 508 Compounds (cont.)
Compound
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexazinone
Merphos
Methoxychlor
Methyl paraoxon
Metolachlor
Metribuzin
Mevinphos
MKG-264
Molinate
Napropamide
Norflurazon
Pebulate
Permethrin, cis-
Permethrin, trans-
Prometon
Prometryn
Pronamide
Propachlor
Propazine
Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
Triademefon
Tricyclazole
Trifluralin
Vernolate
Target
(V9/U
0.2
0.2
0.2
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.05
0.15
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
2.0
0.2
0.1
#
Reps
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
Avg
%
Rec
54
71
80
124
119
15
122
81
49
98
64
64
89
108
72
76
80
78
98
73
92
77
81
82
101
114
104
162
83
208
150
68
54
91
SD
12
10
16
12
15
3
11
5
14
14
4
13
7
7
19
20
20
58
16
7
11
5
8
34
8
14
6
19
12
84
77
13
14
15
MS
MDL
0.074
0.061
0.102
0.038
0.049
0.020
0.036
0.015
0.045
0.044
0.013
0.041
0.021
0.023
0.061
0.031
0.095
0.181
0.050
0.021
0.071
0.015
0.026
0.108
0.025
0.044
0.019
0.060
0.038
0.264
4.861
0.082
0.045
0.16
EC-NPD
MDL
0.010
0.015
0.008
0.760
0.250
0.050
2.500
0.750
0.150
5.000
0.500
0.150
0.250
0.500
0.130
0.500
0.500
0.300
0.190
0.760
0.500
0.130
0.075
0.250
0.760
1.300
4.500
0.500
0.250
0.650
1.000
0.025
0.130
0.67
232
-------
Method 525.1
Table 9. Method 525.1 -Laboratory Fortified Blank
Compound
Alachlor
Aldrin
Ametryn
Atraton
Atrazine
BHC, a
BHC, 0
BHC, 6
BHC, 7
Bromacil
Butachlor
Butyl ate
Carboxin
Chlordane, a
Chiordane, 7
Chlorneb
Chlorobenzilate
Chlorothalonil
Chlorpropham
Cycloate
DCPA
ODD, 4,4'-
DDE, 4,4'-
DDT, 4,4'-
Diaznion
Dichlorvos
Dieldrin
Diphenamid
Disulfoton
Disulfoton sulfone
Disulfoton sulfoxide
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
EPTC
Ethoprop
Etridiazole
Fenamiphos
Fenarimol
Fluridone
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexazinone
Merphos
Methoxychlor
Methyl paraoxon
Target (pg/L)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
# Reps
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
9
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
Mean
Obser.
1.039
1.114
0.850
0.313
0.932
1.070
1.075
1.136
1.105
0.879
1.126
0.843
0.757
1.017
1.144
1.018
1.211
1.211
1.121
0.639
0.822
1.084
1.221
1.128
1.335
0.749
0.819
1.383
0.978
0.880
0.490
1.229
1.358
1.194
1.223
1.331
1.159
1.134
0.911
1.088
1.074
1.108
1.102
1.218
1.024
0.988
0.955
1.182
2.334
Data, 1 Liter
SD
0.15
0.58
0.09
0.10
0.13
0.64
0.61
0.66
0.67
0.21
0.27
0.23
0.07
0.69
0.61
0.60
0.65
0.65
0.67
0.24
0.16
0.63
0.60
0.66
0.84
0.09
0.12
0.79
0.12
0.11
0.47
1.47
0.67
0.56
0.66
0.77
0.70
0.19
0.11
0.58
0.47
0.20
0.22
0.63
0.65
0.60
0.28
0.31
0.83
%RSD
13
15
13
13
14
12
13
12
12
13
9
18
16
9
9
10
11
13
15
10
11
9
13
13
13
28
11
12
16
17
19
24
12
15
11
10
14
29
12
17
11
14
14
12
9
24
10
20
34
Accuracy
104
111
85
31
93
107
108
114
110
88
113
84
76
102
114
102
121
121
112
64
82
108
122
113
134
75
82
138
98
88
49
123
136
119
122
133
116
113
91
109
107
111
110
122
102
99
95
118
233
233
-------
Method 525.1
Table 9. Method 525.1 —Laboratory Fortified Blank Data, 1 Liter (cont.)
Compound
Metolachlor
Metribuzin
Mevinphos
MKG-264
Molinate
Napropamide
Norflurazon
Pebulate
Permethrin, cis-
Permethrin, trans-
Prometon
Prometryn
Pronamide
Propachlor
Propazine
Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
Triademefon
Tricyctazole
Trifluralin
Vernolate
Target ffjg/U
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
# Reps
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
Avg
Mean
Obser.
0.888
1.043
0.727
0.822
0.751
0.827
0.972
1.043
1.123
0.860
1.124
0.562
1.118
0.932
2.179
0.853
0.708
1.043
1.036
1.119
0.913
0.908
1.172
0.931
2.215
0.901
1.05
SO
0.18
0.15
0.13
0.13
0.19
0.18
0.12
0.15
0.24
0.96
0.22
0.11
0.13
0.13
0.90
0.20
0.06
0.21
0.23
0.18
0.13
0.10
0.19
0.42
0.78
0.18
0.40
%RSD
14
13
18
21
12
12
12
11
16
18
11
8
18
14
14
14
9
13
30
17
13
13
15
54
15
16
15
Accuracy
89
104
73
82
75
83
97
104
112
86
112
56
112
93
218
85
71
104
104
112
91
91
117
93
221
90
105
234
-------
Method 531.1
Measurement of N-Methylcarbamoyloximes
and N-Methylcarbamates in Water
by Direct Aqueous Injection HPLC
with Post-Column Derivatization
Revision 3.0 - EPA EMSL-Ci
D. L. Foerst - Method 531, Revision 1.(1985)
T. Engels (Battelle Columbus Laboratories) — National Pesticide
Survey Method 5, Revision 2.0 (1987)
R. L. Graves - Method 531.1, Revision 3.0 (1989)
-------
-------
Method 531.1
Measurement of N-Methylcarbamoyloximes
and N-Methylcarbamates in Water by Direct Aqueous Injection HPLC
with Post Column Derivatization
1. SCOPE AND APPLICA TION
1.1 This is a high performance liquid chromatographic (HPLC) method applicable to the determi-
nations of certain N-methylcarbamoyloximes and N-methylcarbamates in ground water and
finished drinking water.' The following compounds can be determined using this method:
Analyte CAS No.
Aldicarb 116-06-3
Aldicarb sulfone 1646-88-4
Aldicarb sulfoxide 1646-87-3
Baygon 114-26-1
Carbaryl 63-25-2
Carbofuran 1563-66-2
3-Hydroxycarbofuran 16655-82-6
Methiocarb 2032-65-7
Methomyl 16752-77-5
Oxamyl 23135-22-0
1.2 This method has been validated in a single laboratory and estimated detection limits (EDLs)
have been determined for the analytes above (Sect. 12). Observed detection limits may vary
between ground waters, depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.3 This method is restricted to use by or under the supervision of analysts experienced in the use
of liquid chromatography and in the interpretation of liquid chromatograms. Each analyst
must demonstrate the ability to generate acceptable results with this method using the proce-
dure described in Sect. 10.3.
1.4 When this method is used to analyze unfamiliar samples for any or all of the analytes above,
analyte identifications should be confirmed by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 The water sample is filtered and a 400-/iL aliquot is injected into a reverse phase HPLC
column. Separation of the analytes is achieved using gradient elution chromatography. After
elution from the HPLC column, the analytes are hydrolyzed with 0.05 N sodium hydroxide
(NaOH) at 95°C. The methyl amine formed during hydrolysis is reacted with o-phtalaldehyde
(OPA) and 2-mercaptoethanol to form a highly fluorescent derivative which is detected by a
fluorescence detector.2
237
-------
Method 531.1
3. DEFINITIONS
3.1 Internal standard: A pure analyte(s) added to a solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of
the same solution. The internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte: A pure analyte(s), which is extremely unlikely to be found in any sample,
and which is added to a sample aliquot in known amount(s) before extraction and is measured
with the same procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates: (LDl and LD2): Two sample aliquots taken in the analytical laborato-
ry and analyzed separately with identical procedures. Analyses of LDl and LD2 give a
measure of the precision associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field reagent blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.7 Laboratory performance check solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the labora-
tory is capable of making accurate and precise measurements at the required method detection
limit.
3.9 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock standard solution: A concentrated solution containing a single certified standard that is a
method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
238
-------
Method 531.1
an assayed reference compound. Stock standard solutions are used to prepared primary
dilution standards.
3.11 Primary dilution standard solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL solu-
tions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality control sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in liquid
chromatograms. Specific sources of contamination have not been identified. All reagents and
apparatus must be routinely demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks as described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.2 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and through rinsing with tap and reagent water. Drain
dry, and heat in an oven or muffle furnace at 450°C for 1 hour. Do not heat volu-
metric ware. Thermally stable materials might not be eliminated by this treatment.
Through rinsing with acetone may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any accumulation
of dust or other contaminants. Store inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required. WARN-
ING: when a solvent is purified, stabilizers added by the manufacturer are removed,
thus potentially making the solvent hazardous. Also, when a solvent is purified,
preservatives added by the manufacturer are removed, thus potentially reducing the
shelf-life.
4.2 Interfering contamination may occur when a sample containing low concentrations of analytes
is analyzed immediately following a sample containing relatively high concentrations of
analytes. A preventive technique is between-sample rinsing of the sample syringe and filter
holder with two portions of reagent water. After analysis of a sample containing high concen-
trations of analytes, one or more laboratory method blanks should be analyzed.
4.3 Matrix interference may be caused by contaminants that are present in the sample. The extent
of matrix interference will vary considerably from source to source, depending upon the water
sampled. Positive identifications must be confirmed.
239
-------
Method 531.1
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
ing the safe handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available and have been identified4"6
for the information of the analyst.
5.2 WARNING: When a solvent is purified, stabilizers added by the manufacturer are removed,
thus potentially making the solvent hazardous.
6. APPARATUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1 Sampling Equipment
6.1.1 Grab sample bottle: 60-mL screw cap vials (Pierce No. 13075 or equivalent) and
caps equipped with a PTFE-faced silicone septa (Pierce No. 12722 or equivalent).
Prior to use, wash vials and septa as described in Sect. 3.1.1.
6.2 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.3 Filtration Apparatus
6.3.1 Microfiltration: To filter derivatization solutions and mobile phases used in HPLC.
Recommend using 47 mm filters (Millipore Type HA, 0.45 ftm for water and Millip-
ore Type FH, 0.5 pm for organics or equivalent).
6.3.2 Microfiltration: To filter samples prior to HPLC analysis. Use 13 mm filter holder
(Millipore stainless steel XX300/200 or equivalent), and 13 mm diameter 0.2 /xm
polyester filters (Nuclepore 180406 or equivalent).
6.4 Syringes and Syringe Values
6.4.1 Hypodermic syringe: 10-mL glass, with Luer-Lok tip.
6.4.2 Syringe value: 3-way (Hamilton HV3-3 or equivalent).
6.4.3 Syringe needle: 7- to 10-cm long, 17-gauge, blunt tip.
6.4.4 Micro syringes: various sizes.
6.5 Miscellaneous
6.5.1 Solution storage bottles: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-
Hned screw cap.
6.5.2 Helium, for degassing solutions and solvents.
6.6 High Performance Liquid Chromatograph (HPLC)
6.6.1 HPLC system capable of injecting 200- to 400-/uL aliquots, and performing binary
linear gradients at a constant flow rate. A data system is recommended for measuring
peak areas. Table 1 lists retention times observed for method analytes using the
columns and analytical conditions described below.
240
-------
Method 531.1
6.6.2 Column 1 (Primary column): 150 mm long x 3.9 mm I.D. stainless steel packed
with 4 pm NovaPak CIS. Mobil Phase is established at 10:90 methanol:water, hold 2
min., then program as a linear gradient to 80:20 methanol:water in 25 min. Alterna-
tive columns may be used in accordance with the provisions described in Sect. 10.4.
6.6.3 Column 2 (Alternative column):* 250 mm long x 4.6 mm I.D. stainless steel
packed with 5 jtm Beckman Ultrasphere ODS. Mobile phase is established at 1.0
mL/min as a linear gradient from 15:85 methanol:water to methanol in 32 min. Data
presented in this method were obtained using this column.
6.6.4 Column 3 (Alternative column): 250 mm long x 4.6 mm I.D. stainless steel packed
with 5 /*m Supelco LC-1. Mobile phase is established at 1.0 mL/min as a linear
gradient from 15:85 methanol:water to methanol in 32 min.
6.6.5 Post—column reactor: Capable of mixing reagents into the mobile phase. Reactor
should be constructed using PTFE tubing and equipped with pumps to deliver 0.1 to
1.0 mL/min of each reagent; mixing tees; and two 1.0-mL delay coils, one thermosta-
ted at 95°C (ABI URS 051 and URA 100 or equivalent).
6.6.6 Fluorescence detector: Capable of excitation at 230 run and detection of emission
energies greater than 418 nm. A Schoffel Model 970 fluorescence detector was used
to generate the validation data presented in this method.
7. REAGENTS AND CONSUMABLE MATERIALS
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed, thus potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed, thus potentially redu-
cing the shelf-life.
7.1 Reagent Water: Reagent water is defined as water that is reasonably free of contamination that
would prevent the determination of any analyte of interest. Reagent water used to generate the
validation data in this method was distilled water obtained from the Magnetic Springs Water
Co., 1801 Lone Eagle St., Columbus, Ohio 43228.
7.2 Methanol: Distilled-in-glass quality or equivalent.
7.3 HPLC Mobile Phase
7.3.1 Water: HPLC grade (available from Burdick and Jackson).
7.3.2 Methanol: HPLC grade. Filter and degas with helium before use.
7.4 Post-Column Derivatization Solutions
7.4.1 Sodium hydroxide, 0.05 N: Dissolve 2.0 g of sodium hydroxide (NaOH) in reagent
water. Dilute to 1.0 L with reagent water. Filter and degas with helium just before
use.
*Newer manufactured columns have not been able to resolve aldicarb sulfone from oxamyl.
241
-------
Method 531 1
7.4.2 2-Mercaptoethanol (1 + 1): Mix 10.0 mL of 2-mercapto-ethanol and 10.0 mL of
acetonitrile. Cap. Store in hood (CAUTION—stench).
7.4.3 Sodium borate (0.05 N): Dissolve 19.1 g of sodium borate (Na2B4O7 10H2O) in
reagent water. Dilute to 1.0 L with reagent water. The sodium borate will complete-
ly dissolve at room temperature if prepared a day before use.
7.4.4 OPA reaction solution: Dissolve 100 + 10 mg of o-phthalaldehyde (mp 55-58°C) in
10 mL of methanol. Add to 1.0 L of 0.05 N sodium borate. Mix, filter, and degas
with helium. Add 100 ^L of 2-mercaptoethanol (1 +1) and mix. Make up fresh
solution daily.
7.5 Monochloroacetic Acid Buffer (pH3): Prepare by mixing 156 mL of 2.5 M monochloroacetic
acid and 100 Ml 2.5 M potassium acetate.
7.6 4-Bromo-3,5-Dimethylphenyl N-Methycarbamate (BDMC): 98% purity, for use as internal
standard (available from Aldrich Chemical Co.).
7.7 Stock Standard Solutions (1.00 /*g//xL): Stock standard solutions may be purchased as cer-
tified solutions or prepared from pure standard materials using the following procedure:
7.7.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
pure material. Dissolve the material in HPLC quality methanol and dilute to volume
in a 10-mL volumetric flask. Larger volumes may be used at the convenience of the
analyst. If compound purity is certified at 96% or greater, the weight may be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.7.2 Transfer the stock standard solutions into TFE-fluoro-carbon-sealed screw cap vials.
Store at room temperature and protect from light.
7.7.3 Stock standard solutions should be replaced after two months or sooner if comparison
with laboratory fortified blanks, or QC samples indicate a problem.
7.8 Internal Standard Solution: Prepare an internal standard fortification solution by accurately
weighing approximately 0.0010 g of pure BDMC. Dissolve the BDMC in pesticide-quality
methanol and dilute to volume in a 10-mL volumetric flask. Transfer the internal standard
fortification solution to a TFE-fluorocarbon-sealed screw cap bottle and store at room tempera-
ture. Addition of 5 ^L of the internal standard fortification solution to 50 mL of sample
results in a final internal standard concentration of 10 /xg/L. Solution should be replaced when
ongoing QC (Sect. 10) indicates a problem. Note: BDMC has been shown to be an effective
internal standard for the method analytes,1 but other compounds may be used, if the quality
control requirements in Sect. 9 are met.
7.9 Laboratory Performance Check Solution: Prepare concentrate by adding 20 /^L of the 3-
hydroxycarbofuran stock standard solution, 1.0 mL of the aldicarb sulfoxide stock standard
solution, 200 ^L of the methiocarb stock standard solution, and\mL of the internal standard
fortification solution to a 10-mL volumetric flask. Dilute to volume with methanol. Thor-
oughly mix concentrate. Prepare check solution by placing 100 pL of the concentrate solution
into a 100-mL volumetric flask. Dilute to volume with buffered reagent water. Transfer to a
TFE-fluorocarbon-sealed screw cap bottle and store at room temperature. Solution should be
replaced when ongoing QC (Sect. 10) indicates a problem.
242
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Method 531.1
8. SAMPLE COLLECTION, PRESERVATION AND HANDLING
8.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should
be followed; however, the bottle must not be prerinsed with sample before collection.
8.2 Sample Preservation/PH Adjustment: Oxamyl, 3-hydroxycarbofuran, aldicarb sulfoxide, and
carbaryl can all degrade quickly in neutral and basic waters held at room temperature.6'7 This
short term degradation is of concern during the time samples are being shipped and the time
processed samples are held at room temperature in autosampler trays. Samples targeted for
the analysis of these three analytes must be preserved at pH 3. The pH adjustment also
minimizes analyte biodegradation.
8.2.1 Add 1.8 mL of monochloroacetic acid buffer to the 60-mL sample bottle. Add buffer
to the sample bottle at the sampling site or in the laboratory before shipping to the
sampling site.
8.2.2 If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
the sample bottle prior to collecting the sample.
8.2.3 After sample is collected in bottle containing buffer, seal the sample bottle and shake
vigorously for 1 min.
8.2.4 Samples must be iced or refrigerated at 4°C from the time of collection until storage.
Samples must be stored at -10°C until analyzed. Preservation study results indicate
that method analytes are stable in water samples for at least 28 days when adjusted to
pH 3 and stored at -10°C. However, analyte stability may be effected by the matrix;
therefore, the analyst should verify that the preservation technique is applicable to the
samples under study.
9. CALIBRATION
9.1 Establish HPLC operating parameters equivalent to those indicated in Sect. 6.6. The HPLC
system may be calibrated using either the internal standard technique (Sect. 9.2) or the external
standard technique (Sect. 9.3).
9.2 Internal Standard Calibration Procedure. The analyst must select one or more internal stan-
dards similar in analytical behavior to the analytes of interest. The analyst must further
demonstrate that the measurement of the internal standard is not affected by method or matrix
interferences. BDMC has been identified as a suitable internal standard.
9.2.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest by adding volumes of one or of the more stock
standards to a volumetric flask. To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to volume with buffered reagent
water. To prepare buffered reagent water, add 10 mL of 1.0 M monochloroacetic
acid buffer to 1 L of reagent water. The lowest standard should represent analyte
concentrations near, but above, their respective EDLs. The remaining standards
should bracket the analyte concentrations expected in the sample extracts, or should
define the working range of the detector.
9.2.2 Analyze each calibration standard according to the procedure (Sect. 11.2). Tabulate
peak height or area responses against concentration for each compound and internal
243
-------
Method 531.1
standard. Calculate response factors (RF) for each analyte, surrogate and internal
standard usino Rniiatinn 1
r
standard using Equation 1.
Equation 1
CU (Q
Where:
As = Response for the analyte to be measured.
AK = Response for the internal standard.
Ca = Concentration of the internal standard
Cs = Concentration of the analyte to be measured /x/L.
9.2.3 If the RF value over the working range is constant (20% RSD or less) the average RF
can be used for calculations. Alternatively, the results can be used to plot a calibra-
tion curve of response ratios (A,/A1S) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ±20%, the test must be repeated
using a fresh calibration standard. If the repetition also fails, a new calibration cure
must be generated for that analyte using freshly prepared standards.
9.2.5 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards. The single point standards
should be prepared at a concentration that deviates from the sample extract response
by no more than 20%.
9.3 External Standard Calibration Procedure
9.3.1 Prepare calibration standards at a minimum of three (recommend five) concentration
levels for each analyte of interest by adding volumes of one or more stock standards
to a volumetric flask. Dilute to volume with buffered reagent water. The lowest
standard should represent analyte concentrations near, but above, the respective
EDLs. The remaining standards should bracket the analyte concentrations expected in
the sample extracts, or should define the working range of the detector.
9.3.2 Starting with the standard of lowest concentration, analyze each calibration standard
according to Sect. 11.2 and tabulate responses (peak height or area) versus the con-
centration in the standard. The results can be used to prepare a calibration curve for
each compound. Alternatively, if the ratio of response to concentration (calibration
factor) is a constant over the working range <20% relative standard deviation),
linearity through the origin can be assumed and the average ratio or calibration factor
can be used in place of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be verified on each working
day by the measurement of a minimum of two calibration check standards, one at the
beginning and one at the end of the analysis day. These check standards should be at
244
-------
Method 531.1
two different concentration levels to verify the concentration curve. For extended
periods of analysis (greater than 8 hr), it is strongly recommended that check stan-
dards be interspersed with samples at regular intervals during the course of the analy-
ses. If the response for any analyte varies from the predicted response by more than
±20%, the test must be repeated using a fresh calibration standard. If the results still
do not agree, generate a new calibration curve or cue a single point calibration stan-
dard as described in Sect. 9.3.3.
9.3.4 Single point calibration is a viable alternative to a calibration curve. Prepare single
point standards from the secondary dilution standards. The single point standards
should be prepared at a concentration that deviates from the sample extract response
by no more than 20%.
9.3.5 Verify calibration standards periodically, recommended at least quarterly, by analyz-
ing a standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check calibra-
tion.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
monitoring internal standard peak area or height in each sample and blank (when internal
standard calibration procedures are being employed), analysis of laboratory reagent blanks,
laboratory fortified samples, laboratory fortified blanks and QC samples.
10.2 Laboratory Reagent Blanks: Before processing any samples, the analyst must demonstrate that
all glassware and reagent interferences are under control. Each time a set of samples is
extracted or reagents are changed, a laboratory reagent blank (LRB) must be analyzed. If
within the retention time window of any analyte of interest the LRB produces a peak that
would prevent the determination of that analyte, determine the source of contamination and
eliminate the interference before processing samples.
10.3 Initial Demonstration of Capability
10.3.1 Select a representative concentration (about 10 times EDL) for each analyte. Prepare
a sample concentrate (in methanol) containing each analyte at 1000 times selected
concentration. With a syringe, add 50 /xL of the concentrate to each of at least four
50-mL aliquots of reagent water, and analyze each aliquot according to procedures
beginning in Sect. 11.
10.3.2 For each analyte the recovery value of all four of these samples must fall in the range
of R + 30% (or within R ± 3SR if broader) using the values for R and SR for reagent
water in Table 2. For those compounds that meet the acceptance criteria, perfor-
mance is judged acceptable and sample analysis may begin. For those compounds
that meet the acceptance criteria, performance is judged acceptable and sample analy-
sis may begin. For those compounds that fail these criteria, this procedure must be
repeated using four fresh samples until satisfactory performance has been demonstrat-
ed.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
245
-------
Method 531.1
experience with it. It is expected that as laboratory personnel gain experience with
this method the quality of data will improve beyond those required here.
10.4 The analyst is permitted to modify HPLC columns, HPLC conditions, internal standards or
detectors to improve separations or lower analytical costs. Each time such method modifica-
tions are made, the analyst must repeat the procedures in Sect. 10.3.
10.5 Assessing the Internal Standard
10.5.1 When using the internal standard calibration procedure, the analyst is expected to
monitor the IS response (peak area or peak height) of all samples during each analysis
day. The IS response for any sample chromatogram should not deviate from the daily
calibration check standard's IS response by more than 30%.
10.5.2 If >30% deviation occurs with an individual extract, optimize instrument perfor-
mance and inject a second aliquot.
10.5.2.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
10.5.2.2 If a deviation of greater than 30% is obtained for the reinjected extract,
analysis of the sample should be repeated beginning with Sect. 11, provid-
ed the sample is still available. Otherwise, report results obtained from the
reinjected extract, but annotate as suspect.
10.5.3 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
calibration check standard.
10.5.3.1 If the check standard provides a response factor (RF) within 20% of the
predicted value, then follow procedures itemized in Sect. 10.5.2 for each
sample failing the IS response criterion.
10.5.3.2 If the check standard provides a response factor which deviates more than
20% of the predicted value, then the analyst must recalibrate, as specified
in Sect. 9.
10.6 Assessing Laboratory Performance—Laboratory Fortified Blanks
10.6.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
every 20 samples or one per sample set (all sample analyzed within a 24-h period)
whichever is greater. The fortification concentration of each analyte in the LFB
should be 10 times EDL or the MCL, whichever is less. Calculate accuracy as
percent recovery (Xj). If the recovery of any analyte falls outside the control limits
(see Sect. 10.7.2), that analyte is judged out of control, and the source of the problem
must be identified and resolved before continuing analyses.
10.6.2 Until sufficient data become available from within their own laboratory, usually a
minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
performance against the control limits in Sect. 10.3.2 that are derived from the data in
Table 2. When sufficient internal performance data becomes available, develop
control limits from the mean percent recovery (X) and standard deviation (S) of the
246
-------
Method 531.1
percent recovery. These data are used to establish upper and lower control limits as
follows:
Upper Control Limit = X + 3S
Lower Control Limit = X - 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points. These calculated control
limits should never exceed those established in Sect. 10.3.2.
10.6.3 It is recommended that the laboratory periodically determine and document its detec-
tion limit capabilities for analytes of interest.
10.6.4 At least quarterly, analyze a QC sample from an outside source.
10.6.5 Laboratories are encouraged to participate in external performance evaluation studies
such as the laboratory certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as independent checks
on the analyst's performance.
10.7 Assessing Analyte Recovery—Laboratory Fortified Sample Matrix
10.7.1 The laboratory must add a known concentration to a minimum of 5% of the routine
samples or one sample concentration per set, whichever is greater. The concentration
should not be less than the background concentration of the sample selected for
fortification. Ideally, the concentration should be the same as that used for the labora-
tory fortified blank (Sect. 10.6). Over time, samples from all routine sample sources
should be fortified.
10.7.2 Calculate the percent recovery, P, of the concentration for each analyte, after correct-
ing the analytical result, X, from the fortified sample for the background concentra-
tion, b, measured in the unfortified sample, i.e.,:
p = 100 (X - b)
fortifying concentration
and compare these values to control limits appropriate for reagent water data collected
in the same fashion. If the analyzed unfortified sample is found to contain NO back-
ground concentrations, and the added concentrations are those specified in Sect. 10.7,
then the appropriate control limits would be the acceptance limits in Sect. 10.7. If,
on the other hand, the analyzed unfortified sample is found to contain background
concentration, b, estimate the standard deviation at the background concentration, sb,
using regressions or comparable background data and, similarly, estimate the mean,
xa, and standard deviation, sa, of analytical results at the total concentration after for-
tifying. Then the appropriate percentage control limits would be P ± 3SP, where:
247
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Method 531.1
P =
100 X
(b + fortifying concentration)
,,-100 _
fortifying concentration
For example, if the background concentration for Analyte A was found to be 1 pig/L
and the added amount was also 1 /*g/L, and upon analysis the laboratory fortified
sample measured 1.6 p/L, then the calculated P for this sample would be (1.6 /xg/L
minus 1.0 Mg/L)/l ftg/L or 60%. This calculated P is compared to control limits
derived from prior reagent water data. Assume it is known that analysis of an inter-
ference free sample at 1 /xg/1 yields an s of 0.12 jig/L and similar analysis at 2.0 jig/L
yields X and s of 2.01 /tg/L and 0.20 /ig/L, respectively. The appropriate limits to
judge the reasonableness of the percent recovery, 60%, obtained on the fortified
matrix sample is computed as follows:
100 (2.01 pg/L)
2.0
K°-12Pg/L)2 + (O-20
1.0 ftgIL
100.5% + 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
10.7.3 If the recovery of any such analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control (Sect. 10.6), the
recovery problem encountered with the dosed sample is judged to be matrix related,
not system related. The result for that analyte in the unfortified sample is labeled
suspect/matrix to inform the data user that results are suspect due to matrix effects.
