SEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/4-82-057 July 1982
Test Methods
Methods for Organic Chemical
Analysis of Municipal and
Industrial Wastewater
James E. Longbottom and James J. Lichtenberg, Editors
Distribution Record for Methods for
Organic Chemical Analysis of Municipal and
Industrial Wastewater
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Disclaimer
This report has been reviewed by the Environmental Monitoring and Support
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
U.S. Environmental Protection Agency
Environmental Monitoring and Support Laboratory
26 W. St. Clair Street
Cincinnati, OH 45268
ATTN: MOCAW Distribution Record
-------
Foreword
Environmental measurements are required to determine the quality of ambient
waters and the character of waste effluents. The Environmental Monitoring and
Support Laboratory—Cincinnati, conducts research to:
• Develop and evaluate techniques to measure the presence and concentration
of physical, chemical, and Radiological pollutants in water, wastewater,
bottom sediments, and solid waste.
• Investigate methods for the concentration, recovery, and identification of
viruses, bacteria and other microbiological organisms in water; and to
determine the responses of aquatic organisms to water quality.
• Develop and operate an Agency-wide quality assurance program to assure
standardization and quality control of systems for monitoring water and
wastewater.
• Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
Under authority of Sections 304(h) and 501 (a) of the Federal Water Pollution
Control Act of 1972 and the Clean Water Act of 1977, the Environmental
Protection Agency is required to promulgate guidelines establishing test
procedures for the analysis pf pollutants. The test procedures in this manual
have undergone extensive laboratory testing and public review. They represent
the state-of-the-art for the measurement of specific organic analytes in
municipal and industrial wastewater.
Robert L. Booth, Acting Director
Environmental Monitoring and Support Laboratory
-------
Acknowledgments
Throughout the development and refinement of the methods for organic
priority pollutants, the staff of the Physical and Chemical Methods Branch of the
Environmental Monitoring and Support Laboratory—Cincinnati, has operated as
a working committee to test, validate, and edit these methods. The contributions
of the committee members are gratefully acknowledged:
Thomas A. Bellar
Dr. Stephen Billets
Dr. William L Budde
Dr. Denis L. Foerst
Dr. John A. Glaser
Dr. Fred K. Kawahara
Edward H. Kerns
The outstanding efforts of typing, proofreading, and computerized text editing,
performed under a series of critical deadlines by Jean Wilson, Carol Brockhoff,
Beth Casias, and Patricia Hurr, are also gratefully acknowledged.
fv
-------
Contents
Foreword ill
Acknowledgments iv
Introduction vi
Approved Test Procedures for Organic Chemicals ix
Purgeable Halocarbons Method 601
Purgeable Aromatics Method 602
Acrolein and Acrylonitrile Method 603
Phenols Method 604
Benzidines Method 605
Phthalate Esters Method 606
Nitrosamines Method 607
Pesticides and PCBs Method 608
Nitroaromatics and Isophorone Method 609
Polynuclear Aromatic Hydrocarbons Method 610
Haloethers Method 611
Chlorinated Hydrocarbons Method 612
2,3,7,8-Tetrachlorodibenzo-p-dioxin Method 613
Purgeables Method 624
Base/Neutrals, Acids, and Pesticides Method 625
Appendix A A-1
-------
Introduction
In 1976, the U.S. Environmental Protection Agency entered into a Settlement
Agreement requiring it to study and, if necessary, regulate 65 "priority"
pollutants and classes of pollutants (1). This list was later defined to include 129
specific "priority pollutants." In December 1977, Congress passed the Clean
Water Act of 1977 (PL 95-217) declaring the 65 pollutants and classes of
pollutants to be "toxic" under Section 307(a) of the Act.
The Settlement Agreement included a rigid time schedule for the completion
of industrial wastewater analytical surveys and promulgation of effluent
guidelines. To provide the Effluent Guidelines Division of USEPA with a means
of measuring the concentration of pollutants in these wastewaters, the USEPA's
Environmental Monitoring and Support Laboratory in Cincinnati, and the
Environmental Research Laboratory in Athens, Georgia, collaborated on a
research project that resulted in an analytical protocol (2) that was successfully
applied to a variety of wastewaters. The gas chromatography/mass spectrometry
(GC/MS) procedures in that protocol for the measurement of organic pollutants
were the forerunners of methods 624 and 625 that appear in this manual.
In a parallel research project, the Environmental Monitoring and Support
Laboratory in Cincinnati, through a series of research contract and in-house
activities, undertook a systematic study of the analytical behavior of each of the
individual classes of organic compounds to identify or develop non-MS
approaches that could be used for routine monitoring of regulated discharges.
This approach was pursued to minimize the requirements for expensive mass
spectrometer equipment and the skilled operators required to use it. The
resulting test procedures were identified as methods 601 through 612 and were
first published in early 1979, along with a GC/MS method for the measurement
of TCDD (3,4).
All fifteen of the test procedures discussed above were proposed as
amendments to the "Guidelines Establishing Test Procedures for the Analyses of
Pollutants" (40 CFR, Part 136) in December 3, 1979 (5). These guidelines are
required by Section 304(h) of the Clean Water Act. As a result of public comment
from over 200 respondents, extensive editorial revisions were made to the
methods. The majority of the revisions were made either for clarification or to
add additional flexibility for the analyst. These revised methods constitute the
body of this methods manual.
The methods contained herein represent an effort to provide procedures that
are as uniform and cost-effective as practical for a wide cross-section of
chemical compound classes. Due to the variable chemical and physical
properties of the parameters under study, some compromises were made.
Therefore, in some of the methods, the extraction procedures, cleanup
procedures, and determinative steps are not optimum for all parameters.
A distribution list will be established for this manual. The list will be prepared
from the distribution record cards that accompany this package. Future revisions
of existing methods and the addition of new methods will only be sent to
individuals who return these record cards. Correspondence on these methods is
invited.
References
1. Natural Resources Defense Council, Inc., et al., v. Train, 8 ERC 2120 (D.D.C.
1976), modified 12 ERC 1883 (D.D.C. 1979).
2. "Sampling and Analysis Procedures for Screening of Industrial Effluents for
Priority Pollutants," March 1 977 (revised, April 1977). U.S. Environmental
Protection Agency, Effluent Guidelines Division, Washington, D.C. 20460.
3. "Methods for Organic Compounds in Municipal and Industrial Wastewater,"
March 1979, U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
4. "Methods for Organic Compounds in Municipal and Industrial Wastewater,"
April 1979, U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
5. Guidelines Establishing Test Procedures for the Analysis of Pollutants;
Proposed Regulations, 40 Code of Federal Regulations (CFR), Part 136,
Published in Federal Register, 44, 69464.
-------
Approved Test Procedures for
Organic Chemicals
Section 136.3(a) of 40 Code of Federal Regulations cites approved test
procedures through the use of five subtables:
Table 1A - Biological parameters
Table 1 B - Inorganic and physical parameters
Table 1C - Organic chemical parameters
Table 1D - Pesticide parameters
Table 1E - Radiological parameters
The test procedures in this manual are cited in Tables 1C and 1D. These
tables have been reproduced here for the convenience of the user.
-------
40 C.F.R. Part 136, § 136.3
Table 1C. List of approved test procedures for organic compounds
Parameter'1
GC
EPA Method
Number2
GC/MS
HPLC
Other
1. Acenaphthene 610 625
2. Acenaphthalene 610 625
3. Acrolein 603 624*
4. Aerylo nitrite 603 624*
5. Anthracene 610 625
6, Benzene 602 624
7. Benzidine - - 625*
8. Benzofajanthracene 610 625
9, Benzofajpyrene 610 625
JO. Benzo{b)fluoranthene 61O 625
11. Benzo(ghi)perylene 610 625
12, Benzo{k)fluoranthene 610 625
13. Benzyl Chloride
14. Benzyl Butyl Phthatate 606 625
15. Bis!2-chloroethyl) ether 611 625
16. B/s(2-chforoethoxyJ methane 611 625
17, Bis(2-chloroisopropyl) ether 611 625
18. Bis(2-ethylhexyl) phthalate 606 625
19. Bromodichloromethane 601 624
20. Bromoform 601 624
21. Bromoethane 601 624
22. 4-Bromophenylphenyl ether 611 625
23. Carbon tetrachloride 601 624
24. 4-Chloro-3-methylphenol 6O4 625
25. Chlorobenzene 601,602 624
26. Chloroethane 601 624
27. 2-Chloroethylvinyl ether 601 624
28. Chloroform 601 624
29. Chloromethane 601 624
30. 2-Chloronaphthalene 612 625
31. 2-Chlorophenol 604 625
32. 4-Chlorophenylphenyl ether 611 625
33. Chrysene 610 625
34. Dibenzofa.hjanthracene 610 625
35. Dibromochloromethane 6O1 624
36. 1,2-Dichlorobenzene 601, 602, 612 624, 625
37. 1,3-Dichlorobenzene 601, 602, 612 624, 625
38. 1,4-Dichlorobenzene 601, 601, 612 624, 625
39. 3,3'-Dichlorobenzidine - - 625
40. Dichlorodifluoromethane 601 - -
41. 1.1-Dichloroethane 601 624
42. 1,2-Dichloroethane 601 624
43. Dichloroethene 601 624
44. trans- 1,2-Dichloroethene 6O1 624
45. 2.4-Dichlorophenol 6O4 625
46. 1,2-Dichloropropane 601 624
47. cis-1,3-Dichloropropene 601 624
48, trans-1,3-Dichoropropene 6O1 624
49. Diethyl phthalate 606 625
50. 2,4-Dimethylphenol 604 625
51. Dimethyl phthalate 606 625
52. Di-n-butyl phthalate 6O6 625
53. Di-n-octyl phthalate 606 625
54. 2,4-Dinitrophenol 604 625
55. 2,4-Dinitrotoluene 6O9 625
56. 2,6-Dinitrotoluene 6O9 625
610
61O
610
605
610
61O
610
610
610
Note 3, p. 1;
Note 6, p. S48
Note 3, p. 130;
Note 6, p.SI02
Note 3, p. 130;
Note 6, p.S102
Note 3, p. 130;
Note 6, p. SIO2
Note 3, p. 130;
Note 6, p.S102
610
610
605
-------
Table 1 C. Continued
Parameter"1
57. Epichlorohydrin
58. Ethylbenzene
59. Fluoranthene
6O. Fluorene
61. Hexachlorobenzene
62. Hexachlorobutadiene
63. Hexachlorocyclopentadiene
64. Hexachloroethane
65. ldeno(1 ' ,2,3-cdjpyrene
66. Isophorone
67. Methylene chloride
68. 2-Methyl-4,6-dinitrophenol
69. Naphthalene
70. Nitrobenzene
71. 2-Nitrophenol
72. 4-Nitrophenol
73. N-Nitrosodimethylamine
74. N-Nitrosodi-n-propylamine
75. N-Nitrosodiphenylamine
76. PCB-1016
77. PCB-1221
78. PCB-1232
79. PCB-1242
SO. PC B- 1248
81. PCB-1254
82. PCB-1260
83. Pentachlorophenol
84. Phenanthrene
85. Phenol
86. Pyrene
87. 2,3,7,8-Tetrachloro-
dib enzo -p - dioxin
88. 1,1,2,2-Tetrachloroethane
89. Tetrachloroethene
90. Toluene
91. 1 ,2,4-Trichlorobenzene
92. 1,1,1- Trichloroethane
93. 1,1.2-Trichloroethane
94. Trichloroethene
95. Trichlorofluoromethane
96. 2.4,6-Trichlorophenol
97. Vinyl Chloride
GC
- -
602
610
610
612
612
612
612
610
609
601
604
610
609
604
604
6O7
607
607
608
608
6O8
608
608 -
608
608
6O4
610
604
610
. .
601
601
6O2
612
6O1
601
6O1
601
604
601
EPA Method
Number2
GC/MS
- _
624
625
625
625
625
625s
625
625
625
624
625
625
625
625
625
625s
625
6255
625
625
625
625
625
625
625
625
625
625
625
613
624
624
624
625
624
? 624
624
624
625
624
HPLC Other
- - Note 3, p. 13O;
Note 6, p.S102
_
610 - -
610 - -
- -
- -
- -
_ _ _ _
670 - -
_
- - Note 3, p. 130;
Note 6, p.S102
- -
- - _
- -
.
_ _
-
_ _ _ _
- - Note 3, p. 43;
Note 6, p. S78
- - Note 3, p. 43;
Note 6, p. S78
- - Note 3, p. 43;
Note 6, p.S78
- - Note 3, p. 43;
Note 6, p. S78
- - Note 3, p. 43;
Note 6, p. S78
- - Note 3, p. 43;
Note 6, p. S78
- - Note 3, p. 43;
Note 6, p. S78
- - Note 3, p. 140;
Note 6, p. S50
610 - -
- -
610 - -
.
- - Note 3, p. 13O;
Note 6, p. S102
- - Note 3, p. 13O
Note 6, p. S102
- - Note 3, p. 13O;
Note 6, p. S102
- - Note 3, p. 130;
Note 6, p.S102
- -
- -
- -
-------
Table 1C. Notes
1 All parameter concentrations are expressed in micrograms per liter f/jg/LJ.
1 "Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater," USEPA, July 1982.
3 "Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol and Pesticides in Water and Wastewater,"
USEPA, September 1978.
* Method 624 may be extended to screen samples for acrolein and acrylonitrile. However, when they are kno wn to be present,
the preferred method for these two compounds is Method 6O3.
5 Method 625 may be extended to include benzidine, hexachlorocyclopentadiene, N-nitrosodimethylamine, and
N-nitrosodiphenylamine. However, when they are known to be present. Method 605, 612, 607, and 607, respectively, are
the preferred methods for these compound.
s "KalnrtarlAnalytical Methods ApprovedandCitedbvthe UnitedStates EnvironmentalProtection Agency,"Supplement to the
Fifteenth Edition of Standard Methods for the Examination of Water and Wastewater (1981).
40 C.F.R. Part 136, § 136.3
Table 1D. List of approved test procedures for pesticides.1
Parameter
(fJff/LJ
1, Aid r in
2. Ametryn
3. Aminocarb
4. Atraton
5. Atrazine
6. Azinphos methyl
7. Barban
8. a-BHC
9. ft-BHC
10. 6-BHC
1 1. 6-BHC (Lindane)
12, Captan
13. Carbaryl
14. Carbophenothion
15. Chlordane
16. Chlorpropham
17. 2.4-D
18. 4,4'-DDD
19. 4.4'-DDE
20. 4.4'-DDT
21. Demeton-O
Method
GC
GC/MS
GC
TLC
GC
GC .
gc
TLC
GC
GC/MS
GC
GC/MS
GC
GC/MS
GC
GC/MS
GC
TLC
GC
GC
GC/MS
TLC
GC
GC
GC/MS
GC
GC/MS
GC
GC/MS
GC
EPA2
608
625
...
—
---
—
—
608
625s
608
625
608
625s
6O8
625
...
...
—
608
625
...
—
608
625
608
625
6O8
625
...
Std. Methods
15th Ed. ASTM Other
509A D3086 Note 3, p. 7;
Note 4, p. 30
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 94;
Note 6, p. S16
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 25;
Note 6, p.S51
znote 3, p. 104;
Note 6, p. S64
509A D3086 Note 3. p. 7
— — —
D3086
— — —
D3086
— —
509A D3086 Note 3, p. 7;
Note 4, p. 30,
Note 6, p. S73
— — —
505X1 — - Note 3, p. 7
Note 3, p. 94;
Note 6, p. S73
Note 4, p. 30;
Note 6, p. S73
509 A D3086 Note 3, p. 7
— — —
Note 3, p. 104;
Note 6, p.S64
509B — Note 3, p. 1 15;
Note 4. p. 35
509A D3086 Note 3, p. 7;
Note 4, p. 30
— — —
509A D3086 Note 3, p. 7;
Note 4, p. 30
— — —
509A D3O86 Note 3, p. 7;
Note 4, p. 30
— —
Note 3, p. 25;
Note 6, p. S51
-------
, Table 1 D. Continued
Parameter
(fig/L)
22. Demeton-S
i,
23. Diazinon
24. Dicamba
25. Dichlofenthion
26. Dichloran
27. Dicofol
28. Dieldrin
29. Dioxathion
30. Disulfoton
31. Diuron
32. Endosulfan 1
33. Endosulfan II
34. Endosulfan sulfate
35. Endrin
36. Endrin aldehyde
37. Ethion
38. Fenuron
39. Fenuron-TCA
4O. Heptachlor
41 . Heptachlor epoxide
42. Isodrin
43. Linuron
44. Malathion
45. Methiocarb
46. Methoxychlor
47. Mexacarbate
48. Mirex
49. Monuron
.40. Monuron-TCA
Method
GC
GC
GC
GC
GC
GC
GC
GC/MS
GC
GC
TLC
GC
GC/MS
GC
GC/MS
GC
GC/MS
GC
GC/MS
GC
GC/MS
GC
TLC
TLC
GC
GC/MS
GC
GC/MS
GC
TLC
GC
TLC
GC
TLC
GC
TLC
TLC
EPA2
—
—
—
...
.—
—
608
625
...
...
...
608
625*
608
5255
6O8
625
608
625*
608
625
...
...
50S
525
608
625
...
...
...
...
...
...
...
...
...
Std. Methods
15th Ed. ASTM
....
....
509/4
D3OS5
509X1
—
509X1 D3086
— —
509X1 D30S5
:
509X1 £>30S5
.
____
—
509X1 D3086
5O9X1 D3O86
509X1
—
509X1 D3086
5O9X1
Other
Note 3, p. 25;
Note 6, p. S51
Note 3, p. 25;
Note 4, p. 30;
Note 6, p. S51
Note 3, p. 115
Note 4, p. 30;
Note 6, p. S73
Note 3, p. 7
Note 3, p. 7;
Note 4, p. 30
Note 6, p. S73
—
Note 4, p. 30;
Note 6, p. S73
Note 3, p. 25;
Note 6, p. S51
Note 3, p. 104;
Note 6, p.S64
Note 3, p. 7
—
Note 3, p. 7
Note 3, p. 7;
Note 4, p. 3O
, — -
____
Note 4, p. SO;
Note 6, p. S64
Note 3, p. 104;
Note 6, p. S64
Note 3, p. 104;
Note 6, p. S64
Note 3, p. 7;
Note 4, p. 4O
Note 3, p. 7;
Note 4, p. 30;
Note 6, p. S73
Note 4. p. 3O;
Note 6. p. S73
Note 3, p. 1O4;
Note 6, p. S64
Note 3. p. 25;
Note 4, p. 30;
Note 6, p. S51
Note 3, p. 94;
Note 6, p. S60
Note 3, p. 7;
Note 4, p. 30
Note 3, p. 94;
Note 6, p. S60
Note 3, p. 7
Note 3, p. 104;
Note 6, p. S64
Note 3, p. 104;
Note 6, p. S64
-------
Table 1D. Continued
Parameter
(ug/U
51. Neburon
52. Parathion methyl
53. Parathion ethyl
54. PCNB
55. Perthane
56. Prometon
57. Prometryn
58. Propazine
59. Propham
60. Propoxur
61. Secbumeton
62. Siduron
63. Simazine
64. Strobane
65. Swep
66. 2,4,5-T
67. 2,4,5-tp (Silvex)
68. Terbuthylazine
69. Toxaphene
70. Trifluralin
Method EPA2
TLC
GC
GC
GC
GC
GC
GC
GC
TLC
TLC
TLC
TLC
GC
GC
TLC
GC
GC
GC
GC 608
GC/MS 625
GC
Std. Methods
15th Ed. ASTM Other
Note 3, p. 104;
Note 6, p. S64
509A —- Note 3, p. 25;
Note 4, p. 30
509 A — - Note 3, p. 25
509A -— Note 3, p. 7
D3086
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 104;
Note 6, p. S64
Note 3, p. 94;
Note 6, p. S60
Note 3, p. 83;
Note 6, p. S68
Note 3, p. 104;
Note 6, p. S64
Note 3, p. 83; '
Note 6, p. S68
509A —- Note 3, p. 7
Note 3, p. 104;
Note 6, p. S64
509B Note 3, p. 1 15;
Note 4, p. 35
509B Note 3, p. 83;
Note 6, p. S68
Note 3, p. 83;
Note 6, p. S68
509A D3086 Note 3, p. 7;
Note 4, p. 30
— — —
Note 3, p. 7
1 Pesticides are listed in this table by common name for the convenience of the reader. Additional pesticides may be
found under Table 1C, where entries are listed by chemical name.
2 "Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater." USEPA, July 1982.
3 "Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol and Pesticides in Water and Wastewater,"
USEPA, September 1978.
4 Methods for-Analysis of Organic Substances in Water, U.S. Geological Survey Techniques of Water Resources Inv., Book 5,
Ch. A3 (1972), p. 30.
8 The method may be extended to screen samples for a-BHC, and 6-BHC. endosulfan I. endosulfan II. and endrin. However,
when they are known to be present, the referenced gas chromatographic procedures are the preferred methods.
8 "Selected Analytical Methods Approved and Cited by the United States Environmental Protection Agency," Supplement to the
Fifteenth Edition of Standard Methods for the Examination of Water and Wastewater (1981).
-------
SEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Purgeable Halocarbons—
Method 601
1. Scope and Application
1.1 This method covers the determi-
nation of 29 purgeable halocarbons.
The following parameters may be
determined by this method:
Parameter
STORET No.
CAS No.
Bromodichloromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chloroethane
2-Chloroethylvinyl ether
Chloroform
Chloromethane
Dibromochloromethane
1 ,2-Dichlorobenzene
1 ,3-Dichlorobenzene
1 ,4-Dichlorobenzene
Dichlorodifluoromethane
1 , 1 -Dichloroethane
1,2-Dichloroethane
1,1-Dichloroethene
trans-1 ,2-Dichloroethene
1 ,2-Dichloropropane
cis-1 ,3-Dichloropropene
trans-1 ,3-Dichloropropene
Methylene chloride
1 ,1 ,2,2-Tetrachloroethane
Tetrachloroethene
1,1,1 -Trichloroethane
1 , 1 ,2-Trichloroethane
Trichloroethene
Trichlorofluoromethane
Vinyl chloride
32101
32104
34413
32102
34301
34311
34576
32106
34418
32105
34536
34566
34571
34668
34496
34531
34501
34546
34541
34704
34699
34423
34516
34475
34506
34511
39180
34488
39175
75-27-4
75-25-2
74-83-9
56-23-5
108-90-7
75-00-3
100-75-8
67-66-3
74-87-3
124-48-1
95-50-1
541-73-1
106-46-7
75-71-8
75-34-3
107-06-2
75-35-4
1 56-60-5
78-87-5
10061-01-5
10061-02-6
75-09-2
79-34-5
127-18-4
71-55-6
79-00-5
79-01-6
75-69-4
75-01-4
1.2 This is a purge and trap gas
chromatographic method applicable to
the determination of the compounds
listed above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any or
all of the compounds above, compound
identification should be supported by at
least one additional qualitative
601-1
July 1982
-------
technique. This method describes
analytical conditions for a second gas
chromatographic column that can be
used to confirm measurements made
with the primary column. Method 624
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results for
most of the parameters listed above.
1.3 The method detection limit (MDL,
defined in Section 12.1 )<1) for each
parameter is listed in Table 1. The MDL
for a specific wastewater may differ
from those listed, depending upon the
nature of interferences in the sample
matrix.
1.4 Any modification of this method,
beyond those expressly permitted,
shall be considered as major modifica-
tions 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 operation of a purge
and trap system and a gas chromato-
graph and in the interpretation of
chromatograms. Each analyst must
demonstrate the ability to generate
acceptable results with this method
using the procedure described in
Section 8.2.
2. Summary of Method
2,1 An inert gas is bubbled through a
5-mL water sample contained in a
specially-designed purging chamber at
ambient temperature. The halocarbons
are efficiently transferred from the
aqueous phase to the vapor phase. The
vapor is swept through a sorbent trap
where the halocarbons are trapped.
After purging is completed, the trap is
heated and backflushed with the inert
gas to desorb the halocarbons onto a
gas chromatographic column. The gas
chromatograph is temperature pro-
grammed to separate the halocarbons
which are then detected with a halide-
specific detector.'2'3'
2.2 The method provides an optional
gas chromatographic column that may
be helpful in resolving the compounds
of interest from interferences that may
occur.
3. Interferences
3.1 Impurities in the purge gas and
organic compounds out-gassing from
the plumbing ahead of the trap account
for the majority of contamination
problems. The analytical system must
be demonstrated to be free from
contamination under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5. The use of non-TFE plastic tubing,
non-TFE thread sealants, or flow
controllers with rubber components in
the purging device should be avoided.
3.2 Samples can be contaminated by
diffusion of volatile organics (particu-
larly fluorocarbons and methylene
chloride) through the septum seal into
the sample during shipment and
storage. A field reagent blank prepared
from reagent water and carried through
the sampling and handling protocol can
serve as a check on such
contamination.
3.3 Contamination by carry-over can
occur whenever high level and low
level samples are sequentially analyzed.
To reduce carry-over, the purging
device and sample syringe must be
rinsed with reagent water between
sample analyses. Whenever an
unusually concentrated sample is
encountered, it should be followed by
an analysis of reagent water to check
for cross contamination. For samples
containing large amounts of water-
soluble materials, suspended solids,
high boiling compounds or high
organohalide levels, it may be neces-
sary to wash out the purging device
with a detergent solution, rinse it with
distilled water, and then dry it in a
105 °C oven between analyses. The
trap and other parts of the system are
also subject to contamination; there-
fore, frequent bakeout and purging of
the entire system may be required.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified<4-6) for the infor-
mation of the analyst.
4.2 The following parameters
covered by this method have been ten-
tatively classified as known or
suspected, human or mammalian
carcinogens: carbon tetrachloride,
chloroform, 1,4-dichlorobenzene, and
vinyl chloride. Primary standards of
these toxic compounds should be
prepared in a hood. A NIOSH/MESA
approved toxic gas respirator should be
worn when the analyst handles high
concentrations of these toxic
compounds.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
sampling.
5.1.1 Vial—25-mL capacity or larger,
equipped with a screw cap with hole in
center (Pierce #1 3075 or equivalent).
Detergent wash, rinse cap with tap and
distilled water, and dry at 105 °C
before use.
5.1.2 Septum—Teflon-faced silicone
(Pierce #1 2722 or equivalent).
Detergent wash, rinse with tap and
distilled water, and dry at 105 °C for
one hour before use.
5.2 Purge and trap device—The
purge and trap device consists of three
separate pieces of equipment: the
sample purger, trap, and the desorber.
Several complete devices are now
commercially available.
5.2.1 The sample purger must be
designed to accept 5-mL samples with
a water column at least 3 cm deep.
The gaseous head space between the
water column and the trap must have a
total volume of less than 1 5-mL. The
purge gas must pass through the water
column as finely divided bubbles with a
diameter of less than 3 mm at the
origin. The purge gas must be intro-
duced no more than 5 mm from the
base of the water column. The sample
purger, illustrated in Figure 1, meets
these design criteria.
5.2.2 The trap must be at least 25
cm long and have an inside diameter of
at least 0.105 inch. The trap must be
packed to contain the following
minimum lengths of adsorbents: 1.0
cm of methyl silicone coated backing
(Section 6.3.3), 7.7 cm of
2,6-diphenylene oxide polymer
(Section 6.3.2), 7.7 cm of silica gel,
7.7 gm of coconut charcoal (Section
6.3.1). If it is not necessary to analyze
for dichloroclifluroromethane, the char-
coal can be eliminated, and the polymer
section lengthened to 1 5 cm. The
minimum specifications for the trap are
illustrated in Figure 2.
5.2.3 The desorber must be capable
of rapidly heating the trap to 180 °C.
The polymer section of the trap should
601-2
July 1982
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not be heated higher than 180 °C and
the remaining sections should not
exceed 220 °C. The desorber design,
illustrated in Figure 2, meets these
criteria. •
5.2.4 The purge and trap device may
be assembled as a separate unit or be
coupled to a gas chromatograph as
illustrated in Figures 3 and 4.
5.3 Gas chromatograph—An analyti-
cal system complete with a tempera-
ture programmable gas chromatograph
suitable for on-column injection and all
required accessories including syringes,
analytical columns, gases, detector,
and strip-chart recorder. A data system
is recommended for measuring peak
areas.
5.3. 7 Column 1 —8 ft long x 0.1 in
ID stainless steel or glass, packed with
1 % SP-1000 on Carbopack B (60/80
mesh) or equivalent. This column was
used to develop the method perfor-
mance statements in Section 1 2.
Guidelines for the use of alternate
column packings are provided in
Section 10.1.
5.3.2 Column 2 — 6 ft long x 0.1 in
ID stainless steel or glass, packed with
chemically bonded n-octane on Porasil-
C (100/120) mesh or equivalent.
5.3.3 Detector—Electrolytic conduc-
tivity or microcoulometric. These types
of detectors have proven effective in
the analysis of wastewaters for the
parameters listed in the scope. The
electrolytic conductivity detector was
used to develop the method perfor-
mance statements .and MDL listed in
Tables 1 and 2. Guidelines for the use
of alternate detectors are provided in
Section 10.1.
5.4 Syringes— 5-mL glass hypo-
dermic with Luerlok tip (two each), if
applicable to the purging device.
5.5 Micro syringes^-25 juL, 0.006 in
ID needle.
5.6 Syringe valve—2-way, with Luer
ends (three each).
5.7 Syringe—5-mL, gas-tight with
shut-off valve.
5.8 Bottle— 1 5-mL, screw cap, with
Teflon cap liner.
5.9 Balance—Analytical, capable of
accurately weighing 0.0001 g.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an
interferent is not observed at the MDL
of the parameters of interest.
6.1.1 Reagent water can be generated
by passing tap water through a carbon
filter bed containing about 1 Ib. of
activated carbon (Filtrasorb-300 or
equivalent (Calgon Corp.)).
6.1.2 A water purification system
(Millipore Super-Q or equivalent) may
be used to generate reagent water.
6.1.3 Reagent water may also be
prepared by boiling water for 1 5
minutes. Subsequently, while maintain-
ing the temperature at 90 °C, bubble a
contaminant-free inert gas through the
water for one hour. While still hot,
transfer the water to a narrow mouth
screw-cap bottle and seal with a
Teflon-lined septum and cap.