10.8 Assessing Instrument System—Laboratory Performance Check Sample: Instrument perfor-
mance should be monitored on a daily basis by analysis of the LPC sample. The LPC sample
contains compounds designed to indicate appropriate instrument sensitivity, column perfor-
mance (primary column) and chromatographic performance. LPC sample components and
performance criteria are listed in Table 3. Inability to demonstrate acceptable instrument
performance indicates the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method, concentrations of the
instrument QC standard components must be adjusted to be compatible with the laboratory
EDLs.
10.9 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to assess
245
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Method 531.1
the precision of the environmental measurements or field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
7 7. PROCEDURE
11.1 pH Adjustment and Filtration
11.1.1 Add preservative to any samples not previously preserved (Sect. 8). Adjust the pH of
the sample or standard to pH 3 ±0.2 by adding 1.5 mL of 2.5 M monochloroacetic
acid buffer to each 50 mL of sample. This step should not be necessary if sample pH
was adjusted during sample collection as a preservation precaution. Fill a 50-mL
volumetric flask to the mark with the sample. Add 5 /*L of the internal standard
fortification solution (if the internal standard calibration procedure is being employed)
and mix by inverting the flask several times.
11.1.2 Affix the three-way valve to a 10-mL syringe. Place clean filter in the filter holder
and affix the filter holder and the 7- to 10-cm syringe needle to the syringe valve.
Rinse the needle and syringe with reagent water. Prewet the filter by passing 5 mL of
reagent water through the filter. Empty the syringe and check for leaks. Draw 10
mL of sample into the syringe and expel through the filter. Draw another 10 mL of
sample into the syringe, expel through the filter, and collect the last 5 mL for analy- •
sis. Rinse the syringe with reagent water. Discard the filter.
11.2 Liquid Chromatography
11.2.1 Sect. 6.6 summarizes the recommended operating conditions for the liquid chromato-
graph. Table 1 lists retention times observed using this method. Other HPLC col-
umns, chromatographic conditions, or detectors may be used if the requirements of
Sect. 10.4 are met.
11.2.2 Calibrate the system daily as descried in Sect. 10. The standards and samples must be
in pH3 buffered water.
11.2.3 Inject 400 /xL of the sample. Record the volume injected and the resulting peak size
in area units.
11.2.4 If the response for the peak exceeds the working range of the system, dilute the
sample with pH 3 buffered reagent water and reanalyze.
11.3 Identification of Analytes
11.3.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention tune of an unknown compound corre-
sponds, within limits, to the retention time of a standard compound, then identification
is considered positive.
11.3.2 The width of the retention tune window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention tune can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.3.3 Identification requires expert judgement when sample components are not resolved
chromatographically. When peaks obviously represent more than one sample compo-
249
-------
Method 531.1
nent (i.e., broadened peak with shoulder(s) or valley between two or more maxima),
or any time doubt exists over the identification of a peak on a chromatogram, appro-
priate alternate techniques, to help confirm peak identification, need to be employed.
For example, more positive identification may be made by the use of an alterative
detector which operates on a chemical/physical principle different from that originally
used; e.g., mass spectrometry, or the use of a second chromatography column. A
suggested alternative column is described in Sect. 6.6.3.
12. CALCULATIONS
Determine the concentration of individual compounds in the sample using the following equation:
c -
As . RF
where:
Cx = analyte concentration in micrograms per liter,
Ax = response of the sample analyte;
A = response of the standard (either internal or external), in units consistent with those used
for the analyte response;
RF = response factor (with an external standard, RF = 1 , because the standard is the same
compound as the measured analyte);
Qs = concentration of internal standard present or concentration of external standard
that produced As, in micrograms per liter.
13. PRECISION AND A CCURA c Y
13.1 In a single laboratory, analyte recoveries from reagent water were determined at five concen-
tration levels. Results were used to determine analyte EDLs and demonstrate method range.'
Analyte recoveries and standard deviation about the percent recoveries at one concentration are
given in Table 2.
13.2 In a single laboratory, analyte recoveries from two standard synthetic ground waters were
determined at one concentration level. Results were used to demonstrate applicability of the
method to different ground water matrices.' Analyte recoveries from the two synthetic matri-
ces are given in Table 2.
250
-------
Method S31.1
References
1. National Pesticide Survey Method No. 5., "Measurement of N-Methylcarbamoyloximes and
N-Methylcarbamates in Groundwater by HPL with Post Column Derivatization."
2. Moye, H.A., S. J. Sherrer, and P.A. St. John, "Dynamic Labeling of Pesticides for High
Performance Liquid Chromatography: Detection of N-Methylcarbamates and o-Phthaladehy-
de," Anal. Lett. 10. 1049, 1977.
3. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society for Testing and
Materials, Philadelphia, PA, p. 86, 1986.
4. "Carcinogens — Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center For Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, Aug. 1977.
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
7. Foerst, D.L. and H.A. Moye, "Aldicarb in Drinking Water via Direct Aqueous Injection
HPLC with Post column Derivatization," Proceedings of the 12th annual AWWA Water
Quality Technology conference, 1984.
8. Hill, K.M., R.H. Hollowell, and L.A. DalCortevo, "Determination of N-Methylcarbamate
Pesticides in Well Water by Liquid Chromatography and Post Column Fluorescence Derivatiz-
ation," Anal. Chem. 56, 2465 (1984).
9. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, p. 130,
1986.
257
-------
Method 531.1
Table 1. Retention Times for Method Analytes
Retention Time '
(minutes)
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3-Hydroxcarbofuran
Aldicarb
Baygon
Carbofuran
Carbaryl
Methiocarb
BDMC
Primary1
6.80
7.77
8.20
8.94
13.65
16.35
18.86
19.17
20.29
24.74
25.28
Alternative2
15.0
15.2
17.4
18.4
23.3
27.0
29.3
29.6
30.8
34.9
35.5
Alternative3
17.5
12.2
14.6
14.8
19
21.4
24.4
23.4
25.4
28.6
Columns and analytical conditions are described in Sect. 6.6.2 and 6.6.3.
Waters NovaPack C18
Beckman Ultrasphere ODS
Superlco LC-1
252
-------
Table 2. Single Laboratory Accuracy, Precision and Estimated Detection Limits (EDLS)
For Analytes From Reagent Water and Synthetic Ground Waters3
Water 2f EDL
Analyte (pg/Lf
Aldicarb 1.0
Aldicarb sulfone 2.0
Aldicarb sulfoxide 2.0
Baygon 1.0
Carbaryl 2.0
Carbofuran 1.5
3-Hydroxycarbofuran 2.0
Methiocarb 4.0
Methomyl 0.5
Oxamyl 2.0
Concentration Level
V9/L \ ff
5 115
10 101
10 97
5 106
10 97
7.5 102
10 102
20 94
2.5 105
10 100
Reagent Water
SR" ^
3.5 106
4.0 98
4.9 105
3.2 96
5.8 94
5.1 102
4.1 98
1.9 102
4.2 98
4.0 97
Synthetic Water T
SR fl
3.2 102
3.9 95
4.2 94
4.8 97
4.7 104
3.1 100
4.9 101
4.1 112
3.9 105
2.9 102
Synthetic
8.2
9.5
10.3
5.8
10.4
7.0
10.1
3.4
9.5
10.2
Data corrected for amount detected in blank and represent the mean of 7-8 samples.
EDL = Estimated detection limit; defined as either MDL (Appendix B to 40 CFR Part 136 - Definition and Procedure for the
Determination of the Method Detection Limit - Revision 1.11) or a level of compound in a sample yielding a peak in the final extract
with the signal-to-noise ratio of approximately 5, whichever value is higher. The concentration level used in determining the EDL is
not the same as the concentration level presented in this table.
R = Average percent recovery.
SR = Standard deviation of the percent recovery.
Corrected for amount found in blank; Absopure Nature Artesian Spring Water Obtained from the Absopure Water Company in
Plymouth, Michigan.
Corrected for amount found in blank; reagent water fortified with fulvic acid at the 1 mg/L concentration level. A well-characterized
fulvic acid, available from the International Humic Substances Society (associated with the United States Geological Survey in
Denver, Colorado) was used.
I
01
Co
wi
Co
-------
Method 531.1
Table 3. Laboratory Performance Check Solution
Test Analyte
Sensitivity 3-Hydroxycarbofuran
Chromatographic Aldicarb sulfoxide
performance
Column performance Methiocarb
4-Bromo-3,5-dimethylphenyl
N-methylcarbamate (IS)
Cone.
f/jg/mL)
2
100
20
10
Requirements
Detection of analyte; S/N>3
0.90,1.0b
* PGF = peak Gaussian factor. Calculated using the equation:
PGF =
where W 11
W J-
10
is the peak width at half height and W | — | is the peak width at tenth height
1
b Resolution between the two peaks as defined by the equation:
R = -L
w
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
254
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Method 547
Determination of Glyphosate in Drinking
Water by Direct-Aqueous-Injection HPLC,
Post-Column Derivatization, and
Fluorescence Detection
EPA EMSL-Ci
July 1990
T.W. Winfield, W.J. Bashe (Technology Application, Inc.),
T.V. Baker (Technology Applications, Inc.)
-------
-------
Method 547
Determination of Glyphosate in Drinking Water by Direct-Aqueous-
Injection HPLC, Post-Column Derivatization, and Fluorescence
Detection
1. SCOPE AND A PPLICA TION
1.1 This method describes a procedure for the identification and measurement of Glyphosate (N-
phosphonomethyl glycine) in drinking water matrices. Single laboratory accuracy and preci-
sion data have been determined for this method.
1.2 Glyphosate was found to rapidly decompose in chlorinated waters.1 It is therefore unlikely
that the analyte will be evidenced in tap water except as separate glycine and N-phosphonome-
thyl moieties, neither of which is applicable to this method.
Analyte CAS No.
Glyphosphate 1071-83-6
1.3 The method detection limits (MDL, defined in Section 13) for glyposate are listed in Table I.2
The MDLs for a specific sample may differ from those listed.
2. SUMMARY OF METHOD
2.1 A water sample is filtered and a 200 /*L aliquot injected into a cation exchange HPLC column.
Separation is achieved by using an isocratic elution. After elution from the analytical column
at 65°C, the analyze is oxidized with calcium hypochlorite and the product (glycine) coupled
with o-phtaladehyde-2-mercaptoethanol complex at 38° C to give a fluorophor detected by a
fluorometer with excitation at 340 ran and detection of emission measured > 455 nm.1'3
3. DEFINITIONS
3.1 Laboratory Duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.2 Field Duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.3 Laboratory Reagent Blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
257
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Method 547
3.4 Field Reagent Blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.5 Laboratory Performance Check Solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.6 Laboratory Fortified Blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the method is in control, and whether the laboratory is
capable of making accurate and precise measurements at the required method detection limit.
3.7 Laboratory Fortified Sample Matrix (LFM): An aliquot on an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.8 Stock Standard Solution: A concentrated solution containing a single certified standard that is
a method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.9 Calibration Standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL solu-
tions are used to calibrate the instrument response with respect to analyte concentration.
3.10 Quality Control Sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and
other sample processing hardware that lead to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
ferences under the conditions of the analysis by analyzing laboratory reagent blanks as re-
quired by Section 10.2.
4.1.1 Glassware must be scrupulously cleaned." Clean all glassware as soon as possible
after use by rinsing with the last solvent used in it. This should be followed by
detergent washing with hot water, and rinses with tap water and distilled water.
Glassware should then be drained dry, and heated in a laboratory oven at 400°C
for several hours before use. After drying and cooling, glassware should be stored
in a clean environment to prevent any accumulation of dust or other contaminants.
258
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Method 547
4.1.2 The use of high purity reagents and solvents helps to minimize interference prob-
lems. Purification of solvents by distillation in all-glass systems may be required
to achieve necessary purity.
4.2 Samples may become contaminated during shipment or storage. Field blanks must be analyzed
to determine that sampling and storage procedures have prevented contamination.
4.3 The extent of matrix interferences may vary considerably from source to source, depending
upon the nature and diversity of the matrix being sampled. No interferences have been
observed in the matrices studied.
4.4 The extent of interferences that may be encountered using liquid chromatographic techniques
has not been fully assessed. Although the HPLC conditions described allow for a unique
resolution of the compound covered in this method, other matrix components may interfere.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method has not been precisely de-
fined. Each chemical should be treated as a potential health hazard, and exposure to these
chemicals should be minimized. Each laboratory is responsible for maintaining awareness of
OSHA regulations regarding safe handling of chemicals used in this method.5 A reference file
of material data handling sheets should be made available to all personnel involved in the
chemical analysis.
6. APPARA TUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only).
6.1 Sampling Equipment (for discrete or composite sampling)
6.1.1 Grab sample bottle: 60mL screw cap bottles (Pierce No. 13075 or equivalent) and
caps equipped with a PTFE-faced silicone septa (Pierce No. 12722 or equivalent).
Prior to use, wash vials and septa as described in Section 4.1.1.
6.2 Glassware
6.2.1 Autosampler vials: Glass, 3.7 ml, with PTFE-lined septa and screw caps. (Supel-
co, #2-3219, or equivalent)
6.2.2 Volumetric flask: 1000 mL and 100 mL
6.3 Balance: Analytical, capable of accurately weighing 0.0001 g.
6.4 pH Meter: Capable of measuring pH to 0.01 units.
6.5 Filtration Apparatus
6.5.1 Macrofiltration: To filter mobile phase derivatization solutions used in HPLC
system. Membrane filter, 0.2 /x mesh, 47 mm diameter, Nylon 66 (Alltech, #2034
or equivalent)
6.5.2 Microfiltration: To filter samples prior to HPLC analysis. Use 0.45 \t. filters
(Gelman Acrodisc—CR or equivalent)
6.5.3 Helium, for degassing solutions and solvents.
259
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Method 547
6.6 Syringes
6,6.1 One 250 yiL glass syringe, with blunt tip needle for manual injection.
6.6.2 3-5 mL disposable hypodermic syringes with Luer-Lok tip.
6.6.3 Micro syringes, various sizes.
6.7 Instrumentation: A schematic diagram of the analytical system is shown in Figure 1.
6.7.1 A high performance liquid chromatograph (HPLC) capable of injecting 200 /xL
aliquots and utilizing and isocratic pumping system with constant flow rate of 0.5
mL/min.
6.7.2 Column: 250 x 4 mm, Bio-Rad, Aminex A-9. Column specifications: K+ form,
packed at 65 °C, pH = 1.9. This column was used to generate the method perfor-
mance statements in Section 13. Different HPLC columns may be used if require-
ments described in Section 10.3 are met. Use of guard columns is recommended.
6.7.3 Guard Column: C18 packing: (Dupont, Zorbax Guard Column or equivalent).
6.7.4 Column Oven, (Fiatron, Model CH-30 and controller, Model TC-50, or equiva-
lent).
6.7.5 Post Column Reactor (PCR): Capable of mixing reagents into the mobile phase.
Reactor to be equipped with pumps to deliver 0.5 mL/min of each reagent; mixing
tees; two 1.0 mL delay coils, both thermostated at 38°C; and constructed using
PTFE tubing. (Kratos Model URS 051 and URA 200 or equivalent).
6.7.6 Fluorescence Detector: Capable of excitation at 340 nm and detecting of emission
> 455 nm. A Schoeffel Model 970 fluorescence detector was used to generate the
validation data presented in this method.
6.7.7 Data System: A strip chart recording of the detector response must be provided as
a minimum requirement. The use of a data system to calculate retention times and
peak areas is recommended but not required. The system used to generate the data
in Table 1 is as follows.
6.7.7.1 IBM AT computer with 640 KB of RAM, a 20 MB hard disk, and en-
hanced graphics monitor or equivalent.
6.7.7.2 Nelson Analytical Interface and Software Model 2600, Version 4.1 or
equivalent to perform data computations.
6.7.7.3 Printer, Epson FX-286 or equivalent for report generation.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 HPLC Mobile Phase
7.1.1 Reagent Water: Reagent water is defined as water of very high purity, equivalent
to distilled-in-glass solvents.
7.1.2 Mobile Phase: 0.005 M KH2PO4 (0.68 gm) in 960 mL reagent water, add 40 mL
HPLC grade methanol, adjust pH of solution to 1.9 with concentrated phosphoric
acid then filter with 0.22 /* filter and degas with helium before use.
260
-------
Method 547
7.2 Post Column Derivatization Solutions
7.2.1 Calcium hypochlorite solution: Dissolve 1.36 g KH2PO4, 11.6 g NaCl and 0.4 g
NaOH in 500 mL deionized water. Add 15 mg Ca(C10)2 dissolved in 50 mL
deionized water and dilute solution to 1000 mL with deionized water. Filter
solution through 0.22 /x membrane filter and degas with helium before use.
7.2.2 O-phthalaldehyde (OPA) reaction solution
7.2.2.1 2-Mercaptoethanol (1 + 1): Mix 10.0 mL of 2-mercaptoethanol and 10.0
mL of acetonitrile. Cap store in hood. (Caution - stench).
7.2.2.2 Sodium borate (0.05N): Dissolve 19.1 g of sodium borate (Na^O, 10
H20) in 1.0 L of reagent water. The sodium borate will completely dis-
solve at room temperature if prepared a day before use.
7.2.2.3 OPA Reaction Solution: Dissolve 100 + 10 mg of o-phthalaldehyde (mp
55-58°C) in 10 mL of methanol. Add to 1.0 L of 0.05 N sodium borate.
Mix, filter through 0.45 /x membrane filter, and degas. Add 100 n of 2-
mercaptoethanol (1 + 1) and mix. Make up fresh solution daily unless the
reagent solution is protected from atmospheric oxygen. The solution can
be stored in glass bottles under atmospheric conditions at 4°C for up to
two weeks without appreciable increases in background fluorescence or
stored under nitrogen for indefinite periods.
NOTE: Fluoraldehyde (Pierce Chemical), a commercially formulated OPA reaction
solution, may be substituted for Steps 7.2.2.1 through 7.2.2.3.
7.3 Sample Preservation Reagents
7.3.1 Sodium thiosulfate: Granular, ACS grade or better (Fisher, S-446).
7.4 Stock Standard Solution (1.00 /* g/mL)
7.4.1 Accurately weigh and dissolve 0.1000 g of pure glyphosate in 1000 mL of deion-
ized water. Larger or smaller volumes may be used at the convenience of the
analyst. If compound purity is certified at 96% of greater, the weight may be used
without correction to calculate the concentration of the stock standard.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Collect samples in glass containers (6.1.1). Conventional sampling practices6 are to be
followed.
8.2 Sample Preservation: Treatment of samples to remove residual chlorine will eliminate the
possibility of glyphosate losses due to chlorine during storage. Chlorine is destroyed by
adding 100 mg/L of sodium thiosulfate to the sample.
8.3 Sample Storage: Samples should be stored at 4°C away from light and analyzed within 2
weeks. A preservation study7 has demonstrated the stability of glyphosate in frozen samples
for up to 18 months. The analyst should verify appropriate sample holding times applicable to
the sample under study.
261
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Method 547
9. CALIBRATION
9.1 Establish liquid chromatographic operating conditions indicated in the Table 1.
9.2 Prepare a minimum of three calibration standards of glyphosate by serial dilution of the stock
standard solution in deionized water. One of the calibration standards should correspond to a
glyphosate concentration near to, but above the MDL. The other concentrations should
comprise the range of concentrations expected for the samples, or, otherwise, define the
working range of the detector.
9.3 Analyze each calibration standard and tabulate peak area against concentration (in /xg/L) inject-
ed. The results may be used to prepare a calibration curve for glyphosate.
Alternatively, if the ratio of response to concentration (response factor) is constant over the
working range (< 10% relative standard deviation), linearity through the origin can be
assumed and the average ratio or response factor can be used in place of a calibration curve.
9.4 The working calibration curve must be verified on each working day by the measurement of a
minimum of two calibration check standards, one at the beginning and one at the end of the
analysis day. These check standards should be at two different concentration levels to verify
the calibration curve. For extended periods of analysis (greater than 8 hr), it is strongly
recommended that check standards be interspersed with samples at regular intervals during the
course of the analyses. If the response for the analyte varies from the predicted response by
more than ±20%, the test must be repeated using a fresh calibration standard. If the results
still do not agree, generate a new calibration curve.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
analysis of laboratory reagent blanks, laboratory fortified matrix samples, laboratory fortified
blanks and QC samples.
10.2 Laboratory Reagent Blanks: Before processing any samples, the analyst must demonstrate that
all glassware and reagent interference are under control. Each time a set of samples is extract-
ed or reagents are changed, a laboratory reagent blank (LRB) must be analyzed. If within the
retention time window of the analyte of interest the LRB produces a peak that would prevent
the determination of that analyte, determine the source of contamination and eliminate the
interference before processing samples.
10.3 Initial Demonstration of Capability
10.3.1 Prepare laboratory fortified blanks (LFBs) at an analyte concentration of 250 /xg/L.
With a syringe, add .250 mL of the stock standard (Section 7.4) to at least four - 100
mL aliquots of reagent water and analyze each aliquot according to procedures begin-
ning in Section 11.
10.3.2 The glyphosate recovery (R) values determined in 10.3.1 should be within ± 30% of
the R values listed in Table 2 for at least three of four consecutive samples. The
relative standard deviation (Sr) of the mean recovery (R) should be less than 30%. If
the analyte of interest meets the acceptance criterion, performance is judged accept-
262
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Method 547
able and sample analysis may begin. For analytes that fail this criterion, initial
demonstration procedures should be repeated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples via a new, unfamiliar method prior to obtaining some
experience with it. It is expected that as laboratory personnel gain experience with
this method the quality of the data will improve beyond the requirements stated in
Section 10.3.2.
10.4 The analyst is permitted to modify HPLC column, HPLC conditions, or detectors to improve
separations or lower analytical costs. Each time such method modifications are made, the
analyst must repeat the procedures in Section 10.3.
10.5 Laboratory Fortified Blanks
10.5.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample per
sample set (all samples analyzed within a 24-h period). The fortified concentration of
glyphosate in the LFB should be 10 times the MDL. Calculate accuracy as percent
recovery (R). If R falls outside the control limits (See Section 10.5.2.), the analysis
is judged out of control, and the source of the problem must be identified and re-
solved before continuing analyses.
10.5.2 Until sufficient data become available from within their own laboratory, usually a
minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
performance against the control limits in Section 10.3.2. When sufficient internal
performance data become available, develop control limits from the mean percent
recovery (R) and SR of the percent recovery. These data are used to establish upper
and lower control limits as follows:
Upper Control Limit = R + 3SR
Lower Control Limit = R - 3SR
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points.
10.6 Laboratory Fortified Sample Matrix
10.6.1 The laboratory must add a known fortified concentration to a minimum of 10% of the
routine samples or one fortified sample per set, whichever is greater. The fortified
concentration should not be less than the background concentration of the original
sample. Ideally, the fortified concentration should be the same as that used for the
laboratory fortified blank (Section 10.5). Over time, samples from all routine samples
sources should be fortified.
10.6.2 Calculate the accuracy as R for the analyte, corrected for background concentrations
measured in the original sample, and compare these values to the control limits
established in Section 10.5.2 from the analyses of LFBs.
10.6.3 If recovery of any sample falls outside the designated range, and the laboratory
performance of the analyte is shown to be in control (Section 10.5), the recovery
problem encountered with the dosed sample is judged to be matrix related, not system
263
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Method 547
related. The result for the analyte in the original sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix effects.
10.7 Quality Control Samples (QCS): Each quarter the laboratory should analyze at least one QCS
(if available). If criteria provided with the QCS are not met, corrective action should be taken
and documented.
10.8 The laboratory may adopt additional quality control practices for use with this method. The
specific practices that are most productive depend upon the needs of the laboratory and the
nature of the samples. For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements of field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 Sample Cleanup: The cleanup procedure for this direct aqueous injection HPLC method is
limited to the filtration procedure described in Section 11.2.3. Applying only filtration, no
interferences were evidenced in the analysis of tap water, ground water and municipal effluent.
If particular circumstances demand the use of an alternative cleanup procedure, the analyst
must demonstrate that the recovery of the analyte is within limits specified by the method.
11.2 Analysis
11.2.1 Table 1 details the recommended HPLC-PCR operating conditions. An example of
the chromathography achieved under these conditions is shown in Figure 2.
11.2.2 Calibrate the system daily as described in Section 9.
11.2.3 Filter samples using 0.45 fi Acrodisc filters (6.5.2) and inject 200 jtL of sample into
the HPLC-PCR system for analysis.
11.2.4 Record resulting peak sizes in area units.
11.2.5 If the response for a glyphosate peak in a sample chromatogram exceeds the working
calibration range, dilute the sample with reagent water and reanalyze.
11.3 Identification of Analytes
11.3.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound corre-
sponds, within limits (11.3.2), to the retention time of the standard, then identification
is considered positive.
11.3.2 The width of the retention time window used to make identification should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation in retention time can be used to calculate a
suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.3.3 Identification requires expert judgement when sample components are not resolved
chromatographically. When peaks obviously represent more then one sample compo-
nent (i.e., broadened peak with shoulder(s) or valley between two or more maxima),
or any time doubt exists over the identification of a peak in a chromatogram, appro-
priate confirmatory techniques such as use of an alternative detector which operates on
a physical/chemical principle different from that originally used, e.g., mass spec-
264
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Method 547
trometry, or the use of an alternative separation technology, e.g., anion exchange
chromatography, must be employed.
12. CALCULATIONS
12.1 Determine the concentration (C) of glyphosate in the sample by direct comparison with the
calibration curve described in Section 9, or alternatively, by means of the equation below
derived from the calibration data.
12.2 For samples processed as part of a set where laboratory fortified blank and/or laboratory
fortified matrix recoveries fall outside control limits in Section 10.5 & 10.6, data for the
affected samples must be labeled as suspect.
-
where:
A = Area of glyphosate peak in sample
RF = Response factor derived from calibration data
13. METHOD PERFORMANCE
13.1 Method Detection Limits: The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with 99% confidence that the
value is above the background level.2 The concentrations listed in Table 1 were obtained using
reagent water, ground water and dechlorinated tap water.
13.2 Single-laboratory precision and accuracy results at several concentrations in drinking water
matrices are presented in Table 2.
255
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Method 54 7
References
1. Bashe, W.J., T.V. Baker, "Analysis of Glyphosate in Drinking Water by Direct Aqueous
Injection HPLC with Post Column Derivatization," in preparation, Technology Applications,
Inc., 1988.
2. Glasser, J.A., D.L. Foerst, G.M. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
Wastewaters," Environ. Sci. Technol., 15, 1426, 1981.
3. Cowell, J.E., "Analytical Residue Method for N-phosphonomethyl Glycine and Aminomethyl
Phosphonic Acid in Environmental Water," Monsanto Company, Method Number 86-63-1,
1987.
4. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, PA, p. 679, 1980.
5. "OSHA Safety and Health Standards, General Industry," (29CF/?1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
6. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
7. Cowell, J.E., "Storage Stability of Glyphosate in Environmental Water," Monsanto Company,
1988.
266
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Method 547
Table 1. Analytical Conditions and Method Detection Limits for Glyphosate
Matrix1
RW
GW
TW-T
Retention Time (min)
13.5
13.7
11.8
MDL,3 fjg/L
6.00
8.99
5.99
Conditions:
Column: 250 x 4 mm, Bio-Rad, Aminex A-9 (Specifications as per Subsection 6.7)
thermostated at 65°C.
Mobile Phase: 0.005 M KH2P04 - watermethanol (24:1) buffered at pH = 1.9 (Sec-
tion 7).
Elution Mode: Isocratic
Flow Rate: 0.5 mL/min.
Injection Volume: 200 /yL
PCR: Calcium Hypochlorite Flow rate = 0.5 mL/min., OPA solution flow rate =
0.5 mL/min., reactor temperature = 38°C.
Detector: Excitation wavelength at 340 nm and detection emission at 455 nm.
1 RW = Reagent water, GW = ground water, TW-T = tap water spiked after dechlorination
treatment.
2 All MDL data were generated from spiked samples at 25 fjg/L.
267
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Method 547
Table 2. Recovery of Glyphosate In Representative Drinking Water Matrices
Fortified
Concentration
(ug/U
2500
700
250
25
Matrix1
RW
GW
TW-T
RW
GW
TW-T
RW
GW
TW-T
RW
GW
TW-T
Number
of
Replicates
8
8
8
8
8
8
8
8
8
8
8
8
Mean
Recovery
102
103
99.2
101
98.7
96.4
95.6
101
98.0
96.0
96.0
108
Relative
Standard
Deviation
1.96
1.25
1.74
2.65
2.01
1.80
3.91
1.77
1.75
9.07
12.3
6.57
'RW = Reagent water, GW = Ground Water, TW-T = Tap water spiked after dechlorination
treatment.
268
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Method 547
Buffer
Reservoir
HPLC
Pump
Hypochlorite
Reservoir
/ OPA \
\ Reservoir
\
/
Autoinjector
7
Fluorescence
Detector
Nelson
Analytical
Data
System
Computer
Quantitation
52-015-30
Figure 1. HPLC, Post-Column Reactor System
269
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Method 547
uV
145.367—
130.671 —
115.975 —
101.280—
86.584 —
71.888 —
57.192
1.20 2.08
i i
7.25
i
13.69
21.72 24.35 25.60 26.78 28.10
i i i i i
5.0
10.0
15.0
20.0
25.0
Retention Time (Minutes)
52-015-29
Figure 2. Liquid Chromatogram of Glyphosate at 250
(conditions are as stated in Table 1).
270
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Method 548
Determination of Endothall in Drinking
Water by Aqueous Derivatization,
Liquid-Solid Extractionf
and Gas Chromatography
with Electron-Capture Detection
July 1990 - EPA EMSL-Ci
J.W. Hodgeson
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-------
Method 548
Determination of Endothall in Drinking Water by Aqueous
Derivatization, Liquid-Solid Extraction, and Gas Chromatography
with Electron-Capture Detection
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of endothall in drinking water sources and finished
drinking water. The following analyte can be determine by this method:
Analyte CAS No.
Endothall 145-73-3
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compound
listed above. When this method is used to analyze unfamiliar samples, compound identifica-
tion should be supported by at least one additional qualitative technique. A gas chromato-
graph/mass spectrometer (GC/MS) may be used for the qualitative confirmation of results for
Endothall using the extract produced by this method.
1.3 The method detection limit' (MDL, defined in Section 12) for endothall is listed in Table 1.
The MDL for a specific sample may differ from the listed value, depending upon the nature of
interferences in the sample matrix and the amount of sample used in the procedure.
1.4 Any modification of this method beyond those expressly permitted, shall be considered a major
modification subject to application and approval of alternate test procedures under 40 CFR
136.4 and 136.5.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use
of gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 11.
2. SUMMARY OF METHOD
2.1 A 5.0 mL volume of liquid sample is placed in a Kuderna-Danish tube and the volume is
reduced to less than 0.75 mL using a heating block. The tube is charged with glacial acetic
acid and sodium acetate, followed by a solution of the derivatization reagent, pentafluoro-
phenylhydrazine (PFPH), in glacial acetic acid. After heating at 150°C for 90 minutes the
derivative is extracted by a solid sorbent from the reaction solution, followed by elution with
5.0 mL of methyl-tert-butyl ether (MTBE). The MTBE extract is analyzed by gas chromatog-
raphy with electron capture detection (GC/ECD).
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Method 548
3. DEFINITIONS
3.1 Internal Standard: A pure analyte(s) added to a solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of
the same solution. The internal standard must be analyte that is not a sample component.
3.2 Surrogate Analyte: A pure analyte(s), which is extremely unlikely to be found in any sample,
and which is added to a sample aliquot in known amount(s) before extraction and is measured
with the same procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory Duplicates (LDl and LD2): Two sample aliquots taken in the analytical laboratory
and analyzed separately with identical procedures. Analyses of LDl and LD2 give a measure
of the precision associated with laboratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.4 Field Duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory Reagent Blank (LRB): An aliquot of reagent water that is treated exactly as a
sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field Reagent Blank (FRB): Reagent water placed in a sample container in the laboratory and
treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB is to determine if method
analytes or other interferences are present in the field environment.
3.7 Laboratory Performance Check Solution (LPC): A solution of method analytes, surrogate
compounds, and internal standards used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory Fortified Blank (LFB): An aliquot of reagent water to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the labora-
tory is capable of making accurate and precise measurements at the required method detection
limit.
3.9 Laboratory Fortified Sample Matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock Standard Solution: A concentrated solution containing a single certified standard that is
a method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
274
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Method 548
an assayed reference compound. Stock standard solutions are used to prepare primary dilution
standards.
3.11 Primary Dilution Standard Solution: A solution of several analytes prepared in the laboratory
from stock standard solutions and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 Calibration Standard (CAL): A solution prepared from the primary dilution standard solution
and stock standard solutions of the internal standards and surrogate analytes. The CAL solu-
tions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality Control Sample (QCS): A sample matrix containing method analytes or a solution of
method analytes in a water miscible solvent which is used to fortify reagent water or environ-
mental samples. The QCS is obtained from a source external to the laboratory, and is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interference may be caused by contaminants in solvents, reagents, glassware, and
other sample processing hardware that lead to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
ferences under the conditions of the analysis by running laboratory reagent blanks as described
in Section 10.2.
4.1.1 Glassware must be scrupulously clean.2 Clean all glassware as soon as possible after
used by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water, and rinses with tap water and distilled water. It should then
be drained dry, and heated in a laboratory oven at 40°C for several hours before use.
Solvent rinses with methanol may be substituted for the oven heating. After drying
and cooling, glassware should be stored in a clean environment to prevent any accu-
mulation of dust or other contaminants.
4.1.2 The use of high purity regents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
4.2 Matrix interferences may be caused by contaminants that are coextracted from the sample.
The extent of matrix interferences will vary considerably from source to source, depending
upon the nature and diversity of the matrix being sampled. If significant interferences occur in
subsequent samples, some additional cleanup may be necessary to achieve the MDL listed in
Table 1.
4.3 The extent of interferences that may be encountered using gas chromatographic techniques has
not been fully assessed. Although the GC conditions described allow for a unique resolution
of the specific compound covered by this method, other matrix components may interfere.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound should be treated as a potential health hazard.
From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level
by whatever means available. The laboratory is responsible for maintaining a current aware-
275
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Method 548
ness file of OSHA regulations regarding the safe handling of the chemical specified in this
method. A reference file of material data handling sheets should also be made available to all
personnel involved in the chemical analysis. Additionally references to laboratory safety are
available.
6. APPARA TUS AND MA TERIALS
6.1 Sampling Equipment (for discrete or composite sampling).
6.1.1 Grab sample bottle: Amber glass fitted with screw caps lined with Teflon. If amber
bottles are not available, protect samples from light. The container must be washed,
rinsed with methanol, and dried before use to minimize contamination
6.2 Glassware
6.2.1 Volumetric flasks: 5 mL, 25 mL
6.2.2 Vials: Glass, 1 mL, with Teflon-lined caps
6.2.3 Glass syringes, 250 jtL, 500 /*L
6.2.4 Pipets: 1 mL, 4 mL
6.3 Balance: Analytical, capable of accurately weighing 0.0001 g.
6.4 Solid Sorbent Cartridges: CIS, Baker 7020-6 or equivalent
6.5 Vacuum manifold for extraction using solid sorbent cartridges: Supelco 5-7030 or equivalent
6.6 Kuderna-Danish (K-D) concentrator tubes: 10 or 25 mL graduated (Kontes K-570050-1025 or
K-570050-2525)
6.6.1 Snyder column, Kuderna-Danish: 2-ball micro (Kontes, K-569001-0219)
6.7 Tube heater for 25 mL K-D tubes: Kontes
6.8 Boiling chips: Carborundum, #12 granules (Arthur H. Thomas Co. #1590-033 or equivalent).
Heat at 400°C for 30 minutes prior to use. Cool and stored in dessicator.
6.9 Gas chromatographic system capable of temperature programming
6.9.1 Autosampler
6.9.2 Electron capture detector
6.9.3 Column 1: Supelco SPB-5, 0.25 mm x 30 m or equivalent
Column 2: J&W DB-1, 0.32 mm x 30 mm or equivalent
6.9.4 Strip-chart recorder compatible with detector. Use of a data system with printer for
measuring and recording peak areas and retention times is recommended.
7. REAGENTS AND SOLUTIONS
7.1 Reagent Water: reagent water is defined as a water of very high purity, equivalent to distilled
in glass solvents
7.2 Pentafluorophenylhydrazine(PFPH): Aldrich
7.3 Sodium Acetate: Anhydrous, Aldrich
7.4 Sodium Thiosulfate: Baker
276
-------
Method 548
7.5 Acetic Acid: Glacial, Baker
7.6 Methyl-Tert-Butyl Ether (MTBE): Distilled in glass (Burdick & Jackson)
7.7 Endothall-PFPH Derivative: See Appendix for synthesis procedure
7.8 EndosulfanI: USEPA Repository
7.9 Endothall, monohydrate: USEPA Repository
7.10 Stock Standard Solutions
7.10.1 Endothall: 10 ^g/mL in reagent water
7.10.2 Endothall: 50 ^g/mL in reagent water
7.10.3 Stock standard solutions must be replaced after six months, or sooner, if com-
parison with check standards indicates a problem.
7.11 Reaction Solutions
7.11.1 PFPH solution: 4 mg/mL in glacial acetic acid
7.11.2 Internal standard stock solution: 10 /tg/mL endosulfan I in MTBE
8. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
8.1 Grab samples must be collected in glass containers. Conventional sampling practices should
be followed, except that the bottle must not be prewashed with sample before collection.
Composite samples should be collected in refrigerated glass containers in accordance with the
requirements of the program. Automatic sampling equipment must be as free as possible of
Tygon tubing and other potential sources of contamination.
8.2 The samples must be iced or refrigerated at 4°C from the time of collection until
derivatization. The analyte measured here is not known to be light sensitive, but excessive
exposure to light and heat should be avoided.
8.3 Some samples are likely to be biologically active and the stability of samples upon storage will
be different for each matrix. All samples should be derivatized within 7 days of collection,
and analysis completed within 1 day of derivatization. If these criteria are not met, the analyst
must demonstrate the stability of the stored sample by performing suitable holding time
studies.
9. CALIBRATION
9.1 Establish gas chromatographic operating parameters to produce a retention time equivalent to
that indicated in Table 1. The chromatographic system can be calibrated using the internal
standard technique (Section 9.2)
9.1.1 Due to the complex nature of the sample chromatogram, the analyst should periodical-
ly inject a solution containing only pure endothall-PFPH (See Appendix) to verify the
retention time of the derivative.
9.2 Internal Standard Calibration Procedure:
9.2.1 Use 250 and 500 yiL syringes to add sufficient quantities of 7.10.1 or 7.10.2 stock
solutions to reagent water in 25 mL volumetric flasks to produce endothall standard
277
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Method 548
solutions at the following concentrations in /*g/L: 500 (250 ^L of 7.10.2 stock), 200
(100 jiiL of 7.10.2 stock), 100 (50 /xL of 7.10.2 stock) and 50(125 jiL of 7.10.1
stock).
9.2.2 Process each standard as per Section 11.2. It is recommended that triplicate samples
of each standard be processed.
9.2.3 Before analyzing matrix samples, the analyst must process a series of calibration
standards to validate elution patterns and the absence of interferences from reagents.
9.2.4 Analyze each calibration standard and tabulate the ratio of the peak area of the en-
dothall-PFPH derivative versus that of the internal standard against endothall con-
centration. The results may be used to prepare a calibration curve for endothall.
9.2.5 The working calibration curve must be verified on each working day by processing
and analyzing one or more calibration standards. If the response varies from the
previous response by more than ±20%, the test must be repeated using a fresh
calibration standard. Should the retest fail, a new calibration curve must be gene-
rated.
10. QUALITY CONTROL
10.1 Each laboratory that uses this method is required to operate a formal quality control (QC)
program. The minimum QC requirements are initial demonstration of laboratory capability,
analysis of laboratory reagent blanks, laboratory fortified blanks, laboratory fortified matrix
samples and QC check standards.
10.2 Laboratory Reagent Blanks. Before processing any samples, the analyst must demonstrate that
all glassware and reagents interferences are under control. Each time a set of samples is
analyzed or reagent are changed, a method blank must be analyzed. For this method, the
method blank is filtered reagent water. If within the retention time window of an analyte of
interest, the method blank produces a peak which prevents the measurement of that analyte,
determine the source of contamination and eliminate the interference before processing sam-
ples.
10.3 Initial Demonstration of Capability
10.3.1 Select a representative fortified concentration (about 10 times MDL) for endothall.
Prepare a concentrate (in reagent water) containing the analyte at 10 times the selected
concentration. Using a pipet, add 1.00 mL of the concentrate to each of at least four
10 mL aliquots of reagent water and analyze each aliquot according to procedures
beginning in Section 11.
10.3.2 The recovery value should for at least three out of four consecutively analyzed sam-
ples fall in the range of R ± 30% (or within R ± 3SR, if broader) using the values
for R and SR for reagent water. If the recovery value meets the acceptance criteria,
performance is acceptable and sample analysis may begin. If the recovery value fails
these criteria, initial demonstration of capability should be repeated.
10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples by a new, unfamiliar method prior to evidencing a basal
level of skill at performing the technique. It is expected that as laboratory personnel
278
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Method 543
gain experience with this method the quality of the data will improve beyond the
requirements stated in Section 10.3.2.
10.4 The analyst is permitted to modify GC columns, GC conditions, or detectors to improve
separations or lower analytical costs. Each time such method modifications are made, the
analyst must repeat the procedures in Section 10.3.
10.5 Assessing the Internal Standard: In using the IS calibration procedure, the analyst is expected
to monitor the IS response (peak area or peak height) of all samples during each analysis day.
The IS response for any sample chromatogram should not deviate from the calibration standard
IS response by more than 30%.
10.5.1 If a deviation of greater than 30% is encountered for a sample, re-inject the extract.
10.5.1.1 If acceptable IS response is achieved for the re-injected extract, then report
the results for that sample.
10.5.1.2 If a deviation of greater than 30% is obtained for the re-injected extract,
analysis of the sample should be repeated beginning with Section 11,
provided the sample is still available. Otherwise, report results obtained
from the re-injected extract, but annotate as suspect.
10.5.2 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
calibration check standard.
10.5.2.1 If the check standard provides a response factor (RF) within 20% of the
predicated value, then follow procedures itemized in Section 10.5.1 for
each sample failing the IS response criterion.
10.5.2.2 If the check standard provides a response factor (RF) with deviates more
than 20% of the predicted value, then the analyst must recalibrate, as
specified in Section 9.2.
10.6 Assessing Laboratory Performance
10.6.1 The laboratory must analyze at least one LFB per sample set (all samples analyzed
within a 24 hour period). The fortifying concentration in the LFB should be 10 times
the MDL. Calculate accuracy as percent recovery (X,). If the recovery falls outside
the control limits (See Section 10.6.2), the system is judged out of control, and the
source of the problem must be identified and resolved before continuing analyses.
10.6.2 Until sufficient LFB data become available, usually a minimum of results from 20 to
30 analyses, the laboratory should assess its performance against the control limits
described in Section 10.3.2. When sufficient laboratory performance data becomes
available, develop control limits from the mean percent recovery (X) and standard
deviation (S) of the percent recovery. These data are used to establish upper and
lower control limits as follows:
Upper Control Limit = X + 3S
Lower Control Limit = X - 3S
After each group of five to ten new recovery measurements, control limits should be
recalculated using only the most recent 20 to 30 data points.
279
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Method 548
10.6.3 It is recommended that the laboratory periodically determine and document its detec-
tion limit capabilities for endothall.
10.6.4 Each quarter the laboratory should analyze QCS (if available). If criteria provided
with the QCS are not met, corrective action should be taken and documented.
10.7 Assessing Analyte Recovery
10.7.1 The laboratory must add a known fortified concentration to a minimum of 10% of the
routine samples or one fortified sample per set, whichever is greater. The fortified
concentration should not be less than the background concentration of the sample
selected for spiking. The fortified concentration should be the same as that used for
the LFB (Section 10.6). Over time, samples from all routine sample sources should
be fortified.
10.7.2 Calculate the percent recovery (R,) for each analyte, corrected for background con-
centrations measured in the unfortified sample, and compare these values to the
control limits established in Section 10.6.2 for the analyses of LFBs.
10.7.3 If the recovery of any analyte falls outside the designated range, and the laboratory
performance for that analyte is shown to be in control (Section 10.6), the recovery
problem encountered with that dosed sample is judged to be matrix related, not system
related. The result for that analyte in the unfortified sample must be labelled sus-
pect/matrix to inform the data user that the results are suspect due to matrix effects.
7 7. PROCEDURE
11.1 Cleanup and Separation: Cleanup procedures may not be necessary for a relatively clean
samples matrix. If particular circumstances demand the use of an alternative cleanup proce-
dure, the analyst must demonstrate that the recovery of endothall is within the limits specified
by the method.
11.1.1 If the sample is not clean, or the complexity is unknown, the entire sample should be
centrifuged at 2500 rpm for 10 minutes. The supernatant is decanted from the centri-
fuge bottle and passed through glass fiber filter paper into a container which can be
tightly sealed.
11.1.2 Store all samples at 4°C.
11.2 Sample Extraction and Analysis
11.2.1 Measure out a 5.0 mL aliquot of the sample and place it in a 25 mL K-D tube. Add
boiling chips.
11.2.2 Place on tube heater at maximum setting and concentrate sample to less than 0.5 mL.
11.2.3 Add 4 mL glacial acetic acid, 200 mg sodium acetate and 1 mL of glacial acetic acid
containing 4 mg PFPH. Use glass stirring rod to break-up the sodium acetate solid.
Place a Micro Snyder column on each K-D tube.
11.2.4 Heat at !50°C for 90 minutes.
11.2.5 Dilute the reaction mixture with 50 mL reagent water. Wash the residue in the tube
into the aqueous solution.
280
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Method 548
11.2.6 Assemble the vacuum manifold. Rinse the solid sorbent cartridge by passing 5 mL of
reagent water though the cartridge. Discard the water. Extract the aqueous sample
from 12.5 by passing the sample through the solid sorbent cartridge at a rate of
5-6 mL per minute.
11.2.7 Wash the cartridge with 5 mL reagent water. Elute the cartridge with two 2 mL
aliquots of MTBE. Combine the eluates with .05 mL of the internal standard stock
solution (7.11.2) and dilute to 5 ml in a volumetric flask with MTBE.
11.2.8 Analyze the eluates by GC/ECD using conditions described in Table 1. This table
includes the retention time and MDL that were obtained under these conditions.
Sample chromatograms of an endothall standard and a LRB both with internal stan-
dard are represented in Figures 1 and 2. Other columns, chromatographic conditions,
or detectors may be used if the requirements of Section 10.3 are met.
11.3 Identification of Analytes
11.3.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound corre-
sponds, within limits, to the retention time of a standard compound, then identification
is considered positive. However, positive identifications should be confirmed by
retention time comparisons on the second GC column, or by using GC/MS.
11.3.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.3.3 Identification requires expert judgement when sample components are not resolved
chromatographically, that is, when GC peaks obviously represent more than one
sample component (i.e., broadened peak with shoulder(s) or valley between two or
more maxima, or any time doubt exists over the identification of a peak as a chroma-
togram, appropriate techniques such as use of an alternative detector which operates
on a chemical/physical principle different from that originally used, e.g., mass spec-
trometry, or the use of a second chromatography column must be used.
11.4 If the peak area exceeds the linear range of the calibration curve, a smaller sample volume
should be used. Alternatively, the final solution may be diluted with MTBE and reanalyzed.
11.5 If the peak area measurement is prevented by the presence of interferences, further cleanup is
required.
12. CALCULATIONS
12.1 Determine the peak area ratio for endothall in the injected sample.
12.1.1 Calculate the concentration of endothall injected using the calibration curve in Section
9.2. The concentration in a liquid sample can be calculated from Equation 1:
281
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Method 548
Equation 1
Concentration
(VS)
where:
A = Concentration of endothall in extract, in fig/L
VF = Final volume of MTBE, in mL
VS = Sample volume, in mL
12.2 Report results as micrograms per liter. When duplicate and fortified samples are analyzed,
report all data obtained with the sample results.
12.3 For samples processed as part of a set where the laboratory fortified sample recovery falls
outside of the control limits established in Section 10.3, data must be labeled as suspect.
13. METHOD PERFORMANCE
13.1 Method Detection Limits: The MDL is defined as the minimum concentration of a substance
that can be measured and reported with 99 % confidence that the value is above the background
level. The estimated MDL concentration listed in Table 1 was obtained using reagent water.
Similar results were achieved using representative matrices.
13.2 This method has not been tested for linearity of recovery from fortified reagent water.
13.3 In a single laboratory using dechlorinated tap and reagent water fortified matrices, the average
recoveries presented in Table 2 were obtained. The standard deviation of the percent recovery
is also included in Table 2.
282
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Method 548
References
40 CFR Part 136, Appendix B.
ASTM Annual Book of Standards, Part 31, D3694-78. "Standard Practices for Preparation of
Sample Containers and for Preservation of Organic Constituents", American Society for
Testing and Materials, Philadelphia, PA.
283
-------
Method 548
Table 1. Gas Chromatography Conditions and Method Detection Limits
Analyte Ret. Time (mm.) MDL (pg/L)
Endothall 42.3 11.5
GC conditions: 0.25 mm x 30 m SPB-5 column; 2 //L injection; (Split, 40:1) hold one minute at
60°C, program to 300°C at 4°C/minutes, hold at 300°C for 15 minutes.
284
-------
Method 548
Table 2. Single Operator Accuracy and Precision
Analyte
Endothall
Matrix
Type
Reagent Water
Dechlorinated Tap
Water
Average
Percent
Recovery
120
108
84.0
94.0
Standard
Deviation
{percent}
25.3
15.3
13.8
13.3
Fortified
Cone.
(fjg/L)
15
150
15
150
Number of
Analyses
8
8
8
8
100 mg/L sodium thiosulfate (Na2S203) added to prior to fortifying with endothall
285
-------
Method 548
Appendix
Preparation of Endothall-Pentafluorophenylhydrazine
1. Prepare solution A of endothall by dissolving 0.204 g of endothall monohydrate (1.0 mmol) in
14 mL of methylene chloride and 3.6 mL of dry tetrahydrofuran (THF).
2. Prepare solutions B of dicyclohexylcarbodiimide (DCC) by dissolving 0.206 g (1.0 mmol) in
3.4 mL of dry THF.
3. Mix solution A and B and cover with a watchglass. (Note: a white precipitate will form in 3
to 5 minutes).
4. Gently stir the mixture from Step 3 with a magnetic stirrer for 4.5 hours at ambient tempera-
ture.
5. Prepare solution C by dissolving 0.206 g of DCC and 0.198 g of pentafluorophenylhydrazine
(PFPH) in 18 mL of dry THF.
6. Mix solution C with the mixture from step 4, cover with a watchglass and stir the mixture
overnight (16 hours) at ambient temperature.
7. Filter the mixture and dry the filtrate under reduced pressure to yield a beige powder.
8. Recrystallize the beige powder with 20 mL of warm (40°C) methanol: H2O (8:2 v/v).
9. Filter the solution from Step 8 to remove the insoluble material.
10. Allow the filtrate from Step 9 to cool to room temperature. A precipitate will form immedi-
ately upon cooling.
11. Filter and wash the precipitate formed in Step 10 with two 1 mL portions of cold methanol:
H2O (8:2). Save the filtrate.
12. Allow the filtrate from Step 11 to stand overnight covered with a watchglass at ambient
temperature. A precipitate will form on standing.
13. Filter and wash the precipitate from Step 12 with two 1 mL portions of cold methanol: H20
(8:2).
14. Recrystallize the off white precipitate from Step 13 with 20 mL of warm methanol: H20
(8:2). Filter the warm solution and allow the filtrate to cool, producing a white, crystalline
precipitate.
15. Filter the white precipitate from Step 14, wash with two 1 mL portions of cold methanol: H20
(8:2) and dry under vacuum.
286
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Method 548
16. Determine the melting point of the precipitate of Step 15. The melting point of the endothall-
pentafluorophenylhydrazine derivative is 201.OC. If the melting point of the precipitate is not
within 1.0 C of this melting point, recrystallize again as per Step 14-15.
287
-------
00
00
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I
8-8
m
a. CD
If
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o'
o
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Detector Response
I I I I
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mVolts
984.50
3
a
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00
-------
Method 548
Eo>
Endosulfan I (40.42)
600 —
0)
c
0
Q.
(/)
0>
cc
2
o
s
0
Q
500 —
400 —
300 —
200 —
100 —
1
\
\
\
\
\
\ ^ — ^\.
Elution Time (Minutes)
52-015-34
Figure 2. Representative Chromatogram of a Laboratory Reagent Blank
289
-------
-------
Method 548.1
Determination of Endothall in Drinking
Water by Ion-Exchange Extraction,
Acidic Methanol Methylation
and Gas Chromatography/
Mass Spectrometry
Revision 1.0 - EPA EMSL-Ci
August 1992
Jimmie W. Hodgeson
Jeffrey Collins (Technology Applications, Inc.)
W. J. Bashe (Technology Applications, Inc.)
-------
-------
Method 548.1
Determination of Endothall in Drinking Water by
Ion Exchange Extraction, Acidic Methanol Methylation
and Gas Chromatography/Mass Spectrometry
1. SCOPE AND APPLICA TION
1.1 This method is for the identification and simultaneous measurement of endothall in drinking
water sources and finished drinking water. The following analyte can be determined by this
method:
Analyte CAS No.
Endothall 145-73-3
1.2 This is a gas chromatographic/mass spectrometric (GC/MS) method. However, a flame
ionization detector (FID) may be utilized for the determination, but must be supported by an
additional analysis using a confirmatory gas chromatographic column.
1.3 The method detection limit1 (MDL, defined in Sect. 13) for endothall is listed in Table 1 for
both GC/MS and FID. The MDL may differ from the listed value depending upon the nature
of interferences in the sample matrix. In particular, water sources containing high levels of
dissolved calcium, magnesium and sulfate may require sample dilution before extraction to
obtain adequate endothall recovery. Guidelines (Sect. 4.2 and Sect. 11.2.1) are provided on
levels of these ions above which dilution is recommended, as well as appropriate dilution
factors.