6.2 Sodium thiosulfate—(ACS)
Granular.
6.3 Trap Materials
6.3.1 Coconut charcoal (6/10 mesh
sieved to 26 mesh), (Barnaby Chaney,
CA-580-26 lot # M-2649 or
equivalent).
6.3.2 2,6-Diphenylene oxide
polymer—Tenax, (60/80 mesh),
chromatographic grade or equivalent.
6.3.3 Methyl silicone packing—3%
OV-1 on 60/80 mesh Chromosorb-W
or equivalent.
6.3.4 Silica gel—35/60 mesh,
Davison, grade-1 5 or equivalent.
6.4 Methyl Alcohol—Pesticide quality
or equivalent.
6.5 Stock standard solutions—Stock
standard solutions may be prepared
from pure standard materials or
purchased as certified solutions.
Prepare stock standard solutions in
methyl alcohol using assayed liquids or
gas cylinders as appropriate. Because
of the toxicity of some of the
organohalides, primary dilutions of
these materials should be prepared in a
hood. A NIOSH/MESA approved toxic
gas respirator should be used when the
analyst handles high concentrations of
such materials.
6.5.1 Place about 9.8 mL of methyl
alcohol into a 10-mL ground glass
stoppered volumetric flask. Allow the
flask to stand, unstoppered, for about
10 minutes or until all alcohol wetted
'surfaces have dried. Weigh the flask to
the nearest 0.1 mg.
6.5.2 Add the assayed reference
material:
6.5.2.1 Liquids—Using a 10O-//L
syringe, immediately add two or more
drops of assayed reference material to
the flask, then reweigh. Be sure that
the drops fall directly into the alcohol
without contacting the neck of the
flask.
6.5.2.2 Gases—To prepare standards
for any of the six halocarbons that boil
below 30 °C (bromomethane, chloro-
ethane, chloromethane, dichlorodi-
fluoromethane, trichlorofluoromethane,
vinyl chloride), fill a 5-mL valved gas-
tight syringe with the reference
standard to the 5.0-mL mark. Lower
the needle to 5 mm above the methyl
alcohol meniscus. Slowly introduce the
reference standard above the surface
of the liquid (the heavy gas will rapidly
dissolve into the methyl alcohol).
6.5.3 Reweigh, dilute to volume,
stopper, then mix by inverting the flask
several times. Calculate the concentra-
tion in micrograms per microliter from
the net gain in weight. When compound
purity is assayed to be 96% or greater,
the weight can be used without correc-
tion 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 indepen-
dent source.
6.5.4 Transfer the stock standard
solution into a Teflon-sealed screw-cap
bottle. Store, with minimal headspace,
at -10 to - 20 °C and protect from
light.
6.5.5 Prepare fresh standards weekly
for the six gases and 2-chloroethylvinyl
ether. All other standards must be
replaced after one month, or sooner if
comparison with check standards
indicate a problem.
6.6 Secondary dilution standards-
Using stock standard solutions, prepare
secondary dilution standards in methyl
alcohol that contain the compounds of
interest, either singly or mixed together.
The secondary dilution standards
should be prepared at concentrations
such that the aqueous calibration
standards prepared in Sections 7.3.1
or 7.4.1 will bracket the working range
of the analytical system. Secondary
dilution standards should be stored
with minimal headspace and should be
checked frequently for signs of degrad-
ation or evaporation, especially just
prior to preparing calibration standards
from them. Quality control check
standards that can be used to
determine the accuracy of calibration
standards will be available from the
U.S: Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, in Cincinnati, Ohio.
601-3
July 1982
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7. Calibration
7.1 Assemble a purge and trap device
that meets the specifications in Section
5.2. Condition the trap overnight at
180 °C by backflushing with an inert
gas flow of at least 20 mL/min. Prior to
use, daily condition traps 10 minutes
while backflushing at 180 °C.
7.2 Connect the purge and trap
device to a gas chromatograph. The
gas chromatograph must be operated
using temperature and flow rate param-
eters equivalent to those in Table 1.
Calibrate the purge and trap-gas
chromatographic system using either
the external standard technique
{Section 7.3) or the internal standard
technique (Section 7.4).
7.3 External standard calibration
procedure:
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter by carefully
adding 20.0 pL of one or more second-
ary dilution standards to 100, 500, or
1000 mL of reagent water. A 25-fj.L
syringe with a 0.006 inch ID needle
should be used for this operation. One
of the external standards should be at a
concentration near, but above, the
method detection limit (See Table 1)
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. These aqueous standards can
be stored up to 24 hours, if held in
sealed vials with zero headspace as
described in Section 9.2. If not so
stored, they must be discarded after
one hour.
7.3.2 Analyze each calibration
standard according to Section 10, and
tabulate peak height or area responses
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 t-<10% relative standard devia-
tion, BSD), linearity through the origin
can be assumed and the average ratio
or calibration factor can be used in
place of a calibration curve.
7.3.3 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 any parameter varies
from the predicted response by more
than ± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that parameter.
7.4 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more internal
standards that are similar 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. Because of these
limitations, no internal standard can be
suggested that is applicable to all
samples. The compounds recommended
for use as surrogate spikes in Section
8.7 have been used successfully as
internal standards, because of their
generally unique retention times.
7.4. 7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest as
described in Section 7.3.1 .
7.4.2 Prepare a spiking solution con-
taining each of the internal standards
using the procedures described in
Sections 6.5 and 6.6. It is recom-
mended that the secondary dilution
standard be prepared at a concentra-
tion of 1 5 jug/mL of each internal
standard compound. The addition of
1 0/zL of this standard to 5.0 mL of
sample or calibration standard would
be equivalent to 30
7.4.3 Analyze each calibration stand-
ard, according to Section 1 0, adding
1 0 /iL of internal standard spiking solu-
tion directly to the syringe (Section
1 0.4). Tabulate peak height or area
responses against concentration for
each compound and internal standard,
and calculate response factors (RF) for
each compound using equation 1 .
Eq. 1 RF = (AsCis)/(AisCs)
where:
As = Response for the parameter to
be measured.
AJS = Response for the internal
standard.
Cis = Concentration of, the internal
standard.
Cs = Concentration of the
parameter to be measured.
If the RF value over the working range
is a constant (<1 0% RSD), the RF can
be assumed to be invariant and 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. RF.
7.4.4 The working calibration curve
or RF must be verified pn each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with estab-
lished performance criteria to determine
if the results of analyses are within
accuracy and precision limits expected
of the method.
8.1.1 Before performing any
analyses, the analyst must demonstrate
the ability to generate acceptable
accuracy and precision with this
method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted cer-
tain options to improve the separations
or lower the cost of measurements.
Each time such modifications are made
to the method, the analyst is required
to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing labora-
tory performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to generate
acceptable accuracy and precision, the
analyst must perform the following
operations.
8.2.1 Select a representative spike
concentration for each compound to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in methyl alcohol 500
times more concentrated than the
selected concentrations. Quality
control check sample concentrates,
appropriate for use with this method,
will be available from the U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
8.2.2 Using a syringe, add 10 (J.L of
the check sample concentrate to each
of a minimum of four 5-mL aliquots of
reagent water. A representative waste-
so; -4
July 1982
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water may be used in place of the
reagent water, but one or more addi-
tional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for the
results. Wastewater background cor-
rections must be made before R and s
calculations are performed.
8.2.4 Using Table 2, note the average
recovery (X) and standard deviation (p)
expected for each method parameter.
Compare these to the calculated values
for Rand s. If s>2por |X-R|=-2p,
review potential problem areas and
repeat the test.
8.3 The analyst must calculate
method performance criteria and define
the performance of the laboratory for
each spike concentration;and parameter
being measured.
8.2.5 The U.S. Environmental
Protection Agency plans to establish
performance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3.1 Calculate upper and lower
control limts for method performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
where R and s are calculated as in
Section 3.2.3. The UCL and LCL can
be used to construct control charts!7)
that are useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they become available
from the U.S. Environmental Protection
Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy state-
ments of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcu-
lation of R and s. Alternately, the
analyst may use four wastewater data
points gathered through the require-
ment for continuing quality control in
Section 8.4. The accuracy statements
should be updated regularly.(7)
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample'
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of the
sample must be spiked and analyzed as
described in Section 8.2. If the
recovery for a particular parameter
does not fall within the control limits
for method performance, the results
reported for that parameter in all
samples processed as part of the same
set must be qualified as described in
Section 11.3. The laboratory should
monitor the frequency of data so
qualified to ensure that it remains at or
below 5%.
8.5 Each day, the analyst must
demonstrate through the analysis of
reagent water, that interferences from
the analytical system are under control.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. When doubt
exists over the identification of a peak
on the chromatogram, confirmatory
techniques such as gas chromatography
with a dissimilar column, specific
element detector, or mass spectrom-
eter must be used. Whenever possible,
the laboratory should perform analysis
of standard reference materials and
participate in relevant performance
evaluation studies.
8.7 The analyst should maintain
constant surveillance of both the per-
formance of the analytical system and
the effectiveness of the method in
dealing with each sample matrix by-
spiking each sample, standard and
blank with surrogate halocarbons. A
combination of bromochloromethane,
2-bromo-1-chloropropane, and
1,4-dichlorobutane is recommended to
encompass the range of the tempera-
ture program used in this method. From
stock standard solutions prepared as
above, add a volume to give 7500 /ig
of each surrogate to 45 mL of reagent
water contained in a 50-mL volumetric
flask, mix and dilute to volume (1 5
ng/juL). If the internal standard calibra-
tion procedure is being used, the
surrogate compounds may be added
directly to the internal standard spiking
solution (Section 7.4.2). Add 10/nLof
this surrogate spiking solution directly
into the 5-mL syringe with every sample
and reference standard analyzed.
Prepare a fresh surrogate spiking
solution on a weekly basis.
9. Sample Collection,
Preservation, and Handling
9.1 All samples must be iced or
refrigerated from the time of collection
until extraction. If the sample contains
free or combined chlorine, add sodium
thiosulfate preservative (10 mg/40 mL
is sufficient for up to 5 ppm CI2) to the
empty sample bottle just prior to
shipping to the sampling site. USEPA
methods 330.4 and 330.5 may be
used for measurement of residual
chlorine. <8' Field test kits are available
for this purpose.
9.2 Grab samples must be collected
in glass containers having a total
volume of at least 25 mL. Fill the
sample bottle just to overflowing in
such a manner that no air bubbles pass
through the sample as the bottle is
being filled. Seal the bottle so that no
air bubbles are entrapped in it. If
preservative has been added, shake
vigorously for one minute. Maintain the
hermetic seal on the sample bottle until
time of analysis.
9.3 All samples must be analyzed
within 14 days of collection.
10. Sample Extraction and
Gas Chromatography
10^.1 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. Included in this
Table are estimated retention times and
method detection limits that can be
achieved by this method. An example
of the separations achieved by Column
1 is shown in Figure 5. Other packed
columns, chromatographic conditions,
or detectors may be used -if the
requirements of Section 8.2 are met.
10.2 Calibrate the system daily as
described in Section 7.
10.3 Adjust the purge gas (nitrogen
or helium) flow rate to 40 mL/min.
Attach the trap inlet to the purging
device, and set the device to purge.
Open the syringe valve located on the
purging device sample introduction
needle.
10.4 Allow sample to come to
ambient temperature prior to introduc-
ing it to the syringe. Remove the
plunger from a 5-mL syringe and attach
a closed syringe valve. Open the sample
bottle (or standard) and carefully pour
the sample into the syringe barrel to
just short of overflowing. Replace the
601-5
July 1982
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syringe plunger and compress the
sample. Open the syringe valve and
vent any residual air while adjusting the
sample volume to 5.0 mL. Since this
process of taking an aliquot destroys
the validity of the sample for future
analysis, the analyst should fill a
second syringe at this time to protect
against possible loss of data. Add
10.0 fit of the surrogate spiking solu-
tion (8.7) and 10.0 [J.L of the internal
standard spiking solution (Section
7.4.2), if applicable, through the valve
bore, then close the valve.
10.5 Attach the syringe-syringe
valve assembly to the syringe valve on
the purging device. Open the syringe
valves and inject the sample into the
purging chamber.
10.6 Close both valves and purge the
sample for 11.0 ± .1 minutes at
ambient temperature.
10.7 After the 11 -minute purge time,
attach the trap to the chromatograph,
adjust the device to the desorb mode,
and begin to temperature program the
gas chromatograph. Introduce the
trapped materials to the GC column by
rapidly heating the trap to 180 °C
while backf lushing the trap with an
inert gas between 20 and 60 mL/min
for four minutes. If rapid heating of the
trap cannot be achieved, the gas
chromatographic column must be used
as a secondary trap by cooling it to
30 °C (subambient temperature, if poor
peak geometry or random retention
time problems persist) instead of the
initial program temperature of 45 °C.
10.8 While the trap is being desorbed
into the gas chromatograph, empty the
purging chamber using the sample in-
troduction syringe. Wash the chamber
with two 5-mL flushes of reagent
water.
10.9 After desorbing the sample for
four minutes recondition the trap by
returning the purge and trap device to
the purge mode. Wait 15 seconds then
close the syringe valve on the purging
device to begin gas flow through the
trap. The trap temperature should be
maintained at 180 °C. After approxi-
mately seven minutes turn off the trap
heater and open the syringe valve to
stop the gas flow through the trap.
When cool the trap is ready for the
next sample.
10.10 The width of the retention
time window used to make identifica-
tions should be based upon measure-
ments 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.
10.11 If the response for the peak
exceeds the working range of the
system, prepare a dilution of the
sample with reagent water from the
aliquot in the second syringe and
reanalyze.
11. Calculations
11.1 Determine the concentration of
individual compounds in the sample.
11.1.1 If the external standard
calibration procedure is used, calculate
the concentration of material from the
peak response using the calibration
curve or calibration factor determined
in Section 7.3.2.
7 7.1.2 If the internal standard
calibration procedure was used, calcu-
late the concentration in the sample
using the response factor (RF) deter-
mined in Section 7.4.3 and equation 2.
Eq.2.
Concentration //g/L = (AsCis)/(Ais)(RF)
where:
As = Response for the parameter to
be measured.
Ajs = Response for the internal
standard.
Cs = Concentration of the internal
standard.
11.2 Report results in micrograms
per liter. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
11.3 For samples processed as part
of a set where the spiked sample
recovery falls outside of the control
limits which were established according
to Section 8.3, data for the affected
parameters must be labeled as suspect.
12. Method Performance
12.1 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
zero.d) The MDL concentrations listed
in Table 1 were obtained using reagent
water.<9> Similar results were achieved
using representative wastewaters. The
MDL actually achieved in a given
analysis will vary depending on instru-
ment sensitivity and matrix effects.
12.2 This method is recommended
for use in the concentration range from
the MDL up to 1000 x MDL. Direct
aqueous injection techniques Should be
used to measure concentration levels
above 1000 x MDL.
12.3 In a single laboratory (Monsanto
Research), using reagent water and
wastewaters spiked at or near back-
ground levels, the average recoveries
presented in Table 2 were obtained^).
The standard deviation of the measure-
ment in percent recovery is also
included in Table 2Ol.
12.4 The U.S. Environmental
Protection Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. Bellar, T.A., and Lichtenberg, J.J.
Journal American Water Works
Association, 66, 739, (1974).
3. Bellar, T.A., and Lichtenberg, J.J.
"Semi-Automated Headspace Analysis
of Drinking Waters and Industrial
Waters for Purgeable Volatile Organic
Compounds," Proceedings from
Symposium on Measurement of
Organic Pollutants in Water and
Wastewater, American Society for
Testing and Materials, STP 686, C.E.
Van Hall, editor, 1978.
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 Stand-
ards, 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. "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.
8. "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual,"
Methods for Chemical Analysis of Water
and Wastes, EPA 600/4-79-020, U.S.
Environmental Protection Agency,
601-6
July 1982
-------
Environmental Monitoring and Support Table 1. Chromatographic
Laboratory— Cincinnati, Ohio 45268,
March 1979.
9. "EPA Method Validation Study 23,
Method 601 (Purgeable Halocarbons}," Parameter
Report for EPA Contract 68-03-2856 Chloromethane
(In preparation). Bromomethane
Dichlorodifluoromethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorofluoromethane
1 , 1 -Dichloroethene
1 , 1 -Dichloroethane
trans- 1 ,2-Dichloroethene
Chloroform
1, 2-Dichloroethane
1 , 1 , 1 -Trichloroethane
Carbon tetrachloride
Bromodichloromethane
1 ,2-Dichloropropane
trans- 1 ,3-Dichloropropene
Trichloroethene
Dibromochloromethane
1, 1 ,2-Trichloroethane
cis-1 ,3-Dichloropropene
2-Chloroethylvinyl ether
Bromoform
1 , 1,2,2-Tetrachloroethane
Tetrachloroethene
Chlorobenzene
1 , 3-Dichlorobenzene
1 ,2-Dichlorobenzene
1 ,4-Dichlorobenzene
: Conditions and Method Detection Limits
Retention Time Method
Detection Limit
Column 1
T.5O
2.17
2.62
2.67
3.33
5.25
7.18
7.93
9.30
10.1
10.7
11.4
12.6
13.0
13.7
14.9
15.2
15.8
16.5
16.5
16.5
18.0
19.2
21.6
21.7
24.2
34.0
34.9
35.4
Column 2
5.28
7.05
nd
5.28
8.68
10.1
nd
7.72
12.6
9.38
12.1
15.4
13.1
14.4
14.6
16.6
16.6
13.1
16.6
18. 1
18.0
nd
19.2
nd
15.O
18.8
22.4
23.5
22.3
U.9/L
O.08
1.18
1.81
0.18
O.52
0.25
nd
0.13
O.O7
0.10
0.05
0.03
0.03
O.12
0.10
0.04
0.34
0.12
0.09
O.O2
0.20
0.13
0.20
0.03
0.03
0.25
0.32
O.I 5
O.24
nd = not determined
Column 1 conditions: Carbopack B 6O/8O mesh coated with 1 % SP- 1OOO packed in
an 8 ft x 0.1 in ID stainless steel or glass column with helium carrier gas at 40
mL/min flow rate. Column temperature held at 45 °C for 3 min. then programmed
at 8 °C/min. to 220° and held for 15 min.
Column 2 conditions: Porasil-C 10O/120 mesh coated with n-octane packed in a 6 ft
x 0.1 in ID stainless steel or glass column with helium carrier gas at 40 mL/min
flow rate. Column temperature held at 5O°C for 3 min then programmed at
6 °C/min to 17O° and held for 4 min.
601-7
July 1982
-------
Table 2. Single Operator Accuracy and Precision
Parameter
Bromodichloromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chloroethane
2-Chloroethylvinyl ether
Chloroform
Chloromethane
Dibromochloromethane
1 ,2-Dichlorobenzene
1, 3-Dichlorobenzene
1 ,4-Dichlorobenzene
Dichlorodifluoromethane
1, 1-Dichloroethane
1f 2-Dichloroethane
1, 1 -Dichloroethene
trans- 1 ,2-Dichloroethene
1, 2-Dichloropropane
els- 1 ,3-Dichloropropene
trans- 1 ,3-Dichloropropene
Methylene chloride
1, 1 ,2,2-Tetrachloroethane
Tetrachloroethene
1, 1, 1 -Trichloroethane
1, 1 ,2-Trichloroethane
Trichloroethene
Trlchlorofluoromethane
Vinyl chloride
Average
Percent
Recovery
100.9
89.5
105.O
82.5
93.9
91.5
96.3
101.7
91.4
98.3
10.20
91.6
97.5
87.8
102.3
97.8
101.1
91.O
97.7
86.7
73.5
97.9
91.9
94.1
75.1
91.0
106.1
89.3
101.9
Standard
Deviation
%
5.0
9.0
17.3
25.6
8.9
22.4
9.9
20.6
13.4
6.5
2.0
4.3
9.3
18.0
5.5
4.8
21.7
19.3
8.8
6.0
17.2
2.6
15.0
18.1
12.5
25.1
7.4
13.9
11.4
Spike
Range
(ug/U
0.43-46.
1.45-50
Number
of Matrix
Analyses Types
7
3.39-49.2
0.55-50
2.21-50
3.95-50
4.39-133
0.44-50
0.55-23.
0. 75-93.
9
0
4.89-154
2.94-46.
2.99-51.
2.18-43.
0.44-46.
0.44-46.
0.37-50
7
6
4
7
7
O.44-98.0
0.29-39.0
0.44-46.
0.43-50
0. 73-46.
0.46-46.
7
7
7
0.50-35.0
0.37-29.
0.45-50
0
0.38-46.7
149
0.82-32.3
21
20
21
19
20
21
20
20
21
21
21
21
21
21
21
21
19
2O
21
21
20
21
21
21
21
21
21
14
21
Optional
Foam .
Trap (
\
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
R
^-T"
^
u
__Ex/f V4 in.
"N O.D.
-«- 14mm
'^ Inlet V4 in.
~"~ O.D.
V4 in.
O.D. exit
^•Sample Inlet
2-way Syringe valve
17cm 20 gauge syringe needle
6mm O.D. Rubber Septum
lOmm O.D. V16 in. O.D.
VStainless Steel
Inlet
V4 in. O.D.
13X molecular
sieve purge
gas filter
Purge gas
flow control
601-8
1Omm glass frit
medium porosity
Figure 1. Purging device
July 1982
-------
Packing procedure
Construction
Glass 5mm p
wool J
Activated 7.7cm
charcoal
Grade 15
Silica gel 7. 7 en
n
I
Tenax 7.7ci
3% OV-1
1cm
Glass i
n
wool 5mm
%
i
y/.
%
I
1
^
1
'$.
j.
f$
to;
^
7^ foot
resistance
wire wrapped
solid -<;
(Double layer)
15 cm
7-^/foot
resistance
wire wrapped
solid
(Single layer)
8cm —
C
f-
— — v
— *•('
C
c
c
c
,--
-^
^--
c
,--
c
(^
"^
l^
=_^-
^
^
^-
X \f\JIII}JI Wt3l\JI 1 Illllliy .
>" nut a/?tf ferrules
~)
2
)
jl Thermocouple/
' > controller
'- ^- — • sensor
3
>^^^^^
? ^^^^^g
/
-4^ /
p==:
Electronic
temperature
control
and
pyrometer
Tubing 25 cm.
> /_ 0. 105 in. I.D.
" 7 0. /25 /A? O.O.
-^ / stainless steel
^
1
U_OJ
Trap inlet
Figure 2. Trap packings and construction to include desorb capability
Carrier gas flow control
Pressure regulator
Liquid injection ports
Purge gas
flow control \
13X molecular A
sieve filter \ j
Column oven
Purging
device
\-^- Confirmatory column
To detector
\ ~~~~~—Analytical column
• control
Note:
All lines between
trap and GC
should be heated
to 80°C
Figure 3. Schematic of purge and trap device — purge mode
6O1-9
July 1982
-------
Carrier gas flow control
Pressure regulator
Liquid injection ports• Coiumn oven
Purge gas
flow controlx ,
Confirmatory column
To detector
-Analytical column '
optional 4-port column
selection valve
6-port Trap inlet
valve \ Resistance wire
13X molecular
sieve filter
Purging
device
Heater control
Note:
All lines between
trap and GC
should be heated
to 80°C
Figure 4. Schematic of purge and trap device — desorb mode
Column: 1% SP-1000 on Carbopack-8
Program: 45°C-3 minutes, 8°/'minute to 220°C
Detector; Hall 700-A electrolytic conductivity
02 4 6 8 10 12 14 16 18 2O 22 24 26 28 30 32 34 36
Retention time, minutes
Figura 5. Gas chromatogram of purgeable halocarbons
601-10
July 1982
-------
SEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Purgeable Aromatics—
Method 602
1. Scope and Application
1.1 This method covers the determi-
nation of various purgeable aromatics.
The following parameters may be
determined by this method:
Parameter
STORET No.
CAS No.
Benzene
Chlorobenzene
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
Ethylbenzene
Toluene
1.2 This is a purge and trap gas
chromatographic method applicable to
the determination of the compounds
listed above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any or
all of the compounds above, compound
identifications should be supported by
at least one additional qualitative
technique. This method describes
analytical conditions for a second gas
chromatographic column that can be
used to confirm measurements made
with the primary column. Method 624
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results for
all of the parameters listed above.
1.3 The method deteetion limit (MDL,
defined in Section 12.1<1)) for each
parameter is listed in Table 1. The MDL
for a specific wastewater "may differ
from these listed depending upon the
nature of interferences in the sample
matrix.
34030
34301
34536
34566
34571
34371
34010
71-43-2
108-90-7
95-50-1
541-73-1
1O6-46-7
100-41-4
108-88-3
1.4 Any modification of this method,
beyond those expressly permitted,
shall be considered as major modifica-
tions subject to application and
approval for 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 operation of a purge
and trap system and a gas chromato-
graph and in the interpretation of
chromatograms. Each analyst must
demonstrate the ability to generate
acceptable results with this method
using the procedure described in
Section 8.2.
2. Summary of Method
2.1 An inert gas is bubbled through a
5-mL water sample contained in a
specially-designed purging chamber at
ambient temperature. The aromatics
are efficiently transferred from the
aqueous phase to the vapor phase. The
vapor is swept through a sorbent trap
where the aromatics are trapped. After
602-1
July 1982
-------
purging is completed, the trap is heated
and backflushed with the inert gas to
desorb the aromatics onto a gas
chromatographic column. The gas
chromatograph is temperature pro-
grammed to separate the aromatics
which are then detected with a photo-
ionization detector (2'3>.
2.2 The method provides an optional
gas chromatographic column that may
be helpful in resolving the compounds
of interest from interferences that may
occur.
3. Interferences
3.1 Impurities in the purge gas and
organic compounds out-gassing from
the plumbing ahead of the trap account
for the majority of contamination
problems. The analytical system must
be demonstrated to be free from
contamination under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5. The use of non-TFE plastic tubing,
non-TFE thread sealants, or flow
controllers with rubber components in
the purging device should be avoided.
3.2 Samples can be contaminated by
diffusion of volatile organics through
the septum seal into the sample during
shipment and storage. A field reagent
blank prepared from reagent water and
carried through the sampling and
handling protocol can serve as a check
on such contamination.
3.3 Contamination by carry-over can
occur whenever high level and low
level samples are sequentially
analyzed. To reduce carry-over, the
purging device and sample syringe
must be rinsed with reagent water
batween sample analyses. Whenever
an unusually concentrated sample is
encountered, it should be followed by
an analysis of reagent water to check
for cross contamination. For samples
containing large amounts of water-
soluble materials, suspended solids,
high boiling compounds or high
aromatic levels, it may be necessary to
wash out the purging device with a
detergent solution, rinse it with distilled
water, and then dry it in an oven at
105 °C between analyses. The trap
and other parts of the system are also
subject to contamination; therefore,
frequent bakeout and purging of the
entire system may be required.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified!4-6' for the
information of the analyst.
4.2 The following parameters covered
by this method have been tentatively
classified as known or suspected,
human or mammalian carcinogens:
benzene and 1,4-dichlorobenzene.
Primary standards of these toxic
compounds should be prepared in a
hood. An NIOSH/MESA approved toxic
gas respirator should be worn when the
analyst handles high concentrations of
these toxic compounds.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
sampling.
5.1.1 Vial—25-mL capacity or larger,
equipped with a screw cap with hole in
center (Pierce #13075 or equivalent).
Detergent wash, rinse with tap and
distilled water, and dry at 105 °C
before use.
5.1.2 Septum—Teflon-faced silicone
(Pierce #12722 or equivalent).
Detergent wash, rinse with tap and
distilled water, and dry at 105 °C for
one hour before use.
5.2 Purge and trap device—The
purge and trap device consists of three
separate pieces of equipment: the
sample purger, trap, and the desorber.
Several complete devices are now
commercially available.
5.2.1 The sample purger must be
designed to accept 5-mL samples with
a water column at least 3 cm deep.
The gaseous head space between the
water column and the trap must have a
total volume of less than 1 5 mL. The
purge gas must pass through the water
column as finely divided bubbles with a
diameter of less than 3 mm at the
origin. The purge gas must be intro-
duced no more than 5 mm from the
base of the water column. The sample
purger, illustrated in Figure 1, meets
these design criteria.
5.2.2 The trap must be at least 25
cm long and have an inside diameter of
at least 0.105 inch.
5.2.2. / The trap is packed withjl cm
of methyl silicone and 23 cm
2,6-diphenylene oxide polymer as
shown in Figure 2. This trap was used
to develop the method performance
statements in Section 12.
5.2.2.2 Alternatively, either of the
two traps described in M.ethod 601
may be used, although water vapor will
preclude the measurement of low
concentrations of benzene.
5.2.3 The desorber must be capable
of rapidly heating the trap to 1 80 °C.
The polymer section of the trap should
not be heated higher than 1 80 °C and
the remaining sections should not
exceed 200 °C. The desorber design,
illustrated in Figure 2, meets these
criteria.
5.2.4 The purge and trap device may
be assembled as a separate unit or be
coupled to a gas chromatograph as
illustrated in Figures 3, 4, and 5.
5.3 Gas chromatograph—Analytical
system complete with a temperature
programmable gas chromatograph
suitable for on-column injection and all
required accessories including syringes,
analytical columns, gases, detector,
and stripchart recorder. A data system
is recommended for measuring peak
areas.
5.3.1 Column 1 —6 ft long x 0.082
in ID stainless steel or glass, packed
with 5% SP-1200 and 1.75%
Bentone-34 on Supelcoport (100/1 20
mesh) or equivalent. This column was
used to develop the method perfor-
mance statements and the MDLs listed
in Tables 1 and 2. Guidelines for the
use of alternate column packings are
provided in Section 10.1. .
5.3.2 Column 2 —8 ft long x 0.1 in
ID stainless steel or glass, packed with
5% 1,2,3-Tris(2-cyanoethoxy)propane
on Chromosorb W-AW (60/80 mesh)
or equivalent.