1.4 In this ion exchange liquid-solid extraction procedure, endothall may be esterified directly in
the elution solvent, acidic methanol.
1.5 The method performance data provided in this method were obtained using both a GC/MS
system and a gas chromatograph with a flame ionization detector (FID). Modern GC/MS
instruments have sensitivities at least equivalent to the FID. If the analyst has access to a
GC/MS system meeting the specifications described in Sect. 6.10, it should be as the primary
means of identification and measurement.
2. SUMMARY OF METHOD
2.1 Liquid-solid extraction (LSE) cartridges containing an intermediate strength, primarily tertiary
amine anion exchanger are mounted on a vacuum manifold and conditioned with appropriate
solvents. LSE disks may be used instead of cartridges of all quality control criteria specified
in Sect. 9 are met. A 100-mL sample is extracted and the analyte is eluted with 8-mL of
acidic methanol. After addition of a small volume of methylene chloride as a co-solvent, the
dimethyl ester of endothall is formed within 30 min with modest heating (50°C). After
addition of salted reagent water, the ester is partitioned into 8-10 mL of methylene chloride.
The extract volume is reduced to 1 mL with nitrogen purge for a concentration factor of 100.
The extract is analyzed by GC/MS or GC/FID with a megabore capillary column.
293
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Method 548. 1
3. DEFINITIONS
3.1 Internal Standard (IS): A pure analyte(s) added to a sample, extract, or standard solution in
known amount(s) and used to measure the relative responses of other method analytes and
surrogates that are components of the same sample or solution. The internal standard must be
an analyte that is not a sample component.
3.2 Surrogate Analyte (SA): A pure analyte(s), which is extremely unlikely to be found in any
sample, and which is added to a sample aliquot in known amount(s) before extraction or other
processing and is measured with the same procedures used to measure other sample com-
ponents. The purpose of the SA is to monitor method performance with each sample.
3.3 Laboratory Duplicates (LDl and LD2): Two aliquots of the same sample taken in the labora-
tory and analyzed separately with identical procedures. Analyses of LDl and LD2 indicate the
precision associated with laboratory procedures, but not with sample collection, preservation,
or storage procedures.
3.4 Field Duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory Reagent Blank (LRB): An aliquot of reagent water or other blank matrix that is
treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents,
internal standards, and surrogates that are used with other samples. The LRB is used to
determine if method analytes or other interferences are present in the laboratory environment,
the reagents, or the apparatus.
3.6 Field Reagent Blank (FRB): An aliquot of reagent water or other blank matrix that is placed
in a sample container in the labora-tory and treated as a sample in all respects, including
shipment to the sampling site, exposure to sampling site conditions, storage, preservation, and
all analytical procedures. The purpose of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.7 Instrument Performance Check Solution (IPC): A solution of one or more method analytes,
surrogates, internal standards, or other test substances used to evaluate the performance of the
instrument system with respect to a defined set of method criteria.
3.8 Laboratory Fortified Blank (LFB): An aliquot of reagent water or other blank matrix to which
known quantities of the method analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine whether the methodology is in control,
and whether the laboratory is capable of making accurate and precise measurements.
3.9 Laboratory Fortified Sample Matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
254
-------
Method 548.1
3.10 Stock Standard Solution (SSS): A concentrated solution containing one or more method
analytes prepared in the laboratory using assayed reference materials or purchased from a
reputable commercial source.
3.11 Primary Dilution Standard Solution (PDS): A solution of several analytes prepared in the
laboratory from stock standard solutions and diluted as needed to prepare calibration solutions
and other needed analyte solutions.
3.12 Calibration Standard (CAL): A solution prepared from the primary dilution standard solution
or stock standard solutions and the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality Control Sample (QCS): A solution of method analytes in known concentrations which
is used to fortify an aliquot of LRB or sample matrix. The QCS is obtained from a source
external to the laboratory and different from the source of calibration standards. It is used to
check laboratory performance with externally prepared test materials.
4. INTERFERENCES
4.1 Method interference may be caused by contaminants in solvents, reagents, glassware, and
other sample processing hardware that lead to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
ferences under the analytical conditions by analyzing laboratory reagent blanks as described in
Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned2 as soon as possible after use by rinsing with
the last solvent used in it. This should be followed by detergent washing with hot
water, and rinses with tap water and distilled water. It should then be drained dry,
and heated in a laboratory oven at 400°C for several hours before use. Solvent rinses
with methanol may be substituted for the oven heating. After drying and cooling,
glassware should be stored in a clean environment to prevent any accumulation of dust
or other contaminants.
4.1.2 The use of high purity reagents and solvents is absolutely necessary to minimize
interference problems. Purification of solvents by distillation in all-glass systems
immediately prior to use may be necessary.
4.2 The major potential interferences in this ion-exchange procedure are other naturally occurring
ions in water sources, namely, dissolved calcium, magnesium and sulfate. These are the only
ions thus far demonstrated to be interferences when present at concentrations possibly occur-
ring in drinking water sources. For example, the sources identified in Tables 3 and 4 con-
tained elevated concentrations of these ions and reduced recoveries were observed. Sulfate is
an effective counter ion, and displaces endothall from the column when present at high concen-
trations. On the other hand, both calcium and magnesium complex the endothall anion, which
then is no longer available in ionic form for ion-exchange extraction. Table 4 illustrates that
sample dilution or the addition of ethylenediamine tetraacetic acid for complexing the cations,
or a combination of the two, may be used. Figure 1 illustrates quantitatively the separate
effects of these ions on recovery.
4.3 The extent of interferences that may be encountered using this method has not been fully
assessed. Although the GC conditions described allow for a unique resolution of endothall,
295
-------
Method 548.1
other matrix components may interfere. Matrix interferences may be caused by contaminants
that are coextracted from the sample. Matrix interferences will vary considerably from source
to source, depending on the nature of the matrix being sampled. A distinct advantage of this
method is that the anion exchange cartridge provides an effective clean-up mechanism for
many potential organic matrix interferences. Many neutral and basic organics retained by the
column are removed by the methanol wash step of Sect. 11.2.3. The most probable matrix
interferences are other organic acids or phenols retained by the column. For the cartridge to
effectively serve for both sample clean-up and analyte extraction, it is critical that the condi-
tioning steps described in Sect. 11.2.1 be followed exactly.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound should be treated as a potential health hazard.
From this viewpoint, exposure to these chemicals must be minimized. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations regarding the safe
handling of the chemical specified in this method. A reference file of material data handling
sheets should also be made available to all personnel involved in the chemical analysis.
Additionally references to laboratory safety are available.3"5
6. EQUIPMENT AND SUPPLIES
6.1 Sampling Equipment (for discrete or composite sampling). Amber glass bottles (250 mL or
larger) fitted with screw caps lined with Teflon. If amber bottles are not available, protect
samples from light. The container must be washed, rinsed with methanol, and dried before
use to minimize contamination.
6.2 Separator Funnels: 125 mL, with Teflon stopcocks, ground glass or Teflon stoppers.
6.3 Screw Cap: 125 x 13 mm, culture tubes. Screw caps should have Teflon liners.
6.4 Graduated 15 mL centrifuge tubes with #13 ground glass stoppers
6.5 Pasteur Pipets: Glass, disposable 5-3/4" length
6.6 Balance: Analytical, capable of weighing to .0001 g.
6.7 Six or twelve position analytical concentrator (Organomation, N-EVAP model # 111/6917 or
equivalent).
6.8 pH Meter
6.9 Gas Chromatograph: Analytical system complete with GC suitable for flame ionization
detection, split/splitless capillary injection temperature programming, and all required
accessories including syringes, analytical columns, gases and strip chart recorder. A data
system is recommended for measuring peak areas. An auto injector is recommended for
improved precision of analysis.
6.10 Gas Chromatograph/mass Spectrometer/data System (GC/MS/DS):
6.10.1 The GC must be capable of temperature programming and be equipped for
split/splitless or on-column capillary injection. The injection tube liner should be
296
-------
Method 548.1
quartz and about 3 mm in diameter. The injection system must not allow the analytes
to contact hot stainless steel or other metal surfaces that promote decomposition.
6.10.2 The GC/MS interface should allow the capillary column or transfer line exit to be
placed within a few mm of the ion source. Other interfaces, for example, the open
split interface, are acceptable as long as the system has adequate sensitivity (See Sect.
10 for calibration requirements).
6.10.3 The mass spectrometer must be capable of electron ionization at a nominal electron
energy of 70 eV and of scanning from 45 to 450 amu with a complete scan cycle time
(including scan overhead) of 1.5 sec or less. (Scan cycle time = Total MS data
acquisition time in sec divided by total number of scans in the chromatogram). The
spectrometer must produce a mass spectrum that meets all criteria in Table 5 when 5
to 10 ng of DFTPP is introduced into the GC. An average spectrum across the
DFTPP GC peak may be used to test instrument performance.
6.10.4 An interfaced data system is required to acquire, store, reduce, and output mass
spectral data. The computer software must have the capability of processing stored
data by recognizing a GC peak within any given retention time window, comparing
the mass spectra from the GC peak with spectral data in a user-created data base, and
generating a list of tentatively identified compounds with their retention times and
scan numbers. The software must also allow integration of the ion abundance of any
specific ion between specified time or scan number limits, calculation of response
factors as defined in Sect. 10.3.6 (or construction of a second or third order
regression calibration curve), calculation of response factor statistics (mean and
standard deviation), and calculation of concentrations of analytes as described in
Sect. 12.
6.11 GC Columns
6.11.1 GC/MS: DBS, 30 m x 0.25 mm, 0.25 /mi film thickness
6.11.2 FID Primary: RTX Volatiles, 30 m x 0.53 mm. ID, 2.0 urn film thickness, Restek
Catalog No. 10902.
6.11.3 FID Confirmation: DBS, 30 m x 0.32 mm ID, 0.25 pm film thickness
6.12 Liquid-solid Extraction Vacuum System: May be used.
6.13 8 mL Liquid-solid Extraction Cartridges With Frits: Also available from a number of
commercial suppliers. Appropriate liquid-solid extraction disks may also be used in this
method if equivalent or better quality assurance data can be demonstrated (See Sect. 9).
6.14 Liquid-solid Extraction 70 mL Reservoirs And Adapters: Baxter Catalog # 9442 (adapter
catalog # 9430) or equivalent.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagent Water: Reagent water is defined as water in which an interference is not observed at
the endothall method detection.
7.1.1 A Millipore Super-Q Water System or its equivalent may be used to generate
deionized reagent water. Distilled water that has been charcoal filtered may also be
suitable.
297
-------
Method 548.1
7.2 Methanol: Pesticide quality.
7.3 Methylene Chloride: Pesticide quality or equivalent.
7.4 Sodium Sulfate—ACS Granular: Heat in a shallow tray for 4 hrs at 400°C to remove
phthlates and other interfering organic substances or extract with methylene chloride in a
Soxhlet apparatus for 48 hrs.
7.5 10% Sulfuric Acid In Methanol: Using extreme caution, slowly dissolve reagent grade
sulfuric (10% v/v) acid in methanol.
7.6 Sodium Hydroxide (NAOH) 1 N: Dissolve 4 g ACS grade in reagent water and dilute up to
100 mL in a 100 mL volumetric flask.
7.7 10% Sodium Sulfate In Reagent Water: Dissolve 100 g sodium sulfate in reagent water and
dilute to volume in a 1-L volumetric flask.
7.8 Biorex 5 Anion Exchange Resin: BioRad Laboratories Catalog No. 140-7841.
7.9 Disodium Ethylenediamine Tetraacetate (EDTA): Certified ACS Fisher or equivalent.
7.10 Endothall, Monohydrate: Available as neat material from Ultra Scientific, North Kingston, RI
or as a concentrated solution from NSI Environmental Solutions, Research Triangle Park, NC.
7.11 Acenapthene-dlO: Available from MSD Isotopes or Cambridge Chemicals.
7.12 Stock Standard Solutions
7.12.1 Endothall: 50/xg/mL in methanol
7.12.2 Acenaphthene-dlO: 500 pg/mL in methanol. Dissolve 25 mg (approximately 32.2
/xL) Acenapthnene-dlO in 50 mL methanol. Prepare a working standard at 10 ^g/mL
by a 1:50 dilution of the stock standard.
7.12.3 Decafluorotriphenylphosphine(DFTPP): 5/xg/mL.
7.12.4 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional sampling practices should
be followed, except that the bottle must not be prewashed with sample before collection.
Composite samples should be collected in refrigerated glass containers. Automatic sampling
equipment must be as free as possible of plastic tubing and other potential sources of
contamination.
8.2 Sample Preservation
8.2.1 If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
the sample bottle prior to collecting the sample.
8.2.2 After adding the sample to the bottle containing the sodium thiosulfate, seal the bottle
and shake vigorously for 1 min.
8.2.3 The samples must be iced or refrigerated at 4°C from the time of collection until
extraction and analysis. Endothall is not known to be light sensitive, but excessive
exposure to light and heat should be avoided.
298
-------
Method 548.1
8.2.4 A graphical representation of the results of a 14-day holding stability study on
endothall in three different water matrices is presented in Figure 2. These matrices
were a dechlorinated tap water sample, a filtered river water sample containing
considerable biological activity and the same river water biologically preserved at
pH 2. These data indicate that the samples may be held for 7 days before extraction
under the conditions of Sect. 8.2.3. Endothall appears to be biologically stable over
7 days. However, the chemical and biological stability of endothall may be matrix
dependent. The analyst may verify analyte stability in the matrix of interest by
conducting appropriate holding studies. Samples with unusually high biological
activity should be acidified to pH 1.5 to 2.0 with 1:1 HC1:H20.
8.3 Extract Storage: Sample extracts should be stored in the dark at 4°C or less. A maximum
extract holding time of 14 days is recommended.
9. QUALITY CONTROL
9.1 Each laboratory that uses this method is required to operate a formal quality control (QC)
program. The minimum QC requirements are initial demonstration of laboratory capability,
analysis of laboratory reagent blanks, laboratory fortified blanks, laboratory fortified matrix
samples and QC check standards.
9.2 Laboratory Reagent Blanks: Before processing any samples, the analyst must demonstrate that
all glassware and reagent interferences are under control. Each time a set of samples is
analyzed or reagents are changed, a laboratory reagent blank must be analyzed. For this
method, the blank matrix is filtered reagent water. If within the retention time window of
endothall, the reagent blank produces a peak which prevents the measurement of endothall,
determine the source of contamination and eliminate the interference before processing
samples.
9.3 Initial Demonstration Of Capability
9.3.1 Select a representative fortified concentration for endothall. Prepare a methanol
solution containing endothall at 1000 times the selected concentration. The concen-
trate must be prepared independently from the standards used to prepare the calibra-
tion curve (Sect. 10.2). With a syringe, add 100 /iL of the concentrate to each of
four to seven 100-mL aliquots of reagent water and analyze each aliquot according to
procedures in Sect. 11.
9.3.2 Calculate the mean percent recovery (R), the relative standard deviation of the recov-
ery (RSD in Table 2), and the MDL (1). The mean recovery must fall in the range of
R ± 20% using the values for R (Recovery) for reagent water (Table 2). The stan-
dard deviation should be less than 30%. If these acceptance criteria are met, perfor-
mance is acceptable and sample analysis may begin. If either of these criteria fails,
initial demonstration of capability should be repeated until satisfactory performance
has been demonstrated.
9.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples by a new, unfamiliar method prior to demonstrating a
basic level of skill at performing the technique. As laboratory personnel gain experi-
299
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Method 548.1
ence with this method the quality of the data should improve beyond the requirements
stated in Sect. 9.3.2.
9.4 The analyst is permitted to modify GC columns or GC conditions to improve separations or
lower analytical costs. Each time such method modifications are made, the analyst must repeat
the procedures in Sect. 9.3.
9.5 Assessing the Internal Standard: In using the IS calibration procedure, the analyst is expected
to monitor the IS response (peak area) of all samples during each analysis day. The IS
response for any sample chromatogram should not deviate from the most recent calibration
check standard IS response by more than 30%.
9.5.1 If a deviation of greater than 30% is encountered for a sample, reinject the extract.
9.5.1.1 If acceptable IS response is achieved for the reinjected extract, then report
the results for that sample.
9.5.1.2 If a deviation of greater than 30% is obtained for the reinjected extract,
analysis of the sample should be repeated beginning with Sect. 11, pro-
vided the sample is still available. Otherwise, report results obtained from
the reinjected extract, but annotate as suspect.
9.5.2 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
medium calibration check standard.
9.5.2.1 If the check standard provides a response factor (RF) within 20% of the
predicted value, then follow procedures itemized in Sect. 9.5.1 for each
sample failing the IS response criterion.
9.5.2.2 If the check standard provides a response factor (RF) which deviates more
than 20% from the predicted value, then the analyst must recalibrate, as
specified in Sect. 10.2.
9.6 Assessing Laboratory Performance
9.6.1 The laboratory must analyze at least one laboratory fortified blank (LFB) per sample
set (all samples extracted within a 24-hr period). The fortifying concentration in the
LFB should be 10 to 20 times the MDL. Calculate accuracy as percent recovery (Rj).
If the recovery falls outside the control limits (See Sect. 9.6.2), the system is judged
out of control, and the source of the problem must be identified and resolved before
continuing analyses.
9.6.2 Until sufficient LFB data become available, usually a minimum of results from 20 to
30 analyses, the laboratory should assess its performance against the control limits
described in Sect. 9.3.2. When sufficient laboratory performance data become avail-
able, develop control limits from the mean percent recovery (R) and standard devia-
tion (S) of the percent recovery. These data are used to establish upper and lower
control limits as follows:
Upper Control Limit = R + 3S
Lower Control Limit = R - 3S
After each group of five to ten new recovery measurements, control limits should be
recalculated using only the most recent 20 to 30 data points.
300
-------
Method 548.1
9.6.3 Each laboratory should periodically determine and document its detection limit capa-
bilities for endothall.
9.6.4 Each quarter the laboratory should analyze quality control samples (if available). If
criteria provided with the QCS are not met, corrective action should be taken and
documented.
9.7 Assessing Analyte Recovery
9.7.1 The laboratory must add a known fortified concentration to a minimum of 10% of
samples or one fortified matrix sample per set, whichever is greater. The fortified
concentration should not be less than the background concentration of the sample
selected for fortification. The fortified concentration should be the same as that used
for the LFB (Sect. 9.6). Over time, samples from all routine sample sources should
be fortified.
9.7.2 Calculate the percent recovery for endothall, corrected for background concentrations
measured in the unfortified sample, and compare these values to the control limits
established in Sect. 9.6.2 for the analyses of LFBs.
9.7.3 If the recovery falls outside the designated range and the laboratory performance for
that sample set is shown to be in control (Sect. 9.6), the recovery problem encoun-
tered with the fortified sample is judged to be matrix related, not system related. The
result in the unfortified sample must be labelled suspect/matrix to inform the data user
that the results are suspect due to matrix effects.
1 0. CALIBRA TION AND STANDARDIZA TION
10.1 Preparation Of Calibration Standards
10.1.1 Calibration standards as dimethyl esters are prepared by addition of aliquots of the
endothall stock standard (Sect. 7.12.1) to the esterification reaction mixture,
consisting of 8 mL of 10% H2SO4/methanol and 6 mL of methylene chloride in the
screw cap culture tubes (Sect. 6.3). The standards are then esterified and partitioned
into the organic phase according to Sect. 11.4. Prepare endothall acid standards
equivalent to aqueous standards at 100, 50, 25 and 5 jig/L by addition of the
following aliquots of the stock standard solution (Sect. 7.12) to the esterification
reaction mixture—200 /*L, 100 /*L, 50 /xL and 10 /*L. By way of illustration, 200 /xL
of the 50 fj.g/mL stock contains 10 /ig of endothall. When dissolved in 100 mL of
water, the aqueous concentration is 100 /ig/L.
10.1.2 Process each standard as described in Sect. 11.4.1 and Sect. 11.4.2. The internal
standard is added as described in Sect. 11.4.3. Triplicate samples should be prepared
at each concentration level.
10.2 Demonstration and documentation of acceptable initial calibration are required before any
samples are analyzed and intermittently throughout sample analyses as dictated by results of
continuing calibration checks. After initial calibration is successful, a continuing calibration
check is required at the beginning of each 8-hr period during which analyses are performed.
Additional periodic calibration checks are good laboratory practice.
301
-------
Method 548.1
10.3 Initial Calibration
10.3.1 Calibrate the mass spectrometer with calibration compounds and procedures
prescribed by the manufacturer with any modifications necessary to meet the
requirements in Sect. 10.3.2.
10.3.2 Inject into the GC a 1-or 2-fj.L aliquot of the 5 ng/juL DFTPP solution and acquire a
mass spectrum that includes data for m/z = 45-450. Use GC conditions that produce
a narrow (at least five scans per peak) symmetrical peak. If the spectrum does not
meet all criteria (Table 5), the MS must be retuned to meet all criteria before
proceeding with calibration. An average spectrum across the GC peak may be used to
evaluate the performance of the system.
10.3.3 Inject a 1-/*L aliquot of a medium concentration calibration solution, for example 50
Mg/L, and acquire and store data from m/z 45-450 with a total cycle time (including
scan overhead time) of 1.5 sec or less. Cycle time should be adjusted to measure at
least five or more spectra during the elution of the GC peak. Figure 3 illustrates a
total ion chromatogram and mass spectrum of endothall and the internal standard,
acenaphthene-dlO, using the prescribed conditions.
10.3.4 If all performance criteria are met, inject a l-/xL aliquot of each of the other
calibration solutions using the same GC/MS conditions.
10.3.5 Calculate a response factor (RF) for endothall for each calibration solution by use of
the internal standard response as expressed below. This calculation is supported in
acceptable GC/MS data system software (Sect. 6.10.4), and many other software
programs. The RF is a unitless number, but units used to express quantities of
analyte and internal standard must be equivalent.
Where:
Ax = integrated abundance of the quantitation ion of the analyte (m/z 183).
Ais = integrated abundance of the quantitation ion internal standard (m/z 164).
Qx = quantity of analyte injected in ng or concentration units.
Qis = quantity of internal standard injected in ng or concentration units.
10.3.5.1 Calculate the mean RF from the analyses of the calibration solutions.
Calculate the standard deviation (SD) and the relative standard deviation
(RSD) from each mean: RSD = 100 (SD/M). If the RSD of any analyte
or surrogate mean RF exceeds 30%, either analyze additional aliquots of
appropriate calibration solutions to obtain an acceptable RSD of RFs over
the entire concentration range or take action to improve GC/MS perfor-
mance. See Sect. 10.4.5 for possible remedial actions.
302
-------
Method 548.1
10.3.6 As an alternative to calculating mean response factors and applying the RSD test, use
the GC/MS data system software or other available software to generate a linear or
second order regression calibration curve.
10.4 Continuing calibration check. Verify the MS tune and initial calibration at the beginning of
each 8-hr work shift during which analyses are performed using the following procedure.
10.4.1 Inject a 1-/*L aliquot of the 5 ng//xL DFTPP solution and acquire a mass spectrum that
includes data for m/z 45-450. If the spectrum does not meet all criteria (Table 5), the
MS must be retuned to meet all criteria before proceeding with the continuing
calibration check.
10.4.2 Inject a 1-^L aliquot of a medium concentration calibration solution and analyze with
the same conditions used during the initial calibration.
10.4.3 Determine that the absolute area of the quantitation ion of the internal standard has not
decreased by more than 30% from the area measured in the most recent continuing
calibration check, or by more than 50% from the area measured during initial calibra-
tion. If the area has decreased by more than these amounts, adjustments must be
made to restore system sensitivity. These adjustments may require cleaning of the MS
ion source, or other maintenance as indicated in Sect. 10.4.5, and recalibration. Con-
trol charts are useful aids in documenting system sensitivity changes.
10.4.4 Calculate the RF for endothall from the data measured in the continuing calibration
check. The RF must be within 30% of the mean value measured in the initial
calibration. Alternatively, if a linear or second order regression is used, the
concentration measured using the calibration curve must be within 30% of the true
value of the concentration in the medium calibration solution. If these conditions do
not exist, remedial action must be taken which may require repeating the initial
calibration.
10.4.5 Some possible remedial actions: major maintenance such as cleaning an ion source,
cleaning quadrupole rods, etc. require returning to the initial calibration step.
10.4.5.1 Check and adjust GC and/or MS operating conditions; check the MS
resolution, and calibrate the mass scale.
10.4.5.2 Clean or replace the splitless injection liner; silanize a new injection liner.
10.4.5.3 Flush the GC column with solvent according to manufacturer's
instructions.
10.4.5.4 Break off a short portion (about 1 meter) of the column from the end near
the injector; or replace GC column. This action will cause a slight change
in retention times.
10.4.5.5 Prepare fresh CAL solutions, and repeat the initial calibration step.
10.4.5.6 Clean the MS ion source and rods (if a quadrupole).
10.4.5.7 Replace any components that allow analytes to come into contact with hot
metal surfaces.
10.4.5.8 Replace the MS electron multiplier or any other faulty components.
303
-------
Method 548.1
11. PROCEDURE
11.1 Preparation Of Anion Exchange Cartridges
11.1.1 Prepare a 50% (v/v) slurry of Bio-Rex 5 resin and reagent water.
11.1.2 Attach the required number of 8-mL extraction cartridges (Sect. 6.13) to the vacuum
manifold (Sect. 6.12), and insert bottom fritted disks into each cartridge.
11.1.3 Fill the cartridges completely with Bio-Rex 5 slurry. Draw off excess water with
vacuum. The final wet resin bed height should be 3.5 + 0.1 cm. Adjust the height
by adding more slurry and repeating procedure, or add more reagent water to
reservoir and remove excess resin slurry.
11.1.4 After the bed heights are adjusted to 3.5 cm and with excess water removed under
vacuum, insert a fritted disk on top of the resin bed. The fritted disk should press
firmly into the resin and be horizontal to the reservoir to prevent sample channeling
around the disk. Fill the cartridges with reagent water and draw half of the water into
the resin. Maintain the resin cartridges in this condition until ready for use.
NOTE: The use of liquid-solid extraction disks instead of cartridges is permissible
as long as all the quality control criteria specified in Sect. 9 of this method
are met.
11.2 Sample Preparation
11.2.1 As discussed above (Sect. 1.3 and Sect. 4.2), reduced recoveries will be observed if
the sample contains elevated levels of Call, Mgll or sulfate. If facilities are available,
measure the concentrations of these ions. Figure 1 graphically presents analyte
recovery versus individual ion concentration. Reduced recoveries may be anticipated
when the combined Call -I- Mgll exceeds approximately 100 mg/L or sulfate exceeds
approximately 250 mg/L. If measurement of ion concentration is not feasible,
determine the actual recovery for a laboratory fortified sample matrix as described in
Sect. 9.7. In the event of anticipated or measured low recoveries, treat the sample as
described in Sect. 11.2.2.
11.2.2 For samples containing moderately high levels of these ions, add 186 mg of EDTA
(Sect. 7.9) per 100-mL sample (0.005 M). The treated ground water characterized in
Table 3 is an example of a matrix successfully treated this way. For samples
containing very high levels of sulfate, sample dilution may be required in addition to
the EDTA. The western surface water characterized in Table 3 (ca. 2000 mg/L
sulfate) was successfully analyzed after dilution by a factor of 10 and the addition of
75 mg EDTA per 100 mL of the diluted sample (0.002 M). Samples containing
intermediate levels of sulfate can be analyzed with smaller dilution factors.
Guidelines on dilution factors and EDTA addition are given below.
Added EDTA
Sulfate fmg/U Dilution Factor (mg/100 mL)
< 250 1:1 186
250-500 1:2 125
500-1250 1:5 75
> 1250 1:10 75
304
-------
Method 548.1
NOTE: Dilution should not be employed if adequate recovery is attained by the
addition ofEDTA alone.
11.2.3 The addition of EDTA results in a large reagent peak near the end of the temperature
program. Therefore, complete the entire program described in Table 1.
11.2.4 If the ionic nature of the samples being processed is completely unknown, the analyst
as an option may routinely dilute all samples by a factor of 10 and add EDTA as
above. However, the analyst should be able to demonstrate reagent water MDLs of 2
/ig/L or lower. In this event the MDL will be 20 /tg/L or less for the diluted sample,
still a factor of 5 below the regulated maximum contaminant level.
11.3 Sample Extraction
11.3.1 Attach the 70-mL reservoir to the resin cartridge with the adapter (Sect. 6.14).
11.3.2 Condition the resin cartridge by drawing the following reagents through the cartridge
in the following order:
1. 10 mL methanol
2. 10 mL reagent water
3. 10 mL 10% H2S04 in methanol
4. 10 mL reagent water
5. 20 mL 1 N NaOH
6. 20 mL reagent water
Do not allow the cartridge to become dry between steps. Draw each reagent through
the cartridge at a rate of 10 mL/min. Leave a 1-cm layer of reagent water over the
resin bed.
11.3.3 Fill the 70-mL reservoir with 60 mL of the sample. Adjust sample flow rate to 3
mL/min. Add the balance of sample when needed to prevent the reservoir from going
dry.
11.3.4 After the sample passes through the cartridge, remove the 70-mL reservoir and the
adapter. Draw 10 mL of methanol through the resin cartridge. Make sure that any
visible water inside the cartridge dissolves in methanol. Next draw room air through
the cartridge for 5 min under a vacuum of 10-20 in. Hg. Position the culture tube
(Sect. 6.3) inside the manifold to collect the eluent.
11.3.5 Elute the cartridge with 8 mL of 10% H2S04 in methanol, followed by 6 mL of
methylene chloride under vacuum over a 1 min period.
11.4 Sample Derivatization, Partition And Analysis
11.4.1 Cap the culture tube and hold at 50°C for 1 hr in a heating block or water bath.
Remove from heat and allow the tube to cool for 10 min.
11.4.2 Pour the contents of the culture tube into a 125-mL separatory funnel. Rinse the tube
with two x 0.5 mL aliquots of methylene chloride and add the rinsings to the separa-
tory funnel. Add 20 mL of 10% sodium sulfate in reagent water to the separatory
305
-------
Method 548.1
funnel. Shake the funnel three times vigorously, venting with the stopcock, and then
shake vigorously for an additional 15 sec. After the phases have separated, drain the
lower organic layer into a 15-raL graduated centrifuge tube (Sect. 6.4). Repeat the
extraction procedure above with two additional 2-mL aliquots of methylene chloride,
adding the organic phase to the centrifuge tube each time.
11.4.3 Fortify the extract with 250 jiL of the internal standard working solution (Sect.
7.12.2) and concentrate to a final volume of 1.0 mL, using the N-EVAP (Sect. 6.7)
and dry nitrogen.
11.4.4 Inject 2 p.L of the concentrated extract (Sect. 11.4.3) and analyze by GC/MS using
the conditions described in Table 1. This table includes the retention time and MDL
that were obtained under these conditions. A sample total ion chromatogram of
endothall and d-10 acenaphthene illustrating retention times, and the mass spectrum of
the dimethylated endothall are shown in Figure 3. Other columns, chromatographic
conditions, or detectors may be used if the requirements of Sect. 9.3 are met.
11.4.5 If the peak area exceeds the linear range of the calibration curve, a smaller sample
volume should be used.
11.5 Identification Of The Analyte
11.5.1 Identify endothall by comparison of its mass spectrum (after background subtraction)
to a reference spectrum in a user created spectral library. The GC retention time of
the sample component should be within 10 sec of the retention time of endothall in the
latest calibration standard. If a FID is used, identifications should be confirmed by
retention time comparisons on the second GC column (Table 1).
11.5.2 In general, all ions present above 10% relative abundance in the mass spectrum of the
standard should be present in the mass spectrum of the sample component and should
agree within absolute 20%. For example, if an ion has a relative abundance of 30%
in the standard spectrum, its abundance in the sample spectrum should be in the range
of 10-50%. However, the experience of the analyst should weigh heavily in the
interpretation of spectra and chromatograms.
11.5.3 Identification requires expert judgement when sample components are not resolved
chromatographically, that is, when GC peaks from interferences are present. When
endothall coelutes with an interference, indicated by a broad peak or a shoulder on the
peak, the identification criteria can usually be met, but the endothall spectrum will
contain extraneous ions contributed by the coeluting interfering compound.
12. DATA ANALYSIS AND CALCULATIONS
12.1 When using GC/MS, complete chromatographic resolution is not necessary for accurate and
precise measurements of analyte concentrations if unique ions with adequate intensities are
available for quantitation. However, when using FID, complete resolution is essential.
12.1.1 Calculate endothall concentration.
306
-------
Method 548.1
c =
* (A,)RF V
where:
C = concentration of endothall in \nglL in the water sample.
Ax = integrated abundance of thequantitation ion of endothall (mlz 183) in the sample.
Ais = integrated abundance of the quantitation ion of the internal standard (mlz 164) in the sample.
Qa = total quantity (in micrograms) of internal standard added to the water sample.
V = original water sample volume in liters.
RV = mean response factor endothall the initial calibration.
12.1.2 Alternatively, use the GC/MS data system software or other available proven
software to compute the concentration of the endothall from the linear calibration or
the second order regression curves.
12.1.3 Calculations should utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures (one
digit of uncertainty). Experience indicates that three significant figures may be used
for concentrations above 99 j*g/L, two significant figures for concentrations between
1-99 (j.g/L, and one significant figure for lower concentrations.
13. METHOD PERFORMANCE
13.1 Method Detection Limits: The MDL is defined as the minimum concentration of a substance
that can be measured and reported with 99% confidence that the value is above the background
level.' The MDLs listed in Table 1 were obtained using reagent water for detection by
GC/MS and FID.
13.2 In a single laboratory study on fortified reagent water and ground water matrices, the mean
recoveries and relative standard deviations presented in Table 2 were obtained. Table 3
provides the concentrations of Call, Mgll and sulfate for two high ionic strength drinking
water sources studied. Table 4 presents mean recovery data for these fortified sources with
and without the addition of EDTA and/or sample dilution.
14. POLLUTION PREVENTION
14.1 This method utilizes the new liquid-solid extraction technology which requires the use of very
little organic solvent thereby eliminating the hazards involved with the use of large volumes of
organic solvents in conventional liquid-liquid extractions. It also uses acidic methanol as the
derivatizing reagent in place of the highly toxic and explosive diazomethane. These features
make this method much safer for the analyst to employ and a great deal less harmful to the
environment.
307
-------
Method 548.1
15. WASTE MANAGEMENT
15.1 Due to the nature of this method, there is very little need for waste management. No large
volumes of solvents or hazardous chemicals are used. The matrices are drinking water or
source water, and can be discarded down the sink.
308
-------
Method 548.1
References
1. 40 CFR Part 136, Appendix B.
2. ASTM Annual Book of Standards, Part 31, D3694-78. "Standard Practices for Preparation of
Sample Containers and for Preservation of Organic Constituents," American Society for
Testing and Materials, Philadelphia, PA.
3. "Carcinogens-Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
4. "OSHA Safety and Health Standards, General Industry," (29CF/J1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
309
-------
Method 548.1
Table 1. Retention Times And Method Detection Limits
Retention Time (min.)
Method Detection Limit
frg/U'
Compound \ Column A\ Column B \ Column C GC/MS | FID
Endothall 16.02 19.85 18.32 1.79 0.7
d10-Acenaphthene 14.69
'Based on 7 replicate analyses of a reagent water fortified at 2 //g/L
Column A:DB-5 fused silica capillary for GC/MS, 30 m x 0.25 mm, 0.25 micron film
MS inlet temperature = 200°C
Injector temperature = 200°C
Temperature Program: Hold 5 min at 80°C, increase to 260°C at 10°/min, hold 10 min.
Column B:FID primary column, RTX Volatiles, 30 m x 0.53 mm I.D., 2 micron film thickness.
Detector temperature = 280°C
Injector Temperature = 200°C
Carrier gas velocity = 50 cm/sec.
Temperature program: Same as Column A
Column C:FID confirmation column, DB-5, 30 m x 0.32 mm ID, 0.25 micron film
Carrier Gas velocity = 27 cm/sec
Same injector, detector and temperature program as Column A.
370
-------
Method 548.1
Table 2. Endothall Method Development Data
Cone. Recovery1 RSD2
Matrix frg/L) f%) (%)
Reagent Water 2 101 10
Reagent Water 10 86 10
Reagent Water 100 95 3
Ground Water3 2 91 25
Ground Water 10 82 14
Ground Water 100 88 6
1 Based on analysis of 7 replicates.
2 Relative Standard Deviation.
3 High Humic Content Florida Ground Water.
111
-------
Method 548.1
Table 3. Matrix Analyses1
Western Surface, Eastern Ground
Major Ion (mg/L) (mg/L)
Ca 330 122
Mg 132 33
Na 400 23
Sulfate 1850 102
Determination by inductively coupled plasma - mass spectrometry for cations and ion
chromatography for sulfate.
372
-------
Method 548.1
Table 4. Endothall Method Validation Data
Cone. EOT A1 Recovery1 RSD
Matrix (pg/L) (Mole/L) (%) (%)
WS3 25 0 9 19
WS-1/10* 50 0 66 13
WS-1/10 50 0.002 88 5
EG5 25 0 43 17
EG 25 0.005 97 6
EG-1/5 25 0 97 5
Ethylenediamine Tetraacetic Acid
Based on 7 Replicates
WS - Treated Western Surface Water
Dilution Factor in Reagent Water
WG - Eastern Ground Water
313
-------
Method 548.1
Table 5. Ion Abundance Criteria For Bis(Perfluorophenyl)Phenyl Phosphine
(Decafluorotriphenylphosphine, DFTPP)
Mass
(M/z)
51
51
68
70
127
197
198
199
275
365
441
442
443
Relative Abundance Criteria
10-80% of the base peak
<2% of mass 69
<2% of mass 69
2% of mass 69
10-80% of the base peak
<2% of mass 198
base peak or >50% of 442
5-9% of mass 198
10-60% of the base peak
> 1 % of the base peak
Present and < mass 443
base peak or >50% of 198
15-24% of mass 442
Purpose of Checkpoint1
Low mass sensitvity
Low mass sensitvity
Low mass resolution
Low mass resolution
Low-mid mass sensitivity
Mid-mass resolution
Mid-mass resolution and sensitivity
Mid-mss resolution and isotope ration
Mid-high mass sensitivity
Baseline threshold
High mass resolution
High mass resolution and sensitivity
High mass resolution and isotope ration
All ions are used primarily to check the mass measuring accuracy of the mass spectrometer
and data system, and this is the most important part of the performance test. The three
resolution checks, which include natural abundance isotope ratios, constitute the next most
important part of the performance test. The correct setting of the baseline threshold, as
indicated by the presence of low intensity ions, is the next most important part of the
performance test. Finally, the ion abundance ranges are designed to encourage some
standardization to fragmentation patterns.
314
-------
Method 548.1
25
50
S04
NA2SO4
100 150 200 250 300
Concentration mg/L
350
400
600
A CAII
CACL.2
O MGII
MGCL2
52-015-4A
* SOd
CASO4
Endothall Recovery from Reagent Water
with 804, MGII and CAII Ions Present
Figure 1. Endothall Recovery versus Ion Concentrations
375
-------
Method 548.1
100
84
r
o
8
oc
1 7
Holding Time Days
52-015-5A
+ Tap
Dechlorinated
A River
Preserved
O River
Figure 2. Endothall Sample Holding Study
376
-------
Method 548.1
200000—
150000—
100000—
50000-
TIC: 716ML10.D
D-10Acenaphthene
Endothall
Time 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5
Scan 1737 (16.024 Min): 716ML10.D (•)
3000-
2500-
2000-
0)
c
m
ID
c
^ 1500-
1000-
500-
67
59
•
111
ti
123
113
95
85
81
Illl
I,
hi
nl lid LI
,
1{
27
145
I ll
i:
55
183
I TNI lib
M/Z
60
80
100
120
140
160
180
Figure 3. Endothall GC/MS
Upper: Total Ion Chromatography Endothall: 16.02 Min., 10 ng
Lower: Relative Ion Abundance
52-015-66
317
-------
-------
Method 553
Determination of Benzidines and Nitrogen-
Containing Pesticides in Water
by Liquid-Liquid Extraction
or Liquid-Solid Extraction
and Reverse Phase High Performance
Liquid Chromatography/Particle Beam/
Mass Spectrometry
Revision 1.1 - EPA EMSL-Ci
August 1992
Thomas D. Behymer, Thomas A. Bellar, James S. Ho, William L. Budde
-------
-------
Method 553
Determination of Benzidines and Nitrogen-Containing Pesticides in
Water by Liquid-Liquid Extraction or Liquid-Solid Extraction
and Reverse Phase High Performance Liquid Chromatography/Particle
Beam/Mass Spectrometry
1. SCOPE AND APPLICA TION
1.1 This is a general purpose method that provides procedures for determination of benzidines and
nitrogen-containing pesticides in water and wastewater. The method is applicable to wide
range of compounds that are efficiently partitioned from a water sample into methylene
chloride or onto a liquid-solid extraction device. The compounds must also be amenable to
separation on a reverse phase liquid chromatography column and transferable to the mass
spectrometer with a particle beam interface. Paniculate bound organic matter will not be
partitioned onto the liquid-solid extraction system, and more than trace levels or particulates in
the water may disrupt the partitioning process. The compounds listed below are potential
method analytes and single-laboratory accuracy and precision data have been determined for
the compounds as described in Sect. 13. The specific analytical conditions given in the
method are applicable to those compounds for which accuracy and precision data are given.
Other analytes (Sect. 1.2) may require slight adjustments of analytical conditions. A laborato-
ry may use this method to identify and measure additional analytes after the laboratory obtains
acceptable (defined in Sect.9) accuracy and precision data for each added analyte.
Abbre-
Compound viation MW CAS No.
Benzidine BZ 184 92-87-5
Benzoylprop ethyl BP 365 33878-50-1
Caffeine CF 194 58-08-2
Carbaryl CL 201 63-25-2
o-Chlorophenyl thiourea PT 186 5344-82-1
3,3'-Dichlorobenzidine DB 252 91-94-1
3,3'-Dimethoxybenzidine MB 244 119-90-4
3,3'-Dimethylbenzidin LB 212 119-93-7
Diuron Dl 232 330-54-1
Ethylene thiourea ET 102 96-45-7
Linuron (Lorox) LI 248 330-55-2
Monuron MO 198 150-68-5
Rotenone RO 394 83-79-4
Siduron SI 232 1982-49-6
a Monoisotopic molecular weight calculated from the atomic masses of the
isotopes with the smallest masses.
1.2 Preliminary investigation indicates that the following compounds may be amendable to this
method: Aldicarb sulfone, Carbofuran, Methiocarb, Methomyl (Lannate), Mexacarbate
(Zectran), and N-(l-Naphthyl) thiourea. Caffeine, Ethylene thiourea and o-Chlorophenyl
527
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Method 553
thiourea have been successfully analyzed by HPLC/PB/MS, but have not been successfully
extracted from a water matrix.
1.3 Method detection limit (MDL) is defined as the statistically calculated minimum amount that
can be measured with 99% confidence that the reported value is greater than zero.' The MDL
is compound dependent and is particularly dependent on extraction efficiency and sample
matrix. For analytes listed in Tables 3-5, the estimated MDLs range from 2 to 30 fj.g/L.
2. SUMMARY OF METHOD
2.1 Organic compound analytes and surrogates are extracted from 1 L of water sample by liquid-
liquid extraction (LLE) with methylene chloride or by passing 1 L of sample water through a
cartridge or disk containing a solid inorganic matrix coated with a chemically bonded C
organic phase or a neutral polystyrene/divinylbenzene polymer (liquid-solid extraction, LSE).
If LLE is used, the analytes are concentrated in methanol by evaporation of the methylene
chloride and addition of methanl (solvent exchange). If LSE is used, the analytes are eluted
from the LSE cartridge or disk with a small quantity of methanol and concentrated further by
evaporation of some of the solvent. The sample components are separated, identified and
measured by injecting an aliquot of the concentrated methanol solution into a high performance
liquid chromatograph (HPLC) containing a reverse phase HPLC column and interfaced to a
mass spectrometer (MS) with a particle beam (PB) interface. Compounds eluting from the
HPLC column are identified by comparing their measured mass spectra and retention times to
reference spectra and retention times in a data base. Reference spectra and retention times for
analytes are obtained by measurement of calibration standards under the same conditions used
for samples. The concentration of each identified component is measured by relating the MS
response of the quantitation ion produced by that compound to the MS response of the quan-
titation ion produced by the same compound in a calibration standard (external standard).
Surrogate analytes, whose concentration are known in every sample, are measured with the
same external standard calibration procedure. An optional isotope dilution procedure is
included for samples which contain interfering matrix or coeluting compounds.
3. DEFINITIONS
3.1 External Standard (ES): A pure analyte(s) that is measured in an experiment separate from the
experiment used to measure the analyte(s) in the sample. The signal observed for a known
quantity of the pure external standard(s) is used to calibrate the instrument response for the
corresponding analyte(s). The instrument response is used to calculate the concentrations of
the anayte(s) in the sample.
3.2 Surrogate Analyte (SA): A pure analyte(s), which is extremely unlikely to be found in any
sample, and which is added to a sample aliquot in known amount(s) before extraction and is
measured with the same procedures used to measure other sample components. The purpose
of a surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory Duplicates (LD1 and LD2): Two aliquots of the same sample taken in the labora-
tory and analyzed separately with identical procedures. Analyses of LD1 and LD2 indicate the
precision associated with laboratory procedures, but not with samples collection, preservation,
or storage procedures.
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3.4 Field Duplicates (FD1 and FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associate with sample
collection, preservation and storage, as well as with laboratory procedures.
3.5 Laboratory Reagent Blank (LRB): An aliquot of reagent water or other blank matrix that is
treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents,
and surrogates that are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or the
apparatus.
3.6 Field Reagent Blank (RFB): An aliquot of reagent water or other blank matrix that is placed
in a sample container in the laboratory and treated as a sample in all respects, including
shipment to the sampling site, exposure to sampling site conditions, storage, preservation and
all analytical procedures. The purpose of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.7 Instrument Performance Check Solution (IPC): A solution of one or more method analytes,
surrogates, internal standards, or other test substances used to evaluate the performance of the
instrument system with respect to a defined set of method criteria.
3.8 Laboratory Fortified Blank (LFB): An aliquot of reagent water or other blank matrix to which
known quantities of the method analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine whether the methodology is in control,
and whether the laboratory is capable of making accurate and precise measurements.
3.9 Laboratory Fortified Sample Matrix (LFM): An aliquot of an environmental sample to which
known quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.10 Stock Standard Solution (SSS): A concentration solution containing one or more method
analytes prepared in the laboratory using assayed reference materials or purchased from a
reputable commercial source.
3.11 Primary Dilution Standard Solution (PDS): A solution of several analytes prepared in the
laboratory from the standard solutions and diluted as needed to prepare calibration solutions
and other needed analyte solutions.
3.12 Calibration Standard (CAL): A solution prepared from the primary dilution standard solution
or stock standard solutions and the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13 Quality Control Samples (QCS): A solution of method analytes of known concentrations
which is used to fortify an aliquot of LRB or sample matrix. The QCS is obtained from a
source external to the laboratory and different from the source of calibration standards. It is
used to check laboratory performance with externally prepared test materials.
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Method 553
3.14 Instrument Detection Limit (IDL): The minimum quantity of analyte or the concentration
equivalent which gives an analyte signal equal to three times the standard deviation of the
background signal at the selected wavelength, mass, retention time, absorbance line, etc.
4. INTERFERENCES
4.1 When two compounds coelute, the transport efficiency of both compounds thought the particle
beam interface generally improves and enhanced ion abundances are observed in the mass
spectrometer.2 The degree of signal enhancement by coelution is compound dependent. This
coelution effect invalidates the external calibration curve and, if not recognized, will result in
incorrect concentration measurements. Procedures given in this method to check for coeluting
compounds must be followed to preclude inaccurate measurements (Sect. 10.2.6.5 and Sect.
12.1). An optional isotope dilution calibration procedure has been included for use when
interfering matrix or coeluting compounds are present.
4.2 During analysis, major contaminant sources are reagents, chromatography columns, and liquid-
solid extraction columns or disks. Analyses of field and laboratory reagent blanks provide
information about the presence of contaminants.
4.3 Interfering contamination may occur when a sample containing low concentrations of com-
pounds is analyzed immediately after a sample containing relatively high concentrations of
compounds. Syringes, injectors, and other equipment must be cleaned carefully or replaced as
needed. After analysis of a sample containing high concentrations of compounds, a laboratory
reagent blank should be analyzed to ensure that accurate values are obtained for the next
sample.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method has not been precisely de-
fined; each chemical should be treated as a potential health hazard, and exposure to these
chemicals should be minimized. Each laboratory is responsible for maintaining awareness of
procedures and regulations for safe handling of chemicals uses in this method.3"5
5.2 Some method analytes have been tentatively classified as known or suspected human or
mammalian carcinogens. Pure standard materials and stock standard solutions of all analytes
should be handled with suitable protection to skin, eyes, etc.
6. EQUIPMENT AND SUPPLIES
6.1 All glassware must be meticulously cleaned. This may be accomplished by washing with
detergent and water, rinsing with water, distilled water, or solvents, air-drying, and heating
(where appropriated) in an oven. Volumetric glassware is never heated.
6.2 Sample Containers: 1-L or 1-qt amber glass bottles fitted with a Teflon-lined screw cap.
(Bottles in which high purity solvents were received can be used as sample containers without
additional cleaning if they have been handled carefully to avoid contamination during use and
after use of original contents.)
6.3 Separatory Funnels: 2-L and 100-mL with a Teflon stopcock.
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Method 553
6.4 Liquid Chromatography Column Reservoirs: Pear-shaped 100- or 125-mL vessels without a
stopcock but with a ground glass outlet sized to fit the liquid-solid extraction column. (Lab
Glass, Inc., Part No. ML-700-706S, with a 24/40 top outer joint and a 14/35 bottom inner
joint, or equivalent.) A 14/35 outlet joint fits some commercial cartridges.
6.5 Syringe Needles: No. 18 or 20 stainless steel.
6.6 Vacuum Flasks: 1 or 2 L with solid rubber stoppers.
6.7 Volumetric Flasks: Various sizes.
6.8 Laboratory or Aspirator Vacuum System: Sufficient capacity to maintain a slight vacuum of
13 cm (5 in) of mercury in the vacuum flask.
6.9 Micro Syringes: Various sizes.
6.10 Vials: Various sizes of amber vials with Teflon-lined screw caps.
6.11 Drying Column: 0.6 cm x 40 cm with 10 mL graduated collection vial.
6.12 Concentrator Tube: Kuderna-Danish (K-D) 10 mL graduated with ground glass stoppers.
6.13 Analytical Balance: Capable of weighing 0.0001 g accurately.
6.14 Liquid Chromatograph Column: A 15-25 cm x 2 mm (i.d.) stainless steel tube (e.g., Waters
C—18 Novapak or equivalent) packed with silica particles (4-10 jim) with
octadecyldimethylsilyl (C-18) groups chemically bonded to the silica surface. Residual acidic
sites should be blocked (endcapped) with methyl or other non-polar groups and the stationary
phase must be bonded to the solid support to minimize column bleed. Column selection for
minimum bleeding is strongly recommended. The column must be conditioned over night
before each use by pumping a 75-100% v/v acetonitrile: water solution through it at a rate of
about 0.05 mL/min. Other packings and column sizes may be used if equivalent or better
performance can be achieved.
6.15 Guard column of similar packing used in the analytical column is recommended.
6.16 Liquid Chromatograph/Mass Spectrometer/Data System (LC/MS/DS)
6.16.1 The LC must accurately maintain flow rates between 0.20-0.40 mL/min while perfor-
ming a gradient elution from 100% solvent A to 100% solvent B. Pulse dampening is
recommended but not required. An autoinjector is highly desirable and should be
capable of accurately delivering 1-10 /xL injections without affecting the chromatog-
raphy.
6.16.2 The system should include a post-column mixing tee and an additional LC pump for
post-column addition of acetonitrile at a constant rate of 0.1-0.7 mL/min.
6.16.3 The particle beam LC/MS interface must reduce the system pressure to a level fully
compatible with the generation of classical electron ionization (El) mass spectra, i.e.,
about 1 x 106 to 1 x 104 Torr, while delivering sufficient quantities of analytes to
the conventional El source to meet sensitivity, accuracy, and precision requirements.
All significant background components with mass greater than 62 Daltons should be
removed to a level that does not produce ions greater than a relative abundance of
10% in the mass spectra of the analytes.
6.16.4 The mass spectrometer must be capable of electron ionization at a nominal electron
energy of 70 eV. The spectrometer must be capable of scanning from 45 to 500 amu
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Method 553
with a complete scan cycle time (including scan overhead) of 1.5 sec or less. (Scan
cycle time = Total MS data acquisition time in seconds divided by number of scans in
the chromatogram). The spectrometer must produce a mass spectrum that meets all
criteria in Table 1 when 500 ng or less of DFTPPO (Sect. 7.11) is introduced into the
LC. An average spectrum across the DFTPPO LC peak may be used to test instru-
ment performance.
6.16.5 An interfaced data system is required to acquire, store, reduce, and output mass
spectral data. The computer software should have the capability of processing stored
LC/MS data by integration of the ion abundance of any specific ion between specified
time or scan number limits, construction of a first or second order regression calibra-
tion curves, calculation of response factors as defined in Sect. 10.2.9, calculation of
response factor statistics (mean and standard deviation), and calculation of concentra-
tions of analytes from the calibration curve or the equation in Sect. 12.
6.17 Millipore Standard Filter Apparatus, All Glass: This will be used if the disks are to be used to
carry out the extraction instead of cartridges.
7. REAGENTS AND STANDARDS
7.1 Helium nebulizer/carrier gas as contaminant free as possible.
7.2 Liquid-Solid Extraction (LSE) Materials
7.2.1 Cartridges are inert non-leaching plastic, for example polypropylene, or glass and
must not contain contaminants that leach into methanol. The cartridges are packed
with various amounts of sorbents such as Clg or a neutral polystrene/divinylbenzene
polymer. The packing must have a narrow size distribution and must not leach
organic compounds into methanol. One liter of water should pass through the car-
tridge in about 2 hr with the assistance of a slight vacuum of about 13 cm (5 in) of
mercury. Faster flow rates are acceptable if equivalent accuracy and precision are
obtained. Robotic systems topically pump the sample through a cartridge in less than
2 hr. These systems are also acceptable if equivalent accuracy and precision are
obtained. Sect. 9 and Tables 4 and 5 provide criteria for acceptable LSE cartridges
which are available from several commercial suppliers.
7.2.2 Extraction disks (Empore) are thin filter-shaped materials with C18 modified silica, or
neutral polystyrene/divinylbenzene polymer, impregnated in Teflon or other inert
matrix. As with cartridges, the disks should not contain any organic compounds,
either from the Teflon or the bonded silica, which will leach into the methanol eluant.
One liter of reagent water should pass through the disks in 5-20 min using a vacuum
of about 66 cm (26 in) of mercury. Sect. 9 provides criteria for acceptable LSE disks
which are available commercially.
7.3 Solvents
7.3.1 Acetonitrile, methylene chloride, and methanol: HPLC grade and pesticide quality or
equivalent.
7.3.2 Reagent water: Water in which an interferant is not observed at the MDL of the
compound of interest. Prepare reagent water by passing tap water through a filter bed
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Method 553
containing about 0.5 kg of activated carbon or by using a water purification system.