5.3.3 Detector—Photoionization
detector (h-nu Systems, Inc. Model
PI-51 -02 or equivalent). This type of
detector has been proven effective in
the analysis of wastewaters for the
parameters listed in the scope, and
was used to develop the performance
statements in Section 1 2. Guidelines
for the use of alternate detectors are
provided in Section 1 0.1.
5.4 Syringes—5-mL glass
hypodermic with Luerlok tip (two each),
if applicable to the purge device.
602-2
July 1982
-------
5.5 Micro syringes— 25 MU 0.006 in
ID needle.
5.6 Syringe valve—2-way, with Luer
ends (three each). :
5.7 Bottle— 1 5-mL screw-cap with
Teflon cap liner.
5.8 Balance—Analytical, capable of
accurately weighing 0.0001 g.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an inter-
'ferent is not observed at the MDL of
the parameters of interest.
6.1.1 Reagent water can be
generated by passing tap water
through a carbon filter bed containing
about 1 Ib. of activated carbon.
(Filtrasorb-300 or equivalent (Calgon
Corp.)).
6.1.2 A water purification system
(Millipore Super-Q or equivalent) may
be used to generate reagent water.
6.1.3 Reagent water may also be
prepared by boiling water for 1 5
minutes. Subsequently, while main-
taining the temperature at 90 °C,
bubble a contaminant-free inert gas
through the water for one hour. While
still hot, transfer the water to a narrow
mouth screw-cap bottle and seal with a
Teflon-lined septum and cap.
6.2 Sodium thiosulfate—(ACS)
Granular.
6.3 Hydrochloric acid (1+1)—Add
50 mL of concentrated HCI to 50 mL
of reagent water.
6.4 Trap Materials
6.4.1 2,6-Diphenylene oxide
polymer-Tenax, (60/80 mesh) chroma-
tographic grade or equivalent.
6.4.2 Methyl silicone-3% OV-1 on
Chromosorb-W (60/80 mesh) or
equivalent.
6.5 Methyl alcohol—Pesticide quality
or equivalent.
6.6 Stock standard solutions—Stock
standard solutions may be prepared
from pure standard materials or
purchased as certified solutions.
Prepare stock standard solutions in
methyl alcohol using assayed liquids.
Because benzene and 1,4-dichloro-
benzene are suspected carcinogens,
primary dilutions of these materials
should be prepared in a hood.
8.6.1 Place about 9.8 mL of methyl
alcohol into a 10-mL ground glass
stoppered volumetric flask. Allow the.
flask to stand, unstoppered, for about
10 minutes or until all alcohol wetted
surfaces have dried. Weigh the flask to
the nearest 0.1 mg.
6.6.2 Using a 100-^L syringe,
immediately add two or more drops of
assayed reference material to the flask,
then reweigh. Be sure that the drops
fall directly into the alcohol without
contacting the neck of the flask.
6.6.3 Reweigh, dilute to volume,
stopper, then mix by inverting the flask
several times. Calculate the concentra-
tion in micrograms per microliter from
the net gain in weight. When compound
purity is certified at 96% or greater,
the weight can be used without correc-
tion to calculate the concentration of
the stock standard. Commercially
prepared stock standards can be used,
at any concentration, if they are
certified by the manufacturer or by an
independent source.
6.6.4 Transfer the stock standard
solution into a Teflon-sealed screw-cap
bottle. Store at 4 °C and protect from
light.
6.6.5 All standards must be replaced
after one month, or sooner if compari-
son with check standards indicate a
problem.
6.7 Secondary dilution standards-
Using stock standard solutions, prepare
secondary dilution standards in methyl
alcohol that contain the compounds of
interest, either singly or mixed
together. The secondary dilution
standards should be prepared at
concentrations such that the aqueous
calibration standards prepared in
Sections 7.3.1 or 7.4.1 will bracket
the working range of the analytical
system. Secondary solution standards
must be stored with zero headspace
and should be checked frequently for
signs of degradation or evaporation,
especially just prior to preparing
calibration standards from them.
Quality control check standards that
can be used to determine the accuracy
of calibration standards will be
available from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory, in
Cincinnati, Ohio.
7. Calibration
7.1 Assemble a purge and trap
device that meets the specifications in
Section*5.2. Condition the trap over-
night at 1 80 °C by backf lushing with
an inert gas flow of at least 20 mL/min.
Prior to use, daily condition traps 10
minutes while backf lushing at 180 °C.
7.2 Connect the purge and trap
device to a gas chromatograph. The
gas chromatograph must be operated
using temperature and flow rate
parameters equivalent to those in Table
1. Calibrate the purge and trap-gas
chromatographic system using either
the external.standard technique
(Section 7.3) or the internal standard
technique (Section 7.4.).
7.3 External standard calibration
procedure:
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter by carefully
adding 20.0 juL of one or more second-
ary dilution standards to 100, 500, or
1000 mL of reagent water. A 25-fzL
syringe with a 0.006 inch ID needle
should be used for this operation. One
of the external standards should be at a
concentration near, but above, the
MDL (see Table 1) 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.
These aqueous standards must be
prepared fresh daily.
7.3.2 Analyze each calibration
standard according to Section 10, and
tabulate peak height or area responses
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 (-=10% relative standard devia-
tion, RSD), linearity through the origin
can be assumed and the average ratio
or calibration factor can be used in
place of a calibration curve.
7.3.3 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 any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that parameter.
7.4 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested that
602-3
July 1982
-------
is applicable to all samples. The
compound, a, a,a-trifluorotoluene,
recommended as a surrogate spiking
compound in Section 8.7 has been
used successfully as an internal
standard.
7,4. 1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest as
described in Section 7.3.1 .
7.4.2 Prepare a spiking solution con-
taining each of the internal standards
using the procedures described in Sec-
tions 6.6 and 6.7. It is recommended
that the secondary dilution standard be
prepared at a concentration of 1 5 /zg/mL
of each internal standard compound.
The addition of 1 0 ML of this standard
to 5.0 mL of sample or calibration
standard would be equivalent to
30 j
7.4.3 Analyze each calibration
standard, according to Section 1 0,
adding 10/iL of internal standard
spiking solution directly to the syringe
as indicated in Section 1 0.4. Tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each com-
pound using equation 1 .
Eq. 1 RF = (AsCjs)/(AisCs)
where:
As ** Response for the parameter to
be measured.
Ais « Response for the internal
standard.
Cjs = Concentration of the internal
standard.
Cs — Concentration of the
parameter to be measured.
If the RF value over the working range
is a constant ( -s1 0% RSD), the RF
can be assumed to be invariant and the
average RF can be used for calculations.
Alternatively, the results can be used
to plot a calibration curve of response
ratios, AS/A|S, vs. RF.
7.4.4 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 1 0%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance checks
must be compared with established
performance criteria to determine if the
results of analyses are within accuracy
and precision limits expected of the
method.
8.1.1 Before performing any analyses,
the analyst must demonstrate the
ability to generate acceptable accuracy
and precision with this method. This
ability is established as described in
Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the
separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing
laboratory performance. This procedure
is described in Section 8.4.
8.2 To astablish the ability to
generate acceptable accuracy and
precision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each compound to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in methyl alcohol 500
times more concentrated than the
selected concentrations. Quality
control check sample concentrates,
appropriate for use with this method,
will be available from the U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
8.2.2 Using a syringe, add 10 juL of
the check sample concentrate to each
of a minimum of four 5-mL aliquots of
reagent water. A representative waste-
water may be used in place of the
reagent water, but one or more addi-
tional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for the
results. Wastewater background cor-
rections must be made before R and s
calculations are performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s :=-2p
or |X-R| >2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental
Protection Agency plans to establish
performance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and define
the performance of the laboratory for
each spike concentration and parameter
being measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
where R and s are calculated as in
Section 8.2.3
The UCL and LCL can be used to
construct control charts!7! that are use-
ful in observing trends in performance.
The control limits above must be
replaced by method performance
criteria as they become available from
the U.S. Environmental Protection
Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy state-
ments of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the
calculation of R and s. Alternately, the
analyst may use four wastewater data
points gathered through the requirement
for continuing quality control in Section
8.4. The accuracy statements should
be updated regularly!7'.
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of the
sample must be spiked and analyzed as
described in Section 8.2. If the
recovery for a particular parameter
does not fall within the control limits
for method performance, the results
602-4
July 1982
-------
reported for that parameter in all
samples processed as part of the same
set must be qualified as described in
Section 11.3. The laboratory should
monitor the frequency of data so
qualified to ensure that it remains at or
below 5%.
8.5 Each day, the analyst must
demonstrate through the analysis of
reagent water, that interferences from
the analytical system are under control.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. When doubt
exists over the identification of a peak
on the chromatogram, confirmatory
techniques such as gas chromatography
with a dissimilar column, specific
element detector, or mass spectrometer
must be used. Whenever possible, the
laboratory should perform analysis of
standard reference materials and
participate in relevant performance
evaluation studies.
8.7 The analyst should maintain
constant surveillance of both the per-
formance of the analytical system and
the effectiveness of the method in
dealing with each sample matrix by
spiking each sample, standard and
blank with surrogate compounds (e.g.
a,a,a,-trifluorotoluene). From stock
standard solutions prepared as above,
add a volume to give 7500 fj.g of each
surrogate to 45 mL of organic-free
water contained in a 50-mL volumetric
flask, mix and dilute to volume (1 5
r\g/nD. If the internal standard calibra-
tion procedure is being used, the
surrogate compounds may be added
directly to the internal standard spiking
solution (Section 7.4.2} Dose 10 p.L
of this surrogate spiking solution
directly into the 5-mL syringe with
every sample and reference standard
analyzed. Prepare a fresh surrogate
spiking solution on a weekly basis.
9. Sample Collection,
Preservation, and Handling
9.1 The samples must be iced or.
refrigerated from the time of collection
until extraction. If the sample contains
free or combined chlorine, add sodium
thiosulfate preservative (10 mg/40 mL
is sufficient for up to 5 ppm CI2) to the
empty sample bottles just prior to
shipping to the sampling site. USEPA
Methods 330.4 or 330.5 may be used
to measure residual chlorine!8). Field
Test Kits are available for this purpose.
9.2 Collect about 500 mL sample in
a clean container. Adjust the pH of the
sample to about 2. by adding 1+1 HCI
while stirring gently. Fill the sample
bottle in such a manner that no air
bubbles pass through the sample as the
bottle is being filled. Seal the bottle so
that no air bubbles are entrapped in it.
Maintain the hermetic seal on the
sample bottle until time of analysis.
9.3 All samples must be analyzed
within 14 days of collection.(3)
10. Sample Extraction and
Gas Chromatography
10.1 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. Included in this
table are estimated retention times and
method detection limits that can be
achieved by this method. An example
of the separations achieved by Column
1 is shown in Figure 6. Other packed
columns, chromatographic conditions,
or detectors may be used if the
requirements of Section 8.2 are met.
10.2 Calibrate the system daily as
described in Section 7.
10.3 Adjust the purge gas (nitrogen
or helium) flow rate to 40 mL/min.
Attach the trap inlet to the purging
device, and set the device to purge.
Open the syringe valve located on the
purging device sample introduction
needle.
10.4 Allow sample to come to
ambient temperature prior to introduc-
ing it into the syringe. Remove the
plunger from a 5-mL syringe and attach
a closed syringe valve. Open the
sample bottle (or standard) and care-
fully pour the sample into the syringe
barrel to just short of overflowing.
Replace the syringe plunger and
compress the sample. Open the syringe
valve and vent any residual air while
adjusting the sample volume to 5.0
mL. Since this process of taking an
aliquot destroys the validity of the
sample for future analysis, the analyst
should fill a second syringe at this time
to protect against possible loss of data.
Add 10.0.(iL of the surrogate spiking
solution (Section 8.7) and 10.0 juL of
the internal standard spiking solution
(Section 7.4.2), if applicable, through
the valve bore, then close the valve.
10.5 Attach the syringe-syringe
valve assembly to the syringe valve on
the purging device. Open the syringe
valves and inject the sample into the
purging chamber.
10.6 Close both valves and purge the
sample for 12.0 +0.1 minutes at
ambient temperature.
10.7 After the 1 2-minute purge time,
disconnect the purge chamber from the
trap. Dry the trap by maintaining a flow
of 40 mL/min of dry purge gas through
it for six minutes. See Figure 4. A dry
purger should be inserted into the
device to minimize moisture in the gas.
Attach the trap to the chromatograph,
adjust the device to the desorb mode,
and begin to temperature program the
gas chromatograph. Introduce the
trapped materials to the GC column by
rapidly heating the trap to 1 80 °C
while backflushing the trap with an
inert gas between 20 and 60 mL/min
for four minutes. If rapid heating
cannot be achieved, the gas
chromatographic column must be used
as a secondary trap by cooling it to
30 °C (subambient temperature, if poor
peak geometry and random retention
time problems persist) instead of the
initial program temperature of 50 °C.
10.8 While the trap is being desorbed
onto the GC column, empty the
purging chamber using the sample
introduction syringe. Wash the
chamber with two 5-mL flushes of
reagent water.
10.9 After desorbing the sample for
four minutes, recondition the trap by
returning the purge and trap device to
the purge mode. Wait 1 5 seconds then
close the syringe valve on the purging
device to begin gas flow through the
trap. The trap temperature should be
maintained at 1 80 °C. After approxi-
mately seven minutes, turn off the trap
heater and open the syringe valve to
stop the gas flow through the trap.
When cool, the trap is ready for the
next sample.
10.10 The width of the retention
time window used to make identifica-
tions should be based upon measure-
ments 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.
10.11 If the response for the peak
exceeds the working range of the
system, prepare a dilution of the
sample with reagent water from the
aliquot in the second syringe and
reanalyze.
11. Calculations
11.1 Determine the concentration of
individual compounds in the sample.
602-5
July 1982
-------
11.1.1 If the external standard cali-
bration procedure is used, calculate the
concentration of material from the peak
response using the calibration curve or
calibration factor determined in Section
7.3.2.
11.1.2 If the internal standard cali-
bration procedure was used, calculate
the concentration in the sample using
the response factor (RF) determined in
Section 7.4.3 and equation 2.
Eq. 2.
Concentration uglL = (AsCis)/(Ais)(RF)
where:
As - Response for the parameter to
be measured.
AjS = Response for the internal
standard.
CjS = Concentration of the internal
standard.
11.2 Report results in micrograms
per liter. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
11.3 For samples processed as part
of a set where the spiked sample
recovery falls outside of the control
limits which were described in Section
8.3, data for the affected parameters
must be labeled as suspect.
12. Method Performance
12.1 The method detection limit
(MDL) is defined as the minimum con-
centration of a substance that can be
measured and reported with 99%
confidence that the value is above
zeroll). The MDL concentrations listed
in Table 1 were obtained using reagent
waterO). Similar results were achieved
using representative wastewaters.
12.2 This method has been demon-
strated to be applicable for the concen-
tration range from the MDL up to 1000
x MDLO). Direct aqueous injection
techniques should be used to measure
concentration levels above 1000 x
MDL.
12.3 In a single laboratory (Monsanto
Research), using reagent water and
wastewaters spiked at or near
background levels, the average
recoveries presented in Table 2 were
obtained^!. The standard deviation of
the measurement in percent recovery is
also included in Table 2.
12.4 The Environmental Protection
Agency is in the process of conducting
an interlaboratory method study to
fully define the performance of this
method.
References
1. See Appendix A.
2. Bellar, T.A., and Lichtenberg, J.J.
Journal American Water Works
Association, 66, 739, (1974).
3. Bellar, T.A., and Lichtenberg, J.J.
"Semi-Automated Headspace Analysis
of Drinking Waters and Industrial
Waters for Purgeable Volatile Organic
Compunds," Proceedings of Sym-
posium on Measurement of Organic
Pollutants in Water and Wastewater.
American Society for Testing and
Materials, STP 686, C.E. Van Hall,
editor, 1978.
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, August 1977.
5. "OSHA Safety and Health
Standards, General Industry," (29 CFR
1910), Occupational Safety and
Health Administration, OSHA 2206,
(Revised January 1 976).
6. "Safety in Academic Chemistry
Laboratories," American Chemical
Society Publication, Committee on
Safety, 3rd Edition, 1 979.
7. "Handbook for Analytical Quality
Control in Water and Wastewater
Laboratories," EPA-600/4-79-019,
U.S. Environmental Protection Agency,
Office of Research and Development,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
March 1979.
8. "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual,"
Methods for Chemical Analysis of
Water and Wastes, EPA 600/4-79-020.
U.S. Environmental Protection Agency,
Office of Research and Development,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
March 1979.
9. "EPA Method Validation Study 24,
Method 602 (Purgeable Aromatics),"
Report for EPA Contract 68-03-2856
(In preparation).
502-6
July 1982
-------
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
Benzene
Toluene
Ethylbenzene
Chlorobenzene
1 ,4-Dichlorobenzene
1 ,3-Dichlorobenzene
1,2-Dichlorobenzene
Retention
(min.,
Column 1
3.33
5.75
8.25
9.17
16.8
18.2
25.9
Time
I
Column 2
2.75
4.25
6.25
8.02
16.2
15.0
19.4
Method
Detection Limit
M/L
0.2
0.2
0.2
0.2
0.3
O.4
0.4
Column 1 conditions: Supelcoport 100/120 mesh coated with 5% SP-12OO and
1. 75% Bentone-34 packed in a 6 ft. x 0.085 in ID stainless steel column with
helium carrier gas at 36 cc/min flow rate. Column temperature held at 50 °C for 2
min. then programmed at 6 °C/min to 9O °C for a final hold.
Column 2 conditions: Chromosorb W-AW 60/80 mesh coated with 5%
1,2,3-Tris(2-cyanoethyoxy)propane packed in a 6 ft. x 0.085 in ID stainless
steel column with helium carrier gas at 30 cc/min flow rate. Column temperature
held at 40° C for 2 min then programmed at 2° C/min to 1OO°C fora final hold.
Table 2, Single Operator Accuracy and Precision
Parameter
Benzene
Chlorobenzene
1,2-Dichlorobenzene
1 ,3-Dichlorobenzene
1 , 4-Dichlorobenzene
Ethylbenzene
Toluene
Average
Percent
Recovery
91
97
1O4
97
12O
98
77
Standard
Deviation
%
10.0
9.4
27.7
20.0
2O.4
12.4
12.1
Spike Number
Range of
fp.g/U Analyses
0.5-9.7
0.5-100
O.5-1O.O
0.5-4.8
0.5-1O.O
0.5-9.9
0.5-100
21
21
21
21
21
21
21
Matrix
Types
3
3
3
3
3
3
3
602-7 July 1982
-------
Optional
Foam
Trap
Exit 1A in.
O.D.
-*14mm O.D.
Inlet V4 in.
O.D.
V4//7. _
O.D. exit
. Sample Inlet
- 2-way Syringe valve
~17cm 20 gauge syringe needle
6mm O.D. Rubber Septum
O.D. 1/ie in. O.D.
^Stainless Steel
13X molecular
sieve purge
gas filter
Purge gas
flow control
10mm glass frit
medium porosity
Figure 1. Purging device
Packing procedure
Construction
Glasf5mm
wool i
Tenax 23cm
3% OV-1
Glass wool
1cm
5mm
•di
Trap inlet
Compression fitting
nut and ferrules
14ft 7-*/1oot resistance
wire wrapped solid
Thermocouple/controller
sensor
Electronic
temperature
control
and
pyrometer
Tubing 25 cm.
0.105 in. I.D.
0.125 in. O.D.
stainless steel
Figure 2. Trap packings and construction to include desorb capability
602-8
July 1982
-------
Carrier gas flow control uVu/{f injection ports
Pressure regulator
Purge gas
flow control \
13X molecular
sieve filter —
Column oven
_ Confirmatory column
To detector
Analytical column
Valve-3
optional 4-port column
selection valve
Trap inlet (Tenax end)
Resistance wire
Trap
22°C
Heater control
Va/ve-2
Figure 3. Purge-trap system (Purge-sorb Mode)
Note: All lines between
trap and GC
should be heated
to 8O°C
Carrier gas flow control
Pressure regulator
Liquid injection ports
Purge gas
flow control\
13X molecular
sieve filter
Column oven
_*_ Confirmatory column
To detector
-Analytical column
Valve-3
optional 4-port column
selection valve
Trap inlet (Tenax end)
Resistance wire
Heater control
Valve-2
Figure 4. Purge-trap system (Trap-dry Mode).
Note: All lines between
trap and GC
should be heated
to 80°C
602-9
July 1982
-------
Carrier gas flow control Liquid injection ports
Pressure regulator
\
Purge gas
flow control \
13X molecular I £
sieve filter
L...—
Column oven
Confirmatory column
To detector
*~~ • Analytical column
• Va/ve-3
optional 4-port column
selection valve
... 7 Trap inlet (Tenax end)
^Resistance wire Hegter contro,
Ibt-—Tr"P
180°C
Valve-2
Figure 6. Purge-trap system (Desorb Mode).
Note: All lines between
trap and GC
should be heated
to 80°C
CD
| « Column: 5% SP 1200+
§ gj 7.75% Bentone-34 on
Supelcoport
"o ns S Program: 50°C for 2 min, 6° per min to 90°C
? Q> -S Detector: Photoionization, 10.2
1 s 1
o ?! Is
g *- §§
"5
s
o
^
i^
ti:
a
53
tf
J'
QQ Q)
•*,,
S.
c
II
c
S c
s %
•Q ^
2 £
-S 2
i 1
Q o
4 9
11
, M .
tiii — i 1 1 i__J 1 i i
volts
$
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-------
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Acrolein and Acrylonitrile—
Method 603
1. Scope and Application
1.1 This method covers the determi-
nation of acrolein and acrylonitrile. The
following parameters may be
determined by this method:
Parameter STORET No.
CAS No.
Acrolein
Acrylonitrile
34210
34215
107-02-8
107-13-1
1.2 This is a purge and trap gas
chromatographic method applicable to
the determination of the compounds
listed above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for either or
both of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. This method
describes analytical conditions for a
second gas chromatographic column
that can be used to confirm
measurements made with the primary
column. Method 624 provides gas
chromatograph/mass spectrometer
(GC/MS) conditions appropriate for the
qualitative and quantitative
confirmation of results for the
parameters listed above, if used with
the purge and trap conditions described
in this method.
1.3 The method detection limit (MDL,
defined in Section 12.1) <1 > for each
parameter is listed in Table 1. The MDL
for a specific wastewater may differ
from those listed, depending upon the
nature of interferences in the sample
matrix.
1.4 Any modification of this method,
beyond those expressly permitted,
shall be considered as major modifica-
tions subject to application and
approval of alternate test procedures
under 40 CFR 1 36.4 and 136.5
1.5 This method is restricted to use
by or under the supervision of analysts
experienced in the operation of a purge
and trap system and a gas chromato-
graph (GC) and in the interpretation of
GC chromatograms. Each analyst must
demonstrate the ability to generate
acceptable results with this method
using the procedure described in
Section 8.2.
2. Summary of Method
2.1 An inert gas is bubbled through a
5-mL water sample contained in a
heated purging chamber. Acrolein and
acrylonitrile are transferred from the
aqueous phase to the vapor phase. The
vapor is swept through a sorbent trap
where the analytes are trapped. After
the purge is completed, the trap is
heated and backflushed with the inert
gas to desorb the compounds onto a
gas chromatographic column. The gas
chromatograph is temperature
603-1
July 1982
-------
programmed to separate the analytes
which are then detected with a flame
ionization detector I2-3'.
2.2 The method provides an optional
gas chromatographic column that may
be helpful in resolving the compounds
of interest from the interferences that
may occur.
3. Interferences
3.1 Impurities in the purge gas and
organic compounds out-gassing from
the plumbing ahead of the trap account
for the majority of contamination
problems. The analytical system must
be demonstrated to be free from
contamination under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5. The use of non-TFE plastic tubing,
non-TFE thread sealants, or flow
controllers with rubber components in
the purging device should be avoided.
3.2 Samples can be contaminated by
diffusion of volatile organics through
the septum seal into the sample during
shipment and storage. A field reagent
blank prepared from reagent water and
carried through the sampling and
handling protocol can serve as a check
on such contamination.
3.3 Contamination by carry-over can
occur whenever high level and low
level samples are sequentially
analyzed. To reduce carry-over, the
purging device and sample syringe
must be rinsed out between samples
with reagent water. Whenever an
unusually concentrated sample is
encountered, it should be followed by
an analysis of reagent water to check
for cross contamination. For samples
containing large amounts of water-
soluble materials, suspended solids,
high boiling compounds or high analyte
levels, it may be necessary to wash out
the purging device with a detergent
solution, rinse it with distilled water,
and then dry it in a 105 °C oven
between analyses. The trap and other
parts of the system are also subject to
contamination, therefore, frequent
bakeout and purging of the entire
system may be required.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified<4-6) for the
information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
sampling.
5.1.1 Vial—25-mL capacity or larger,
equipped with a screw cap with a hole
in the center (Pierce #1 3075 or
equivalent). Detergent wash, rinse with
tap and distilled water and dry at
105 °C before use.
5.1.2 Septum—Teflon-faced silicone
(Pierce #1 2722 or equivalent).
Detergent wash, rinse with tap and
distilled water, and dry at 105 °C for
one hour before use.
5.2 Purge and trap device—The
purge and trap device consists of three
separate pieces of equipment: the
sample purger, the trap, and the
desorber. Several complete devices are
now commercially available.
5.2.1 The sample purger must be
designed to accept 5-mL samples with
a water column at least 3 cm deep.
The gaseous head space between the
water column and the trap must have a
total volume of less than 1 5 mL. The
purge gas must pass through the water
column as finely divided bubbles with a
diameter of less than 3 mm at the
origin. The purge gas must be intro-
duced no more than 5 mm from the
base of the water column. The purge
device must be capable of being heated
to 85 °C within 3.0 minutes after
transfer of the sample to the purge
device and being held at 85 ± 2 °C
during purge cycle. The entire water
column in the purge device must be
heated. Design of this modification to
the standard purge device is optional,
however, use of a water bath is
suggested.
5.2.1.1 Heating mantle—To be used
to heat water bath.
5.2.1.2 Temperature controller-
equipped with thermocouple/sensor to
accurately control water bath tempera-
ture to ± 2 °C. The sample purger
illustrated in Figure 1 meets these
design criteria.
5.2.2 The trap must be at least 25
cm long and have an inside diameter of
at least 0.105 inch. The trap must be
packed to contain 1.0 cm of methyl
silicone coated packing (Section 6.5.2)
and 23 cm of 2,6-diphenylene oxide
polymer (Section 6.5.1). The minimum
specifications for the trap are
illustrated in Figure 2.
5.2.3 The desorber must be capable
of rapidly heating the trap to 100 °C.
The polymer section of the trap should
not be heated higher than 180 °C. The
desorber, illustrated in Figure 2, meets
these design criteria.
5.2.4 The purge and trap device may
be assembled as a separate unit as
illustrated in Figure 3 or be coupled to a
gas chromatograph.
5.3 pH paper—Narrow pH range,
about 3.5 to 5.5 (Fisher Scientific
Short Range Alkacid No. 2, #14-837-2
or equivalent).
5.4 Gas chromatograph—An analytical
system complete with a temperature
programmable gas chromatograph
suitable for on-column injection and all
required accessories including syringes,
analytical columns, gases, detector,
and strip-chart recorder. A data system
is recommended for measuring peak
areas.
5.4.7 Column 1—6 ft long x 0.1 in
ID stainless steel or glass, packed with
Durapak-Carbowax 400/Porasil-C
(100/120 mesh) or equivalent. This
column was used to develop the
method performance statements given
in Section 1 2. Guidelines for the use of
alternate column packings are provided
in Section 10.1.
5.4.2 Column 2-6 ft long x 0.1 in
ID stainless steel or glass, packed with
Chromosorb 101 (60/80 mesh) or
equivalent.
5.4.3 Detector—Flame ionization.
This type of detector has proven effec-
tive in the analysis of wastewaters for
the parameters listed in the scope, and
was used to develop the method per-
formance statements in Section 1 2.
Guidelines for the use of alternate
detectors are provided in Section 10.1.
5.5 Syringes—5-mL, glass
hypodermic with Luerlok tip (two
each).
5.6 Micro syringes—25 p.L.
5.7 Syringe valve, 2-way with Luer
ends (three each).
5.8 Bottle— 1 5-mL screw-cap with
Teflon cap liner.
603-2
July 1982
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5.9 Balance—Analytical, capable of
accurately weighing 0.0001 g.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an inter-
ferent is not observed at the MDL of
the parameters of interest.
6.1.1 Reagent water can be gener-
ated by passing tap water through a
carbon filter bed containing about one
pound of activated carbon (Filtrasorb-
300 or equivalent, Calgori Corp).
6.1.2 A water purification system
(Millipore Super-Q or equivalent) may
be used to generate reagent water.
6.1,3 Reagent water may also be
prepared by boiling water for 1 5
minutes. Subsequently, while maintain-
ing the temperature at 90 °C, bubble a
contaminant-free inert gas through the
water for one hour. While still hot,
transfer the water to a narrow mouth
screw cap bottle and seal with a Teflon
lined septum and cap.
6.2 Sodium thiosulfate—(ACS)
Granular.
6.3 Sodium hydroxide solution (10
N) — Dissolve 40 g NaOH in reagent
water and dilute to 100 mL.
6.4 Hydrochloric acid solution (1 +1 )—
Slowly add 50 mL concentrated HCI to
50 mil reagent water.
6.5 Trap Materials
6.5.7 2,6-Diphenylene oxide polymer,
Tenax (6O/80 mesh) chromatographic
grade.
6.5.2 Methyl Silicone packing—3%
OV-1 on Chromosorb-W (60/80 mesh)
or equivalent.
6.6 Stock standard solutions—Stock
standard solutions may be prepared
from pure standard materials or
purchased as certified solutions.
Prepare stock standard solutions in
' reagent water using assayed liquids.
Since acrolein and acrylonitrile are
lachrymators, primary dilutions of
these compounds should be prepared
in a hood. A NIOSH/MESA;approved
toxic gas respirator should be used
when the analyst handles high concen-
trations of such materials.
6.6.1 Place about 9.8 mL of reagent
water into a 10-mL ground glass
stoppered volumetric flask: For acrolein
standards the reagent water must be
adjusted to pH 4 to 5. Weigh the flask
to the nearest 0.1 mg.