Store in clean, narrow-mouth bottles with Teflon-lined septa and screw caps.
7.4 Hydrochloric acid, concentrated.
7.5 Sodium sulfate, anhydrous.
7.6 Reducing Agents for Chlorinated Water: Sodium sulfite, sodium thiosulfate or sodium arse-
nite.
7.7 Ammonium Acetate, Sodium Chloride, and Sodium Hydroxide (IN): ACS reagent grade.
7.8 Stock Standard Solutions (SSS): Individual solution of analytes, surrogates, and isotopically
labelled analogues of the anlytes may be purchased as certified solutions or prepared from pure
materials. To prepare, add 10 mg (weighed on an analytical balance to 0.1 mg) of the pure
material to 1.9 mL of methanol or acetonitrile in a 2-mL volumetric flask, dilute to the mark,
and transfer the solution to an amber glass vial. Certain analytes, such as 3,3' -dimethoxy-
benzidine, may require dilution in 50% v/v acetonitrile or methanol: water solution. If the
analytical standard is available only in quantities smaller than 10 mg, reduce the volume of
solvent accordingly. If compound purity is certified by the supplier at >96%, the weighed
amount can be used without correction to calculate the concentration of the solution (5 /xg//xL).
Store the amber vials in a freezer at < 0°C.
7.8.1 Benzidines as the free base or as acid chlorides may be used for calibration purposes.
However, the concentration of the standard must be calculated as the tree base.
7.9 Primary Dilution Standard Solution (PDS): The stock standard solutions are used to prepare a
primary dilution standard solution that contains multiple analytes. The recommended solvent
for this dilution is a 50% v/v acetonitrile:water mixture. Aliquot of each of the stock standard
solutions are combined to produce the primary dilution in which the concentration of the
analytes is a least equal to the concentration of the most concentrated calibration solution.
Store the primary dilution standard solution in an amber vial in a freezer at < 0°C, and check
frequently for signs of deterioration or evaporation, especially just before preparing calibration
solutions.
7.10 Fortification Solution of Surrogates: The analyst should monitor the performance of the
extraction, cleanup (when used), and analytical system and the effectiveness of the method in
dealing with each sample matrix by spiking each sample, standard, and blank with 1 or 2
surrogates recommended to encompass the range of the gradient elution program used in this
method. The compounds recommended as surrogates for the analysis of benzidines and
nitrogen-containing pesticides are benzidine-D8 (DBZ), caffeine-15N2 (NCF), 3,3'-dichloro-
benzidene-D6 (DCB), and bis-(perfluorophenyl)-phenylphosphine oxide (OD) unless their
unlabelled counterpart is being analyzed or they will be used for isotope dilution calibration
(Abbreviations in parentheses are used in Figure 4). Prepare a solution of the surrogates in
methanol or acetonitrile at a concentration of 5 mg/mL of each. Other surrogates may be
included in this solution as needed. (A 10-/*L aliquot of this solution added to 1L of water
gives a concentration of 50 /ug/L of each surrogate). Store the surrogate fortifying solution in
an amber vial in a freezer at <0°C.
7.11 MS Performance Check Solution: Prepare a 100 ng/p.L solution of bis-(perfluorophenyl)-
phenylphosphine oxide (DFTPPO) in acetonitrile. Store this solution in an amber vial in a
freezer at < 0° C° DFTPPO is not currently commercially available. For this method
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Method 553
development work, DFTPPO was synthesized from bis-(perfluorophenyl) phenyl phosphine
(DFTPP) in solution by adding a slight excess ui hydrogen peroxide (DFTPP + H2O2 -»
DFTPPO + H2O). The solvent was removed and the resulting crystals were thoroughly
washed with water to remove any residual hydrogen peroxide. It is critical to remove all
residual hydrogen peroxide before adding the DFTPPO to the CAL solution. Any residual
hydrogen peroxide will degrade some analytes.
7.12 Calibration Solutions (CAL1-CAL6): Prepare a series of six concentration calibration solu-
tions in acetonitrile which contain all analytes at concentrations of 2, 5, 10, 25, 50 and 100
times the instrument detection limit of each compound with a constant concentration of each
surrogate in each CAL solution. This calibration range may be optimized by the operator, but
each analyte must be bracketed by at least 2 calibration points. CAL1 through CAL6 are
prepared by combining appropriate aliquots of the primary dilution standard solution (Section.
7.9) and the fortification solution of surrogates (Sect. 7.10). DFTPPO may be added to one or
more CAL solutions to verify MS tune (See Sect. 10.3.1.). Store these solutions in amber
vials in a freezer at < 0°C. Check these solutions quarterly for signs of deterioration.
7.12.1 For isotope dilution calibration, prepare the calibration solutions as described above
with the addition of one coeluting isotopically labelled analog for each analyte of
interest. The concentration for each coeluting labelled standard should be approxi-
mately 25 to 50 times the instrument detection limit of the analyte of interest and must
be constant in all calibration solutions (CAL1 through CAL6). These solutions permit
the relative response (unlabelled to labelled) to be measured as a function of the
amount of analyte injected. If more than one labelled compound is used, one spiking
solution containing all labelled compounds should be prepared.
7.13 Mobile Phase: Solvent A is a 75:25 v/v watenacetonitrile solution containing ammonium
acetate at a concentration of 0.01 M. This composition is used to eliminate biological activity
in the A Phase. Solvent B is acetonitrile. Both solvents are degassed in an ultrasonic bath
under reduced pressure and maintained by purging with a low flow of helium.
8. SAMPLE COLLECTION, PRESERVATION AND STORAGE
8.1 Sample Collection: When sampling from a water tap, open the tap and allow the system to
flush until the water temperature has stabilized (usually about 2-5 min). Adjust the flow to
about 500 mL/min and collect samples from the flowing stream. Keep samples sealed from
collection time until analysis. When sampling from an open body of water, fill the sample
container with water from a representative area. Sampling equipment, including automatic
sampler, must be free of plastic tubing, gaskets, and other parts that may leach analytes into
water. Automatic samplers that composite samples over time must use refrigerated glass
sample containers.
8.2 Sample Dechlorination and Preservation: All samples should be iced or refrigerated at 4°C
from the time of collection until extraction. Residual chlorine should be reduced at the
sampling site by addition of a reducing agent. Add 40-50 mg of sodium sulfite or sodium
thiosulfate (these may be added as solids with stirring until dissolved) to each liter of water.
8.3 Holding Time: Samples must be extracted within 7 days and the extracts analyzed within 30
days of sample collection. Extracts should be stored in an amber vial in a freezer at < 0°C.
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Method 553
8.4 Field Blanks
8.4.1 Processing of a field reagent blank (FRB) is recommended along with each sample
set, which is composed of the samples collected from the same general sample site at
approximately the same time. At the laboratory, fill a sample container with reagent
water, seal, and ship to the sampling site along with the empty sample containers.
Return the FRB to the laboratory with filled sample bottles.
9. QUALITY CONTROL
9.1 Quality control (QC) requirements are the initial demonstration of laboratory capability fol-
lowed by regular analyses of LRBs, LFBs, and laboratory fortified matrix samples. The
laboratory must maintain records to document the quality of the data generated. Additional
QC practices are recommended.
9.2 Initial demonstration of low system background and acceptable particle size and packing.
Before any samples are analyzed, or any time a new supply of LSE cartridges or disks is
received from a supplier, or a new column is installed, it must be demonstrated that a LRB is
reasonably free of contamination that would prevent the determination of any analyte of
concern. In this same experiment it must be demonstrated that the particle size and packing of
the LSE cartridge are acceptable. Consistent flow rate is an indication of acceptable particle
size distribution and packing.
9.2.1 A source of potential contamination may be the liquid-solid extraction (LSD) car-
tridges and disks and columns which may contain silicon compounds and other con-
taminants that could prevent the determination of method analytes. Generally, con-
taminants will be leached from the cartridges, disks, or columns into the solvent and
produce a variable background. If the background contamination is sufficient to
prevent accurate and precise analyses, this condition must be corrected before pro-
ceeding with the initial demonstration. Figure 1 show unacceptable background
contamination from a column with stationary phase bleed.
9.2.2 Other sources of background contamination are solvents, reagents, and glassware.
Background contamination must be reduced to an acceptable level before proceeding
with the next section. In general, background for method analytes should be below
the MDL.
9.2.3 One liter of water should pass through the cartridge in about 2 hr (faster flow rates
are acceptable if precision and accuracy are acceptable) with a partial vacuum of about
13 cm (5 in) of mercury. The extraction time should not vary unreasonably among
LSE cartridges. Robotic systems typically pump the sample through a cartridge in
less than 2 hr. These systems are also acceptable if equivalent accuracy and precision
are obtained. Extraction disks may be used at a faster flow rate (See Sect. 7.2.2).
9.3 Initial Demonstration of Laboratory Accuracy and Precision: Analyze 5-7 replicates of a LFB
containing each analyte of concern at a concentration in the range of 10-50 times the instru-
ment detection limits (see regulation and maximum contaminant levels for guidance on appro-
priate concentrations).
9.3.1 Prepare each replicate by adding an appropriated aliquot of the PDS, or another certi-
fied quality control sample, to reagent water. Analyze each replicate according to the
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Method 553
procedures described in Sect. 11 and on a schedule that results in the analyses of all
replicates with 48 hr.
9.3.2 Calculate the measured concentration of each analyte in each replicate, the mean
concentration of each analyte in all replicates, and mean accuracy (as mean percentage
of true value) for each analyte, and the precision (as relative standard deviation, RSD)
of the measurements for each analyte. Calculate the MDL of each analyte using the
referenced procedures.1
9.3.3 For each analyte and surrogate, the mean accuracy, expressed as a percentage of the
true value, should be 70-130% and the RSD should be < 30%. The MDLs should
be sufficient to detect analytes at the regulatory levels. If these criteria are not met
for an analyte, take remedial action and repeat the measurements for the analyte to
demonstrate acceptable performance before samples are analyzed.
9.3.4 Develop and maintain a system of control charts to plot the precision and accuracy of
analyte and surrogate measurements as a function of time. Charting of surrogate
recoveries is an especially valuable activity since these are present in every sample
and the analytical results will form a significant record of data quality.
9.4 Laboratory Reagent Blanks (LRBs): With each batch of samples processed as a group within a
work shift, analyze a laboratory reagent blank to determine the background system contami-
nation. Any time a new batch of LSE cartridges or disks is used, or new supplies of other
reagents are used, repeat the demonstration of low background described in Sect. 9.2.
9.5 With each batch of samples processed as a group within a work shift, analyze a single LFB
containing each analyte of concern at a concentration as determined in Sect. 9.3. Evaluate the
accuracy of the measurements (Sect. 9.3.3), and estimate whether acceptable MDLs can be
obtained. If acceptable accuracy and MDLs cannot be achieved, the problem must be located
and corrected before further samples are analyzed. Add these results to the ongoing control
charts to document data quality.
9.6 Determine that the sample matrix does not contain materials that adversely affect method
performance. This is accomplished by analyzing replicates of laboratory fortified matrix
samples and ascertaining that the precision, accuracy, and MDLs of analytes are in the same
range as obtained with LFBs. If a variety of different sample matrices are analyzed regularly,
for example, drinking water from groundwater and surface water sources, matrix independence
should be established for each.
9.7 With each set of field samples a FRB should be analyzed. The results of these analyses will
help define contamination resulting from field sampling and transportation activities.
9.8 At least quarterly, replicates of LFBs should be analyzed to determine the precision of the
laboratory measurements. Add these results to the ongoing control charts to document data
quality.
9.9 At least quarterly, analyze a QCS from an external source. If measured analyte concentrations
are not of acceptable accuracy (Sect. 9.3.3), check the entire analytical procedure to locate and
correct the problem source.
9.10 Numerous other specific QC measures are incorporated into other parts of this procedure, and
serve to alert the analyst to potential problems.
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Method 553
10. CALIBRA TION AND STANDARDIZA TION
10.1 Demonstration and documentation of acceptable initial calibration and system optimization are
required before any samples are analyzed and is required intermittently during sample analysis
as indicated by results of continuing calibration checks. After initial calibration is successful,
a continuing calibration check is required at the beginning of each 8-hr period during which
analyses are performed. Additional periodic calibration checks are good laboratory practice.
10.2 Initial Calibration
10.2.1 Optimize the interface according to the manufacturer's instructions. This usually is
accomplished on initial installation by flow injection with caffeine or benzidine and
should utilize a mobile phase of 50% v/v acetonitrile:water. Major maintenance
may require reoptimization.
10.2.2 Calibrate the MS mass and abundance scales using the calibration compound and
manual (not automated) ion source tuning procedures specified by the manufactur-
er. Calibration must be accomplished while a 50% v/v acetonitrile:water mixture
is pumped through the LC column and the optimized particle beam interface. For
optimum long-term stability and precision, interface and ion source parameters
should be set near the center of a broad signal plateau rather than at the peak of a
sharp maximum (sharp maxima vary short term with particle beam interfaces and
gradient elution conditions).
10.2.3 Fine tune the interface by making a series of injections into the LC column of a
medium level CAL standard and adjusting the operating parameters until optimum
sensitivity and precision are obtained for the maximum number of target com-
pounds.6 Suggested additional operating conditions are:
mobile phase purge: helium at 30 mL/min continuous,
mobile phase flow rate: 0.3 mL/min through the column,
gradient elution: hold for 1 min at 25 % acetonitrile, then linearly program to
= 70% acetonitrile in 29 min, start data acquisition immediately,
post-column addition: acetonitrile at 0.1-0.7 mL/min, depending on the interface
requirements. Maintain a minimum of 30% acetonitrile in the interface to
improve system precision and possibly sensitivity,
desolvation chamber temperature: 45°-80°C,
ion source temperature: 250°-290°C,
electron energy: 70 eV, and
scan range: 62-465 amu at 1-2 sec/scan.
10.2.4 The medium level standard (CAL) used in Sect. 10.2.3 should contain DFTPPO, or
separately inject into the LC a 5-^.L aliquot of the 100 ng/jtl DFTPPO solution and
acquire a mass spectrum that includes data form M/Z 62-465. Use LC conditions that
produce a narrow (at least ten scans per peak) symmetrical peak. If the spectrum
does not meet all criteria (Table 1), the MS ion source must be returned and adjusted
to meet all criteria before proceeding with calibration. An average spectrum across
the LC peak may be used to evaluate the performance of the system. Figure 2 repre-
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Method 553
sents the average composite spectrum obtained for DFTPPO from a multilaboratory
study involving 5 different particle beam interfaces from 13 laboratories.
10.2.5 Inject a 5 /xL aliquot of a medium concentration calibration solution, for example 50
ng/fiL, and acquire and store data from m/z 62-465 with a total cycle time (including
scan overhead time) of 1.5 sees or less. Cycle time should be adjusted to measure at
least ten spectra during the elution of each LC peak.
10.2.6.1 LC Performance: 3,3'-dimethyl- and 3,3'-dimethoxybenzidine should be
separated by a valley whose height is less than 25% of the average peak
height of these two compounds. If the valley between them exceeds 25%,
modify the gradient. If this fails, the LC column requires maintenance.
(See Sect. 10.3.6)
10.2.6.2 Peak tailing: Examine a total ion chromatogram and determine the degree
of peak tailing. Severe tailing indicates a major problem and system main-
tenance is required to correct the problem. (See Sect. 10.3.6)
10.2.6.3 MS sensitivity: Signal/noise in any analyte mass spectrum should be at
least 3:1.
10.2.6.4 Column bleed: Figure 1 shows an unacceptable chromatogram with
column bleed. Figure 3 is the mass spectrum of dimethyloctadecylsilanol,
a common stationary phase bleed product. If unacceptable column bleed is
present, the column must be changed or conditioned to produce an accept-
able background (Figure 4).
10.2.6.5 Coeluting compounds: Compounds which coelute cannot be measured
accurately because of carrier effects in the particle beam interface. Peaks
must be examined carefully for coeluting substances and if coeluting com-
pounds are present at greater than 10% the concentration of the target
compound, condition must be adjusted to resolve the components, the
target compound must be flagged as positively biased, or isotope dilution
calibration should be used.
10.2.7 If all performance criteria are met, inject a 5 /xL aliquot of each of the other CAL
solutions using the same LC/MS conditions.
10.2.8 The general method of calibration (external) is a second order regression of integrated
ion abundances of the quantitiation ions (Table 2) as a function of amount injected.
For second order regression, a sufficient number of calibration points must be ob-
tained to accurately determine the equation of the curve. For some individual analytes
over a short concentration range, reasonable linearity may be observed and response
factors may be used. Calculate response factors using the equation below. Second
order regressions and response factor calculations are supported in acceptable LC/MS
data system software (Sect. 6.16.5), and may other software programs.
332
-------
Method 553
where:
Ax = integrated abundance of the quantitation ion of the analyte.
Qx = quantity of analyte injected in ng or concentration units.
10.2.9 If response factors are used (i.e., linear calibration with the line going through the
origin), calculate the mean RF from the analyses of the six CAL solutions for each
analyte and surrogate. Calculate the standard deviation (SD) and the relative standard
deviation (RSD) from each mean (M): RSD = 100 (SD/M). If the RSD of any
analyte or surrogate mean RF exceeds 20%, either analyze additional aliquots of
appropriate CAL solutions to obtain an acceptable RSD of RFs over the entire concen-
tration range, take action to improve LC/MS performance, or use the second order
regression calibration. (See Sect. 10.2.8)
10.3 Continuing Calibration Check: Verify the MS tune and initial calibration at the beginning of
each 8-hr work shift during which analyses are performed using the following procedure:
10.3.1 Inject a 5-/iL aliquot of the 100 ng//xL DFTPPO solution (this may be contained in the
medium level CAL solution used in Sect. 10.3.2) and acquire a mass spectrum that
includes data for m/z 62-465. If the spectrum does not meet all criteria (Table 1), the
MS must be returned and adjusted to meet all criteria before proceeding with the
continuing calibration check.
10.3.2 Inject a 5-/xL aliquot of a medium level CAL solution and analyze with the same
conditions used during the initial calibration. One or more additional CAL solutions
should be analyzed.
10.3.3 Demonstrate acceptable performance for the criteria shown in Sect. 10.2.6.
10.3.4 Determine that the absolute areas of the quantitation ions of the external standards and
surrogate(s) have not changed by more than 20% from the areas measured during
initial calibration. If these areas have changed by more than 20%, recalibration and
other adjustment are necessary. These adjustments may require cleaning of the MS
ion source, or other maintenance as indicated in Sect. 10.3.6, and recalibration.
Control charts are useful aids in documenting system sensitivity changes.
10.3.5 Using the previously generated second order regression curve, calculate the concentra-
tions in the medium level CAL solution and compare the results to the known values
in the CAL solution. If calculated concentrations deviate by more than 20% from
known values, recalibration of the system with the 6 CAL solutions is required. If
response factors were used, calculate the RF for each analyte and surrogate from the
data measured in the continuing calibration check. The RF for each analyte and
surrogate must be within 20% of the mean value measured in the initial calibration.
10.3.6 Some possible remedial actions of Major maintenance such as cleaning an ion source,
cleaning quadrupole rods, etc. require returning to the initial calibration step.
333
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Method 553
10.3.6.1 Check and adjust LC and /or MS operating conditions: check the MS
resolution, and calibrate the mass scale.
10.3.6.2 Replace the mobile phases with fresh solvents. Verify that the combined
flow rate from the LC and post-column addition pumps is constant.
10.3.6.3 Flush the LC column with acetonitrile.
10.3.6.4 Replace LC column; this action will cause a change in retention times.
10.3.6.5 Prepare fresh CAL solutions and repeat the initial calibration step.
10.3.6.6 Clean the MS ion source, entrance lens, and rods (if a quadrupole).
10.3.6.7 Replace any component that leak.
10.3.6.8 Replace the MS electron multiplier or any other faulty components.
10.3.6.9 Clean the interface to eliminate plugged components and/or replace skim-
mers according to the manufacturer's instructions.
10.3.6.10 If automated peak areas are being used, verify values by manual integra-
tion.
10.3.6.11 Increasing ion source temperature can reduce peak tailing, but excessive
ion source temperature can affect the quality of the spectra for some com-
pounds.
10.3.6.12 Air leaks into the interface may affect the quality of the spectra (e.g.
DFTPPO), especially when ion source temperatures are operated in excess
of 280°.
10.4 Calibration with Isotope Dilution (Optional): For samples with interfering matrix or coeluting
peaks, the most reliable method for quantitiation is the use of coeluting isotope labelled
internal standards.7 Isotope dilution calibration will be limited by the availability and cost of
the labelled species and the requirement that each analyte must coelute with the labelled
internal standard. Because the labelled internal standard must coelute with the analyte, the
quantitation ion for the internal standard must be larger than that of the analyte and not present
in the anlayte's mass spectrum. In addition, it must be verified that the labelled internal
standard is not contaminated by its unlabelled counterpart.
10.4.1 A calibration curve encompassing the concentration range is prepared for each com-
pound to be determined. The relative response (analyte integrated ion abundances to
labelled integrated ion abundance) vs. amount of analyte injected is plotted using
linear regression. A minimum of five data points are employed for this type of
calibration.
10.4.2 To calibrate, inject a 5.0 p.L aliquot of each of the calibration standards (Sect. 7.12.1)
and compute the relative response (analyte integrated ion abundances to labelled
compound integrated ion abundance). Plot this versus the amount of analyte injected
by linear regression. This plotted line or the equation of this line should be used for
quantitative calculations. Unless this line goes through the origin, the response factors
at each point will not constant and therefore, average response factors cannot be used.
These calculations are supported in acceptable LC/MS data system software (Sect.
6.15.5), and in many other software programs.
334
-------
Method 553
10.4.3 Following Sect. 10.3 to verify calibration at the beginning of each 8-hr work shift by
injecting a 5.0 /xL aliquot of a medium CAL solution and analyze it with the same
conditions used during the initial calibration. Using the previously generated first
order regression line (relative response versus amount of analyte injected), calculate
the concentrations in the medium level CAL solution and compare the results to the
known values in the CAL solution. If calculated concentrations deviate by more than
20% from know values, recalibration of the system with the CAL solutions, contain-
ing the isotopically labelled analogues, is required.
11. PROCEDURE
11.1 The extraction procedure depends on the anlaytes selected and the nature of the sample. LSE
(cartridge or disk) is limited to particulate-free water, e.g., drinking water. Consult Table 3-5
to determine which analytes are amenable to liquid-solid and liquid-liquid extraction. Sect.
11.2 provides the LSE procedure using cartridges and Sect. 11.3 provides the LSE procedure
using disks. Sect. 11.4 provides the procedure for LLE. After the extraction is complete,
proceed to Sect. 11.5 to continue with the method.
11.2 Liquid-Solid Extraction (LSE) Procedure Using Cartridges (This procedure may be manual or
automated).
11.2.1 Set up the extraction apparatus shown in Figure 5. The reservoir is not required but
recommended for convenient operation. Water drains from the reservoir through the
LSE cartridge and into a syringe needle which is inserted through a rubber stopper
into the suction flask. A slight vacuum of 13 cm (5 in) of mercury is used during all
operations with the apparatus. The pressure used is critical as a vacuum greater than
13 cm may result in poor precision. About 2 hr is required to draw a liter of water
through the cartridge, but faster flow rates are acceptable if precision and accuracy
are acceptable. The use of robotic extraction systems is acceptable if equivalent
MDLs, precision and accuracy are obtained.
11.2.2 Mark the water meniscus on the side of the sample bottle for later determination of
the sample volume. A 1-L sample is recommended. Pour the water sample into the
2-L separator funnel with the stopcock closed. Adjust the pH to 7.0 by the dropwise
addition of hydrochloric acid or 1 N sodium hydroxide. Residual chlorine must not
be present, as a reducing agent should have been added at the time of sampling. For
extractions using C,8 cartridges, add 0.01 M ammonium acetate (0.77 g in 1 L) to the
water sample and mix until homogeneous. Add a 10-/xL aliquot of the fortification
solution for surrogates, and mix until homogeneous. The concentration of surrogates
in the water should be 10-50 times the instrument detection limit.
11.2.3 Flush each cartridge with two 10-mL aliquots of methanol, letting the cartridge drain
dry after each flush. This solvent flush may be accomplished by adding methanol
directly to the solvent reservoir in Figure 5. Add 10 mL of reagent water to the
solvent reservoir, but before the reagent water level drops below the top edge of the
packing in the LSE cartridge, open the stopcock of the separatory funnel and begin
adding sample water to the solvent reservoir. Close the stopcock when an adequate
amount of sample is in the reservoir.
335
-------
Method 553
11.2.4 Periodically open the stopcock and drain a portion of the sample water into the solvent
reservoir. The water sample will drain into the cartridge, and from the exit into the
suction flask. Maintain the packing material in the cartridge immersed in water at all
times. Wash the separatory funnel and cartridge with 10 mL of reagent water, and
draw air through the cartridge for 10 min.
11.2.5 Transfer the LSE cartridge to the elution apparatus shown in Figure 5B. Wash the
2-L separatory funnel with 15 mL of methanol, close the stopcock of the 100-mL
separatory funnel of the elution apparatus, and elute the cartridge with two 7.5-mL
aliquots of the methanol washings. Concentrate the extract to the desired volume
under a gentle stream of nitrogen. Record the exact volume of the extract.
11.2.5.1 If isotope dilution calibration is used, spike the extract with the isotopically
labelled standards prior to solvent evaporation. The concentration of these
isotopically labelled compounds after the desired extract volume is reached
should be the same as the concentration in each CAL solution.
11.2.6 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume
to the nearest 5 mL.
11.3 Liquid-Solid Extraction (LSE) Procedure Using Disks (This procedure may be manual or
automated).
11.3.1 Mark the water meniscus on the side of the sample bottle for later determination of
the sample volume. A 1-L sample is recommended. Pour the water sample into the
2-L separatory funnel with the stopcock closed. Adjust the pH to 7.0 by the dropwise
addition of hydrochloric acid or 1 N sodium hydroxide. Residual chlorine must not
be present because a reducing agent should have been added at the time of sampling.
For extractions using C18 disks, add 0.01 M ammonium acetate (0.77 g in 1 L) to the
water sample and mix until homogeneous. Add a 10-/*L aliquot of the fortification
solution for surrogates, and mix until homogeneous. The concentration of surrogates
in the water should be 10-50 times the instrument detection limit.
11.3.2 Preparation of Disks
11.3.2.1 Inset the disk into the 47 mm filter apparatus (See Figure 6). Wash and
pre-wet the disk with 10 mL methanol (MeOH) by adding the MeOH to
the disk and allowing it to soak for about a min, then pulling most of the
reaming MeOH through. A layer of MeOH must be left on the surface of
the disk, which should not be allowed to go dry from this point until the
end of the sample extraction. THIS IS A CRITICAL STEP FOR A UNI-
FORM FLOW AND GOOD RECOVERY.
11.3.2.2 Rinse the disk with 10 mL reagent water by adding the water to the disk
and pulling most through, again leaving a layer of water on the surface of
the disk.
11.3.3 Add the water sample to the reservoir and turn on the vacuum to begin the extraction.
Full aspirator vacuum may be used. Particulate-free water may pass through the disk
in as little as 10 minutes or less. Extract the entire sample, draining as much water
from the sample container as possible.
336
-------
Method 553
11.3.4 Remove the filtration top from the flask, but do not disassemble the reservoir and
fritted base. Empty the water from the flask, and insert a suitable sample tube to
contain the eluant. The only constraint on the sample tube is that it must fit around
the drip tip of the fritted base. Reassemble the apparatus.
11.3.5 Add 5 mL MeOH to the sample bottle, and rinse the inside walls thoroughly. Allow
the MeOH to settle to the bottom of the bottle, and transfer to the disk with a dis-
posable pipet, rinsing the sides of the glass filtration reservoir in the process. Pull
about half of the MeOH through the disk, release the vacuum, and allow the disk to
soak for a minute. Pull the remaining MeOH through the disk.