6.6.2 Using a 1 00-juL syringe,
immediately add two or more drops of
assayed reference material to the flask,
then reweigh. Be sure that the drops
fall directly into the water without
contacting the neck of the flask.
6.6.3 Reweigh, dilute to volume,
stopper, then mix. by inverting the flask
several times. Calculate the concentra-
tion in micrograms per microliter from
the net gain in weight. When com-
pound purity is assayed to be 96%,or
greater, the weight can 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.
6.6.4 Transfer the stock standard
solution into a Teflon-sealed screw-cap
bottle. Store at 4 °C and protect from
light.
6.6.5 Prepare fresh standards
weekly.
6.7 Secondary dilution standards-
Using stock standard solutions, prepare
secondary dilution standards in reagent
water that contain the compounds of
interest, either singly or mixed together.
The secondary dilution standards
should be prepared at concentrations
such that the aqueous calibration stan-
dards prepared in Sections 7.3.1 or
7.4.1 will bracket the working range of
the analytical system. Secondary
dilution standards should be prepared
weekly and stored at 4 °C. They should
be checked" frequently for signs of
degradation or evaporation. Quality
control check standards that can be
used to determine the accuracy of
calibration standards will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, in Cincinnati,
Ohio.
7. Calibration
7.1 Assemble a purge and trap
device that meets the specifications in
Section 5.2. Condition the trap over-
night at 180 °C by backflushing with
an inert gas flow of at least 20 mL/min.
Prior to use, daily condition traps 10
minutes while backflushing at 180 °C.
7.2 Connect the purge and trap
device to a gas chromatograph. The
gas chromatograph must be operated
using temperature and flow rate
parameters equivalent to those in Table
1. Calibrate the purge and trap-gas
chromatographic system using either
the external standard technique
(Section 7.3) or the internal standard
technique (Section 7.4).
7.3 External standard calibration
procedure:
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter by carefully
adding 20.0 fii. of one or more
secondary dilution standards to 100,
500, or 1000 mL of reagent water. A
25-jnL syringe should be used for this
operation. One of the external
standards should be at a concentration
near, but above, the method detection
limit 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. These standards
must be prepared fresh daily.
7.3.2 Analyze each calibration
standard according to Section 10, and
tabulate peak height or area responses
versus the concentration of 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 (-=10% relative standard devia-
tion, RSD), linearity through the origin
can be assumed and the average ratio
or calibration factor can be used in
place of a calibration curve.
7.3.3 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 any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.4 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested that
is applicable to all samples.
7.4.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest as
described in Section 7.3.1.
7.4.2 Prepare a spiking solution
containing each of. the internal
standards using the procedures
described in Sections 6.6 and 6.7. It is
recommended that the secondary
603-3
July 1982
-------
dilution standard be prepared at a
concentration of 1 5 (JLgfrnL of each
internal standard compound. The
addition of 1 0 ML of this standard to
5.0 mL of sample or calibration
standard would be equivalent to 30
7.4.3 Analyze each calibration
standard according to Section 1 0
adding 10 L of internal standard
spiking solution directly to the syringe
(Section 10.4). Tabulate peak height
or area responses against concentra-
tion for each compound and internal
standard, and calculate response
factors (RF) for each compound using
equation 1 .
Eq. 1 RF = (AsCis)/(AisCs)
where:
As — Response for the parameter to
be measured.
Ais = Response for the internal
standard.
CIs « Concentration of the internal
standard.
Cs = Concentration of the
parameter to be measured.
If the RF value over the working range
is a constant ( -=:1 0% RSD), the RF
can be assumed fo be invariant and 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. RF.
7.4.4 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 1 0%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with
established performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
8. 1. 1 Before performing any analyses,
the analyst must demonstrate the
ability to generate acceptable accuracy
and precision with this method. This
ability is established as described in
Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the separa-
tions or lower the cost of measure-
ments. Each time such modifications
are made to the method, the analyst is
required to repeat the procedure in
Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing labora-
tory performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and pre-
cision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each compound to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in reagent water 500
times more concentrated than the
selected concentrations. Quality
control check sample concentrates,
appropriate for use with this method,
will be available from the U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
8.2.2 Using, a syringe, add 10 ML of
the check sample concentrate to each
of a minimum of four 5-mL aliquots of
reagent water. A representative waste-
water may be used in place of the
reagent water, but one or more addi-
tional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for the
results. Wastewater background
corrections must be made before R and
s calculations are performed.
8.2.4 Using Table 2, note the average
recovery (X) and standard deviation (p)
expected for each method parameter.
Compare these to the calculated values
for R and s. If s :=- 2p or |X - R| =- 2p,
review potential problem areas and
repeat the test.
8.2.5 The U.S. Environmental
Protection Agency plans to establish
performance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and define
the performance of the laboratory for
each spike concentration and parameter
being measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
where R and s are calculated as in
Sections 8.2.3.
The UCL and LCL can be used to
construct control charts!7' that are
useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they become avail-
able from the U.S. Environmental
Protection Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy state-
ments of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcu-
lation of R and s. Alternately, the
analyst may use four wastewater data
points gathered through the require-
ment for continuing quality control in
Section 8.4. The accuracy statements
should be updated regularly*7'.
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of the
sample must be spiked and analyzed as
described in Section 8.2. If the
recovery for a particular parameter
does not fall within the control limits
for method performance, the results
reported for 'that parameter in all
samples processed as part of the same
set must be qualified as described in
Section 11.3. The laboratory should
monitor the frequency of data so
qualified to ensure that it remains at or
below 5%.
8.5 Each day, the analyst must
demonstrate through the analysis of
reagent water, that interferences from
the analytical system are under control.
8.6 It is recommended that the
laboratory adopt additional quality
603-4
July 1982
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assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. When doubt
exists over the identification of a peak
on the chromatogram, confirmatory
techniques such as gas chromatography
with a dissimilar column, specific ele-
ment detector, or mass spectrometer
must be used. Whenever possible, the
laboratory should perform analysis of
standard reference materials and
participate in relevant performance
evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 The samples must be iced or
refrigerated from the time of collection
until extraction. If the sample contains
free or combined chlorine, add sodium
thiosulfate preservative (10 mg/40 mi-
is sufficient for up to B.ppm CI2) to the
empty sample bottles just prior to
shipping to the sampling site. EPA
methods 330.4 and 330.5 may be
used for measurement of chlorine
residual!8). Field test kits are available
for this purpose.
9.2 If acrolein is to be analyzed,
collect about 500 ml_ sample in a clean
glass container. Adjust the pH of the
sample to 4 to 5 using acid or base,
measuring with narrow range pH paper.
Samples for acrolein analysis receiving
no pH adjustment must be analyze
within three days of sampling.
9.3. Fill a sample bottle just to
overflowing in such a manner that no
air bubbles pass through the sample as
the bottle is being filled. Seal the bottle
so that no air bubbles aretentrapped in
it. If preservative has been added,
shake vigorously for one minute.
Maintain the hermetic seal on the
sample bottle until time of analysis.
9.4 All samples must be analyzed
within 14 days of collection^).
10. Sample Extraction and
Gas Chromatography
10.1 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. Included in this
Table are estimated retention times and
MDL that can be achieved by this
method. An example of the separations
achieved by Column 1 is shown in
Figure 4. Other packed columns,
chromatographic conditions, or
detectors may be used if the require-
ments of Section 8.2 are met.
10.2 Calibrate the system daily as
described in Section 7.
10.3 Adjust the purge gas (nitrogen
or helium) flow rate to 20 mL/min.
Attach the trap inlet to the purging
device, and set the device to purge.
Open the syringe valve located on the
purging device sample introduction
needle.
10.4 Remove the plunger from a
5-mL syringe and attach a closed
syringe valve. Open the sample bottle
(or standard) and carefully pour the
sample into the syringe barrel to just
short of overflowing. Replace the
syringe plunger and compress the
sample. Open the syringe valve and
vent any residual air while adjusting the
sample volume to 5.0 ml. Since this
process of taking an aliquot destroys
the validity of the sample for future
analysis, the analyst should fill a
second syringe at this time to protect
against possible loss of data. Add 10.0
uL of the internal standard spiking
solution (Section 7.4.2), if applicable,
through the valve bore then close the
valve.
10.5 Attach the syringe-syringe
valve assembly to the syringe valve on
the purging device. Open the syringe
valves and inject the sample into the
purging chamber.
10.6 Close both valves and purge the
sample for 15.0 ±0.1 minutes while
heating at 85 ± 2 °C.
10.7 After the 1 5-minute purge time,
attach the trap to the chromatograph,
and adjust the device to the desorb
mode. Begin the temperature program
for the gas chromatograph. Introduce
the trapped materials to the GC column
by rapidly heating the trap to 100 ±
10 °C while backflushing the trap with
an inert gas between 20 and 60 mL/min
for two minutes.
10.8 While the trap is being desorbed
into the gas chromatograph, empty the
purging chamber using the sample
introduction syringe. Wash the
chamber with two 5-mL flushes of
reagent water.
10.9 After desorbing the sample for
2.0 minutes recondition the trap by
returning the purge and trap device to
the purge mode. Wait 1 5 seconds then
close the syringe valve on the purging
device to begin gas flow through the
trap. The trap temperature should be
maintained at 100 °C. After approxi-
mately seven minutes turn off the trap
heater and open the syringe valve to
stop the gas flow through the trap.
When cool the trap is ready for the
next sample.
10.10 The width of the retention
time window used to make identifica-
tions should be based upon measure-
ments 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. Calculations
11.1 Determine the concentration of
individual compounds in the sample.
11.1.1 If the external sample calibra-
tion procedure is used, calculate the
concentration of material from the peak
response using the calibration curve or
calibration factor determined in Section
7.3.2.
11.1.2 If the internal standard cali-
bration procedure was. used, calculate
the concentration in the sample using
the response factor (RF) determined in
Section 7.4.2 and the equation 2:
Eq. 2
Concentration /zg/L = (AsCis)/(Ais)(RF)
where:
As = Response for the parameter to
be measured.
AJS = Response for the internal
standard.
CjS = Concentration of the internal
standard.
11.2 Report results in micrograms
per liter. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
11.3 For samples processed as part
of a set where the spiked sample
recovery falls outside of the control
limits in 8.3, data for the affected
parameters must be labeled as suspect.
12. Method Performance
12.1 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
zero'1). The MDL concentrations listed
in Table 1 were obtained using reagent
waterO). Similar results were achieved
using representative wastewaters.
12.2 This method is recommended
for the concentration range from the
MDL up to 1000 x MDL. Direct
aqueous injection techniques should be
used to measure concentration levels
above 1000 x MDL.
603-5
July 1982
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12.3 In a single laboratory (EMSL-CI),
using spiked wastewater, the average
recoveries presented in Table 2 were
obtained'3'. Seven replicate spiked
samples were analyzed for each
parameter. The relative standard devia-
tion of the measurement is also
Included in Table 2.
12.4 The U.S. Environmental
Protection Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. Bellar, T.A., and Lichtenberg, J.J.
Journal American Waterworks
Association, 66, 739, (1974).
3. Kerns, E.H., et al, "Determination of
Acrolein and Acrylonitrile in Water by
Heated Purge and Trap Technique,"
1980, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
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. "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 19 79.
8: "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual,"
Methods for Chemical Analysis of
Water and Wastes, EPA 60O/4-79-020,
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory—Cincinnati, Ohio 45268.
March 1979.
9. "EPA Method Validation Study 25,
Method 603 (Acrolein and Acryloni-
trile)," Report for EPA Contract
68-03-2856 (In preparation).
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time
(mln.)
Method
Detection Limit
Parameter
Column 1
Column 2
Acrolein
Acrylonitrile
9.2
13.5
8.2
9.8
0.6
0.5
Method detection limit based upon recovery of 5.0 ug/L dose into tap water.
Column 1 conditions: Durapak Carbowax 400/Porasil C, (WO/12O mesh) packed in
a 6 ft x. 0.1 in. ID stainless steel or glass column with helium carrier gas at 30
mL/min flow rate. Column temperature held at 45° C for 2 min, then programmed
at8°C/min to 85 °C and held for 12 min. Thecolumn temperature should then be
raised to 120 °C for 7 minutes to bake out water. Failure to dry the column may
lead to irreproducable retention times'3'.
Column 2 conditions: Chromosorb 101, (6O/80 mesh) packed in a 6 ft x 0.1 in ID
stainless steel, glass column with helium carrier gas at 40 mL/min flow rate.
Column temperature held at 80°C for 4 min. then programmed at 15°C/min to
120°C and held 12 min.
Table 2. Single Operator Accuracy and Precision
Parameter
Average
Percent
Recovery
Standard
Deviation
Spike
Range
((J.B/U
Number
of Matrix
Analyses Types
Acrolein
Acrylonitrile
96
107
11. 16
5.6
20
20
7
7
603-6
July 1982
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Optional
Foam
Trap
V4 in.
O.D.
-*- 14mm
O.D.
Inlets/4 in.
O.D.
11
11
i'
^ Sample Inlet
l°)-«- 2-way Syringe valve
17cm 20 gauge syringe needle
O.D. Rubber Septum
O.D. Vie in. O.D.
\/Stainless Steel
13X molecular
sieve purge
gas filter
Purge gas
flow control
10mm glass frit
medium porosity
Figure 1. Purging device
Packing procedure Construction
Glass 5mm
wool
Tenax 23cm
3% OV-1
Glass
wool
1cm
5mm t
Compression fitting
nut and ferrules
14ft 7Vfoot resistance
wire wrapped solid
Thermocouple/controller
sensor
Electronic
temperature
control
and
pyrometer
Tubing 25 cm.
0.105 in. I.D.
0.125 in. O.D.
stainless steel
Trap inlet
Figure 2. Trap packings and construction to include desorb capability
603-7 juiy 1982
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Purge Mode
13X molecular
sieve filter
GC Injection
port
2-Position
6-port valve,
clockwise
rotation
Trap
Heated
water bath
Desorb Mode
13X molecular
sieve filter
GC injection
port
Purge
flow
controller
2-position
6-port valve
clockwise
rotation
Trap
Column: Porasil C/Carbowax 400
Program: 45°C-2 min.
8°C/min. to 85°C
Detector: Flame ionization
water bath
Figure 3. Schematic of heated purge and trap device
J 4 8 12 16 20
Retention time, minutes
Figure 4. Gas chromatogram of
acrolein and acrylonitrile
603-8
July 1982
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vvEPA
United States
Environmental Protection
Agency
Environmental (Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Phenols —
Method 604
1. Scope and Application
1.1 This method covers the determi-
nation of phenol and certain substituted
phenols. The following parameters may
be determined by this method:
Parameter
STORET No.
CAS No.
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dichlorophenol
2,4-Dimethylphenol
2,4-Dinitrophenol
2-Methyl-4,6-dinitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2,4,6-Trichlorophenol
34452
34586
34601
34606
34616
34657
34591
34646
39032
34694
34621
59-50-7
95-57-8
120-83-2
105-67-9
51-28-5
534-52-1
88-75-5
100-02-7
87-86-5
108-95-2
88-06-2
1.2 This is a gas chromatographic
(GC) method applicable to the
determination of the compounds listed
above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any or
all of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. This method
describes analytical conditions for
derivatization, cleanup and electron
capture gas chromatography that can
be used to confirm measurements
made by flame ionization. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions ap-
propriate for the qualitative and
quantitative confirmation of results for
all of the parameters listed above, us-
ing the extract produced by this
method,
1.3 The method detection limit
(MDL, defined in Section 14.1) "'for
each parameter is listed in Table 1. .
The MDL for a specific wastewater
may differ from those listed,
depending upon the nature of
interferences in the sample matrix.
The MDL listed in Table 1 for each
parameter was achieved with a flame
ionization detector. Comparable
results were achieved when the
derivatization cleanup and the
electron capture detector were
employed (See Table 2).
1.4 Any modification of this method,
beyond that expressly permitted, shall
be considered a major modification
subject to application and approval of
alternate test procedures under 40
CRF 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
604-1
July 1982
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ability to generate acceptable results
with this method using the procedure
described in Section 8.2.
2. Summary of Method
2.1 A 1 -liter sample of wastewater
is acidified and extracted with
methylene chloride using separatory
funnel techniques. The extract is dried
and concentrated to a volume of 10
mL or less. During the concentration
step, the solvent is exchanged to
2-propanol. Flame ionization gas
chromatographic conditions are
described which allow for the
measurement of the compounds in
the extract e>.
2.2 A preliminary sample wash
under basic conditions can be
employed for samples having high
general organic and organic base
interferences.
2.3 The method also provides for the
preparation of pentafluorobenzyl
bromide (PFBB) derivatives for
electron capture gas chromatography
as an additional cleanup procedure to
aid in the elimination of inter-
ferences l2'31.
3. Interferences
3.1 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 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 as described in Section
8.5.
3.1.1 Glassware must be
scrupulously cleaned141. 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. It should then be drained dry,
and heated in a muffle furnace at
400°C for 15 to 30 minutes. Some
thermally stable materials, such as
PCBs, may not be eliminated by this
treatment. Solvent rinses with
acetone and pesticide quality hexane
may be substituted for the muffle
furnace heating. Volumetric ware
should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and
stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted or
capped with aluminum foil.
3.1.2 The use of high purity
reagents and solvents helps to
minimize interference problems.
Purification of solvents by distilla-
tion in all-glass systems may be
required.
3.2 Matrix interferences may be
caused by contaminants that are
co-extracted from the sample. The
extent of matrix interferences will
vary considerably from source to
source, depending upon the nature
and diversity of the industrial complex
or, municipality being sampled. The
cleanup procedure in Section 12 can
be used to overcome many of these
interferences, but unique samples
may require additional cleanup
approaches to achieve the method
detection limits listed in Tables 1 and
2.
3.3 The basic sample wash (Section
10.2) may cause significantly
reduced recovery of phenol and
2,4-dimethylphenol. The analyst must
recognize that results obtained under
these conditions are minimum
concentrations.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional
references to laboratory safety are
available and have been identified (s~7'
for the information of the analyst.
4.2 Special care should be taken in
handling pentafluorobenzyl bromide,
which is a lachrymator, and 18 crown
6 ether, which is highly toxic.
5. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling.
5.1.1 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with Tef-
lon. Foil may be substituted for Teflon
if the sample is not corrosive. If amber
bottles are not available, protect
samples from light. The container and
capliner must be washed, rinsed with
acetone or methylene chloride, and
dried before use to minimize con-
tamination.
5.1.2 Automatic sampler (optional)
- The sampler must incorporate glass
containers for the collection of a
minimum of 250 mL of sample.
Sample containers must be kept
refrigerated at 4°C and protected from •
light during compositing. If the
sampler uses a peristaltic pump, a
minimum length of compressible
silicone rubber tubing may be used.
Before use, however, the
compressible tubing must be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.7 Separatory funnel - 2000-mL,
with Teflon stopcock.
5.2.2. Drying column -
Chromatographic column 400-mm
long x 19-mm ID with coarse frit.
5.2.3. Chromatographic column -
100-mm long x 10-mm ID, with Teflon
stopcock.
5.2.4. Concentrator tube,
Kuderna-Danish - 10-mL, graduated
(Kontes K-570050-1025 or
equivalent). Calibration must be
checked at the volumes employed in
the test. Ground glass stopper is used
to prevent evaporation of extracts.
5.2.5 Evaporative flask,
Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach
to concentrator tube with springs.
5.2.6 Snyder column,
Kuderna-Danish - three-ball macro
(Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column,
Kuderna-Danish - two-ball micro
{Kontes K-569001 -0219 or
equivalent).
5.3 Vials - Amber glass, 10- to 15-
mL capacity, with Teflon-lined
screwcap.
5.4 Reaction flask - Pyrex glass, 15-
to 25-mL round bottom flask with
standard tapered joint, fitted with a
water cooled condenser and U-shaped
drying tube containing granular
calcium chloride.
5.5 Boiling chips - Approximately
10/40 mesh. Heat to 400°C for 30
minutes or Soxhlet extract with
methylene chloride.
5.6 Water bath - Heated, with
concentric ring cover, capable of
604-2
July 1982
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temperature control (± 2°C). The bath
.should be used in a hood.
5.7 Balance - Analytical, capable of
accurately weighing 0.0001 g.
5.8 Gas chromatograph - An
analytical system complete with a
temperature programmable gas
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical columns,
gases, detector, and strip-chart
recorder. A data system is
recommended for measuring peak
areas.
5.8.1 Column for underivatized
phenols - 1.8 m long x 2 mm ID
glass, packed with 1% SP-1240 DA
on Supelcoport (80/100 mesh) or
equivalent. This column was used to
develop the method performance
statements in Section 14. Guidelines
for the use of alternate column
packings are provided in Section
11-.1.
5.8.2 Column for derivatized
phenols, - 1.8 m long x 2 mm ID glass
column packed with 5% OV-17 on
Chromosorb W-AW-DMCS (80/100
mesh). The carrier gas is'5%
methane/95% Argon at a flow rate of
30 mL/min. The column temperature
is 200°C. This column has proven
effective in the analysis of
wastewaters for derivatization
products of the parameters listed in
the scope (Section 1.1), and was used
to develop the method performance
statements in Section 14. Guidelines
for the use of alternate columns are
provided in Section 11.1.
5.8.3 Detectors - flame ionization
and electron capture. The flame
ionization is used when determining
the parent phenols. The electron cap-
ture is used when determining the
derivatized phenols. Guidelines for
use of alternate detectors are provided
in Section 11.1.
6. Reagents
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Sodium hydroxide solution (10
N) - (ACS) Dissolve 40g NaOH in
reagent water and dilute to 100 ml.
6.3 Sodium hydroxide solution (1 N)
- (ACS) Dissolve 4g NaOH in reagent
water and dilute to 100 mL.
6.4 Sodium sulfate - (ACS)
Granular, anhydrous. Purify by
heating at 400°C for four hours in a
shallow tray.
6.5 Sodium thiosulfate - (ACS)
Granular.
6.6 Sulfuric acid solution (1 + 1) -
(ACS) Slowly, add 50 mL H2SO4 (sp.
gr. 1.84) to 50 mL of reagent water.
6.7 Sulfuric acid (1 N) - (ACS)
Slowly, add 29 mL H2SO4 (ACS, sp.
gr. 1.84) to reagent water and dilute
to one liter.
6.8 Potassium carbonate - (ACS)
powdered.
6.9 Pentafluorobenzyl bromide (a-
Bromopentafluorotoluene) - 97%
minimum purity. NOTE: This chemical
is a lachrymator. (See Section 4.2.)
6.10 18-crown-6 ether
(1,4,7,10,13,16 -
Hexaoxacyclooctadecane) - 98%
minimum purity. NOTE: This chemical
is highly toxic.
6.11 Derivatization reagent - Add
one mL pentafluorobenzyl bromide
and one gram 18 crown 6 ether to a
50-mL volumetric flask and-dilute to
volume with 2-propanol. Prepare fresh
weekly. This operation should be car-
ried out in a hood. Store 4°C and pro-
tect from light.
6.12 Acetone, hexane, methanol,
methylene chloride, 2-propanol,
hexane, toluene - Pesticide quality or
equivalent.
6.13 Silica gel - Davison chemical,
grade 923 (100/200 mesh) or
equivalent. Activate at 130°C
overnight and store in a desiccator.
6.14 Stock standard solutions (1.00
/ug/yt/L) - Stock standard solutions may
be prepared from pure standard
materials or purchased as certified
solutions.
6.14.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 grams of pure material.
Dissolve the material in pesticide
quality 2-propanol and dilute to
volume in a 10-mL volumetric flask.
Larger volumes may be prepared at
the convenience of the analyst. If
compound purity is certified at 96% or
greater, the weight can be used
without correction to calculate the
concentration of the stock standard.
Commercially prepared stock
standards can be used at any
concentration if they are certified by
the manufacturer or by an
independent source.
6.14.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards, will be available from the
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio, 45268.
6.14.3 Stock standard solutions
must be replaced after six months, or
sooner if comparison with check
standards indicates a problem.
7. Calibration
7.1 To calibrate the FIDGC for the
analysis of underivatized phenols,
establish gas chromatographic
operating parameters equivalent to
those indicated in Table 1. The gas
chromatographic system can be
calibrated using the external standard
technique (Section 7.2) or the internal
standard technique (Section 7.3),
7.2 External standard calibration
procedure for FIDGC.
7.2.7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with 2-
propanol. One of the external
standards should be at a
concentration near, but above, the
MDL 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.
7.2.2 Using injections of 2 to 5 //L
of each calibration standard, 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
calibration curve.
7.2.3 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 any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
6O4-3
July 1982
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7.3 Internal standard calibration
procedure for FIDGC. To use this
approach, the analyst must select one
or more internal standards that are
similar 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. Because of these
limitations, no internal standard can
be suggested that is applicable to all
samples.
7.3.7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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 internal standards, and dilute to
volume with 2-propanol. One of the
standards should be at a
concentration near, but above, the
MDL 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.
7.3.2 Using injections of 2 to 5 pL
of each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1 RF = {A,C»)/(Ai. C,)
where:
A> = Response for the parameter to be
measured.
Ata = Response for the internal
standard.
Cu = Concentration of the internal
standard, Oug/L).
Ct = Concentration of the parameter
to be measured, (/ug/L).
If the RF value over the working range
is a constant « 10% RSD), the RF can
be assumed to be nonvariant and the
averge RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
of response ratios, As/As, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 To calibrate the ECGC for the
analysis of phenol derivatives,
establish gas chromatographic
operating parameters equivalent to
those indicated in Table 2.
7.4.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with 2-
propanol. One of the external
standards should represent a
concentration near but above the MDL
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.
7.4.2 Each time samples are to be
derivatived, simultaneously treat a
one-mL aliquot of each calibration
standard as described in Section 12.
7.4.3 After derivatization, inject 2 to
5 //L of each column eluate collected
and tabulate peak height or area
responses against the calculated
equivalent mass of underivatized
phenol injected. The results can be
used to prepare a calibration curve for
each compound.
7.5 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of
interferences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist
of an initial demonstration of
laboratory capability and the analysis
of spiked samples as a continuing
check on performance. The laboratory
is required to maintain performance
records to define the quality of data
that is generated. Ongoing
performance checks must be
compared with established
performance criteria to determine if
the results of analyses are within
accuracy and precision limits expected
of the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method. This ability is
established as described in Section
8.2.
8.1.2 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyse a minimum of 10% of all
samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate in 2-propanol 1000 times
more concentrated that the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be
used in place of the reagent water,
but one or more additional aliquots
must be analyzed to determine
background levels, and the spike level
must exceed twice the background
level for the test to be valid. Analyze
the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater
background corrections must be made
before R and s calculations are
performed.
8.2.4 Using Table 3, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or |X-R| > 2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agencyplans to establish per-
formance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter being
measured.
604-4
July 1982
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8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in
Section 8.2.3. The UCL and LCL can
be used to construct control charts'8'
that are useful in observing trends in
performance. The control limits above
must be replaced by method
performance criteria as they become
available from the U.S. Environmental
Protection Agency.
5.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined
as R ± S. The accuracy statement
should be developed by the analysis of
four aliquots of wastewater as
described in Section 8.2.2, followed
by the calculation of R and s.
Alternately, the analyst may use four
wastewater data points gathered
through the requirement for
continuing quality control in Section'
8.4. The accuracy statements should
be updated regularly'8'.
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample'per month,
whichever is greater. One aliquot of
the sample must be spiked and
analyzed as described in Section 8.2.
If the recovery for a particular
parameter does not fall'within the
control limits for method performance,
the results reported for that parameter
in all samples processed as part of the
same set must be qualified as
described in Section 13-3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagents interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as
a safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may
be analyzed to monitor the precision
of the sampling technique. When
doubt exists over the identification of
a peak on the chromatogram,
confirmatory techniques such as gas
chromatography with a dissimilar
column, specific element detector, or
mass spectrometer must be used.
Whenever possible, the laboratory
should perform analysis of standard
reference materials and participate in
relevant performance evaluation
studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices'9' 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.
9.2 The samples must be iced or
refrigerated at 4°C from the time of
collection until extraction. Fill the
sample bottle and at time of collection
if residual chlorine is present, add 80
mg of sodium thiosulfate and mix
well. U.S. Environmental methods
330.4 and 330.5 may be used for
measurement of residual chlorine1101
Field test kits are available for this
purpose.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction'21.
10. Sample Extraction
10.1 Mark the water meniscus on
the side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel.
10.2 For samples high inorganic
content, the analyst may solvent wash
the sample at a basic pH as
prescribed in Section 10.2.1 and
10.2.2 to remove potential method
interferences. Prolonged or exhaustive
contact with solvent during the wash
may result in low recovery of some of
the phenols, notably phenol and 2,4-
dimethyl phenol. For relatively clean
samples, the wash should be omitted
and the extraction, beginning with
Section 10.3, should be followed.
10.2.1 Adjust the pH of the sample
to 12.0 or greater with 10 N sodium
hydroxide.
10.2.2 Add 60 mL of methylene
chloride to the sample by shaking the
funnel for one minute with periodic
venting to release vapor pressure.
Discard to solvent layer. The wash
can be repeated up to two additional
times if significant color is being
removed.
10.3 Adjust the sample to a pH of 1
to 2 with sulfuric acid (1 + 1).
10.4 Add 60 mL of methylene
chloride to the sample bottle, seal,
and shake 30 seconds to rinse the
inner walls. Transfer the solvent to
the separatory funnel and shake for
two minutes. Allow the solvent to
separate from the sample and collect
the methylene chloride in a 250-mL
Erlenmeyer flask. If the emulsion
interface between layers is more than
one-third the size 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, or
centrifugation.
10.5 Add a second 60-mL volume of
methylene chloride to the sample
bottle and complete the extraction
procedure a second time, combining
•the extracts in the Erlenmeyer flask.
Perform a third extraction in the same
manner.
10.6 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 KD if the requirements of
Section 8.2 are met.
10.7 Pour the combined extract
through a drying column containing
three to four inches of anhydrous
sodium sulfate, and collect in the K-D
concentrator. Rinse the Erlenmeyer
flask and column with 20 to 30 mL
methylene chloride to complete the
quantitative transfer.
10.8 Add one to two clean boiling
chips to the flask and attach a three-
ball 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 (60 to 65°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
in vapor. Adjust the vertical position
of the apparatus and the water
temperature as required to complete
the concentration in 15 to 20
minutes. At the proper rate of distilla-
tion the balls of the column will
actively chatter but the chambers will
604-5
July 1982
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not flood. When the apparent volume
of liquid reaches 1 mL, remove the K-
D apparatus and allow it to drain for
at least 10 minutes while cooling.
10.9 Increase the temperature of
the hot water bath to 95 to 100°C.
Remove the Snyder column and rinse
the flask and its lower joint into the
concentrator tube with 1 to 2 mL of 2-
propanol. A 5-mL syringe is
recommended for this operation.
Attach a micro-Snyder column to the
concentrator tube and prewet the
column by adding about 0.5 mL of 2-
propanol 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
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 reached 2.5 mL,
remove the K-D apparatus and allow
it to drain for at least 10 minutes
while cooling. Add an additional 2 mL
of 2-propanol through the top of the
micro-Snyder column and resume
concentrating as before. When the
apparent volume of liquid reaches 0.5
mL, remove the K-D apparatus and
allow it to drain for at least 10
minutes while cooling. Remove the
micro-Snyder column and rinse its
lower joint into the concentrator tube
with a minimum amount of 2-
propanol. Adjust the extract volume to
1.0 mL. Stopper the concentrator tube
and store in refrigerator at 4°C, if
further processing will not be per-
formed immediately. If the sample ex-
tract requires no further cleanup, pro-
ceed with flame ionization gas
chromatographic analysis (Section
11). If the sample requires further
cleanup, .proceed to Section 12. If the
extracts will be stored longer than two
days, they should be transferred to
Teflon-sealed screw-cap vials.
10.10 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, Gas Chromatography -
Flame Ionization Detector
11.1 Table 1 summarizes the
recommended gas chromatographic
column and operating conditions. This
Table includes retention times and
MDL obtained under these conditions.
An example of the parameter separa-
tion achieved by this column is shown
in Figure 1. Other packed columns,
chromatographic conditions, or de-
tectors may be used if the require-
ments of Section 8.2 are met.
Capillary (open-tubular) columns may
also be used if the relative standard
deviations of responses for replicate
injections are demonstrated to be less
than 6% and the requirements of Sec-
tion 8.2 are met.
11.2 Calibrate the system daily as
described in Section 7.1.
11.3 If the internal standard
approach is used, the standard must
be added to the sample extract and
mixed thoroughly immediately before
injection into the instrument.
11.4 Inject 2 to 5 //L of the sample
extract using the solvent-flush
technique . Smaller (1.0 ^L) volumes
may be injected if automatic injectors
are employed. Record the volume
injected to the nearest 0.05 //L and
the resulting responses in peak area
or peak height units. If the response
for the peak exceeds the working
range of the system, dilute the extract
and reanalyze.
11.5 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 may
be used to calculate a suggested
window size; however, the experience
of the analyst should weigh heavily in
the interpretation of chromatograms.
11.6 If the measurement of the peak
response is prevented by the presence
of interferences, an alternate gas
chromatographic procedure is
required. Section 12 describes a
derivatization and column
chromatographic procedure which has
been tested and found to be a
practical means of analyzing phenols
in complex extracts.
12. Derivatization and
Electron Capture Gas
Chromatography
12.1 Pipet a 1.0-mL aliquot of the 2-
propanol solution of standard or
sample extract into a glass reaction
vial. Add 1.0-mL of derivatizing
reagent (Section 6.11). This is a
sufficient amount of reagent to
derivatize a solution whose total
phenolic content does not exceed 0.3
mg/mL.
12.2 Add about 3 mg of potassium
carbonate to the solution and shake
gently.
1 2.3 Cap the mixture and heat it for
four hours at 80°C in a hot water
bath.
12.4 Remove the solution from the
hot water bath and allow it to cool.
12.5 Add 10 mL of hexane to the
reaction flask and shake vigorously for
one minute. Add 3.0 mL of distilled,
deionized water to the reaction flask
and shake for two minutes. Decant a
portion of the organic layer into a
concentrator tube and cap with a
glass stopper.
12.6 Pack a 10-mm ID
chromatographic column with 4.0
grams of activated silica gel. After
settling the silica gel by tapping the
column, add about two grams of
anhydrous sodium sulfate to the top.
12.7 Pre-elute the column with 6
mL of hexane. Discard the eluate and
just prior to exposure of the sulfate
layer to air, pipet onto the column 2.0
mL of the hexane solution (Section
12.5) that contains the derivatized
sample or standard. Elute the column
with 10.0 mL of hexane (Fraction 1)
and discard this fraction. Elute the
column, in order, with: 10.0 mL of
15% toluene in hexane (Fraction 2);
10.0 mL of 40% toluene in hexane
(Fraction 3); 10.0 mL 75% toluene in
hexane (Fraction 4); and 10.0 mL 15%
2-propanol in toluene (Fraction 5). All
elution mixtures are prepared on a
volumeivolume basis. Elution patterns
for the phenolic derivatives are shown
in Table 2. Fractions may be combined
as desired, depending upon the
specific phenols of interest or level of
interferences.
12.8 Analyze the fractions by
electron capture gas Chromatography.
Table 2 summarizes the
recommended gas chromatographic
column and operating conditions. This
Table includes retention times and
MDL obtained under these conditions.
An example of the parameter separa-
tion achieved by this column is shown
in Figure 2.
12.9 Calibrate the system daily with
a minimum of three aliquots of
calibration standards, containing each
of the phenols of interest that are
derivatized according to the procedure
(See Section 7.4).
12.10 Inject 2 to 5//L of the column
fractions using the solvent-flush tech-
nique. Smaller (1.0 /jL) volumes can
be injected if automatic devices are
employed. Record the volume injected
to the nearest 0.05 /c/L, and the result-
ing peak size, in area units or height.
If the peak response exceeds the
604-6
July 1982
-------
linear range of the system, dilute the
extract and reanalyze.
13. Calculations
1 3.1 Calculate the concentration of
individual compounds in the sample
determined by the flame ionization
procedure (without derivatization) as
indicated below.
13. 1.1 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 in Section 7.2.2. The
concentration in the sample can be
calculated from equation 2:
Eq.2.Concentration,//g/L =
(V.) (Vs)
where: ,
A = Amount of material injected, in
nanograms.
Vj = Volume of extract injected (fjL).
Vt = Volume of total extract (fjL).
Vs = Volume of water extracted (ml_).
13.1 .2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
equation 3.
Eq. 3. Concentration,/^/!. =
-------
Table 1. Chromatographic Conditions and
Method Detection Limit
Parameter
Retention Method
Time Detection Limit
(min.)
2-Chlorophenol
2-Nitrophenol
Phenol
2,4-Dimethylphenol
2,4-Dichlorophenol
2.4, 6- Trichlorophenol
4-Chloro-3-methylphenol
2,4-Dinitrophenol
2-Methyl-4, 6-dinitmphenol
Pentachlorophenol
4-Nitrophenol
1.70
2.00
3.01
4.03
4.3O
6.05
7.50
10.00
10.24
12.42
24.25
O.31
0.45
0.14
0.32
0.39
0.64
0.36
13.0
16.0
7.4
2.8
Column conditions: Supelcoport (80/100 mesh) coated
With 1% SP-1240 DA in 1.8 m long x 2 mm ID glass
column with nitrogen carrier gas at a flow rate of 30
mL/min flow rate. Column temperature was 80° C at
injection, programmed immediately at 8°C/min to
1503C final temperature. Method detection limits were
determined with a flame ionization detector.
Table 2. Silica Gel Fractionation and Electron Capture
Gas Chromatography of PFBB Derivatives
Electron Capture
Recovery P/o) by Fraction"
Parent Compound 1
2'Chlorophenol
2-Nitroptienol
Phenol
2,4'Dimethylphenol
2,4-Dichlorophenol
2.4,6-Trichtorophenol
4-Chloro-2-methyphenol
Pentachlorophenol
4-Nitrophenol
2
-
-
-
-
-
50
-
75
-
3
90
-
90
95
95
50
84
20
-
4 5
1
9 90
10
7
1
-
14
-
J 90
Retention
Time
(min.)
3.3
9.1
1.8
2.9
5.8
7.0
4.8
28.8
14.0
Method
Detection
Limit t/jg/L)
0.58
0.77
2.2
0.63
0.68
0.58
1.8
0.59
0.70
'Eluting solvent compositions as given in Section 12.7.
Column conditions: Chromosorb W-AW-DMCS (8O/100 mesh) coated with 5% OV-17 packed in a 1.8
m long x 20 mm ID glass column with 5% methane/95% argon carrier gas at a flow rate of 30
mL/min. Column temperature isothermal at 200° C.
Table 3. Single Operator Accuracy and Precision
Parameter
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dichtorophenol
S.4-Dimethylphenol
2,4-Dinitrophenol
2'Methyl-4.6-dinitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2.4.6-Trichlorophenol
Average
Percent
Recovery
82
67
74
51
74
86
67
45
79
41
71
Standard
Deviation
%
15.0
14.8
11.4
14.0
16.5
12.4
12.9
7.9
8.8
8.4
14.5
Spike
Range
(pg/L)
0.70 - 3.5
0.74- 3.7
1.03 - 5.2
0.82 - 4. 1
28.7
34.6
0.80 - 4.0
15.9
21.0
0.76 - 3.8
1.20 - 6.0
Number
of
Analyses
21
21
21
21
14
21
21
21
21
21
21
Matrix
Types
3
3
3
3
2
3
3
3
3
3
3
604-8
July 1982
-------
o
-~ 2
^ -c
c 5.
•c -c
i
g- « Column: 1% SP-1240DA on Supelcoport
.§ ^ -1 Program: 80°C.-0 minutes 8°/minute to 150°C.
§ § 9 Detector: Flame ionization
T QJ ^
u'tt
,
\
^
^
^
*g
tZ
!
o
•C ^
p Q)
fe ' -c
5^-9-
C
0)
c
Q.
g
ts
C
Ifll1?
01 S ,1 g.
2*
1 -g o S 5
I 'l-a:s
I , 5 9
II *^ ' ^^
1 ^^^
"u VIUV.
• i i i i
^
^
0| ^
o
2
-c
9.
o
,g ^
•5 §
I t
« 5
*• .4s
a=?
ri
^
1^1 1 1 1 1 1 1 1
0
8 12 16 20 24
Retention time, minutes
28
Figure 1. Gas chromatogram of phenols
I
•S-
i
1
Column: 5% OV-17 in Chromosorb W-AW
Temperature: 200°C.
Detector: Electron capture
i
a
604-9
O 4 8 12 16 20 24 28 32
Retention time, minutes
figure 2. Gas chromatogram of PFB derivatives of phenols.
July 1982
-------
-------
SEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Benzidines -
Method 605
1. Scope and Application
1.1 This method covers the
determination of certain benzidines.
The following parameters can be
determined by this method:
Parameter
STORET No.
CAS No.
Benzidine
3,3'-Dichlorobenzidine
1.2 This is a high performance liquid
chromatography (HPLC) method
applicable to the determination of the
compounds listed above in municipal
and industrial discharges as provided
under 40 CFR 136.1. When
this method is used to analyze
unfamiliar samples for the compounds
above, identifications should be
supported by, at least, one additional
qualitative technique. This method
decribes electrochemical conditions at
a second potential which can be used
to confirm measurements made with
this method. Method 625 provides
gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results of
the parameters listed above, using the
extract produced by this method.
1.3 The method detection limit (MDL
defined in Section 14)m for each
parameter is listed in Table 1. The
MDL for a specific wastewater may
differ depending upon the nature of
the interferences in the sample
matrix.
1.4 Any modification of this method,
beyond those expressly permitted,
shall be considered as major
modifications subject to application
and approval of alternate test
39120
34631
92-87-5
91-94-1
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
HPLC instrumentation and in the
interpretation of liquid
chromatograms. Each analyst must
demonstrate the ability to generate
acceptable results with this method
using the procedure described in
Section 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is extracted
with chloroform using liquid-liquid
extractions in a separatory funnel. The
chloroform extract is back-extracted
with acid, neutralized, and extracted
with chloroform. The chloroform is
'exchanged to methanol and
concentrated using a rotary
evaporator and nitrogen blowdown. It
is then brought to a volume of 5 mL
with an acetate buffer. HPLC
conditions are described which permit
the separation and measurement of
the benzidine compounds using an .
electrochemical detector (2).
2.2 The acid back extraction step
acts as a general purpose cleanup to
aid in the elimination of interferences.
605-1
July 1982
-------
3. Interferences
3.1 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 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 as described in Section
8.5,
3.1.1 Glassware must be
scrupulously cleaned131. 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 reagent
water. It should then be drained dry,
and heated in a muffle furnace at
400°C for 15 to 30 minutes. Some
thermally stable materials, such as
PCBs, may not be eliminated by this
treatment. Solvent rinses with
acetone and pesticide quality hexane
may be substituted for the muffle
furnace heating. Volumetric ware
should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and
stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted or
capped with aluminum foil.
3.1.2 The use of high purity
reagents and solvents helps to
minimize interference problems. Puri-
fication of solvents by distillation in
all-glass systems may be required.
3.2 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 industrial complex
or municipality being sampled. The
cleanup procedures that are inherent
in the extraction step are used to
overcome many of these inter-
ferences, however unique samples
may require additional cleanup
approaches to achieve the MDL listed
in Table 1.
3.3 Some dye plant effluents
contain large amounts of components
with retention times close to
benzidine. In these cases, it has been
found useful to reduce the electrode
potential in order to eliminate
interferences yet still detect benzidine.
(See Section 12.7.)
4. Safety
4.1 The toxicity or carcinogenicity of
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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional
references to laboratory safety are
available and are identified for the
benefit of the analyst.
4.2 Benzidine, 3,3'-
dichlorobenzidine and chloroform,
used for extraction of samples have
been tentatively classified as known
or suspected, human or mammalian
carcinogens.
5. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling.
5.1.1 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with Tef-
lon. Foil may be substituted for Teflon
if the sample is not corrosive. If amber
bottles are not available, protect
samples from light. The container
must be washed, rinsed with acetone,
or methylene chloride, and dried be-
fore use to minimize contamination.
5.1.2 Automatic sampler (optional) -
Must incorporate glass sample
containers for the collection of a
minimum of 250 mL. Sample
containers must be kept refrigerated
at 4°C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however,
the compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested.)
5.2.1 Separatory funnels - 2000,
1000, and 250-mL, with Teflon
stopcock.
5.2.2 Vials - Amber glass, 10- to 15-
mL capacity, with Teflon-lined screw
cap.
each reagent used in this method has 5.2.3 Rotary evaporator.
5.2.4 Flasks, round bottom, 100-mL,
with 24/40 joints.
5.2.5 Centrifuge tubes - conical,
screw-capped, graduated, equipped
with Teflon lined caps.
5.2.6 Pipettes'- Pasteur, with bulbs. :;.'
5.3 Balance - Analytical, capable of
accurately weighing 0.0001 g.
5.4 HPLC - An analytical system
complete with column supplies,
compatible recorder, high pressure
syringes and the following com-
ponents.
5.4.1 Solvent delivery system - with
pulse damper, Altex 110A, or
equivalent.
5.4.2 Injection valve (Optional) -
Waters U6K or equivalent.
5.4.3 Electrochemical detector -
Bioanalytical Systems LC-2A with
glassy carbon electrode, or equivalent.
This detector has proven effective in
the analysis of wastewaters for the
parameters listed in the scope, and
was used to develop the method
performance statements in Section
14. Guidelines for the use of alternate
detectors are provided in Section
12.1.
5.4.4 Electrode polishing kit -
Princeton Applied Research Model
9320 or equivalent.
5.4.5 Column - Lichrosorb RP-2, 5
micron particle diameter, in a 25 cm x
4.6 mm ID stainless steel column. This
column was used to develop the
method performance statements in
Section 14. Guidelines for the use of
alternate column packings are
provided in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Sodium hydroxide solution (5 N)
- (ACS) Dissolve 20g NaOH in reagent
water and dilute to 100 ml.
6.3 Sodium hydroxide (1 M)-(ACS) -
Dissolve 40 g NaOH in reagent water
and dilute to one liter.
6.4 Sodium thiosulfate - (ACS)
Granular.
6.5 Sodium tribasic phosphate (0.4
M) - (ACS) Dissolve 160 g of trisodium
phosphate decahydrate in reagent
water and dilute to one liter.
6.6 Sulfuric acid solution (1 + 1) -
(ACS) Slowly, add 50 mL H2SO4 (sp.
gr. 1.84) to 50 mL of reagent water.
605-2
July 1982
-------
6.7 Sulfuric acid (1 M) - (ACS)
Slowly add 58 mL H2SC>4 (sp. gr. 1.84)
to reagent water and dilute to one
liter.
6.8 Acetate buffer (0.1 M, pH-4.7)
.Dissolve 5.8 mL glacial acetic acid
(ACS) and 13.6 g of sodium acetate
trihydrate (ACS) in reagent water
which has been purified by filtration
through a RO-4 Millipore System or
equivalent and dilute to one liter.
6.9 Acetonitrile, chloroform
(preserved with 1% ethanol),
methanol - Pesticide quality or
equivalent.
6.10 Mobile phase - Place equal
volumes of filtered acetonitrile
(Millipore type FH filter or equivalent)
and filtered acetate buffer (millipore
type GS filter or equivalent) in a
narrow-mouth glass container and
mix thoroughly. Prepare fresh weekly.
Degas daily by sonicating under
vacuum, or by heating and stirring, or
by purging with helium.
6.11 Stock standard solutions (1.00
yug//uL) - Stock standard ,solutions may
be prepared from pure standard
materials or purchased as certified
solutions.
6.11.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 g of pure material.
Dissolve the material in pesticide
quality methanol and dilute to volume
in a 10-mL volumetric flask. Larger
volumes can be used at the
convenience of the analyst. If
compound purity is certified at 96% or
greater, the weight can be used
without correction to calculate the
concentration of the stock standard.
Commercially prepared stock
standards can be used at any
concentration if they are certified by
the manufacturer or by an
independent source. '
6.11.2 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. Quality control
check samples that can be used to
determine the accuracy bf calibration
standards will be available from the
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio 45268.
6.11.3 Stock standard solutions
must be replaced after six months, or
sooner if comparison with quality
control check samples indicate a
problem.
7. Calibration
7.1 Establish chromatographic
operating parameters equivalent to
those given in Table 1. The HPLC
system can be calibrated using the
external standard technique (Section
7.2) or the internal standard
technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with mobile
phase. One of the external standards
should be at a concentration near, but
above, the MDL 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.
7.2.2 Using syringe injections of 5
to 25 fjL or a constant volume
injection loop, inject each calibration
standard, 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
calibration curve.
7.2.3 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 any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound. If serious loss of
sensitivity occurs, polish the electrode
and recalibrate.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested
that is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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 internal standards, and dilute to
volume with mobile phase. One of the
standards should be at a
concentration near, but above, the
MDL 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.
7.3.2 Using 5 to 25 fjL aliquots or a
constant volume injection loop, inject
each calibration standard, and
tabulate peak height or area
responses against concentration for
each compound and internal standard,
and calculate response factors (RF) for
each compound using equation 1.
Eq. 1 RF = (AsCis)/(Ais Cs)
where:
As = Response for the parameter to be
measured.
Ais = Response for the internal
standard.
Cis = Concentration of the internal
standard, (/ug/L).
Cs = Concentration of the parameter
to be measured, (/jg/L).
If the RF value over the working
range is a constant (< 10% RSD), the
RF can be assumed to be nonvariant
and 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. RF.
7.3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
If serious loss of response occurs,
polish the.electrode and recalibrate.
7.4 Before using any alternate
cleanup procedure, the analyst must
process a series of calibration
standards through the procedure to
validate elution patterns and the
absence of interferences from the
reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a
formal quality control program. The
605-3
July 1982
-------
minimum requirements of this
program consist of an initial
demonstration of laboratory capability
and the analysis of spiked samples as
a continuing check on performance.
The laboratory is required to maintain
performance records to define the
quality of data that is generated.
Ongoing performance checks must be
compared with established
performance criteria to determine if
the results of analyses are within
accuracy and precision limits expected
of the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method. This ability is
established as described in Section
8,2.
8.1.2 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
'generate acceptable accuracy and
precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate in methanol 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be
used in place of the reagent water,,
but one or more additional aliquots
must be analyzed to determine
background levels, and the spike level
must exceed twice the background
level for the test to be valid. Analyze
the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater
background corrections must be made
before R and s calculations are
performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or IX-Rl > 2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish per-
formance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter being
measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in
Section 8.2.3. The UCL and LCL can
be used to construct control charts171
that are useful in observing trends in
performance. The control limits above
must be replaced by method
performance criteria as they become
available from the U.S. Environmental
Protection Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy state-
ments of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined
as R ± s. The accuracy statement
should be developed by the analysis of
four aliquots of wastewater as de-
scribed in Section 8.2.2, followed by
'the calculation of R and s. Alternately,
the analyst may use four wastewater
data points gathered through the
requirement for continuing quality
control in Section 8.4. The accuracy
statements should be updated
regularly C7).
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of
the sample must be spiked and
analyzed as described in Section 8.2.
If the recovery for a particular
parameter does not fall within the
control limits for method performance,
the results reported for that parameter
in all samples processed as part of the
same set must be qualified as
described in Section 13.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagents interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as
a safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may
be analyzed to monitor the precision
of the sampling technique. When
doubt exists over the identification of
a peak on the chromatogram,
confirmatory techniques such as
HPLC with a dissimilar column, gas
chromatography, or mass
spectrometry must be used. Whenever
possible, the laboratory should
perform analysis of standard
reference materials and participate in
relevant performance evaluation
studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices'8' 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 free as possible of Tygon and other
potential sources of contamination.
9.2 The samples must be iced or
refrigerated at 4°C and stored in the
dark from the time of collection until
extraction. Both benzidine and 3,3'-
dichlorobenzidine are easily oxidized.
Fill the sample bottle and, at time of
collection, if residual chlorine is
present, add 80 mg of sodium
thiosulfate per liter of sample, and
mix thoroughly. U.S. Environmental
Protection Agency methods 330.4 and
330.5 may be used for measurement
605-4
July 1982
-------
of residual chlorine191. Field test kits
are available for this purpose. After
mixing, adjust the pH ofthe sample to
a range of 2 to 7 with sulfuric acid.
9.3 If 1,2-diphenyl hydrazine is likely
to be present, adjust the pH of the
sample to 4.0 ± 0.2 to prevent
rearrangement to benzidine.
9.4 All samples must be extracted
within seven days. Extracts may be
held up to seven days before analysis,
if stored under an inert (oxidant free)
atmosphere121. The extract should be
protected from light.
10. Sample Extraction
10.1 Mark the water meniscus on
the side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a |two-liter
separatory funnel. Check the pH of
the sample with wide-range pH paper
and adjust to within the range of 6.5
to 7.5 with sodium hydroxide or
sulfuric acid solutions. •
10.2 Add 100 ml_ chloroform to the
sample bottle, seal, and shake 30
seconds to rinse the inner walls.
(Caution: Handle chloroform in a well
ventilated area.) Transfer the solvent
to the separatory funnel and extract
the sample by snaking ttie funnel for
two minutes with periodic venting to
release excess pressure. Allow the
organic layer to separate from the
water phase for a minimum of
minutes. 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 chloroform
extract in a 250-mL separatory funnel.
1O.3 Add a 50-mL volume of
chloroform to the sample bottle and
repeat the extraction procedure a
second time, combining ihe extracts
in the separatory funnel. Perform a
third extraction in the same manner.
10.4 Separate and discard any
aqueous layer remaining in the 250-
mL separatory funnel after combining
the organic extracts. Add 25 mL of 1
M sulfuric acid and extract the sample
by shaking the funnel for two
minutes. Transfer the aqueous layer
to a 250-mL beaker. Extract with two
additional 25-mL portions of 1 M
sulfuric acid and combine the acid
extracts in the beaker.
10.5 Place a stirbar in the 250-mL
beaker and stir the acid extract while ,
carefully adding 5 mL of 0.4 M
sodium tribasic phosphate. While
monitoring with a pH meter,
neutralize the extract to a pH between
6 and 7 by dropwise addition of 5 N
NaOH while stirring the solution
vigorously. Approximately 25 to 30 mL
of 5 N NaOH will be required and it
should be added over at least a two-
minute period. Do not allow the
sample pH to exceed 8.
10.6 Transfer the neutralized extract
into a 250-mL separatory funnel. Add
30 mL of chloroform and shake the
funnel for two minutes. Allow the
phases to separate, and transfer the
organic layer to a second 250-mL
separatory funnel.
10.7 Extract the aqueous layer with
two additional 20-mL aliquots of
chloroform as before. Combine the
extracts in the 250-mL separatory
funnel.
10.8 Add 20 mL of reagent water to
the combined organic layers and
shake for 30 seconds.
10.9 Transfer the organic extract
into a 100-mL round bottom flask.
Add 20 mL of methanol and
concentrate to 5 mL with a rotary
evaporator at reduced pressure and
35°C. An aspirator is recommended
for use as the source of vacuum. Chill
the receiver with ice. This operation
requires approximately 10 minutes.
Other concentration techniques may
be employed, if the requirements of
Section 8.2 are met.
10.10 Using a 9-inch Pasteur
pipette, transfer the extract to a 15
mL conical screw-capped centrifuge
tube. Rinse the flask, including the
entire side wall, with 2-mL portions of
methanol and combine with the
original extract.
10.11 Carefully concentrate the
extract to 0.5 mL using a gentle
stream of nitrogen while heating in a
30°C water bath. Dilute to 2 mL with
methanol, reconcentrate to 1 mL, and
dilute to 5 mL with acetate buffer. Mix
extract thoroughly. Cap the centrifuge
tube and store refrigerated and
protected from light if further
processing will not be performed
immediately.
10.12 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. Cleanup and Separation
11.1 Additional cleanup procedures
may not be necessary for a relatively
clean sample matrix. The single
operator precision and accuracy data
in Section 14 were gathered using
only those cleanup procedures that
are inherent in the extraction
procedures of this method. If
particular circumstances demand the
use of an alternative cleanup
procedure, the analyst must
determine the elution profile and
demonstrate that the recovery of each
compound of interest is no less than
85%.
12. Liquid Chromatography
12.1 Table 1 summarizes the
recommended operating conditions for
the HPLC. This Table includes
retention times, capacity factors, and
MDL that were obtained under these
conditions. An example of the para-
meter separation achieved by the
HPLC column is shown in Figure 1.
Other HPLC columns, chroma-
tographic conditions, or detectors may
be used if the requirements of Section
8,2 are met. When the HPLC is idle, it
is advisable to maintain a 0.1 mL/min
flow through the column to, prolong
column life. •
12.2 Calibrate the system daily as
described in Section 7.
12.3 If the internal standard
approach is being used, the standard
must be added to the sample extract,
and mixed thoroughly immediately,
before injection into the instrument.
12.4 Inject 5 to 25 fsL of the sample
extract. If constant volume injection
loops are not used, record the volume
injected to the nearest 0.05 /mL, and
the resulting peak size in area or peak
height units.
12.5 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.
12.6 If the response for the peak
exceeds the working range of the
system, dilute the extract with mobile
phase and reanalyze.
12.7 If the measurement of the peak
response for benzidine is prevented by
the presence of interferences, reduce
the electrode potential to +0.6 volts
and reanalyze. If the benzidine peak is
still obscured by interferences, further
cleanup is required.
605-5
July 1982
-------
13. Calculations
13.1 Determine the concentration of
individual compounds in the sample.
13,1,1 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 in Section 7.2.2. The
concentration in the sample can be
calculated from the equation 2:
(AXVJ
Eq. 2. Concentration, //g/L = (Vi){Vs)
where:
A = Amount of material injected, in
nanograms.
V, = Volume of extract injected
OuL).
Vt = Volume of total extract 0"L).
Vs = Volume of water extracted (mL).
13.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
equation 3.
e o „ • /, (As)ds)
Eq. 3. Concentration, jug/L = (Ais)(RF)(V0)
where:
As - Response for the parameter to
be measured.
Ais = Response for the internal
standard.
I9 = Amount of internal standard
added to each extract (/ug).
V0 = Volume of water extracted, in
liters.
13.2 Report results in micrograms
per liter without correction for
recovery data. When duplicate and
spiked samples are analyzed, report
all data obtained with the sample
results.
13.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in Section 8.4, data for
the affected parameters must be
labeled as suspect.
14. Method Performance
14.1 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
zero111. The MDL concentrations listed
in Table 1 were obtained using
reagent water'101. Similar results were
achieved using representative
wastewaters.
14.2 This method has been tested
for linearity of analyte recovery from
reagent water and has been
demonstrated to be applicable over
the concentration range from 7 x MDL
to 3000 x MDL110'.
14.3 In a single laboratory (Battelle,
Columbus Laboratories), using spiked
wastewater samples, the average
recoveries presented in Table 2 were
obtained'2'. Each spiked sample was
analyzed in triplicate on two separate
days. The standard deviation of the
percent recovery is also included in
Table 2.
14.4 The U.S. Environmental Protec-
tion Agency is in the process of
conducting an interlaboratory method
study to fully-define the performance
of this method.
References
1. See Appendix A.
2. "Determination of Benzidines in
Industrial and Municipal Waste-
waters," Report for EPA Contract 68-
03-2624 (In preparation).
3. ASTM Annual Book of Standards,
Part 31, D3694, "Standard Practice
for Preparation of Sample Containers
and for Preservation," American
Society for Testing and Materials,
Philadelphia, PA, p. 679, 1980.
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. 1 977.
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. "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.
8. ASTM Annual Book of Standards,
Part 31, D3370, "Standard Practice
for Sampling Water," American
Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual,"
Methods for Chemical Analysis of
Water and Wastes, EPA 600/4-79-
020, U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory - Cincinnati,
Ohio 45268, March 1979.