11.3.6 Repeat the above step twice. Concentrate the combined extracts to the desired volume
under a gentle stream of nitrogen. Record the extract volume of the extract. (Prelim-
inary investigation indicates that acetonitrile is a better extraction solvent for rotenone
when extracting water, containing high levels of particulate matter, with LSE disks.)
11.3.6.1 If isotope dilution calibration is used, spike the extract with the isotopically
labelled standards prior to solvent evaporation. The concentration of these
isotopically labelled compounds after the desired extract volume is reached
should be the same as the concentration in each GAL solution.
11.3.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume
to the nearest 5 mL.
11.4 Liquid-Liquid Extraction (LLE) Procedure
11.4.1 Mark the water meniscus on the side of the sample bottle for later determination of
the sample volume. A 1-L sample is recommended. Pour the water sample into a
2-L separatory funnel with the stopcock closed. Residual chlorine should not be
present as a reducing agent should have been added at the time of sampling. Add a
10-jtL aliquot of the fortification solution for surrogates, and mix until homogeneous.
The concentration of surrogates in the water should be 10-50 times the instrument
detection limit.
11.4.2 Adjust the pH of the sample to 7.0 by dropwise addition of hydrochloric acid or 1 N
sodium hydroxide. Add 100 g of sodium chloride to the sample and shake to dissolve
the salt.
11.4.3 Add 60 mL of methylene chloride to the sample bottle, shake, and transfer the solvent
to the separatory funnel and extract the sample by vigorously shaking the funnel for 2
min with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 min. If the emulsion interface
between layers is more than one-third the volume of the solvent layer, mechanical
techniques must be employed to complete the phase separation. The optimum tech-
nique depends on the sample, but may include stirring, filtration of the emulsion
through glass wool, centrifuging, etc. Collect the methylene chloride extract in a 500-
mL Erlenmeyer flask.
11.4.4 Add a second 60-mL volume of methylene chloride and repeat the extraction a second
time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in
the same manner.
337
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Method 553
11.4.5 Assemble a K-D concentrator by attaching a 10-mL concentrator tube to a 500-mL
evaporative flask. Dry the extract by pouring it through a solvent-rinsed drying
column containing about 10 cm of anhydrous sodium sulfate. Collect the extract in
the K-D concentrator, and rinse the column with 20-30 mL of methylene chloride.
11.4.6 Add 1 or 2 clean boiling stones to the evaporative flask and attach a macro Snyder
column. Pre-wet the Snyder column by adding about 1 mL of methylene chloride to
the top. Place the K-D apparatus on a hot water bath, 65-70°C, so that the con-
centrator tube is partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the concentration in
15-20 minutes. At the proper rate of distillation, the balls of the column will actively
chatter, but the chambers will not flood. When the apparent volume of the liquid
reaches 2 mL, add 20 mL of methanol through the Snyder column using a syringe and
needle. Raise the temperature of the hot water bath to 90°C, and concentrate the
sample to about 2 mL. Concentrate the extract to the desired volume under a gentle
stream of nitrogen. Record the exact volume of the concentrated extract.
11.4.6.1 If isotope dilution calibration is used, spike the extract with the isotopically
labelled standards prior to solvent evaporation. The concentration of these
isotopically labelled compounds after the desired extract volume is reached
should be the same as the concentration in each CAL solution.
11.4.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1000-mL gradulated cylinder. Record the sample volume
to the nearest 5 mL.
11.5 Liquid Chromatography/Mass Spectrometry (LC/MS)
11.5.1 Analyze a 5-/tL aliquot with the LC/MS system under the same conditions used for
the initial and continuing calibrations (Sect. 10.2).
11.6 Identification of Analytes
11.6.1 At the conclusion of data acquisition, use the system software to display the chro-
matogram, mass spectra and retention times of the peaks in the chromatogram.
11.6.2 Identify a sample component by comparison of its mass spectrum (after background
subtraction) to a reference spectrum in the user-created data base. The LC retention
time of the sample component should be within 10 sec of the time observed for that
same compound when a calibration solution was analyzed. In general, all ions that
are present above 10% relative abundance in the mass spectrum of the standard should
be present in the mass spectrum of the sample component and should agree within
absolute 20%. For example, if an ion has a relative abundance of 30% in the stan-
dard spectrum, its abundance in the sample spectrum should be in the range of 10 to
50%. Some ions, particularly the molecular ion, are of special importance, and
should be evaluated even if they are below 10% relative abundance.
11.6.3 Use the data system software to examine the ion abundances of components of the
chromatogram. If any ion abundance exceeds the system working range, dilute the
sample aliquot and analyze the diluted aliquot.
338
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Method 553
11.6.4 Identification is hampered when sample components are not resolved chromatographi-
cally and produce mass spectra containing ions contributed by more than one analyte.
When LC peaks obviously represent more than one sample component (i.e., broad-
ened peak with shoulder(s) or valleys between two or more maxima), appropriate
analyte spectra and background spectra can be selected by examining plots of charac-
teristic ions for tentatively identified components. When analytes coelute (i.e., only
one LC peak is apparent), the identification criteria can be met but each analyte
spectrum will contain extraneous ions contributed by the coeluting compound.
11.6.5 Structural isomers that produce very similar mass spectra can be explicitly identified
only if they have sufficiently different LC retention times. (See Sect. 10.2.6.1.)
Acceptable resolution is achieved if the height of the valley between two isomer peaks
is less than 25% of the average height of the two peak heights. Otherwise, structural
isomers are identified as isomeric pairs.
11.6.6 Background components appear in variable quantities in laboratory and field reagent
blanks, and generally subtraction of the concentration in the blank from the concentra-
tion in the sample is not recommended because the concentration of the background in
the blank is highly variable. If method analytes appear in the blank, then resample.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Complete chromatographic resolution is necessary for accurate and precise measurements of
analyte concentrations. Compounds which coelute cannot be measured accurately because of
carrier effects in the particle beam interface.2 Peaks must be examined carefully for coeluting
substances and if coeluting compounds are present at greater than 10% the concentration of the
target compound, either conditions must be adjusted to resolve the components, or the target
compound must be removed from the list of quantitative analytes.
12.2 Use the LC/MS system software or other available proven software to compute the concentra-
tions of the analytes and surrogates from the second order regression curves. Manual verifica-
tion of automated integration is recommended.
12.2.1 For isotope dilution calculations, use the first order plot of relative response (analyte
integrated ion abundances to labelled integrated ion abundance) vs. amount of analyte
injected or the equation of the line to compute concentrations. If the plotted line does
not go through the origin, response factors will not be constant at each calibration
point; therefore, average response factors cannot be used.
12.3 If appropriate, calculate analyte and surrogate concentrations from response factors and the
following equation.
339
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Method 553
c .
RF V V
where:
CA = Concentration of analyte or surrogate in pglL in the water sample.
Ax = integrated abundance of the quantitation ion of the analyte in the sample.
V = original water sample volume in liters.
RF = mean response factor of analyte the initial the initial calibration.
Ve = volume of final extract in pL
V = injection volume in \iL
13. METHOD PERFORMANCE
13.1 Single laboratory accuracy and precision data (Tables 3-5) for each listed analyte were ob-
tained. Five to seven 1-L aliquots of reagent water containing approximately 5 times the MDL
of each analyte were analyzed with this procedure. (For these experiments, the final extract
volume was 0.5 mL.)
13.1.2 With these data, MDLs were calculated using the formula:
.,.-0=099,
where'.
l(n-\ i-a = 099) = sfudent's t value for the 99% confidence level with n-\ degrees of freedom
n = number of replicates
S = standard deviation of replicate analyses.
13.2 A multilaboratory (12 laboratories) validation of the determinative step was done for four of
the analytes: benzidine (BZ), 3,3'-dimethoxybenzidine (MB), 3,3'-dimethylbenzidine (LB),
and 3,3'-dichlorobenzidine (DB). Table 6 gives the results from this study for single labora-
tory precision, overall laboratory precision, and overall laboratory accuracy. The two con-
centration levels shown represent the two extremes of the concentration range studied.
14. POLLUTION PREVENTION
14.1 Although this method allows the use of either LLE or LSE, LSE is highly recommended
whenever possible. Only small amounts of methanol are used with this procedure as compared
to much larger amounts of methylene chloride used for LLE. All other compounds used are
neat materials used to prepare standards and sample preservatives. All compounds are used in
small amounts and pose minimal threat to the environment if properly disposed.
14.2 For information about pollution prevention that may be applicable to laboratory operations,
consult "Less Is Better: Laboratory Chemical Management for Waste Reduction" available
from the American Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street, N.W., Washington, D.C., 20036.
340
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Method 553
15. WASTE MANAGEMENT
15.1 There are generally no waste management problems involved with discarding spent or left over
samples in this method since most often the sample matrix is drinking water. If a sample is
analyzed which appears to be highly contaminated with chemicals, analyses should be carried
out to assess the type and degree of contamination so that the samples may be discarded
properly. All other expired standards should be discarded properly. It is the laboratory's
responsibility to comply with all applicable regulations for waste disposal. The Agency
requires that laboratory waste management practices be conducted consistent with all applicable
rules and regulations, and that laboratories protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Also, compliance is required
with any sewage discharge permits and regulations, particularly the hazardous waste identi-
fication rules and land disposal restrictions. For further information on waste management,
consult "The Waste Management Manual for Laboratory Personnel" also available from the
American Chemical Society at the address in Sect. 14.2.
341
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Method 553
References
1. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
Wastewaters," Environ. Sci. Techno!. 1981, 15, 1426-1435.
2. Bellar, T.A., T.D. Behymer, and W.L. Budde, "Investigation of Enhanced Ion Abundances
from a Carrier process in High-Performance Liquid Chromatography Particle Beam Mass
Spectrometry," J. Am. Soc. Mass Spectrom.. 1990, 1, 92—98.
3. "Carcinogens—Working With Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29CFK1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Com-
mittee on Chemical Safety, 3rd Edition, 1979.
6. Behymer, T.D., T.A. Bellar, and W.L. Budde, "Liquid Chromatography/Particle Beam/Mass
Spectrometry of Polar Compounds of Environmental Interest," Anal. Chem.. 1990, 62,
1686-1690.
7. Ho, J.S., T.D. Behymer, W.L. Budde, and T.A. Bellar, "Mass Transport and Calibration in
Liquid Chromatography/Particle Beam/Mass Spectrometry," J. Am. Soc. Mass Spectrom..
1992, 3, 662-671.
342
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Method 553
Table 1. Ion Abundance Criteria for BlS(Perfluorophenyl) Phenylphosphine Oxide
(Decafluorotriphenylphosphine Oxide, DFTPPO)
Mass Relative Abundance
(M/z) Criteria Purpose of Checkpoint
77 Present, major ion Low mass sensitivity
168 Present, major ion Mid-mass sensitivity
169 4-10% of 168 Mid-mass resolution and isotope ratio
271 Present, major ion Base peak
365 5-10% of base peak Baseline threshold check
438 Present Important high mass fragment
458 Present Molecular ion
459 15-24% of mass 458 High mass resolution and isotope ratio
All ions are used primarily to check the mass measuring accuracy of the mass spectrometer and a
data system, and is the most important part of the performance test. There resolution checks,
which include natural abundance isotope rations, constitute the next most important part of the
performance test. The correct setting of the baseline threshold, as indicated by the presence of
low intensity ions, is the next most important part of the performance test. Finally, the ion abun-
dance ranges are designed to encourage some standardization of fragmentation patterns.
343
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Method 553
Table 2. Retention Time Data and Quantitation Ions for Method Analytes
Retention
Time (min: sec)
Quantitation
Compound A' Bf Ion (m/z)
Benzidine 4.3 4.9 184
Benzoylprop ethyl 24.8 31.3 105
Caffeine 1.4 1.6 194
Carbaryl 10.1 14.7 144
o-Chlorophenyl thiourea 2.7 3.0 151
3,3'-Dichlorobenzidine 16.6 22.7 252
3,3'-Dimethoxybenzidine 8.1 11.5 244
3,3'-Dimethylbenzidine 8.5 12.4 212
Diuron 11.0 16.1 72
Ethylene thiourea 1.2 1.4 102
Linuron 16.0 21.9 161
Rotenone 21.1 27.4 192
Siduron 14.8 20.6 93
Surrogates:0
Benzidine-D8 4.2 4.8 192
Caffeine-15N2 1.3 1.6 196
3,3'-Dichlorobenzidine-D6 16.5 22.6 258
Bis(perfluorophenyl)-phenylphosphine 22.0 28.9 271
oxide
a These retention times were obtained on a Hewlett-Packard 1090 liquid chromatograph with a
Waters C18 Novapak 15 cm x 2 mm column using gradient conditions given in Sect. 10.2.3.
h These retention times were obtained o a Waters 600 MS liquid chromatograph with a Waters
C18 Novapak 15 cm x 2 mm column using gradient conditions given in Sect. 10.2.3.
c These compounds cannot be used if unlabelled compounds are presents (See Sect. 4.1)
344
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Table 3. Accuracy and Precis
Using Liquid-Liquid Extraction
Compound
Benzidine
Benzoylprop ethyl
Caffeine
Carbaryl
o-Chlorophenyl thiourea
3,3'-Dichlorobenzidine
3,3'-Dimethoxybenzindine
3,3'-Dimethylbenzindine
Diuron
Ethylene thiourea
Linuron
Monuron
Rotenone
Siduron
Ol
ata from Six
True Cone.
(ug/U
22.9
32.5
14.4
56.6
32.6
24.8
31.6
31.7
25.0
32.0
95.0
31.2
50.3
27.9
nge (See Sect.
Determinations
Mean
Observed
Cone.
(ug/U
20.5
33.0
10.5
52.2
15.3
21.7
29.2
31.8
26.2
0.0
89.5
31.8
44.9
29.6
9.3.3)
of the
Method Analytes
in Reagent
Pel. Mean Method
Std.
Dev.
(ug/U
0.8
1.1
0.9
2.9
2.2
0.7
2.3
1.0
1.3
0.0
3.9
1.2
9.4
1.4
Std.
Dev. 1
(%)
3.3
3.3
6.3
5.1
6.8
2.9
7.3
3.1
5.1
0.0
4.1
3.8
18.8
5.2
Accuracy
% of True
Cone.)
89.6
101.6
72.6
92.3
47.0
89.6
92.3
100.4
104.8
0.0
94.2
101.9
89.3
106.3
Water
Method
Detection
Limit (MDU
(ug/U
2.5
3.7
3.1
9.8
7.4*
2.4
7.7
3.3
4.4
*
13.1
4.0
31.6
4.7
1
Ul
01
Co
-------
Co
Table 4. Accuracy and Precision Data from Seven Determinations of the Method Analytes In Reagent Water
Using Liquid-Solid Extraction (C18 LSE Cartridge)
Compound
Benzidine
Benzoylprop ethyl
Caffeine
Carbaryl
o-Chlorophenyl thiourea
3,3'-Dichlorobenzidine
3,3'-Dimethoxybenzindine
3,3'-Dimethylbenzindine
Diuron
Ethylene thiourea
Linuron
Monuron
Rotenone
Siduron
True Cone.
(ug/U
22.9
32.5
14.4
56.6
32.6
5.0
31.6
31.7
25.0
32.0
95.0
31.2
50.3
27.9
Mean
Observed
Cone.
(ug/L)
12.2
29.3
6.4
53.9
0.0
4.4
25.5
31.4
24.4
0.0
88.9
30.5
45.0
24.8
Std.
Dev.
(ug/U
1.7
2.0
1.4
1.8
0.0
0.4
1.8
1.0
1.4
0.0
4.8
2.9
2.4
2.0
Pel.
Std.
Dev.
(%)
13.7
6.9
21.4
3.3
0.0
10.0
7.1
3.1
5.6
0.0
5.4
9.6
5.4
7.9
Mean Method Method
Accuracy
(% of True
Cone.)
53.2
90.2
44.2
95.2
0.0
89.6
80.8
99.0
97.6
0.0
93.6
97.8
89.6
88.9
Detection
Limit (MOD
(ug/U
5.3*
6.3
4.4*
5.7
*
1.4
5.7
3.0
4.4
*
15.1
9.1
7.5
6.3
I
o
Q.
Wl
01
Co
'Recovery was not in the 70-130% range (See Sect. 9.3.3)
-------
Table 5. Accuracy and
Precision Data from
Six Determinations of the Method Analytes in Reagent Water
Using Liquid-Solid Extraction (Neutral Polystrene/Divinylbenzene Polymer
Compound
Benzidine
Benzoylprop ethyl
Caffeine
Carbaryl
o-Chlorophenyl thiourea
3,3'-Dichlorobenzidine
3,3'-Dimethoxybenzindine
3,3'-dimethylbenzindine
Diuron
Ethylene thiourea
Linuron
Monuron
Rotenone
Siduron
True Cone.
(ug/U
22.9
32.5
14.4
56.6
32.6
5.0
31.6
31.7
25.0
32.0
95.0
31.2
50.3
27.9
•Recovery was not in the 70-130% range (See Sect
Mean
Observed
Cone.
(ug/U
24.7
31.1
0.7
59.5
0.0
5.0
32.8
31.5
26.1
0.0
97.9
34.4
40.5
26.8
. 9.3.3)
Std.
Dev.
(ug/U
2.4
3.0
0.5
4.7
0.0
0.5
2.2
2.1
1.8
0.0
8.7
2.5
6.0
1.0
LSE Disk)
Pel.
Std.
Dev.
(%)
9.8
9.6
72.5
7.9
0.0
9.4
6.7
6.7
7.0
0.0
9.0
7.3
14.8
3.6
Mean Method
Accuracy
(% of True
Cone.)
108.0
95.8
5.2
105.1
0.0
101.7
103.8
99.4
104.5
0.0
103.0
110.4
80.5
96.1
Method
Detection
Limit (MDL)
(ug/U
8.1
10.1
1.8*
15.8
*
1.6
7.4
7.1
6.1
*
29.3
8.4
20.2
3.4
(6
O
Q.
Wl
Co
-------
Method 553
Table 6. Mean Recoveries, Multilaboratory Precision and Estimates of Single
Analyst Precision for the Measurements of Four Benzidines by LC/PB/MS
Compound
10 ug/mL
Recovery
(%)
RSD
Multi-Lab
RSD
Single
Analyst
100 ug/mL
Recovery
<%)
RSD
Multi-Lab
RSD
Single
Analyst
BZ 96 10 5.6 97 10 9.1
MB 104 20 18 95 10 7.0
LB 98 14 10 97 8.6 4.9
DB 96 18 9.4 97 9.1 4.6
348
-------
Method 553
50000 -
45000 -
40000 -
35000 -
30000 -
25000 -
20000 -
15000-
10000-
5000-
0-
Dimethyloctadecylsilanol
(Major Ions — M/Z, 75,313)
r
12
T
16
l
20
I
24
I
28
4 8
C18 Column Following Exposure to Ammonium Acetate
l
32
100
-90
-80
-70
-60
-50
-40
-30
-20
- 10
-0
24000
20000 -
16000-
12000-
8000-
4000-
0-
i
8
l
16
i
20
I
24
4 8 12
C18 Column Maintained with Acetonitrile Flushing
n
28
i
32
- 100
-90
-80
-70
-60
-50
-40
-30
-20
- 10
-0
52-015-35B
Figure 1. Unacceptable Chromatogram with Column Bleed and
Acceptable Chromatogram Following Column Flushing
349
-------
Method 553
100-
90-
80
70-
60-
50-
40-
30-
20-
10-
o
271
77
69
168
99
117
I
20
I ' I
6 22
255
>4
F F o ^ F
/ \ II / \
F\_fP\_/f
F F Jv F F
Hl MH
Hi^jH
H
438
291
45
365
|
I ' I ' I ' I ' I
100 150 200 250 300 350 400 450
52-015-37A
Mass
Figure 2. Average Spectrum of DFTPPO from Multilaboratory Study
350
-------
Method 553
100%
75
INT_
61
50
CH3
I
HO- Si-
CHo
Dimethyloctadecylsilanol
M-Methyl
313
281
295 I
—lU. ttJk_
100
150
200
M/Z
250
300
52-015-38A
Mass Spectrum of C18 Column Bleed
Figure 3. Mass Spectrum of Dimethyloctadecylsilanol,
A Common Stationary Phase Bleed Product
351
-------
Method 553
1200000
CB + DCB
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
0.3 mL/min 0.01 M Ammonium Acetate
Post Column 0.1 mL/min Acetonitrile
Figure 4. Total Ion Chromatogram of Analytes and Surrogates
(140-950 ng injected)
352
-------
Method 553
HD
2 Liter
Separatory Funnel
HD
\7
HD
125mL
Solvent
Reservoir
Ground Glass
Stopper 14/35
LSE Cartridge
Rubber Stopper
No. 18-20 Luer-lok
Syringe Needle
HD
125ml_
Solvent
Reservoir
Ground Glass
Stopper 14/35
LSE Cartridge
100mL
Separatory
Funnel
Drying Column
(Na2SO4)
1.2 cm x 40 cm
10 mL
Graduated
Vial
A. Extraction Apparatus
B. Elution Apparatus
Figure 5. Schematic Diagram of a Liquid-Solid Extraction (LSE) Apparatus
353
-------
Method 553
Source
Vacuum
1 Liter
Suction Flask
Pinch Clamp
52-015-41
Figure 6. Schematic Diagram of Liquid-Solid Disk Extraction Apparatus
354
-------
Method 555
Determination of Chlorinated Acids in Water
by High Performance Liquid
Chromatography with a Photodiode Array
Ultraviolet Detector
Revision 1.0 - EPA EMSL-Ci
August 1992
James W. Eichelberger, Winslow J. Bashe (Technology Applications, Inc.)
-------
-------
Method 555
Determination of Chlorinated Acids in Water
by High Performance Liquid Chromatography
with a Photodiode Array Ultraviolet Detector
1. SCOPE AND APPLICA TION
1.1 This is a high performance liquid chromatographic (HPLC) method for the determination of
certain chlorinated acids in ground water and finished drinking water. The following com-
pounds can be determined by this method:
Analyte CAS No.
Acifluorfen 50594-66-6
Bentazon 25057-89-0
Chloramben3 133-90-4
2,4-D 94-75-7
2,4-DB 94-82-6
Dicamba 1918-00-9
3,5-Dichlorobenzoic acid 51-36-5
Dichlorprop 120-36-5
Dinoseb 88-85-7
5-Hydroxydicambaa 7600-50-2
MCPA 94-74-6
MCPP 7085-19-0
4-Nitrophenola 100-02-7
Pentachlorophenol" (PCP) 87-86-5
Picloram" 1918-02-1
2,4,5-T 93-76-5
2,4,5-TP 93-72-1
8 Analytes measurable from 20 ml sample volume only.
b Use a 100 mL sample for pentachlorophenol in order to attain a MDL of
0.3 /yg/L. The MLC for this compound is 1.0 jt/g/L
1.2 This method is applicable to the determination of salts and esters of analyte acids. The form
of each analyte is not distinguished by this method. Results are calculated and reported for
each listed analyte as the total free acid.
1.3 This method has been validated in a single laboratory and method detection limits (MDLs)
have been determined from a 20-mL sample for the analytes above.1 Observed MDLs may
vary among ground waters, depending on the nature of interferences in the sample matrix and
the specific instrumentation used.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use
of HPLC and in the interpretation of chromatograms. Each analyst must demonstrate the abi-
lity to generate acceptable results with this method using the procedure described in Sect. 9.3.
357
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Method 555
1.5 Analytes that are not separated chromatographically cannot be individually identified and
measured in the same calibration mixture or water sample unless an alternative technique for
identification and quantitation exists (Sect. 11.3).
1.6 When this method is used to analyze unfamiliar samples, analyte identifications must be con-
firmed by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured sample volume of approximately 100 mL is adjusted to pH 12 with 6 N sodium
hydroxide, shaken, and allowed to set for 1 hr to hydrolyze chlorinated esters. The sample is
acidified with H3PO4, filtered, and the chlorinated acids are extracted from a 20-mL aliquot.
The 20-mL aliquot is pumped through an HPLC cartridge (containing Clg-silica), trapping the
chlorinated acids. The concentrator cartridge is valved in-line with the C,8 analytical column
following extraction. The analytes are separated and measured by photodiode array-ultraviolet
detection (PDA-UV).
NOTE: A liquid-solid extraction disk is perfectly acceptable for use in the in-line
extraction of the analytes providing all quality control (QC) criteria in Sect. 9 are met
or exceeded.
2.2 The method measures the analytes from 20-mL volumes. Volumes of up to 100 mL may be
analyzed by this procedure for certain analytes. The analytes which may not be determined in
a larger volume are indicated in Sect. 1.1.
3. DEFINITIONS
3.1 Laboratory duplicates (LD1 AND LD2): Two aliquots of the same sample taken in the
laboratory and analyzed separately with identical procedures. Analyses of LD1 and LD2
indicate the precision associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.2 Field duplicates (FD1 AND FD2): Two separate samples collected at the same time and place
under identical circumstances and treated exactly the same throughout field and laboratory
procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory procedures.
3.3 Laboratory reagent blank (LRB): An aliquot of reagent water or other blank matrix that is
treated exactly as a sample including exposure to all glassware, equipment, solvents, and
reagents that are used with other samples. The LRB is used to determine if method analytes
or other interferences are present in the laboratory environment, the reagents, or the apparatus.
3.4 Field reagent blank (FRB): An aliquot of reagent water or other blank matrix that is placed in
a sample container in the laboratory and treated as a sample in all respects, including shipment
to the sampling site, exposure to sampling site conditions, storage, preservation, and all
analytical procedures. The purpose of the FRB is to determine if method analytes or other
interferences are present in the field environment.
358
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Method 555
3.5 Laboratory fortified blank (LFB): An aliquot of reagent water or other blank matrix to which
known quantities of the method analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine whether the methodology is in control,
and whether the laboratory is capable of making accurate and precise measurements.
3.6 Laboratory fortified sample matrix (LFM): An aliquot of an environmental sample to which
know quantities of the method analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.7 Stock standard solution (SSS): A concentrated solution containing one or more method
analytes prepared in the laboratory using assayed reference materials or purchased from a
reputable commercial supplier.
3.8 Primary dilution standard solution (PDS): A solution of several analytes prepared in the
laboratory from stock standard solutions and diluted as needed to prepare calibration solutions
and other needed analyte solutions.
3.9 Calibration standard (CAL): A solution prepared from the primary dilution standard solution
or stock standard solutions and the internal standards and surrogate analytes. The CAL
solutions are used to calibrate the instrument response with respect to analyte concentration.
3.10 Quality control sample (QCS): A solution of method analytes of known concentrations which
is used to fortify an aliquot of LRB or sample matrix. The QCS is obtained from a source
external to the laboratory and different from the source of calibration standards, It is used to
check laboratory performance with externally prepared test materials.
3.11 Method detection limit (MDL): The minimum concentration of an analyte that can be identi-
fied, measured and reported with 99% confidence that the analyte concentration is greater than
zero.
3.12 External standard (ES): A pure analyte(s) that is measured in an experiment separate from the
experiment used to measure the analyte(s) in the sample. The signal observed for a known
quantity of the external standard(s) is used to calibrate the instrument response for the corre-
sponding analytes(s). The instrument response is used to calculate the concentrations of the
analyte(s) in the sample.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and
other sample processing apparatus that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by analyzing laboratory reagent blanks as
described in Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible
after use by thoroughly rinsing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with dilute acid, tap and reagent
water. Drain dry, and heat in an oven or muffle furnace at 400°C for 1 hr. Do not
359
-------
Method 555
heat volumetric ware. Thermally stable materials such as PCBs might not be elimi-
nated by this treatment. Thorough rinsing with acetone may be substituted for the
heating. After drying and cooling, seal and store glassware in a clean environment to
prevent any accumulation of dust or other contaminants. Store inverted or capped
with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
WARNING: When a solvent is purified, stabilizers added by the manufacturer
are removed, thus potentially making the solvent hazardous. Removal of preservatives
by distillation may also reduce the shelf-life of the solvent.