10. "Determination of Method
Detection Limit and Analytical Curve
for EPA Method 605 - Benzidines,"
Special letter report for EPA Contract
68-03-2624, Environmental
Monitoring and Support Laboratory -
Cincinnati, Ohio 45268.
505-6
July 1982
-------
Table 1. Chromatographic Conditions and Method
Detection Limits
Retention Column Method
Parameter time Capacity Detection Limit
(min) Factor (fjg/i-J
Benzidine 6.1 1.44 0.08
3.3'-dichlorobenzidine 12.1 3.84 0.13
HPLC Column conditions: Lichrosorb RP-2, 5 micron
particle size, in a 25 cm x 4.6 mm ID stainless steel
column. Mobile Phase: O.'S mL/min of 50%
acetonitrile/50% 0.1M pH 4.7 acetate buffer. The method
detection limit was determined using an electrochemical
detector operated at + 0.8 volts.
Table 2.
Parameter
Single Operator Accuracy and Precision
Standard Spike
Average
\Percent
Recovery
Deviation
range
Number
of
Analyses
Matrix
Types
Benzidine 65
3,3'-Dichlorobenzidine 64
11.4
9.6
1.0-50
1.0-50
30
30
5
5
Column: Lichrosorb RP-2
Mobile phase: 50% Acetonitrile in
acetate 'buffer
Detector: Electrochemical at
+0.8 volts
I
•8
CO
6 12
Retention time, minutes
Figure 1. Liquid chromatogram
of benzidines
605-7
July 1982
-------
-------
SEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Phthalate Esters —
Method 606
1. Scope and Application
1.1 This method covers the
determination of certain phthalate
esters. The following parameters can
be determined by this method:
Parameter
STORET No.
CAS No.
Bis(2-ethylhexyl) phthalate
Butyl benzyl phthalate
Di-n-butyl phthalate
Diethyl phthalate
Dimethyl phthalate
Di-n-octyl phthalate
1.2 This is a gas chromatographic
(GC) method applicable to the
determination of the compounds listed
above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any or
all of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. This method
describes analytical conditions for a
second gas chromatographic column
that can be used to confirm measure-
ments made with the primary column.
Method 625 provides gas chromato-
graph/mass spectrometer (GC/MS)
conditions appropriate for the qualita-
tive and quantitative confirmation of
results for all of the parameters listed
above, using the extract produced by
this method.
1.3 The method detection limit
(MDL, defined in Section 14)'1' for
each parameter is listed in Table 1.
The MDL for a specific wastewater
may differ from those listed,
depending upon the nature of
interferences in the sample matrix.
39100
34292
39110
34336
34341
34596
117-81-7
85-68-7
84-74-2
84-66-2
131-11-3
117-84-0
1.4 The sample extraction and
concentration steps in this method are
essentially the same as in methods
608, 609, 611 and 612. Thus, a single
sample may be extracted to measure
the parameters included in the scope
of each of these methods. When
cleanup is required, the concentration
levels must be high enough to permit
selecting aliquots, as necessary to
apply appropriate cleanup procedures.
The analyst is allowed the latitude,
under Gas Chromatography (Section
1 2), to select chromatographic
conditions appropriate for the
simultaneous measurement of
combinations of these parameters.
1.5 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 CRF 136.4 and 136.5.
1.6 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
606-1
July 1982
-------
ability to generate acceptable results
with this method using the procedure
described in Section 8.2.
2, Summary of Method
2.1 A measured volume of sample,
approximately 1 -liter, is solvent
extracted with methylene chloride
using a separatory funnel. The
methylene chloride extract is dried
and exchanged to hexane during
concentration to a volume of 10 mL or
less. Gas chromatographic conditions
are described which permit the
separation and measurement of the
compounds in the extract by electron
capture gas chromatography121'
2.2 Analysis for phthalates is
especially complicated by their
ubiquitous occurrence in the
environment. This method provides
Florisil and alumina column cleanup
procedures to aid in the elimination of
interferences that may be
encountered.
3. Interferences
3.1 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 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 as described in Section
8.5.
3.1.1 Glassware must be
scrupulously cleaned131. 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. It should then be drained dry,
and heated in a muffle furnace at
400°C for 15 to 30 minutes. Some
thermally stable materials, such as
PCBs, may not be eliminated by this
treatment. Solvent rinses with
acetone and pesticide quality hexane
may be substituted for the muffle
furnace heating. Volumetric ware
should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and
stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted or
capped with aluminum foil.
3.1.2 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.
3.2 Phthalate esters are
contaminants in many products
commonly 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.
Serious phthalate contamination can
result at any time, if consistent quality
control is not practiced. Great care
must be experienced to prevent such
contamination. Exhaustive cleanup of
reagents and glassware may be
required to eliminate background
phthalate contamination14'51.
3.3 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 industrial complex
or municipality being sampled. The
cleanup procedures in Section 11 can
be used to overcome many of these
interferences, but unique samples
may require additional cleanup
approaches to achieve the MDL listed
in Table 1.
4. Safety
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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional
references to laboratory safety are
available and have been identified (6 8)
for the information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling.
5.7.7 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with Tef-
lon. Foil may be substituted for Teflon
if the sample is not corrosive. If amber
bottles are not available, protect
samples from light. The container and
cap liner must be washed, rinsed with
acetone or methylene chloride, and
dried before use to minimize con-
tamination.
5.1.2 Automatic sampler (optional) -
Must incorporate glass sample
containers for the collection of a
minimum of 250 mL Sample
containers must be kept refrigerated
at 4°C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however,
the compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.7 Separatory funnel - 2000-mL,
with Teflon stopcock.
5.2.2 Drying column -
Chromatographic column 400 mm
long x 19 mm ID with coarse frit.
5.2.3 Chromatographic column -
300 mm long x 10 mm ID with coarse
fritted disc at bottom and Teflon
stopcock (Kontes K-420540-0213 or
equivalent).
5.2.4 Concentrator tube, Kuderna-
Danish - 10-mL, graduated (Kontes K-
570050-1025 or equivalent).
Calibration must be checked at the
volumes employed in the test. Ground
glass stopper is used to prevent
evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-
Danish - 500-mL (Kontes K-570001-
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.6 Snyder column, Kuderna-
Danish - three-ball macro (Kontes K-
503000-0121 or equivalent).
5.2.7 Snyder column, Kuderna-
Danish - two-ball micro (Kontes K-
569001-0219 or equivalent).
5.2.8 Vials - Amber glass, 10-to 15-
mL capacity, with Teflon-lined screw-
cap.
5.3 Boiling chips - approximately
10/40 mesh. Heat to400°C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath - Heated, with
concentric ring cover, capable of
temperature control,(+ 2°C). The bath
should be used in a hood.
5.5 Balance - Analytical, capable of
'accurately weighing 0.0001 g.
5.6 Gas chromatograph - Analytical
system complete with gas
606-2
July 1982
-------
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical columns,
gases, detector, and strip-chart
recorder. A data system is
recommended for measuring peak
areas.
5.6.1 Column 1 - 180cm long x 4
mm ID glass, packed with 1.5% SP-
2250/1.95% SP-2401 o'n Supelcoport
(100/120 mesh) or equivalent. This
column was used to develop the ,
method performance statements in
Section 14. Guidelines for the use of
alternate column packings are
provided in Section 1 2.1.
5.5.2 Column 2 - 180 cm long x 4
mm ID glass, packed with 3% OV-1 on
Supelcoport (TOO/120 mesh) or
equivalent.
5.6.3 Detector - Electron capture.
This detector has proven effective in
the analysis of wastewaters for the
parameters listed in the:scope and
was used to develop the! method
performance statements in Section
1.4. Guidelines for the use of alternate
detectors are provided in Section
12.1.
6. Reagents :
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Acetone, hexane, isooctane,
methylene chloride, methanol -
Pesticide quality or equivalent.
6.3 Ethyl ether - Nanograde,
redistilled in glass if necessary.
6.3.1 Must be free of peroxides as
indicated by EM Quant test strips.
(Available from Scientific Products Co.,
Cat. No. P1126-8 and others.)
6.3.2 Procedures recommended for
removal of peroxides are provided
with the test strips. After cleanup, 20
ml_ ethyl alcohol preservative must be
added to each liter of ether.
6.4 Sodium sulfate - (ACS)
Granular, anhydrous. Several levels of
purification may be required in order to
reduce background phthalate levels to
an acceptable level: 1) Heat four
hours at 400°C in a shallow tray, 2)
Heat 1 6 hours at 450-50p°C in a
shallow tray, 3) Soxhlet extract with
methylene chloride for 48 hours.
6.5 Florisil - PR grade (60/100
mesh). Purchase activated at 1250°F
and store in dark in glass container
with ground glass stopper or foil-lined
screw cap. To prepare for use, place
10Og of Florisil into a 500-mL beaker
and heat for approximately 16 hours
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
minutes and let it stand for at least
two hours. Keep the bottle sealed
tightly.
6.6 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 1 6 hours 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 minutes
and let it stand for at least two hours.
Keep the bottle sealed tightly.
6.7 Stock standard solutions (T.OO
jug/jt/L) - Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.7.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 grams of pure material.
Dissolve the material in pesticide
quality isooctane, dilute to volume in
a 10-mL volumetric flask. Larger
volumes can be used at the
convenience of the analyst. If
compound purity is certified at 96% or
greater, the weight can be used
without correction to calculate the
concentration of the stock standard.
Commercially prepared stock
standards can be used at any
concentration if they are certified by
the manufacturer or by an
independent source.
5.7.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards will be available from the
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio, 45268
6.7.3 Stock standard solutions must
be replaced after six months, or
sooner if comparison with check
standards indicate a problem.
7. Calibration
7.1 Establish gas chromatographic
operating parameters equivalent to
those indicated in Table 1. The gas
chromatographic system can be
calibrated using the external standard
technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with isooctane.
One of the external standards should
be at a concentration near, but
above, MDL and the other concentra-
tions should correspond to the ex-
pected range of concentrations found
in real samples or should define the
working range of the detector.
7.2.2 Using injections of 2 to 5/uL
of each calibration standard, tabulate
peak height or area responses against
the mass injected. The results can be
used to prepare a calibration curve for
each parameter. 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
calibration curve.
7.2.3. 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 any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that parameter.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested
that is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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 internal standards, and dilute to
606-3
July 1982
-------
volume with isooctane. One of the
standards should be at a
concentration near, but above, the
MDL 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.
7.3.2 Using injections of 2 to
of each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1 RF = (A»Cis)/{Ai, Cs)
where:
A» = Response for the parameter to
be measured.
A,s = Response for the internal
standard.
C»= Concentration of the internal
standard, (jjg/L).
Cs = Concentration of the parameter
to be measured, (/ug/L).
If the RF value over the working range
is a constant (<10% RSD), the RF can
be assumed to be invariant and the
average RF can be used for calcula-
tions. Alternatively, the results can be
used to plot a calibration curve of re-
sponse ratios, As/A,,, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of
interferences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist
of an initial demonstration of
laboratory capability and the analysis
of spiked samples as a continuing
check on performance. The laboratory
is required to maintain performance
records to define the quality of data
that is generated. Ongoing
performance checks must be
compared with established
performance criteria to determine if
the results of analyses are within
accuracy and precision limits expected
of the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method. This ability is
established as described in Section
8.2.
8.1.2 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method will be available
from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
8.2.2 Using a pipet, add 1.00 ml_ of
the check sample concentrate to each
of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be
used in place of the reagent water,
but one or more additional aliquots
must be analyzed to determine
background levels, and the spike level
must exceed twice the background
level for the test to be valid. Analyze
the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater
Background corrections must be made
before R and s calculations are
performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or |X-R| > 2p, review potential
problem areas and repeat the test.
5.2.5 The U. S. Environmental Pro-
tection Agency plans to establish per-
formance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter being
measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in
Section 8.2.3. The UCL and LCL can
be used to construct control charts'9'
that are useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they b'ecome avail-
able from the U.S. Environmental Pro-
tection Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An
accuracy statement for the method is
defined as R ± s. The accuracy
statement should be developed by the
analysis of four aliquots of
wastewater as described in Section
8.2.2, followed by the calculation of R
and s. Alternately, the analyst may
use four wastewater data points
gathered through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly191.
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of
the sample must be spiked and
analyzed as described in Section 8.2.
If the recovery for a particular
parameter does not fall within the
control limits for method performance,
the results reported for that parameter
in all samples processed as part of the
same set must be qualified as described
in Section 13.3. The laboratory should
monitor the frequency of data so
qualified to ensure that it remains at
or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
606-4
July 1982
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glassware and reagents interferences
are under control. Each'time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as
a safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may
be analyzed to monitor the precision
of the sampling technique. When
doubt exists over the identification of
a peak on the chromatogram,
confirmatory techniques such as gas
chromatography with a dissimilar
column, specific element detector, or
mass spectrometer must be used.
Whenever possible, the laboratory
should perform analysis of standard
reference materials and participate in
relevant performance evaluation
studies.
9. Sample Collection,
Preservation, and Handling
t
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices {10) should be
followed, except that the bottle must
not be prerinsed with sample before
collection. Composite samples should
be collected in refrigerated glass
containers in accordance with the
requirements of the pro'gram.
Automatic sampling equipment must
be as free as possible of Tygon and
other potential sources pf
contamination.
9.2 The samples must be iced or
refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction'2'.
10. Sample Extraction
10.1 Mark the water meniscus on
the side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel.
10.2 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner walls.
Transfer the solvent to the separatory
funnel and extract the sample by
shaking the funnel for two minutes
with periodic venting to release
excess pressure. Allow the organic
layer to separate from the water
phase for a minimum of 10 minutes.
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 250-mL
Erlenmeyer flask.
10.3 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 extractionln the same
manner.
10.4 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 if the requirements
of Section 8.2 are met.
10.5 Pour the combined extract
through a drying column containing
about 10 cm of anhydrous sodium
sulfate, and collect the extract in the
K-D concentrator. Rinse the
Erlenmeyer flask and column with 20
to 30 mil of methylene chloride to
complete the quantitative transfer.
10.6 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 1 mL 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 im-
mersed 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 concentra-
tion in 1 5 to 20 minutes. At the
proper rate of distillation the balls of
the column will actively chatter but
the chambers will not flood with con-
densed solvent. When the apparent
volume of liquid reaches 1 mL,
remove the K-D apparatus and allow
it to drain and cool for a least 10
minutes.
10.7 Increase the temperature of
the hot water bath to about 80°C.
Momentarily remove the Snyder
column, add 50 mL of hexane and a
new boiling chip and reattach the
Snyder column. Pour about 1 mL of
hexane into the top of the Snyder
column and concentrate the solvent
extract as before. Elapsed time of con-
centration should be 5 to 10 minutes.
When the apparent volume of liquid
reaches 1 mil, remove the K-D ap-
paratus and allow it to drain and cool
for at least 10 minutes.
10.8 Remove the Snyder column and
rinse the flask and its lower joint into
the concentrator tube with 1 to 2 mL
of hexane and adjust the volume to
10 mL. A 5-mL syringe is
recommended for this operation.
Stopper the concentrator tube and
store refrigerated if further processing
will not be performed immediately. If
the extracts will be stored longer than
two days, they should be transferred
to Teflon-sealed screw-cap bottles. If
the sample extract requires no further
cleanup, proceed with gas
chromatographic analysis. If the
sample requires cleanup, proceed to
Section 11.
10.9 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. Cleanup and Separation
11.1 Cleanup procedures may not
be necessary for a relatively clean
sample matrix. The cleanup
procedures recommended in this
method have been used for the
analysis of various clean waters and
industrial effluents. If particular
circumstances demand the use of an
alternative cleanup procedure, the
analyst must determine the elution
profile and demonstrate that the
recovery of each compound of interest
is no less than 85%.
11.2 If the entire extract is to be
cleaned up by one of the following
two procedures, it must be
concentrated to about 2 mL. To the
concentrator tube in Section 10.8,
add a clean boiling chip and attach a
two-ball micro-Snyder column.
Prewet the column by adding about
0.5 ,mL hexane to the top. Place the
K-D apparatus on a hot water bath
(80°C) so that the concentrator tube is
partially immersed in hot water.
Adjust the vertical position of the
apparatus and the water temperature
as required to complete the
concentration in 5 to 10 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 liquid reaches
about 0.5 mL, remove the K-D
apparatus and allow it to drain and
606-5
July 1982
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cool for at least 10 minutes. Remove
the micro-Snyder column and rinse
its lower joint into the concentrator
tube with 0,2 mL of hexane. Proceed
with one of the following cleanup
procedures. Proper use of either
procedure should yield acceptable
results.
11.3 Florisil column cleanup for
phthalate esters
11.3.1 Place 10g of Florisil into a
10 mm ID chromatography column
and tap the column to settle the
Florisil. Add 1 cm of anhydrous
sodium sulfate to the top of the
Florisil.
11.3.3 Preelute the column with 40
mL of hexane. Discard this eluate and
just prior to exposure of the sodium
sulfate layer to the air, transfer the 2
mL sample extract onto the column,
using an additional 2 mL of hexane to
complete the transfer.
11.3.4 Just prior to exposure of the
sodium sulfate layer to the air, add 40
mL hexane and continue the elution
of the column. Discard this hexane
eluate.
7 7.5.5 Next elute the phthalate
esters with 100 mL of 20% ethyl
ether/80% 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 by standard K-D
technique. No solvent exchange is
necessary. After concentration and
cooling, adjust the volume of the
cleaned up extract to 10 mL in the
concentrator tube and analyze by gas
chromatography.
11.4 Alumina column cleanup for
phthalate esters.
77.4.7 Place 10g of alumina into a
10 mm ID chromatography column
and tap the column to settle the
alumina. Add 1 cm of anhydrous
sodium sulfate to the top of the
alumina.
11.4.3 Preelute the column with 40
mL of hexane. Discard this eluate and
just prior to exposure of the sodium
sulfate layer to the air, transfer the 2
mL sample extract onto the column,
using an additional 2 mL of hexane to
complete the transfer.
11.4.4 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.
7 7.4.5 Next elute the column with
140 mL of 20% ethyl ether/80%
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 by
standard K-D technique. No solvent
exchange is necessary. After con-
centration and cooling, adjust the
volume of the cleaned up extract to
10 mL in the concentrator tube and
analyze by gas chromatography.
12. Gas Chromatography
12.1 Table 1 summarizes the
recommended operating conditions .
for the gas chromatograph. Included
in this table are estimated retention
times and MDL that can be achieved
by this method. Examples of the
separations achieved by column 1 are
shown in Figures 1 and 2. Other
packed columns, chromatographic
conditions, or detectors may be used
if the requirements of Section 8.2 are
met. Capillary (open-tubular) columns
may also be used if the relative
standard deviations of responses for
replicate injections are demonstrated
to be less than 6% and the require-
ments of Section 8.2 are met.
12.2 Calibrate the system daily as
described in Section 7.
12.3 If the internal standard
approach is being used, the analyst
must not add the internal standard to
.sample extracts until immediately
before injection into the instrument.
Mix thoroughly.
12.4 Inject 2 to 5 /uL of the sample
extract using the solvent-flush
technique1"1. Smaller (1.0/uL)
volumes can be injected if automatic
devices are employed. Record the
volume injected to the nearest 0.05
fjL, and the resulting peak size in area
or peak height units.
12.5 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.
12.6 If the response for the peak
exceeds the working range of the
system, dilute the extract and
reanalyze.
12.7 If the measurement of the
peak response is prevented by the
presence of interferences, further
cleanup is required.
13. Calculations
13.1 Determine the concentration of
individual compounds in the sample.
13.1.1 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 in Section 7.2.2. The
concentration in the sample can be
calculated from equation 2:
Eq. 2. Concentration, /ug/L=
(A) (Vt)
(Vi) (Vs)
where:
A = Amount of material injected,
in nanograms.
V, = Volume of extract injected G"L).
V( = Volume of total extract (/uL).
Vs = Volume of water extracted (mL).
13.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
equation 3.
„
Eq. 3. Concentration, /ug/L= (Als)(RF)(V0)
where:
As = Response for the parameter to
be measured.
Ais = Response for the internal
standard.
U = Amount of internal standard
added to each extract (/ug).
V0 = Volume of water extracted, in
liters.
73.2 Report results in micrograms
per liter without correction for
recovery data. When duplicate and
spiked samples are analyzed, report
all data obtained with the sample
results.
73.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in Section 8.3, data for
the affected parameters must be
labeled as suspect.
14. Method Performance
14.1 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
zero111. The MDL concentrations listed
in Table 1 were obtained using
reagent water'121. Similar results were
achieved using representative
wastewaters.
14.2 This method has been tested
for linearity of recovery from spiked
reagent water and has been
demonstrated to be applicable over
606-6
July 1982
-------
the concentration range from 5 X
MDL to 1000 X MDL with the
following exceptions: dimethyl and
diethyl phthalate recoveries at 1000 X
MDL were low (70%); bis-2-ethylhexyl
and di-n-octyl phthalate recoveries at
5 X MDL were low (60%)(12).
14.3 In a single laboratory
(Southwest Research Institute), using
spiked wastewater samples, the
average recoveries presented in Table
2 were obtained. Each spiked sample
was analyzed in triplicate on two
separate days. The standard deviation
of the percent recovery is also
included in Table 2<2).
14.4 The U.S. Environmental Protec-
tion Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. "Determination of Phthalates in
Industrial and Municipal
Wastewaters." Report for EPA
Contract 68-03-2606 (In preparation).
3. ASTM Annual Book of Standards,
Part 31, D3694, "Standard Practice
for Preparation of Sample Containers
and for Preservation," American
Society for Testing and Materials,
Philadelphia, PA, p. 679, 1980.
4. Giam, C.S., Chan, H:S. and Neff,
G.S., "Sensitive Method for
Determination of Phthalate Ester
Plasticizers in Open-Ocean Biota
Samples," Analytical Chemistry, 47,
2225, (1975).
5. Giam, C.S., Chan, HIS., "Control
of Blanks in the Analysis of
Phthalates in Air and Ocean Biota
Samples," National Bureau of
Standards (U.S.), 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, 1.979.
9. "Handbook for Analytical Quality
Control in Water and Wastewater
Laboratories," EPA-600/4-79-01 9,
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory - Cincinnati,
Ohio 45268, March 1979.
10. ASTM Annual Book of
Standards, Part 31, D3370, "Standard
Practice for Sampling Water,"
American Society for Testing and
Materials, Philadelphia, PA, p. 76,
1980.
11. Burke, J.A., "Gas
Chromatography For Pesticide Residue
Analysis; Some Practical Aspects,"
Journal of the Association of Official
Analytical Chemists, 48, 1037 (1965).
12. "Method Detection Limit and
Analytical Curve Studies, EPA
Methods 606, 607, and 608." Special
letter report for EPA Contract 68-03-
2606. Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio
45268.
Table 1 .
Chromatographic Conditions and Method
Detection Limits
Retention Time
(min.)
Method
Detection Limit
Parameter Column
Dimethyl phthalate 2.O3
Diethyl phthalate 2.82
Di-n-butyl phthalate 8.65
Butyl benzyl phthalate 6.94
Bis(2-ethylhexyl) phthalate 8.92
Di-n -octyl phthalate 16.2
1 Column 2
0.95
1.27
3.50
* 5.11*
* 1O.5 *
* 18.0 *
ffjg/L)
O.29
O.49
0.36
0.34
2.0
3.0
Column 1 conditions: Supelcoport (100/120 mesh)
coated with 1.5% SP-225O/1.95% SP-2401 packed in a-
1.8 m long x 4 mm ID glass column with 5%
methane/95% argon carrier gas at a flow rate of 6O
mL/min. Column temperature, isothermal at 180°C,
except where otherwise indicated.
Column 2 conditions: Supelcoport (100/120 mesh)
coated with 3% OV-1 in a 1.8 m long x 4 mm ID glass
column with 5% methane/95% argon carrier gas at 60
mL/min flow rate. Column temperature isothermal
200°C except where otherwise indicated.
*Retention time based upon isothermal oven
temperature of 22O°C.
Table 2. Single Operator Accuracy and Precision
Average
Percent
Parameter Recovery
Bis(2-ethylhexyl)
phthalate
Butyl benzyl phthalate
Di-n-butyl phthalate
Diethyl phthalate
Dimethyl phthalate
Di-n-octyl phthalate
85
82
80
94
94
86
Standard
Deviation
%
4.2
6.5
6.2
1.3
3.4
4.9
Spike
Range
ffjg/L}
24-1000
3-100
20-1500
15-50
15-50
4O-15O
Number
of
Analyses
24
24
24
18
18
24
Matrix
Types
4
4
4
3
3
4
606-7
July 1982
-------
Column: 1.5% SP-22SO+
1.95% SP^2401 on
Supelcoport
Temperature: 180°C.
Detector: Electron capture
Column: 1.5% SP-2250+
1.95% SP-2401 on
Supelcoport
Temperature: 220° C.
Detector: Electron capture
s
-S
CO
"§.
"5.
0 2 4 6 8 10 12
Retention time, minutes
Figure 1. Gas chromatogram of
phthalates.
4 8 12 16 18
Retention time, minutes
Figure 2. Gas chromatogram of
phthalates.
606-8
July 1982
-------
SrEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Nitrosamines
Method 607
1. Scope and Application
1.1 This method covers the
determination of certain nitrosamines.
The following parameters can be
determined by this method:
Parameter
STORET No.
CAS No.
N-Nitrosodimethylamine
N-Nitrosodiphenylamine
N-Nitrosodi-n-propylamine
1.2 This is a gas chromatographic
(GC) method applicable to the determi-
nation of the parameters listed above in
municipal and industrial discharges as
provided under 40 CFR 1 36.1. When
this method is used to analyze unfa-
miliar samples for any or all of the
compounds above, compound identifi-
cations should be supported by at least
one additional qualitative technique.
This method describes analytical
conditions for a second gas
chromatographic column that can be
used to confirm measurements made
with the primary column. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results for
N-nitrosodi-n-propylamine. In order to
confirm the presence of N-nitrosodi-
phyenylamine, the cleanup procedure
specified in Section 11.3 or 11.4 must
be used. In order'to confirm the pre-
sence of N-nitrosodimethylamine by
GC/MS, chromatographic column 1 of
this method must be substituted for
the column recommended in method
625. Confirmation of these parameters
using GC-high resolution mass
spectrometry or a Thermal Energy
Analyzer is also recommended
practice^,2).
34438
34433
34428
62-75-9
86-30-6
621-64-7
1.3 The method detection limit (MDL
defined in Section 14.1 )<3> for each
parameter is listed in Table 1. The MDL
for a specific wastewater may differ
from those listed, depending upon the
nature of interferences in the sample
matrix.
1.4 Any modification of this method,
beyond those expressly permitted,
shall be considered as major
modifications subject to application
and approval of alternate test
procedures under 40 CFR 1 36.4 and
136.5.
1.5 This method is restricted to use
by or under the supervision of analysts
experienced in the use of gas chroma-
tography 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 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one liter, is solvent
extracted with methylene chloride
using a separatory funnel. The
methylene chloride extract is washed
with dilute HCI to remove free amines,
dried and concentrated to a volume of
607-1
July 1982
-------
10 mL or less. Gas chromatographic
conditions are described which permit
the separation and measurement of the
compounds in the extract after it has
been exchanged to methanol.<4>
2.2 The method provides Florisil and
alumina column cleanup procedures to
separate diphenylamine from the nitro-
samines and to aid in the elimination of
interferences that may be encountered.
3. Interferences
3.1 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 gas 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 8.5.
3.1.1 Glassware must be scrupulously
cleaned!5'. 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. It should then be
drained dry, and heated in a muffle
furnace at 400 °C for 15 to 30
minutes. Some thermally stable
materials, such as PCBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Volumetric
ware should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and stored
in a clean environment to prevent any
accumulation of dust or other
contaminants. Store inverted or capped
with aluminum foil.
3.1.2 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.
3.2 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 industrial complex or
municipality being sampled. The
cleanup procedures (Section 11) can
be used to overcome many of these
interferences, but unique samples may
require additional cleanup approaches
to achieve the MDL listed in Table 1.
3.3 N-Nitrosodiphenylamine is
reported!6-9* to undergo transnitrosation
reactions. Care must be exercised in
the heating or concentrating of
solutions containing this compound in
the presence of reactive amines.
3.4 The sensitive and selective
Thermal Energy Analyzer and the
reductive Hall detector may be used in
place of the nitrogen-phosphorus
detector when interferences are
encountered. The Thermal Energy
Analyzer offers the highest selectivity
of the non-mass spectrometric
detectors.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been Identifiedno-i2) for the
information of the analyst.
4.2 These nitrosamines are known
carcinogens!13-17)f therefore, utmost
care must be exercised in the handling
of these materials. Nitrosamine
reference standards and standard
solutions should be handled and
prepared in a ventilated glove box
within a properly ventilated room.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
or composite sampling.
5.1.1 Grab sample bottle—Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If
amber bottles are not available, protect
samples from light. The container must
be washed, rinsed with acetone or
methylene chloride, and dried before
use to minimize contamination.
5.1.2 Automatic sampler (optional) —
Must incorporate glass sample
containers for the collection of a mini-
mum of 250 mL. Sample containers
must be kept refrigerated at 4 °C and
protected from light during compositing.
If the sampler uses a peristaltic pump,
a minimum length of compressible
silicone rubber tubing may be used.
Before use, however, the compressible
tubing should be thoroughly rinsed
with methanol, followed by repeated
rinsings with distilled water to minimize
the potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.7 Separatory funnel—2000-mL
and 250-mL, with Teflon stopcock.
5.2.2 Drying column—Chroma-
tographic column approximately 400
mm long x 19 mm ID with coarse frit.
5.2.3 Concentrator tube, Kuderna-
Danish—10-mL, graduated (Kontes K-
570050-1025 or equivalent). Calibra-
tion must be checked at the volumes
employed in the test. Ground glass
stopper is used to prevent evaporation
of extracts.
5.2.4 Evaporative flask, Kuderna-
Danish—500-mL (Kontes K-570001-
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.5 Snyder column, Kuderna-
Danish—three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Snyder column, Kuderna-
Danish—two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.7 Vials—Amber glass, 10-to
15-mL capacity, with Teflon-lined
screw cap.
5.2.8 Chromatographic column—
Pyrex, approximately 400 mm long x
22 mm ID, with coarse fritted disc at
bottom and Teflon stopcock (Kontes
K-420540-0234 or equivalent), for
use in Florisil column cleanup
procedure.
5.2.9 Chromatographic column—
Pyrex, approximately 300 mm long x
10 mm ID, with coarse fritted disc at
bottom and Teflon stopcock (Kontes
K-420540-021 3 or equivalent), for
use in alumina column cleanup
procedure.
5.3 Boiling chips—approximately
10/40 mesh. Heat to 400 °C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath—Heated, with
concentric ring cover, capable of
temperature control (± 2 °C). The bath
should be used in a hood.
5.5 Balance—Analytical, capable of
accurately weighing 0.0001 g.
607-2
July 1982
-------
5.6 Gas chromatograph —An
analytical system complete with gas
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical columns,
gases, detector, and strip-chart
recorder. A data system is
recommended for measuring peak
areas.
5.6.1 Column 1 — 1.8 rri long x 4
mm ID Pyrex glass, packed with
Chromosorb W AW (80/1 00 mesh)
coated with 10% Carbowax 20 M/2%
KOH or equivalent. This column was
used to develop the method perfor-
mance statements in Section 14.
Guidelines for the use of alternate
column packings are provided in
Section 12.2.
5.6.2 Column 2—1.8 rri long x 4
mm ID, Pyrex glass packed with
Supelcoport (100/1 20 mesh) coated
with 10% SP-2250.
5.6.3 Detector—Nitrogen-
Phosphorus, reductive Hall or Thermal
Energy Analyzerd/2). TheSe detectors
have proven effective in the analysis of
wastewaters for the parameters listed
in the scope. A "nitrogen-phosphorus
detector was used to develop the
method performance statements in
Section 14. Guidelines for the use of
alternate detectors are provided in
Section 12.2.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an inter-
ferent is not observed at the MDL of
each parameter of interest.
6.2 Sodium hydroxide solution (10
N)-(ACS) Dissolve 40g NaOH in
reagent water and dilute tp 100 mL.
6.3 Sodium thiosulfateT (ACS)
Granular.
6.4 Sulfuric acid solution (1 + 1 ) —
(ACS) Slowly, add 50 mL H2SO4 (sp.
gr. 1.84) to 50 mL of reagent water.
6.5 Sodium sulfate—(ACS) Granular,
anhydrous. Purify by heating at 400 °C
for 4 hours in a shallow tray.
6.6 Hydrochloric acid (1 +9) —(ACS)
Add one volume of cone. HCI to nine
volumes of reagent water.
6.7 Acetone, methanol, imethylene
chloride and pentane—Pesticide quality
or equivalent.
6.8 Ethyl ether—Nanograde,
redistilled in glass if necessary.
6.8.1 Must be free of peroxides as
indicated by EM Quant test strips.
(Test strips are available from Scientific
Products Co., Catalog No. P1126-8
and others.)
6.8.2 Procedures recommended for
removal of peroxides are provided with
the test strips. After cleanup, 20 mL
ethyl alcohol preservative must be
added to each liter of ether.
6.9 Florisil-PR grade (60/100
mesh); purchase activated at 1250 °F
and store in dark in glass containers
with glass stoppers or foil-lined screw
caps. Before use, activate each batch
at least 1 6 hours at 130 °C in a foil
covered glass container.
6.10 Alumina—Basic activity Super I,
W 200 series (ICN Life Sciences
Group, No. 404571). Place 100 g of
alumina, as it comes from the manu-
facturer, into a 500-mL reagent bottle
and add 2 mL of reagent water. Mix
the alumina preparation thoroughly by
shaking or rolling for 10 minutes and
let it stand for at least two hours. The
preparation should be homogenous
before use. Keep the bottle sealed
tightly to ensure proper activity.
6.11 Stock standard solutions (1.00
ng/^L)—Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.11.1 Prepare stock standard
solutions by accurately weighing about
0.0100 grams of pure material.
Dissolve the material in pesticide
quality methanol, dilute to volume in a
10-mL volumetric flask. Larger volumes
can be used at the convenience of the
analyst. If 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 prepared stock
standards can be used at any
concentration if they are certified by
the manufacturer or by an independent
source.
6.11.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards will be available from the
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
6.11.3 Stock standard solutions
must be replaced after six months, or
sooner if comparison with check
standards indicate a problem.
7. Calibration
7.1 Establish gas chromatographic
operating parameters equivalent to
those indicated in Table 1. The gas
chromatographic system can be
calibrated using the external standard
technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding volumes of one or more stock
standards to a volumetric flask and
diluting to volume with methanol. One
of the external standards should be at a
concentration near, but above, the
MDL 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.
7.2.2 Using injections of 2 to 5 pL of
each calibration standard,.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 calibration
curve.
7.2.3 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 any parameter varies
from the predicted response by more
than ± 10%, the test must be repeated
using a fresh calibration.standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested that,
is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
607-3
July 1982
-------
levels for each parameter 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
internal standards, and dilute to'volume
with methanol. One of the standards
should be at a concentration near, but
above, the MDL 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.
7.3.2 Using injections of 2 to 5 f/L of
each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1. RF = (AsCis)/(AisCs)
where:
As = Response for the parameter to
be measured.
AJS ** Response for the internal
standard.
CjS = Concentration of the internal
standard, (f*g/L).
Cs = Concentration of the param-
eter to be measured, (//g/L).
If the RF value over the working
range is a constant «10% RSD), the
RF can be assumed to be nonvariant
and the average RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
of response ratios, AJA\S, vs. RF.
7,3.3 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elutio'n
patterns and the absence of interfer-
ences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with
established performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
8.1.1 Before performing any analyses,
the analyst must demonstrate the
ability to generate acceptable accuracy
and precision with this method. This
ability is established as described in
Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the separa-
tions or lower the cost of measurements.
Each time such modifications are made
to the method, the analyst is required
to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze, a minimum of 10% of all
samples to monitor continuing labora-
tory performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and pre-
cision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each compound to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in methanol 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL aliquots
of reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
.the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for the
results. Wastewater background
corrections must be made before R and
s calculations are performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the cal-
culated values for R and s. If s > 2p or
|X-R| > 2p, review potential problem
areas and repeat the testi
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and define
the performance of the laboratory for
each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in
Section 8.2.3. The UCL and LCL can
be used to construct control charts!18)
that are useful in observing trends in
performance. The control limits above
must be replaced by method
performance criteria as they become
available from the U.S. Environmental
Protection Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcula-
tion of R and s. Alternately, the analyst
may use four wastewater data points
gathered through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly!18).
8.4. The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample analysis
must be at least 1 0% of all samples or
one sample per month, whichever is
greater. One aliquot of the sample must
be spiked and analyzed as described in
Section 8.2. If the recovery for a
particular parameter does not fall
within the control limits for method
performance, the results reported for
that parameter in all samples processed
as part of the same set must be quali-
fied as described in Section 13.3. The
laboratory should monitor the frequency
of data so qualified to ensure that it
remains at or below 5%.
607-4
July 1982
-------
8.5 Before processing any samples,
the analyst should demonstrate through
the analysis of a one-liter aliquot of
reagent water, that all glassware and
reagent interferences are under control.
Each time a set of samples is extracted
or there is a change in reagents, a
laboratory reagent blank should be
processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. When doubt
exists over the identification of a peak
on the chromatogram, confirmatory
techniques such as gas-chromatography
with a dissimilar column, specific
element detector, or mass spectrometer
must be used. Whenever possible, the
laboratory should perform analysis of
standard reference materials and parti-
cipate in relevant performance
evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must ,be collected
in glass containers. Conventional
sampling practicesdS) should be
followed, except that the bottle must
not be prerinsed 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 and other potential ,
sources of contamination!
9.2 The samples must be iced or
refrigerated at 4 °C from the time of
collection until extraction- Fill the
sample bottle and at time'of collection,
if residual chlorine is present, add 80
mg of sodium thiosulfate per liter of
sample. U.S. Environmental Protection
Agency methods 330.4 and 330.5
may be used for measurement of
residual chlorine(20). Field:test kits are
available for this purpose. If diphenylni-
trosamine is to be determined, adjust
the sample pH to 7 to 10 with sodium
hydroxide or sulfuric acid.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction^).
9.4 Nitrosamines are known to be
light sensitive!7'. Samples should be
stored in amber or foil-wrapped bottles
in order to minimize photblytic
decomposition.
•10. Sample Extraction
10.1 Mark the water meniscus on the
side of the sample bottle for later deter-
mination of sample volume. Pour the
entire sample into a two-liter separatory
funnel. Check the pH of the sample
with wide-range pH paper and adjust to
within the range of 5 to 9 with sodium
hydroxide or sulfuric acid.
10.2 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner surface.
Transfer the solvent to the separatory
funnel and extract the sample by
shaking the funnel for two minutes
with periodic venting to release excess
pressure. Allow the organic layer to
separate from the water phase for a
minimum of 10 minutes. If the emulsion
interface between layers is more than
one-third the volume of the solvent
layer, the analyst must employ me-
chanical techniques to complete the
phase separation. The optimum tech-
nique depends upon the sample, but
may include stirring, filtration of the
emulsion through glass wool, centrifu-
gation, or other physical methods.
Collect the methylene chloride extract
in a 250-mL Erlenmeyer flask.
10.3 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.
10.4 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 if the requirements of
Section 8.2 are met.
10.5 Add 10mLof HCI (1 + 1)
solution to the combined extracts and
shake for two minutes. Allow the layers
to separate. Transfer the combined
extract to a drying column containing
10 cm of anhydrous sodium sulfate
and collect it in the K-D concentrator.
Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride
to complete the quantitative transfer.
10.6 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 1 mL 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 1 5 to 20 minutes.
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 1 mL, remove
the K-D apparatus and allow it to drain
and cool for at least 10 minutes.
Remove the Snyder column and rinse
the flask and its lower joint into the
concentrator tube with 1 to 2 mL of
methylene chloride. A 5-mL syringe is
recommended for this operation.
Stopper the concentrator tube and
store refrigerated if further processing
will not be performed immediately. If
the extracts will be stored longer than
two days, they should be transferred to
Teflon-sealed screw-cap bottles.
10.7 If N-nitrosodiphenylamine is to
be measured by gas chrornatography,
the analyst must first use a cleanup
column to eliminate diphenylamine
interference (Section 11). If N-nitroso-
diphenylamine is of no interest, the
analyst may proceed directly with gas
chromatographic analysis (Section 12).
10.8 Determine the original sample
volume by refilling the sample bottle to
the mark with water and measure it in
a 1000-mL graduated cylinder. Record
the sample volume to the nearest 5 mL.
11. Cleanup and Separation
11.1 Cleanup procedures may not be
necessary for a relatively clean sample
matrix. The cleanup procedures recom-
mended in this method have been used
for the analysis of various clean waters
and industrial effluents. If particular
circumstances demand the use of an
alternative cleanup procedure, the
analyst must determine the elution
profile and demonstrate that the
recovery of each compound of interest
is no less than 85%. Diphenylamine, if
present in the original sample extract
must be separate from the nitros-
amines if N-nitrosodiphenylamine is to
be determined by this method..
11.2 If the entire extract is to be
cleaned up by one of the following
procedures, it must be concentrated to
2.0 mL. To the concentrator tube in
Section 10.6, add a clean boiling chip
and attach a two-ball micro-Snyder
column. Prewet the column by adding
about 0.5 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. Adjust the
vertical position of the apparatus and
the water temperature as required to
607-5
July 1982
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complete the concentration in 5 to 10
minutes. At the proper rate of distilla-
tion the balls of the column will
actively chatter but the chambers will
not flood. When the apparent volume
of liquid reaches about 0.5 mL, remove
the K-D apparatus from the hot water
bath and allow it to drain for at least
10 minutes while cooling. Remove the
micro-Snyder column and rinse its
lower joint into the concentrator tube
with 0.2 mL of methylene chloride.
Adjust the final volume to 2.0 mL and
proceed with one of the following
cleanup procedures.
11.3 Florisil column cleanup for
nitrosamines:
71.3.1 Place 22 g of activated
Florisil in a 22 mm ID chromatographic
column. After settling the Florisil by
tapping the column, add about a 5-mm
layer of anhydrous granular sodium
sulfate to the top.
17.3.2 Preelute the column, after
colling, with 40 mL of ethyl ether/
pentane (15 + 85HV/V). Discard the
eluate and just prior to exposure of the
sodium sulfate layer to air, quantita-
tively transfer a 2.0 mL aliquot of the
sample extract into the column by
decantation using an additional 2 mL of
psntane to complete the transfer.
7 7.3.3 Perform the first elution with
90 mL of ethyl ether/pentane (15 +
85KV/V) and discard the eluate. This
fraction will contain the diphenylamine,
if it is present in the extract.
7 7.3.4 Perform the second elution
with 100 mL of acetone/ethyl ether (5
+ 95HV/V) and collect the eluate in a
500-mL K-D flask equipped with a
10-mL concentrator tube. This fraction
will contain all of the nitrosamines
listed in the scope of the method.
7 7.3.5 Add 1 5 mL of methanol to
the collected eluate and concentrate as
in Section 10.6 at 70 to 75 °C, using
pentane for the diluting and rinsing
solvent.
7 7.3.6 Analyze by gas
chromatography.
11.4 Alumina column cleanup for
nitrosamines:
71.4.1 Place 12 g of the alumina
preparation into a 10 mm ID chromato-
graphic column and tap the column to
settle the alumina. Add 1 to 2 cm of
anhydrous sodium sulfate to the top of
the alumina.
11.4.2 Preelute the column with 10
mLof ethyl ether/pentane"(3 + 7}(V/V).
Discard the eluate (about 2 mL) and.
just prior to exposure of the sodium
sulfate layer to air, transfer a 2.0 mL
aliquot of the sample extract onto the
column by decantation using an addi-
tional 2 mL of pentane to complete the
transfer.
11.4.3 Just prior to exposure of the
sodium sulfate layer to the air, add 70
mL of ethyl ether/pentane (3 + 7)(V/V).
Discard the first 10 mL of eluate.
Collect the remainder of the eluate in a
500-mL K-D flask equipped with a
10-mL concentrator tube. This fraction
contains N-nitrosodiphenylamine and
probably a small amount of N-nitrosodi-
n-propylamine.
7 7.4.4 Next elute the column with
60 mL of ethyl ether/pentane (1 + 1)
(V/V), collecting the eluate in a second
K-D flask equipped with a 10-mL
concentrator tube. Add 1 5 mL of
methyl alcohol to the K-D flask. This
fraction will contain N-nitrosodimethyl-
amine, most of the N-nitrosodi-n-
propylamine and any diphenylamine
that is present.
77.4.5 Concentrate both fractions as
in Section 10.6 using pentane as the
diluting and rinsing solvent.
11.4.6 Analyze the fractions by gas
chromatography.
12. Gas Chromatography
12.1 N-nitrosodiphenylamine com-
pletely reacts to form diphenylamine at
the normal operating temperatures of a
GC injection port (200 to 250 °C).
Thus, N-nitrosodiphenylamine is
chromatographed and detected as
diphenylamine. Accurate determination
depends on removal of diphenylamine
that may be present in the original
extract prior to GC (See Section 11).
12.2 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. This Table
includes retention times and MDL that
were obtained under these conditions.
Examples of the parameter separations
achieved by these columns are shown
in Figures 1 and 2. Other packed
columns, chromatographic conditions,
or detectors may be used if the
requirements of Section 8.2 are met.
Capillary (open-tubular) columns may
also be used if the relative standard
deviations of responses for replicate
injections are demonstrated to be less
than 6% and the requirements of
Section 8.2 are met.
12.3 Calibrate the system daily as
described in Section 7.
12.4 If the extract has not been
submitted to one of the cleanup
procedures in Section 11, it is
necessary to exchange the solvent
from methylene chloride to methyl
alcohol before the thermionic detector
can be used. To a 1-10 mL volume of
methylene chloride extract in a concen-
trator tube, add 2 mL methyl alcohol,
and a clean boiling chip. Attach a two-
ball micro-Snyder column. Prewet the
column by adding about 0.5 mL
methylene chloride through the top.
Place the K-D apparatus on a boiling
water bath so that the concentrator
tube is partially immersed in the hot
water. Adjust the vertical position and
insulate the apparatus as necessary to
complete the concentration in 5 to 1 0
minutes. At the proper rate of distilla-
tion the balls of the column will
actively chatter but the chambers will
not flood. When the apparent volume
of liquid reaches about 0.5 mL, remove
the K-D and allow It to drain for at least
10 minutes while cooling. Remove the
micro-Snyder column and rinse its
lower joint into the concentrator tube
with 0.2 mL of methyl alcohol. Adjust
the final volume to 2.0 mL.
12.5 If the internal standard
approach is being used, add the
internal standard to sample the extract
and mix thoroughly, immediately
before injection into the instrument.
12.6 Inject 2 to 5 \A. of the sample
extract using the solvent-flush
technique!211. Smaller (1.0 \A.) volumes
can be injected if automatic devices are
employed. Record the volume injected
to the nearest 0.05 yL, and the
resulting peak size in area or peak
height units.
12.7 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.
12.8 If the response exceeds the
working range of the system, dilute the
extract and reanalyze.
12.9 If the measurement of the peak
response is prevented by the presence
of interferences, further cleanup is
required.
13. Calculations
13.1 Determine the concentration of
individual compounds in the sample.
13.1.1 If the external standard
calibration procedure is used, calculate
607-6
July 1982
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the amount of material injected from
the peak response using the calibration
curve or calibration factor in Section
7.2.2. The concentration in the sample
can be calculated from equation 2:
(A)(Vt)
Eq. 2. Concentration, pig/L = i\/.\i\/ \
where:
A = Amount of material injected, in
nanograms. '
Vj = Volume of extract injected
Vt = Volume of total extract (/d_).
Vs = Volume of water extracted
(mL).
13.1.2 If the internal standard cali-
bration procedure was used, calculate
the concentration in the sample using
the response factor (RF) determined in
Section 7.3.2 and equation 3.
Eq. 3
(As)(ls)
Concentration, wj/L = ; (A.S)(RF)(VO)
where:
As = Response for the parameter to
be measured.
Ais = Response for the internal
standard.
ls = Amount of internal standard
added to each extract (/^g).
V0 = Volume of water extracted, in
liters.
13.2 Report results in micrograms
per liter without correction for recovery
data. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
13.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in Section 8.3, data for
the affected parameters must be
labeled as suspect.
14. Method Performance
14.1 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
zero<3>. The MDL concentrations listed
in Table 1 were obtained using reagent
water'22'. Similar results were achieved
using representative wastewaters.
14.2 This method has been tested
for linearity of analyte recovery from
reagent water and has been
demonstrated to be applicable over the
concentration range from 4 x MDL to
1000 x MDL<22>.
14.3 In a single laboratory (South- '
west Research Institute), using spiked
wastewater samples, the average
recoveries presented in Table 2 were
obtained!4'. Each spiked sample was
analyzed in triplicate on two separate
days. The standard deviation of the
percent recovery is also included in
Table 2.
14.4 The U.S. Environmental Protec-
tion Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. Fine, D.H., Lieb, D. and Rufeh, R.,
"Principle of Operation of the Thermal
Energy Analyzer for the Trace Analysis
of Volatile and Non-volatile N-nitroso
Compounds;" Journal of Chromatog-
raphy, 107, 351, (1 975).
2. Fine, D.H. Hoffman, F., Rounbehler,
D.P. and Belcher, N.M., "Analysis of
N-nitroso Compounds by Combined
High Performance Liquid Chromatog-
raphy and Thermal Energy Analysis,"
Walker, E.A., Bogovski, P. and
Griciute, L., editors, N-Nitroso
Compounds—Analysis and Formation
Lyon, International Agency for
Research on Cancer (IARC Scientific
Publications No. 14), pp. 43-50,
(1976)
3. See Appendix A.
4. "Determination of Nitrosamines in
Industrial and Municipal Wastewaters."
Report for EPA Contract 68-03-2606
(In preparation).
5. ASTM Annual Book of Standards,
Part 31, D 3694. "Standard Practice
for Preparation of Sample Containers
and for Preservation," American
Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
6. Buglass, A.J., Challis, B.C. and
Osborn, M.R. "Transnitrosation and
Decomposition of Nitrosamines."
Bogovski, P. and Walker, E.A., editors,
N-Nitroso Compounds in the Environ-
ment, Lyon, International Agency for
Research on Cancer (IARC Scientific
Publication No. 9), pp. 94-100,
(1974).
7. Burgess, E.M. and Lavanish, J.M.,
"Photochemical Decomposition of
N-nitrosamines," Tetrahedon Letters,
1221, (1964).
8. Druckrey, H., Preussmann, R.,
Ivankovic, S. and Schmahl, D.,
"Organotrope Carcionogene
Wirkungen bei 65 Verschiedenen
N-Nitroso-Verbindungen an
BD-Ratten." Z. Krebsforsch., 69, 103
(1967).
9. Fiddler, W. "The Occurrence and
Determination of N-nitroso
Compounds," Toxicol. Appl. Pharma-
col., 31, 352, (1975).
10. "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.
11. "OSHA Safety and Health
Standards, General Industry,"
(29CFR1 910), Occupational Safety
and Health Administration, OSHA
2206, (Revised, January 1 976).
12. "Safety in Academic Chemistry
Laboratories," American Chemical
Society Publication, Committee on
Chemical Safety, 3rd Edition, 1 979.
13. Lijinsky, W., "How Nitrosamines
Cause Cancer." New Scientist, 73,
216, (1977)
14. Mirvish, S.S. "N-Nitroso
compounds: Their Chemical and in vivo
Formation and Possible Importance as
Environmental Carcinogens." J.
Toxicol. Environ. Health, 3, 1 267
(1977).
1 5. "Reconnaissance of Environmental
Levels of Nitrosamines in the Central
United States." National Enforcement
Investigations Center, Environmental
Protection Agency, Report No.
EPA-330/1-77-001, (1977)
16. "Atmospheric Nitrosamine Assess-
ment Report." Office of Air Quality
Planning and Standards, Environmental
Protection Agency, Research Triangle
Park, North Carolina, (1976).
1 7. "Scientific and Technical Assess-
ment Report on Nitrosamines." Office
of Research and Development, Environ-
mental Protection Agency, Report No.
EPA-660/6-7-001, (1976).
18. "Handbook of Analytical Quality
Control in Water and Wastewater
Laboratories," EPA-600/4-79-019,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268,
March 1979.
19. ASTM Annual Book of Standards,
Part 31, D 3370, "Standard Practice
for Sampling Water-," American
Society for Testing and Materials,
Philadelphia, PA, p. 679, 1980.
20. "Methods 330.4 (Titrimetric,
DPD-FAS) and 330.5 (Spectrophoto-
metric, DPD) for Chlorine, Total
Residual," Methods for Chemical
Analysis of Water and Wastes, EPA
600-4/79-020, U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1979.
21. Burke, J.A., "Gas Chromatography
for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the
Association of Official Analytical
Chemists, 48, 1037 (1965).
607-7
July 1982
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22. "Method Detection Limit and
Analytical Curve Studies EPA Methods
606, 607, 608," special letter report
for EPA Contract 68-03-2606.
Environmental Monitoring and Support
Laboratory—Cincinnati, Ohio 45268.
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
N-Nitrosodimethylamine
N'Nitrosodl-n-propylamine
N-Nitrosodiphenylamine3
neieniion lime
(min.)
Column 1 Column 2
4. 1 O.88
12. 1 4.2
12.8b 6.4<=
Method
Detection Limit
O.15
0.46
0.81
Column 1 conditions: Chromosorb W AW (80/100 meshl coated with 1O% Carbo-
wax 20 M/2% KOH packed in a 1.8m long x 4 mm ID glass column with helium
carrier gas at a flow rate of 40 mL/min. column temperature Isothermal, at
110°C, except as otherwise indicated.
Column 2 conditions: Supelcoport (1OO/12O meshl coated with 10% SP-225O
packed in a 1.8 m long x 4 mm ID glass column with helium carrier gas at a flow
rate of 40 mL/min. column temperature. Isothermal at 12O°C, except as other-
wise indicated.
"Measured as diphenylamine
^Determined Isothermally at 220 °C.
^Determined isothermally at21O °C.
Tablo 2. Single Operator Accuracy and Precision
Average Standard Spike Number
Percent Deviation Range of Matrix
Parameter Recovery % (\ig/L) Analyses Types
N-Nitrosodimethylamine 32 3.7 0.8 29 5
N-Nitrosodiphenylamine 79 7.1 1.2 29 5
N-Nitrosodi-n-propylamine 61 4.2 9.O 29 5
607-8 July 1982
-------
Column: 10% Carbowax 20M + 2%
KOH on Chromosorb W-AW
Temperature: 110°
Detector: Phosphorus/Nitrogen
Column: 10% Carbowax 20M + 2% KOH on
Chromosorb W-AW
Temperature: 220°C.
Detector: Phosphorus/Nitrogen
2 4 6 8 10\ 12 14
Retention time, minutes
Figure 1. Gas chromatogram of
nitrosamines.
0
6 8 10 12 14 16 18
Retention time, minutes
Figure 2. Gas chromatogram of N-nitrosodiphenylamine
as diphenylamine.
607-9
July 1982
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-------
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA
Research and Development
Test Method
Organochlorine Pesticides
and PCBs —Method 608
1. Scope and Application
1.1 This method covers the
determination of certain Organochlorine
pesticides and PCBs. The following
parameters can be determined by this
method:
Parameter
STORET No.
CAS No.
Aldrin
a-BHC
/3-BHC
d-BHC
y-BHC
Chlordane
4,4 '-ODD
4,4 '-DDE
4,4 '-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1 01 6
PCB-1221
PCB-1232
PCB-1242
PCB-1 248
PCB-1254
PCB-1 260
39330
39337
39338
34259
39340
39350
39310
39320
39300
39380
34361
34356
34351
39390
34366
39410
39420
39400
34671
39488
39492
39496
39500
39504
39508
309-00-2
319-84-6
319-85-7
319-86-8
58-89-9
57-74-9
72-54-8
72-55-9
50-29-3
60-57-1
959-98-8
33212-65-9
1031-07-8
72-20-8
7421-93-4
76-44-8
1024-57-3
8001-35-2
12674-11-2
1 1 1 04-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
1.2 This is a gas chromatographic
(GC) method applicable to the determi-
nation of the compounds listed above
in municipal and industrial discharges
as provided under 40 CFR 1 36.1.
When this method is used to analyze
unfamiliar samples for any or all of the
compounds above, compound identifi-
cations should be supported by at least
one additional qualitative technique.
This method describes analytical
conditions for a second gas
chromatographic column that can be
used to confirm measurements made
with the primary column. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
608-1
July 1982
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quantitative confirmation of results for
all of the parameters listed above,
using the extract produced by this
method.
1.3 The method detection limit (MDL,
defined in Section 14.D'1) for each
parameter is listed in Table 1. The MDL
for a specific wastewater may differ
from those listed, depending upon the
nature of interferences in the sample
matrix.
1.4 The sample extraction and
concentration steps in this method are
essentially the same as in methods
606, 609, 611 and 612. Thus, a
single sample may be extracted to
measure the parameters included in the
scope of each of these methods. When
cleanup is required, the concentration
levels must be high enough to permit
selection of aliquots as necessary to
apply appropriate cleanup procedures.
The analyst is allowed the latitude to
select gas chromatographic conditions
appropriate for the simultaneous
measurement of combinations of these
parameters.
1.5 Any modification of this method,
beyond those expressly permitted,
shall be considered as major
modifications subject to application
and approval of alternate test
procedures under 40 CFR 136.4 and
136.5.
1.6 This method is restricted to use
by or under the supervision of analysts
experienced in the use of gas chroma-
tography 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 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is solvent
extracted with methylene chloride
using a separatory funnel. The
methylene chloride extract is dried and
exchanged to hexane, during
concentration to a final volume of 10
mL or less. Gas chromatographic
conditions are described which permit
the separation and measurement of the
parameters in the extract by electron
capture GC<2).
2.2 The method provides a Florisil
column procedure and elemental sulfur
removal procedure to aid in the
elimination of interferences that may
be encountered.
3. Interferences
3.1 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 gas 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 8.5.
3.1.1 Glassware must be scrupulously
cleaned'3). 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. It should then be
drained dry and heated in a muffle
furnace at 400 °C for 1 5 to 30
minutes. Some thermally stable
materials, such as RGBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Thorough
rinsing with such solvents usually
elminates PCB interference. Volumetric
ware should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and stored
in a clean environment to prevent any
accumulation of dust or other
contaminants. Store inverted or capped
with aluminum foil.
3.1.2 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.
3.2 Interferences by phthalate esters
can pose a major problem in pesticide
analysis when using the elution capture
detector. These compounds generally
appear in the chromatogram as large
eluting peaks, especially in the 1 5 and
50% fractions from Florisil. Common
flexible plastics contain varying
amounts of phthalates. These phtha-
lates are easily extracted or leached
from such materials 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(4,5). The
interferences from phthalate esters can
be avoided by using a microcoulometric
or electrolytic conductivity detector.
3.3 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 industrial complex or
municipality being sampled. The
cleanup procedures in Section 11 can
be used to overcome many of these
interferences, but unique samples may
require additional cleanup approaches
to achieve the MDL listed in Table 1.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified!6-8' for the
information of the analyst.
4.2 The following parameters
covered by this method have been
tentatively classified as known or
suspected, human or mammalian
carcinogens: 4,4'-DDT,4,4'-DDD, the
BHCs, and the PCBs. Primary
standards of these toxic compounds
should be prepared in a hood.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
or composite sampling.
5.1.1 Grab sample bottle—Arnber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If
amber bottles are not available, protect
samples from light. The container must
be washed, rinsed with acetone or
methylene chloride, and dried before
use to minimize contamination.
5.1.2 Automatic sampler (optional) —
Must incorporate glass sample
containers for the collection of a mini-
mum of 250 mL. Sample containers
must be kept refrigerated at 4 °C and
protected from light during compositing.
If the sampler uses a peristaltic pump,
a minimum length of compressible
608-2
July 1982
-------
silicone rubber tubing may be used.