4.2 The acid forms of the analytes are strong organic acids which react readily with alkaline
substances and can be lost during sample preparation. Glassware must be acid-rinsed with 1 N
hydrochloric acid prior to use to avoid analyte losses due to adsorption.
4.3 Matrix interferences may be caused by contaminants that are coextracted from the sample.
Also, note that all method analytes are not resolved from each other on a single column, i.e.,
one analyte of interest may interfere with another analyte of interest. The extent of matrix
interferences will vary considerably from source to source, depending upon the water sampled.
The procedures in Sect. 11 can be used to overcome many of these interferences. Tentative
identifications should always be confirmed (Sect. 11.3).
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
ing the safe handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in the chemical
analysis.
5.2 WARNING: When a solvent is purified, stabilizers added by the manufacturer are removed,
thus potentially making the solvent hazardous. Therefore, storage of large volumes of purified
solvents may be hazardous. Therefore, only small volumes of solvents should be purified just
before use.
6. EQUIPMENT AND SUPPLIES
6.1 Ssmple bottle: Borosilicate, 125-mL volume, graduated, fitted with Teflon-lined screw cap.
Protect samples from light. The container must be washed and dried as described in Sect.
4.1.1 before use to minimize contamination. Cap liners may be cut to fit from Teflon sheets
and extracted with methanol overnight prior to use.
6.2 Glassware
6.2.1 Volumetric flask, Class A: 100 mL, with ground glass stoppers.
360
-------
Method 555
6.2.2 Graduated cylinder: 100 mL
6.2.3 Disposable pipets, Transfer: borosilicate glass
6.2.4 Glass syringe: 50 mL, with Luer-Lok fitting
6.2.5 Volumetric pipette, Class A: 20 mL
6.3 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.4 Liquid chromatograph: Analytical system complete with gradient programmable HPLC
suitable for use with analytical HPLC columns and all required accessories including an
injector, analytical column, semi-prep guard column, and photodiode array UV detector. A
data system is necessary for measuring the peak areas and for assessing the confirmation of the
peak identification. A personal computer (PC) of at least the AT-class is generally needed to
control and collect data from the photodiode array UV detector. Table 1 lists the retention
times observed for the method analytes using the column and analytical conditions described
below. Figure 1 is a schematic drawing of the analytical system including the sample concen-
trator column (semi-prep guard column).
6.4.1 Primary Column: 250 mm x 4.6 mm I.D. ODS-AQ, 5 jon spherical (YMC Ltd.).
Any column may be used if equivalent or better performance (better peak shape,
better analyte efficiency, or more complete separation of analytes) can be demonstrat-
ed. Mobile phase flow rate is established at 1.0 mL/min (linear velocity of 6.0
cm/min). Two mobile phase components are used: A—0.025 M H3PO4; B—Aceto-
nitrile. A gradient solvent program is used to separate the analytes: 90:10 A:B to
10:90 A:B in 30 min, linear ramp / hold at 10:90 for 10 min. Reverse the gradient
and establish initial conditions: 10:90 A:B to 90:10 A:B in 10 min, linear ramp.
Allow column back pressure to restablize for 5 to 10 min before beginning the next
analysis. Total restabilization time will be determined by each analyst.
6.4.2 Confirmation Column: 300 mm x 3.9 mm I.D. Nova-Pak C18, 4 /mi spherical (Wa-
ters Chromatography Division, Millipore). Any column may be used if equivalent or
better performance (better peak shape, better analyte efficiency, or more complete
separation of analytes) can be demonstrated. Mobile phase and conditions same as
primary column.
6.4.3 Sample Concentrator Column: 30 mm x 10 mm I.D. ODS-AQ, 5 pm spherical
(YMC Ltd). An alternative concentrator column may be used if all QC criteria in
Sect. 9 can be equalled or improved. Also, a liquid-solid extraction disk may be used
if all QC criteria in Sect. 9 can be equalled or improved.
6.4.4 6-port Switching Valve: Rheodyne Model 7000 (Rheodyne Corp).
6.4.5 Sample Delivery Pump: A piston-driven pump capable of delivering aqueous sample
at a flow rate of 5.0 mL/min. An analytical HPLC pump may serve as the sample
delivery pump. A Waters Model 6000A was used to generate the data presented in
this method.
6.4.6 Detector: Photodiode Array-Ultraviolet (PDA-UV), LKB-Bromma Model 2140
Rapid Spectral Detector or equivalent. Detector parameters: Scan Range - 210 to
310 nm at 1 scan/sec, detector integration—1 sec.
361
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Method 555
6.4.7 Data Handling System: DOS-based Personal Computer, AT-class machine or machine
of greater capability with 640 K RAM or more, an 80 Mb hard disk or larger, VGA
monitor or equivalent.
7. REAGENTS AND STANDARDS
7.1 Acetonitrile: HPLC Grade or equivalent.
7.2 Sodium sulfite, granular, anhydrous: ACS Grade.
7.3 Sodium hydroxide (NaOH), pellets: ACS Grade.
7.3.1 NaOH, 6 N: Dissolve 216 g NaOH in 900 mL reagent water.
7.4 Phosphoric acid, 85% AR,: ACS grade.
7.4.1 0.025 M: Mix 2.0 mL of H3PO4 in 998 mL of reagent water.
7.5 Stock standard solutions (1.00 /ig/jtL): Stock standard solutions may be purchased as certified
solutions or prepared from pure standard materials using the following procedure:
7.5.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
pure material. Dissolve the material in acetonitrile and dilute to volume in a 10-mL
volumetric flask. Larger volumes may be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially pre-
pared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.5.2 Transfer the stock standard solutions into Teflon-lined sealed screw cap amber vials.
Store at room temperature and protect from light.
7.5.3 Stock standard solutions should be replaced after two months or sooner if comparison
with laboratory fortified blanks, or QC samples indicate a problem.
7.6 Hydrochloric acid: ACS grade.
7.6.1 HCl, 1 N : Dilute 50 mL in 600 mL of reagent water.
7.7 Filters, 0.45 /mi, Nylon, 25 mm i.d. (Gelman Sciences)
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional sampling practices should
be followed; however, the bottle must not be prerinsed with sample before collection.2
8.2 Sample Preservation and Storage
8.2.1 Add hydrochloric acid (1:1) to the sample to produce a pH of 2. The pH may be
measured in the field using pH indicator strips.
8.2.2 Residual chlorine should be reduced at the sampling site by the addition of a reducing
agent. Add 4-5 mg of sodium sulfite (this may be added as a solid with shaking until
dissolved) to each 100 mL of water.
8.2.3 The samples must be iced or refrigerated at 4°C away from light from the time of
collection until extraction. The samples must be analyzed within 14 days of collec-
tion. However, analyte stability may be affected by the matrix. Therefore, the analyst
should verify that the preservation technique is applicable to the samples under study.
362
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Method 555
If the 14-day holding time is exceeded, the data should be flagged so that the data
user is aware of possible analyte degradation.
8.2.4 Field reagent blanks (FRB): Processing a (FRB) is recommended along with each set,
which is composed of the samples collected from the same general sample site at
approximately the same time. At the laboratory, fill a sample container with reagent
water, seal, and ship to the sampling site along with the empty sample containers.
During sample collection, open the FRB and add HC1 (Sect. 8.2.1) and sodium sulfite
(Sect. 8.2.2) Return the FRB to the laboratory with filled sample bottles.
9. QUALITY CONTROL
9.1 Minimum QC requirements are initial demonstration of laboratory capability, analysis of
laboratory reagent blanks, laboratory fortified samples, laboratory fortified blanks, and QC
samples.
9.2 Laboratory reagent blanks (LRB): Before processing any samples, the analyst must demon-
strate that all glassware and reagent interferences are under control. Each time a set of
samples is extracted or reagents are changed, a LRB must be analyzed. If within the retention
time window of any analyte the LRB produces a peak that would prevent the determination of
that analyte, determine the source of contamination and eliminate the interference before
processing samples.
9.3 Initial demonstration of capability
9.3.1 Select a representative fortified concentration for each analyte. Prepare a sample
concentrate (in acetonitrile) containing each analyte at 1000 times the selected concen-
tration. With a syringe, add 100 fiL of the concentrate to each of at least four 100-
mL aliquots of reagent water, and analyze each aliquot according to procedures
beginning in Sect. 11.
9.3.2 Calculate the recoveries, the relative standard deviation, and the MDLs.5 For each
analyte the recovery value for all four of these samples must fall in the range of R ±
30%, using the value for R for reagent water in Table 2. As the calibration proce-
dure employs a fortified reagent water blank for the determination of the calibration
curves or factors, the recovery values for the analytes should, by definition, be within
this range. If the mean recovery of any analyte fails this demonstration, repeat the
measurement of that analyte to demonstrate acceptable performance.
9.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
analyzing unknown samples using a new, unfamiliar method prior to obtaining some
experience with it. As laboratory personnel gain experience with this method the
quality of data should improve beyond what is required here.
9.4 The analyst is permitted to modify LC columns, LC conditions, and detectors. Each time such
method modifications are made, the analyst must repeat the procedures in Sect. 9.3.
NOTE: The LC column and guard cartridge used to generate the data in this method
were found to be unique C/g-silica columns. Before substituting other CI8 columns, a
careful review of the literature is recommended.
363
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Method 555
9.5 Assessing laboratory performance: Laboratory fortified blank
9.5.1 The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
every 20 samples or one per sample set (all samples analyzed within a 24-hr period)
whichever is greater. The concentration of each analyte in the LFB should be 10
times the MDL or the MCL, whichever is less. Calculate accuracy as percent recov-
ery (X,). If the recovery of any analyte falls outside the control limits (See Sect.
9.5.2), that analyte is judged out of control, and the source of the problem should be
identified and resolved before continuing analyses.
9.5.2 Until sufficient data become available from within the laboratory, usually a minimum
of results from 20 to 30 analyses, the laboratory should assess laboratory performance
against the control limits in Sect. 9.3.2 that are derived from the data in Table 2.
When sufficient internal performance data become available, develop control limits
from the mean percent recovery (X) and standard deviation (S) of the percent recov-
ery. These data are used to establish upper and lower control limits as follows:
Upper Control Limit = X + 3S
Lower Control Limit = X - 3S
After each five to ten new recovery measurements, new control limits should be
calculated using only the most recent 20-30 data points. These calculated control
limits should never exceed those established in Sect. 9.3.2.
9.5.3 It is recommended that the laboratory periodically determine and document its detec-
tion limit capabilities for the analytes of interest.
9.5.4 At least quarterly, analyze a QC sample from an outside source.
9.5.5 Laboratories are encouraged to participate in external performance evaluation studies
such as the laboratory certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as independent checks
on the analyst's performance.
9.6 Assessing analyte recovery: Laboratory fortified sample matrix
9.6.1 The laboratory should add a known concentration to a minimum of 10% of the routine
samples or one sample per set, whichever is greater. The concentration should not be
less than the background concentration of the sample selected for fortification. Ideal-
ly, the concentration should be the same as that used for the laboratory fortified blank
(Sect. 9.5). Over time, samples from all routine sample sources should be fortified.
9.6.2 Calculate the percent recovery, P of the concentration for each analyte, after correct-
ing the analytical result, X, from the fortified sample for the background concentra-
tion, b, measured in the unfortified sample,
p = 100 (X - b}
fortifying concentration
364
-------
Method 555
and compare these values to control limits appropriate for reagent water data collected
in the same fashion. If the analyzed unfortified sample is found to contain NO back-
ground concentrations, and the added concentrations are those specified in Sect. 9.5,
the appropriate control limits would be the acceptance limits in Sect. 9.5. If, on the
other hand, the analyzed unfortified sample is found to contain background concentra-
tion, b, estimate the standard deviation at the background data, sb, using regressions
or comparable background data and similarly, estimate the mean, Xa, and standard
deviation, sa, of analytical results or the total concentration after fortifying. Then the
appropriate percentage control limits would be P + 3sp, where:
P = 100 x
(b + fortifying concentration)
and
sp = 100 _ ^L^
fortifying concentration
For example, if the background concentration for Analyte A was found to be 1
/xg/L and the added amount was also 1 /xg/L, and upon analysis the laboratory
fortified sample measured 1.6 /*g/L, then the calculated P for this sample would
(1.6 /xg/L minus 1.0 /tg/L)/ 1.0 /xg/L or 60%. This calculated P is compared to
control limits derived from prior reagent water data. Assume it is known that
analysis of an interference free sample at 1.0 /ig/L yields an s of 0.12 /xg/L and
similar analysis at 2.0 /xg/L yields X and S of 2.01 /xg/L and 0.20 /xg/L, respec-
tively. The appropriate limits to judge the reasonableness of the percent recovery,
60%, obtained on the fortified matrix sample is computed as follows:
100 (2.01 /xg/L)
2.0 Mg/L
+ 3 (100) [(Q-12 WW + (0.20 /xg/L)2]* _
1.0/xg/L
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
9.6.3 If the recovery of any such analyte falls outside the designated range, and the labora-
tory performance for that analyte is shown to be in control (Sect. 9.5), the recovery
problem encountered with the fortified sample is judged to be matrix related, not
system related. The result for that analyte in the unfortified sample is labeled sus-
pect/matrix to inform the data user that the results are suspect due to matrix effects.
365
-------
Method 555
9.7 The laboratory may adopt additional QC practices for use with this method. The specific
practices that are most productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be analyzed to assess the
precision of the environmental measurements. The field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
10. CALIBRA TION AND STANDARDIZA TION
10.1 Establish HPLC operating parameters equivalent to those indicated in Sect. 6.4.1. The HPLC
system should be calibrated using the external standard technique (Sect. 10.2).
NO TE: Calibration standard solutions must be prepared such that no unresolved
analytes are mixed together. The method analytes have been separated into two cali-
bration solutions (See Table 1 for Groups A and B). The analytes in these solutions
have been found to be resolved under the LC conditions listed. Mixtures of these
analytes at concentration levels of 100 pg/mL (in acetonitrile) are suggested as a
possible secondary dilution standard. Figures 2 and 3 are typical chromatograms of
Groups A and B as separated on the primary HPLC column.
10.2 External standard calibration procedure
10.2.1 Prepare calibration standards (CAL) at a minimum of three (five are recommended)
concentration levels for each analyte of interest by adding volumes of one or more
stock standards to volumetric flasks. Alternatively, add various volumes of a primary
dilution standard solution of Group A or B (Sect. 10.1) to a volumetric flask. Dilute
to volume with the aqueous mobile phase (0.025 M H3P04). The lowest standard
should contain analyte concentrations near, but above, the respective MDL. The
remaining standards should bracket the analyte concentrations expected in the sample
extracts, or should define the working range of the detector.
10.2.2 Starting with the standard of the lowest concentration, process each calibration stan-
dard according to Sect. 11.1 and tabulate response (peak area) versus injected quantity
in the standard. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (response factor) is
a constant over the working range (20% RSD or less), linearity through the origin can
be assumed and the average ratio or response factor can be used in place of a calibra-
tion curve.
10.2.3 The working calibration curve or response factor must be verified on each working
day by the measurement of a CAL, analyzed at the beginning of the analysis day. It
is highly recommended that an additional check standard be analyzed at the end of the
analysis day. For extended periods of analysis (greater than 8 hr), it is strongly
recommended that check standards be interspersed with samples at regular intervals
during analyses. If the response for any analyte varies from the predicted response by
more than ±25%, the test must be repeated using a fresh calibration standard. If the
results still do not agree, generate a new calibration curve.
366
-------
Method 555
10.2.4 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
standard prepared from reference material obtained from an independent source.
Results from these analyses must be within the limits used to routinely check calibra-
tion.
7 7. PROCEDURE
11.1 Hydrolysis, preparation, and extraction
11.1.1 Add preservative to blanks and QC check standards. Mark the water meniscus on the
side of the sample bottle for later determination of sample volume (Sect. 11.1.5).
11.1.2 Add 1.7 mL of 6 N NaOH to the sample, seal, and shake. Check the pH of the
sample with pH paper; if the sample does not have a pH greater than or equal to 12,
adjust the pH by adding more 6 N NaOH. Let the sample sit at room temperature for
1 hr, shaking the sample bottle and contents periodically.
11.1.3 Add 2 mL of concentrated H3PO4 to the sample, seal, and shake to mix. Check the
pH of the sample with pH paper; if the sample does not have a pH less than or equal
to two, adjust the pH by adding more H3PO4.
11.1.4 From the homogeneous sample, remove a 20-mL aliquot for analysis. Filter the
aliquot through a 0.45 /*m filter into a graduated cylinder or other convenient graduat-
ed container. Using an HPLC pump (or HPLC reagent delivery pump), pump the 20-
mL aliquot through the on-line concentrator column at a flowrate of 5.0 mL/min (See
Figure 1). The use of a liquid-solid extraction disk is perfectly acceptable providing
all QC criteria in Sect. 9 are met or exceeded. After passing the sample through the
concentrator column, follow with an additional 10-mL of the aqueous mobile phase
(0.025 M H3PO4).
11.1.5 After analysis is completed, determine the original sample volume by refilling the
sample bottle to the mark and transferring the water to a 100-mL graduated cylinder.
Record the sample volume to the nearest 1 mL.
11.2 High performance liquid chromatography
11.2.1 Sect. 6.4.1 summarizes the recommended operating conditions for the HPLC. Includ-
ed in Table 1 are retention times observed using this method. Other HPLC columns,
chromatographic conditions, or detectors may be used if the requirements of Sect. 9.3
are met.
11.2.2 Calibrate the system daily as described in Sect. 10.
11.2.3 After loading the sample (or calibration standard) onto the concentrator column, valve
the sample into the analytical stream, backflushing the concentrator column. The
photodiode array detector (PDA-UV) is set to scan and record from 210 to 310 nm, 1
scan per second during the entire chromatographic run (40 min). Extract the 230 ran
trace from the stored data and record the resulting peak size in area units for all
analytically significant peaks.
11.2.4 If the responses for the peaks exceed the working range of the system, dilute an
additional 20-mL aliquot of the sample with reagent water, adjust the pH to 12 with
NaOH, and reanalyze according to Sect. 11.1.2.
367
-------
Method 555
11.3 Identification of analytes
11.3.1 Identify a sample component by comparison of its retention time to the retention time
of a reference chromatogram. If the retention time of an unknown compound corre-
sponds, within limits, to the retention time of a standard compound, then identification
is considered positive.
11.3.2 The width of the retention time window used to make identifications should be based
upon measurements of actual retention time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
11.3.3 Identification requires expert judgment when sample components are not resolved
chromatographically. When peaks obviously represent more that one sample compo-
nent i.e., broadened peak with shoulder(s) or vollies between two or more maxima, or
any time doubt exists over the identification or a peak on a chromatogram, appropriate
alternative techniques, to help confirm peak identification, should be used. For this
method, the use of the PDA-UV detector affords the analyst the option of using a
secondary wavelength for the analysis of the questionable identification. The response
ratio for a compound of interest at two wavelengths may be determined from stan-
dards of known purity. If the wavelength response ratio and the retention time match-
es a given unknown to a method analyte, more certainty may be assigned to the
identification of the unknown. If this method of compound confirmation is employed,
each analyst will need to determine the wavelength response ratio for each analyte.
Table 3 lists suggested alternative wavelengths for each analyte in the scope of the
method. An alternative LC column may be used to separate and confirm the identifi-
cation of unknown peaks. A suggested alternative column is described in Sect. 6.4.2.
12. DA TA ANAL YSIS AND CALCULA TIONS
12.1 Calculate analyte concentrations in the sample from the response for the analyte using the
calibration procedure described in Sect. 10.
12.2 Calculate the amount of sample analyte injected from the peak response using the calibration
curve or calibration response factor determined in Sect. 10.2. The concentration (C) in the
sample can be calculated from Equation 1.
Equation 1
Concentration (ng/L) =
^ /'^ S?
Where:
A = Amount of material injected (ng).
V = Volume of standard injected (mL).
V( = Volume of sample injected (mL).
V = Volume of water sample (mL).
368
-------
Method 555
13. METHOD PERFORMANCE
13.1 In a single laboratory, analyte recoveries from reagent water were determined at two concen-
tration levels. Results were used to determine analyte MDLs and demonstrated method range.5
Analyte MDLs and analyte recoveries and standard deviations about the percent recoveries at
one concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from dechlorinated tap water and ground waters were
determined at one concentration level, 10 /*g/L. Results were used to demonstrate appli-
cability of the method to different tap and ground water matrices. Analyte recoveries from tap
water and ground water are given in Table 4. MDLs calculated from results of analyses of six
100 mL reagent water samples at 0.5 /tg/L concentrations for each analyte are listed in
Table 5.
14. POLLUTION PREVENTION
14.1 This method utilizes the new in-line liquid-solid extraction technology which requires the use
of very small quantities of organic solvents. This feature eliminates the hazards involved with
the use of large volumes of potentially harmful organic solvents needed for conventional
liquid-liquid extractions. Also, this method uses no derivatizing reagents, which are toxic or
explosive, to form gas chromatographable derivatives. These features make this method much
safer for use by the analyst in the laboratory and a great deal less harmful to the environment.
14.2 For information about pollution prevention that may be applicable to laboratory operations,
consult "Less is Better: Laboratory Chemical Management for Waste Reduction," available
from the American Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 Due to the nature of this method, there is little need for waste management. No large volumes
of solvents or hazardous chemicals are used. The matrices of concern are finished drinking
water or source water. However, the Agency requires that laboratory waste management
practices be consistent with all applicable rules and regulations, and that laboratories protect
the air, water, and land by minimizing and controlling all releases from fume hoods and bench
operations. Also, compliance is required with any sewage discharge permits and regulations,
particularly the hazardous waste identification rules and land disposal restrictions. For further
information on waste management, consult "The Waste Management Manual for Laboratory
Personnel," also available from the American Chemical Society at the address in Sect. 14.2.
369
-------
Method 55o
References
1. Giazer, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., Environ. Sci.
Technol. 15, 1981, pp. 1426-1435.
2. "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No. 68-03-2697.
Available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268.
3. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society for Testing and
Materials, Philadelphia, PA, p. 86,1986.
4. Giam, C.S., H.S. Chan, and G.S. Nef. "Sensitive Method for Determination of Phthalate
Ester Plasticizers in Open-Ocean Biota Samples," Analytical Chemistry. 47, 2225 (1975).
5. 40 CFR, Part 136, Appendix B.
370
-------
Method 555
Table 1. Retention Times for Method Analytes
Retention Times'
(minutes)
Group
(A)
(A)
(A)
(B)
(A)
(A)
(B)
(A)
(B)
(B)
(A)
(B)
(B)
(A)
(A)
(B)
(B)
Primary Column
19.0
19.7
21.1
21.6
24.0
25.2
25.5
25.6
26.7
27.2
27.3
27.5
28.0
29.2
30.7
32.8
33.4
Confirmation
Column
12.8
13.5
14.8
5.0
18.2
19.5
20.1
20.1
21.3
21.8
21.8
22.4
22.8
23.9
25.5
27.7
28.3
Analyte
Picloram
5-Hydroxydicamba
Chloramben
4-Nitrophenol
Dicamba
Bentazon
MCPA
2,4-D
3,5-Dichlorobenzoic acid
MCPP
Dichloroprop
2,4,5-T
2,4-DB
2,4,5-TP
Acifluorfen
Dinoseb
Pentachlorophenol
Columns and analytical conditions are described in Sect. 6.4.1 and Sect. 6.4.2.
377
-------
Method 555
Table 2. Single Laboratory
(MDLs) for Analytes From
Accuracy,
Precision and Method
Detection Limits
Reagent Water a
MDL
Analyte d
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic
acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol
(PCP)
Picloram
2,4,5-T
2,4,5-TP
ig/Lf
1.7
4.6
3.1
1.3
1.9
2.1
2.1
1.7
1.5
2.2
0.8
1.7
1.2
1.6
0.5
1.3
1.8
Concentra tion
(fjg/U
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
Reagent Water
ff
104
126
83
112
92
104
94
108
97
132
93
95
95
99
104
93
90
S d
1.7
14.6
10.0
4.2
5.9
6.6
6.7
5.4
4.8
7.0
2.5
5.5
4.0
5.2
1.7
4.1
5.8
Data represent the average of 6-7 samples. Sample volume = 20 mL.
MDL = method detection limit; defined in Appendix B to 40 CFR Part 136 —Definition and
Procedure for the Determination of the Method Detection Limit—Revision 1.11.
R = average percent recovery.
S, = standard deviation of the percent recovery
372
-------
Method 555
Table 3. Confirmation Wavelengths and Area Response Ratios for Method
Confirmation Area Response
Wavelength (nm) Ratio'
Acifluorfen 293 1 .72
Bentazon 240 1 .08
Chloramben 214 0.61
2,4-D 285 4.02
2,4-DB 285 5.93
Dicamba 220 0.66
3,5-Dichlorobenzoic acid 285 5.15
Dichlorprop 285 4.07
Dinoseb 268 0.48
5-Hydroxydicamba 293 1 .89
MCPA 285 6.66
MCPP 285 6.49
4-Nitrophenol 310 0.56
Pentachlorophenol (PCP) 290 5.65
Picloram 223 0.82
2,4,5-T 290 . 4.00
2,4,5-TP 293 3.84
Area Response Ratio = Peak Area for 230 nm / Peak Area for Conf . Wavelength
373
-------
Method 555
Table 4. Single Laboratory Precision and Accuracy Data From Tap Water And
Ground Water3
Dechlorina ted
Analyte
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol
-------
Method 555
Table 5. Single Laboratory Recovery and Precision Data and Method Detection
Limits (MDLs) for Analytes from Reagent Water3
Analyte
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxdicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol (PCP)
Picloram
2,4,5-T
2,4,5-TP
MDL
dig/if
0.40
0.12
NR
0.34
0.31
0.24
0.38
0.33
0.26
NR
0.35
0.19
NR
0.15
NR
0.21
0.37
Concentration
(tig/U
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Reagent Water
ff
114
91
NR
121
99
80
105
110
99
NR
124
125
NR
93
NR
80
77
S,<
23.7
7.3
NR
20.2
18.5
14.1
22.5
19.4
15.5
NR
21.0
11.1
NR
8.6
NR
12.7
21.7
a Data represent the average of six samples. Sample Volume = 100 ml
b MDL= Method detection limit; defined in Appendix B to 40 CFR 136—Definition and
Procedure for the Determination of the Method Detection Limit—Revision 1.11.
c R = Average percent recovery.
d S, = Standard deviation of the percent recovery.
NR = Not Recovered.
375
-------
Method 555
Precolumn Extraction Hardware
Analysis Mode
Sample Pump
Extraction Mode
Precolumr
Waste
Column
Rheodyne 7000 Valve
(6-Port)
Precolumn
Analytical Analytical Pump(s)
Waste
Analytical
Column
Analytical Pump(s)
Detector
L
Detector
52-015-8
Figure 1. Schematic Diagram of Sample Concentration and
Analytical HPLC Hardware
376
-------
Method 555
0200-
0 150-
0.100-
0.050 -
c
0)
.O
E
6
S
a
o
'S.
g
|
S
Q
I
-0050
1 1 1 1 1 1 1 1 1 1
0.0 5.0 10.0 15.0 20.0 25.0
C1 (Minutes)
300
Figure 2. Typical HPLC Chromatogram of Group AAnalytes
35!o 4OO
52-015-9
377
-------
Method 555
Method 555
0.20 -
0.15-
0.10-
0.05-
0-!
-0.05
o
c
------- |