Before use, however, the compressible
tubing should be thoroughly rinsed
with methanol, followed by repeated
rinsings with distilled water to minimize
the potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.1 Separatory funnel — 2000-mL,
with Teflon stopcock.
5.2.2 Drying column—Chroma-
tographic column approximately 400
mm long x 19 mm ID, with coarse frit.
5.2.3 Chromatographic icolumn—
Pyrex, 400 mm long x 22 mm ID,
with coarse fritted plate and Teflon
stopcock (Kontes K-42054 or
equivalent).
5.2.4 Concentrator tube, Kuderna-
Danish—10-mL, graduated (Kontes K-
570050-1025 or equivalent). Calibra-
tion must be checked at the volumes
employed in the test. Ground glass
stopper is used to prevent evaporation
of extracts.
5.2.5 Evaporative flask, Kuderna-
Danish-500-mL (Kontes K-570001 -
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.6 Snyder column, Kuderna-
Danish—three-ball macro !(Kontes
K-503000-0121 or equivalent).
5.2.7 Vials—Amber glass, 10-to
1 5-mL capacity, with Teflon-lined
screw cap.
5.3 Boiling chips—approximately
10/40 mesh. Heat to 400 °C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath — Heated, with
concentric ring cover, capable of
temperature control (± 2 °C). The bath
should be used in a hood.
5.5 Balance—Analytical,' capable of
accurately weighing 0.0001 g.
5.6 Gas chromatograph—An
analytical system complete with gas
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical columns,
gases, detector, and strip-chart
recorder. A data system is
recommended for measuring peak
areas. '
5.6.1 Column 1—1.8 m long x 4
mm ID glass, packed with 1.5%
SP-2250/1.95% SP-2401 on
Supelcoport (100/1 20 mesh) or
equivalent. Column 1 was used to
develop the method performance
statements in Section 14. Guidelines
for the use of alternate column
packings are provided in Section 12.1.
5.6.2 Column 2—1.8 m long x 4
mm ID glass, packed with 3% OV-1 on
Supelcoport (100/1 20 mesh) or
equivalent.
5.6.3 Detector—Electron capture.
This detector has proven effective in
the analysis of wastewaters for the
parameters listed in the scope, and
was used to develop the method
performance statements in Section 14.
Guidelines for the use of alternate
detectors are provided in Section 1 2.1.
6. Reagents
6.1 Reagent water— Reagent water is
defined as a water in which an inter-
ferent is not observed at the MDL of
each parameter of interest.
6.2 Sodium hydroxide solution (10
N)-(ACS). Dissolve 40g NaOH in
reagent water and dilute to 100 mL.
6.3 Sodium thiosulfate—(ACS).
Granular.
6,4 Sulfuric acid solution (1 +1 ) —
(ACS). Slowly, add 50 ml H2SO4 (sp.
gr. 1.84) to 50 mL of reagent water.
6.5 Acetone, hexane, isooctane
(2,2,4-trimethylpentane), methylene
chloride—Pesticide quality or
equivalent.
6.6 Ethyl ether—Pesticide quality or
equivalent, redistilled in glass if
necessary.
6.6.1 Must be free of peroxides as
indicated by EM Laboratories Quant
test strips (Available from Scientific
Products Co., Cat. No. P11 26-8, and
others suppliers.)
6.6.2 Procedures recommended for
removal of peroxides are provided with
the test strips. After cleanup, 20 mL
ethyl alcohol preservative must be
added to each liter of ether.
6.7 Sodium sulfate—(ACS) Granular,
anhydrous. Purify by heating at 400 °C
for 4 hours in a shallow tray.
6.8 Florisil - PR grade (60/100
mesh); purchase activated at 1 250 °F
and store in dark in glass containers
with glass stoppers or foil-lined screw
caps. Before use, activate each batch
at least 1 6 hours at 130 °C in a foil
covered glass container.
6.9 Mercury—Triple distilled.
6.10
6.11
Copper powder— Activated .
Stock standard solutions ( 1 .00
— Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.11.1 Prepare stock standard
solutions by accurately weighing about
0.01 00 grams of pure material.
Dissolve the material in isooctane,
dilute to volume in a 1 0-mL volumetric
flask. Larger volumes can be used at
the convenience of the analyst. If
compound purity is certified at 96% or
greater, the weight can be used
without correction to calculate the
concentration of the stock standard,
Commercially prepared stock standards
can be used at any concentration if
they are certified by the manufacturer
or by an independent source.
6.11.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards will be available from the
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
6. 1 1.3 Stock standard solutions
must be replaced after six months, or
sooner if comparison with check
standards indicate a problem.
7. Calibration
7.1 Establish gas chromatographic
operating parameters which produce
retention times equivalent to those
indicated in Table 1 . The gas
chromatographic system may be
calibrated using the external standard
technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure: :
7.2. 1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding volumes of one or more stock
standards to a volumetric flask and
diluting to volume with isooctane. One
of the external standards should be at a
concentration near, but above, the
MDL 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.
608-3
July 1982
-------
7.2.2 Using injections of 2 to 5 \A. of
each calibration standard, 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 calibration
curve.
7.2.3 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 any parameter varies
from the predicted response by more
than ± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested that
is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding volumes of pne or 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 isooctane. One of the standards
should be at a concentration near, but
above, the MDL and the other concen-
trations should correspond to the
expected range of concentrations
found in real samples or should define
the working range of the detector.
7.3.2 Using injections of 2 to 5 f*L of
each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1. RF = (AsCis)/(AisCs)
where:
As = Response for the parameter to
be measured.
Ajs - Response for the internal
standard.
Cis = Concentration of the internal
standard, (ng/L).
Cs = Concentration of the param-
eter to be measured, tfig/L).
If the RF value over the working
range is a constant «10% RSD), the
RF can be assumed to be invariant and
the average RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
of response ratios, As/Ajs, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 10%, the test must be. repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 The cleanup procedure in Section
11 utilizes Florisil chromatography.
Florisil from different batches or
sources may vary in absorptive
capacity. To standardize the amount of
Florisil which is used, the use of lauric
acid value<9> is suggested. The refer-
enced procedure determines the
adsorption from hexane solution of
lauric acid (mg) per gram Florisil. The
amount of Florisil to be used for each
column is calculated by dividing this
factor into 110 and multiplying by 20
g-
7.5 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of interfer-
ences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with
established performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
8.1.1 Before performing any analyses,
the analyst must demonstrate the
ability to generate acceptable accuracy
and precision with this method. This
ability is established as described in
Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the separa-
tions or lower the cost of measurements.
Each time such modifications are made
to the method, the analyst is required
to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing labora-
tory performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and pre-
cision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each compound to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
8.2.2 Using a pipet, add 1.00 ml_ of
the check sample concentrate to each
of a minimum of four 1000-mL aliquots
of reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for the
results. Wastewater background cor-
rections must be made before R and s
calculations are performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the cal-
culated values for R and s. If s > 2p or
|X-R| > 2p, review potential problem
areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and's based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and define
608-4
July 1982
-------
the performance of the laboratory for.
each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
where R and s are calculated as in
Section 8.2.3. The UCL' and LCL can
be used to construct control charts' 10>
that are useful in observing trends in
performance. The control limits above
be replaced by method performance
criteria as they become available from
the U.S. Environmental Protection
Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed-by the calcula-
tion of R and s. Alternately, the analyst
may use four wastewater data points
gathered through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly! 10).
8.4. The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked :sample analysis
must be at least 10% of all samples or
one sample per month, whichever is
greater. One aliquot of the sample must
be spiked and analyzed as described in
Section 8.2. If the recovery for a
particular parameter does not fall
within the control limits [for method
performance, the results reported for
that parameter in all samples processed
as part of the same set must be quali-
fied as described in Section 13.5. The
laboratory should monitor the frequency
of data so qualified to ensure that it
remains at or below 5 % I
8.5 Before processing any samples,
the analyst should demonstrate through
the analysis of a one-liter aliquot of
reagent water, that all glassware and
reagent interferences are under control.
Each time a set of samples is extracted
or there is a change in reagents, a
laboratory reagent blank should be
processed as a safeguard against
laboratory contamination.
8.6 It is recommended 'that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. When doubt
exists over the identification of a peak
on the chromatogram, confirmatory
techniques such as gas chromatography
with a dissimilar column, specific
element detector, or mass spectrometer
must be used. Whenever possible, the
laboratory should perform analysis of
standard reference materials and parti-
cipate in relevant performance
evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practicesd D 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.
9.2 The samples must be iced or
refrigerated at 4 °C from the time of
collection until extraction. If the
samples will not be extracted within
72 hours of collection, the sample
should be adjusted to a pH range of
5.0 to 9.0 with sodium hydroxide or
sulf uric acid. Record the volume of acid
or base used. If aldrin is to be
determined, add sodium thiosulfate
when residual chlorine is present. U.S.
Environmental Protection Agency
methods 330.4 and 330.5 may be
used to measure chlorine residual!12).
Field test kits are available for this
purpose.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction!2).
10. Sample Extraction
10.1 Mark the water meniscus on the
side of the sample bottle for later deter-
mination of sample volume. Pour the
entire sample into a two-liter separatory
funnel.
10.2 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner surface.
Transfer the solvent to the separatory
funnel and extract the sample by
shaking the funnel for two minutes
with periodic venting to release excess
pressure. Allow the organic layer to
separate from the water phase for a
minimum of 10 minutes. If the emulsion
interface between layers is more than
one-third the volume of the solvent
layer, the analyst must employ me-
chanical techniques to complete the
phase separation. The optimum tech-
nique depends upon the sample, but
may include stirring, filtration of the
emulsion through glass wool, centrifu-
gation, or other physical methods.
Collect the methylene chloride extract
in a 250-mL Erlenmeyer flask.
10.3 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.
10.4 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 Kuderna Danish if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract
through a drying column containing
about 10 cm of anhydrous sodium
sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer
flask and column with 20 to 30 mL of
methylene chloride to complete the
quantitative transfer.
10.6 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 1 mL 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 1 5 to 20 minutes.
At the proper rate of distillation the
balJs of the column will actively chatter
but the chambers will not flood with
condensed solvent. 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 minutes.
10.7 Increase the temperature of the
hot water bath to about 80 °C.
Momentarily remove the Snyder
column, add 50 mL of hexane and a
new boiling chip and reattach the
Snyder column. Prewet the column by
adding about 1 mL of hexane to the
top. Concentrate the solvent extract as
before. The elapsed time of concentra-
tion should be 5 to 10 minutes. When
the apparent volume of liquid reaches 1
mL, remove the K-D apparatus and
allow it to drain and cool at least 10
minutes.
608-5
July 1982
-------
10.8 Remove the Snyder column and
rinse the flask and its lower joint into
the concentrator tube with 1 to 2 mL
of hexane. A 5-mL syringe is recom-
mended for this operation. Stopper the
concentrator tube and store
refrigerated if further processing will
not be performed immediately. If the
extracts will be stored longer than two
days, they should be transferred to
Teflon-sealed screw-cap bottles. If the
sample extract requires no further
cleanup, proceed with gas chromato-
graphic analysis. If the sample requires
cleanup proceed to Section 11.
10.9 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. Cleanup and Separation
11.1 Cleanup procedures may not be
necessary for a relatively clean sample
matrix. The cleanup procedures recom-
mended in this method have been used
for the analysis of various clean waters
and industrial effluents. If particular
circumstances demand the use of an
alternative cleanup procedure, the
analyst must determine the elution
profile and demonstrate that the
recovery of each compound of interest
is no less than 85%. The Florisil
column allows for a select f ractionation
of the compounds and will eliminate
polar materials. Elemental sulfur
interferes with the electron capture gas
chromatography of certain pesticides,
but can be removed by the techniques
described below.
11.2 Florisil column cleanup:
11.2.1 Add a weight of Florisil
{nominally 21 g) predetermined by cali-
bration (Section 7.4 and 7.5), to a
chromatographic column. Settle the
Florisil by tapping the column. Add
sodium sulfate to the top of the Florisil
to form a layer 1 to 2 cm deep. Add 60
mL of hexane to wet and rinse the
sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to air,
stop the elution of the hexane by
closing the stopcock on the chroma-
tography column. Discard the eluate.
11.2.2 Adjust the sample extract
volume to 10 mL with hexane and
transfer it from the K-D concentrator
tube to the Florisil column. Rinse the
tube twice with 1 to 2 mL hexane,
adding each rinse to the column.
7 7,2.3 Place a 500-mL K-D flask and
clean concentrator tube under the
chromatography column. Drain the
column into the flask until the sodium
sulfate latyer is nearly exposed. Elute
the column with 200 mL of 6% ethyl
ether in hexane (V/V) (Fraction 1) using
a drip rate of about 5 mL/min. Remove
the K-D flask and set aside for later
concentration. Elute the column again,
using 200 mL of 1 5% ethyl ether in
hexane (V/V)(Fraction 2), into a second
K-D flask. Perform the third elution
using 200 mL of 50% ethyl ether in
hexane (V/V)(Fraction 3). The elution
patterns for the pesticides an PCB's are
shown in Table 2.
7 7.2.4 Concentrate the eluates by
standard K-D techniques (Section
10.6), substituting hexane for the
glassware rinses and using the water
bath at about 85 °C. Adjust final
volume to 10 mL with hexane. Analyze
by gas chromatography.
11.3 Elemental sulfur will usually
elute entirely in Fraction 1 of the Florisil
column cleanup. To remove sulfur
interference from this fraction or the
original extract, pipet 1.00 mL of the
concentrated extract into a clean con-
centrator tube or Teflon-sealed vial.
Add one to three drops of mercury and
sea|d3). Agitate the contents of the
vial for 1 5 to 30 seconds. Prolonged
shaking (two hours) may be required. If
so, this may be accomplished with a
reciprocal shaker. Alternatively,
activated copper powder may be used
for sulfur removal
-------
Table 7. Chromatographic Conditions and Method
Detection Limits
Retention Time Method
(min.) Detection Limit
Parameter
o-BHC
f-BHC
P-BHC
Heptachfor
6-BHC
Aldrin
Hepachlor epoxide
Endosulfan 1
4,4 '-DDE
Diefdrin
Endrin
4,4'-DDD
Endosulfan II
4,4'-DDT
Endrin aldehyde
Endosulfan sulfate
Chlordane
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Column 1
1.35
.70
1.90
2.00
2.15
2.40
3.50
4. SO
5.13
5.45
6.55
7.83
8.0O
9.40
11.82
14.22
mr
mr
mr
mr
mr
mr
mr
mr
mr
Column 2
1.82
2.13
1.97
3.35
2.2O
4.10
5.00
6. 2O
7.15
7.23
8.10
9.08
8.28
11.75
9.3O
10.70
mr
mr
mr
mr
mr
mr
mr
mr
mr
vofl-
0.003
0.004
0.006
0.003
O.OO9
0.004
0.083
O.O14
O.004
0.002
0.006
O.011
0.004
0.012
0.023
O.O66
0.014
0.24
nd
nd
nd
0.065
nd
nd
nd
Column 1 conditions: Supelcoport (1OO/12Omesh) coated
with 1.5%SP-225O/1.95%SP-24O1 packed ina 1.8m
long x 4 mm ID glass column with 5% Methane/95%
Argon carrier gas at a flow rate of 6O mL/min. Column
temperature isothermal at 200 °C, except for PCB-1016
through PCB-1248, which should be measured at
160°C.
Column 2 conditions: Supelcoport (100/120 mesh) coated
with 3% OV-1 in a 1.8 m long x 4 mm ID glass column
with 5% Methane/95% Argon carrier gas at a flow rate of
60 mL/min. Column temperature, isothermal at 200 °C,
for the pesticides; 140°C for PCB-1221 and 1232;
170°C for PCB-1016 and 1242 to 1268.
mr — Multiple peak response. See Figures 2 thru 10.
nd — Not determined.
Table 2. Distribution of Chlorinated Pesticides andPCBs
into Florisil Column Fractions^
Percent Recovery
by Fraction
Parameter
Aldrin
a-BHC
P-BHC
6-BHC
Y-BHC
Chlordane
4,4'-DDD
4,4' -DDE
4,4'-DDT
Dieldrin
Endosulfan 1
•Endosulfan
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Fraction
1
100
100
97
98
100
100
99
98
100
0
37
0
0
4
0
100
100
96
97
97
95
97
103
90
95
Fraction
2
100
64
7
0
96
68
4
Fraction
3
91
106
26
Eluant composition by fraction:
Fraction 1—6% ethyl ether in hexane
Fraction 2— 15% ethyl ether in hexane
Fraction 3—50% ethyl ether in hexane
608-8
July 1982
-------
Table 3. Single
Parameter
Aldrin
a-BHC
P-BHC
6-BHC
f-BHC
Chlorane
4-4' -ODD
4, 4' -DDE
4, 4' -DDT
Dieldrin
Endosulfan 1
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptach/or
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Operator Accuracy and Precision
Average Standard Spike
< Percent Deviation Range
Recovery % (ng/L)
89
89
88
86
97
93
92
89
92
95
96
97
99
95
87
88
93
95
94
96
88
92
90
92
: 91
2.5
2.0
1.3
3.4
3.3
4.1
1.9
2.2
3.2
2.8
2.9
2.4
4.1
2.1
2.1
3.3
1.4
3.8
1.8
4.2
2.4
2.0
1.6
3.3
5.5
2.0
1.0
2.0
2.0
1.0
20
6.0
3.0
8.0
3.0
3.0
5.0
15
5.O
12
1.O
2.0
200
25
55-1 10
110
28-56
40
4O
8O
Number
of
Analyses
15
15
15
15
15
21
15
15
15
15
12
14
15
12
11
12
15
18
12
12
12
12
12
18
18
Matrix
Types
3
3
3
3
3
4
3
3
3
2
2
3
3
2
2
2
3
3
2
2
2
2
2
Column: 1.5%SP-2250+
1.95% SP-2401 on
Supelcoport
Temperature: 200°C.
Detector: Electron capture
I
I/
I I A
P Aft
u y iw/v\
vVl/ ^ — ^_
f
3 0 4 8 . 12 16
Rfttantinn tima, minutas
Column: 1.5% SP-225O+
1.95% SP-2401 on Supelcoport
Temperature: 200°C.
Detector: Electron capture
Figure 2.
Gas chromatogram
of chlordane.
O 4 8 12
Retention time, minutes
Figure 1. Gas chromatogram of pesticides.
16
608-9
July 1982
-------
Column: 1.5%SP-2250+
1.95% SP-2401 on
Supelcoport
Temperature: 200°C.
Detector: Electron capture
2 6 10 14 18 22
Retention time, minutes
Figure 3. Gas chromatogram of toxaphene.
Column: 1,5% SP-2250+ 1.95% SP-2401 on
Supelcoport
Temperature: 16O°C.
Detector: Electron capture
26
Column: 1.5% SP-2250+ 1.95% SP-2401 on
Supelcoport
Temperature: 160°C.
Detector: Electron capture
2 6 10 14 18
Retention time, minutes
Figure 4. Gas chromatogram of PCB-1016.
608-10
22
2 6 10 14 18
Retention time, minutes
Figure 5. Gas chromatogram of PCB-1221.
Column: 1.5% SP-2250+ 1.95% SP-2401 on
Supelcoport
Temperature: 160°C.
Detector: Electron capture
22
2 6 10 14 18
Retention time, minutes
Figure 6. Gas chromatogram of PCB-1232.
July 1982
22
24
-------
Column: 1.5% SP-2250+ 1.95% SP-2401 on
Supelcoport
Temperature: 160°C.
Detector: Electron capture
2 6 10 14 18
Retention time, minutes
Figure 7. Gas chromatogram of PCB-1242.
22
Column: 1.5% SP-2250+ 1.95% SP-24O1 on
Supelcoport
Temperature: 200°C.
Detector: Electron capture
26 10 14
Retention time, minutes
Figure 9. Gas chromatogram of PCB-1254.
18
22
Column: 1.5% SP-2250+ 1.95% SP-24O1 on
Supelcoport
Temperature: 160°C.
Detector: Electron capture
2 6 10 14 18 22
Retention time, minutes
Figure 8. Gas chromatogram of PCB-1248.
608-11
26
Column: 1.5% SP-2250+ 1.95% SP-2401 on
Supelcoport
Temperature: 200°C.
Detector: Electron capture
Figure 10.
July 1982
• JO 14 18 22
Retention time, minutes
Gas chromatogram of PCB-1260.
26
-------
-------
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Nitroaromatics and
Isophorone — Method 609
1. Scope and Application
1.1 This method covers the
determination of certain nitroaromatics
and isophorone. The following
parameters may be determined by this
method:
Parameter STORET No.
CAS No.
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Isophorone
Nitrobenzene
1.2 This is a gas chromatographic
(GC) method applicable to the determi-
nation of the compounds listed above
in municipal and industrial discharges
as provided under 40 CFR 136.1.
When this method is used to analyze
unfamiliar samples for any or all of the
compounds above, compound identifi-
cations should be supported by at least
one additional qualitative technique.
This method describes analytical
conditions for a second gas
chromatographic column that can be
used to confirm measurements made
with the primary column. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results for
all of the parameters listed above,
using the extract produced by this
method.
1.3 The method detection limit (MDL
defined in Section 14.1 MD for each
parameter is listed in Table 1. The MDL
for a specific wastewater may differ
from those listed, depending upon the
nature of interferences in the sample
matrix.
34611
34626
34408
34447
121-14-2
606-20-2
78-59-1
98-95-3
1.4 The sample extraction and
concentration steps in this method are
essentially the same as in method 606,
608, 611 and 612. Thus, a single
sample may be extracted to measure
all of the parameters included in the
scope of each of these methods. When
cleanup is required the concentration
levels must be high enough to permit
selection of aliquots of the extract, as
necessary, to apply appropriate
cleanup procedures. The analyst is
allowed the latitude, under Gas
Chromatography (Section 12), to
select chromatographic conditions
appropriate for the simultaneous
measurement of combinations of these
parameters.
1.5 Any modifications of this
method, beyond those expressly
permitted, shall be considered as major
modifications subject to application
and approval of alternate test
procedures under 40 CFR 1 36.4 and
136.5.
1.6 This method is restricted to use
by or under the supervision of analysts
experienced in the use of gas chroma-
609-1
July 1982
-------
tography 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 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is extracted
with methylene chloride using
separatory funnel techniques. The
extract is dried and exchanged to
hexane during concentration to 1.0 mL
by evaporation. Isophorone and
nitrobenzene are measured by flame
ionization gas chromatography
(FIDGC). The dinitrotoluenes are
measured by electron capture GC
(ECGC)(2).
2.2 The method provides a Florisil
chromatographic cleanup procedure to
aid in the elimination of interferences
that may be encountered.
3. Interferences
3.1 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 gas 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 8.5.
3.1.1 Glassware must be scrupulously
cleaned'3). 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. It should then be
drained dry, and heated in a muffle
furnace at 400 °C for 15 to 30
minutes. Some thermally stable
materials, such as PCBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Thorough
rinsing with such solvents usually
eliminates PCB interferences.
Volumetric ware should not be heated
in a muffle furnace. After drying and
cooling, glassware should be sealed
and stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted or
capped with aluminum foil.
3.1.2 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.
3.2 Matrix interferences ma.y 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 industrial complex or
municipality being sampled. The
cleanup procedures in Section 11 can
be used to overcome many'of these
interferences, but unique samples may
require additional cleanup approaches
to achieve the MDL listed in Table 1.
4. Safety
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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified<4-6) for the
information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
or composite sampling.
5.1.1 Grab sample bottle—Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If
amber bottles are not available, protect
samples from light. The container must
be washed, rinsed with acetone or
methylene chloride, and dried before
use to minimize contamination.
5.1.2 Automatic sampler (optional) —
Must incorporate glass sample
containers for the collection of a mini-
mum of 250 mL. Sample containers
must be kept refrigerated at 4 °C and
protected from light during compositing.
If the sampler uses a peristaltic pump,
a minimum length of compressible
silicone rubber tubing may be used.
Before use, however, the compressible
tubing should be thoroughly rinsed
with methanol, followed by repeated
rinsings with distilled water to minimize
the potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.7 Separatory funnel—2000-mL,
with Teflon stopcock.
5.2.2 Drying column—Chroma-
tographic column approximately 400
mm long x 1 9 mm ID with coarse frit.
5.2.3 Concentrator tube, Kuderna-
Danish— 10-mL, graduated (Kontes K-
570050-1025 or equivalent). Calibra-
tion must be checked at the volumes
employed in the test. Ground glass
stopper is used to prevent evaporation
of extracts.
5.2.4 Evaporative flask, Kuderna-
Danish-500-mL (Kontes K-570001-
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.5 Snyder column, Kuderna-
Danish—Three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Snyder column, Kuderna-
Danish—Two-ball micro (Kontes
K-569001 -021 9 or equivalent).
5.2.7 Vials—Amber glass, 10-to
1 5-mL capacity, with Teflon-lined
screw-cap.
5.2.8 Chromatographic column—
100 mm long x 10 mm ID, Teflon
stopcock.
5.3 Boiling chips—approximately
10/40 mesh. Heat to 400 °C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath—Heated, with
concentric ring cover, capable of
temperature control ( ± 2 °C). The bath
should be used in a hood.
5.5 Balance—Analytical, capable of
accurately weighing 0.0001 g.
5.6 Gas chromatograph—Analytical
system complete with gas chromato-
graph suitable for on-column injection
and all required accessories including
syringes, analytical columns, gases,
detector, and strip-chart recorder. A
data system is recommended for
measuring peak areas.
5.6.7 Column 1-1.2m (4 ft.) long
x 2 mm or 4 mm ID, Pyrex glass,
packed with Gas-Chrom Q (80/100
mesh) coated with 1.95% QF-1/1.5%-
OV-1 7. This column was used to
develop the method performance
statements in Section 14. Guidelines
for the use of alternate column
packings are provided in Section 12.1.
5.6.2 Column 2 —3.0m (10ft.) long
x 2 or 4 mm ID, Pyrex glass, packed
609-2
July 1982
-------
with Gas-Chrom Q (80/100 mesh)
coated with 3% OV-101.
5.5.3 Detector—Flame ionization.
This detector has proven effective in
the analysis of wastewaters for
isophorone and nitrobenzene, and was
used to develop the method perfor-
mance statements in Section 14.
Guidelines for the use of alternate
detectors are provided in Section 1 2.1.
'5.6.4 Detector—Electron capture.
This detector has proven effective in
the analysis of wastewaters for the
dinitrotoluenes, and was used to
develop the method performance
statements in Section 1 4. Guidelines
for the use of alternate detectors are
provided in Section 1 2.1.
6. Reagents
6.1 Reagent water— Reagent water is
defined as a water in whiph an inter-
ferent is not observed at the MDL of
each parameter of interest.
6.2 Sodium hydroxide solution (10
N)-(ACS) Dissolve 40g NaOH in
reagent water and dilute to 100 mL.
6.3 Sulfuric acid solution (1+1) —
(ACS) Slowly, add 50 mL H2SO4 (sp.
gr. 1.84) to 50 mL of reagent water.
6.4 Acetone, hexane, methanol,
methylene chloride, —Pesticide quality
or equivalent.
6.5 Sodium sulfate—(ACS) Granular,
anhydrous. Purify by heating at 400 °C
for 4 hours in a shallow tray.
6.6 Florisil-PR grade (60/100
mesh), purchase activated at 1 250 °F
and store in dark in glass containers
with glass stoppers or foil-lined screw
caps. Before use, activate each batch
overnight at 200 °C in glass containers
loosely covered with foil.
6.7 Stock standard solutions (1.00
Hg/fjL) — Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
5.7.1 Prepare stock standard
solutions by accurately weighing about
0.0100 g of pure mate.rial. Dissolve
the material in pesticide quality
hexane, dilute to volume in a 10-mL
volumetric flask. Larger volumes can
be used at the convenience of the
analyst. If compound purity is certified
at 96% or greater, the weight can be
used without correction t6 calculate
the concentration of the stock
standard. Commercially prepared stock
standards can be used at any
concentration if they are certified by
the manufacturer or by an independent
source.
6.7.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards, will be available from the
U.S. Environmental Protection Agency,
Environmental Monitoring and Systems
Laboratory, Cincinnati, Ohio 45268.
5.7.3 Stock standard solutions must
be replaced after six months, or sooner
if comparison with check standards
indicate a problem.
7. Calibration
7.1 Establish gas chromatographic
operating conditions to produce
resolution of the parameters equivalent
to those indicated in Table 1. The gas
chromatographic system may be
calibrated using the external standard
technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest'by
adding volumes of one or more stock
standards to a volumetric flask and
diluting to volume with hexane. One of
the external standards should be at a ,
concentration near, but above, the
MDL limit 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.
7.2.2 Using injections of 2 to 5 pL of
each calibratiorvstandard, 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 calibration
curve.
7.2.3' 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 any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested that
is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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
internal standards, and dilute to volume
with hexane. One of the standards
should be at a concentration near, but
above, the MDL and the other concen-
trations should correspond to the
expected range of concentrations
found in real samples or should define
the working range of the detector.
7.3.2 Using injections of 2 to 5 pL of
each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1. RF = (AsCis)/(AisCs)
where:
As = Response for the parameter to
be measured.
Ais = Response for the internal
standard.
Cis = Concentration of the internal
standard (f*g/L).
Cs = Concentration of the param-
eter to be measured (f/g/L).
If the RF value over the working
range is a constant «10% RSD), the
RF can be assumed to be invariant and
the average RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
of response ratios, As/Ajs, vs. RF.
7.3.3 The working calibration curve
or RF mustbe verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
609-3
July 1982
-------
Alternatively, a new calibration curve
must be prepared for that compound. •
7.4 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of interfer-
ences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with
established performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
8.1.1 Before performing any analyses,
the analyst must demonstrate the
ability to generate acceptable accuracy
and precision with this method. This
ability is established as described in
Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the separa-
tions or lower the cost of measurements.
Each time such modifications are made
to the method, the analyst is required
to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing labora-
tory performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and pre-
cision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each compound to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL aliquots
of reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for the
results. Wastewater background cor-
rections must be made before R and s
calculations are performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the cal-
culated values for R and s. If s > 2p or
|X-R| > 2p, review potential problem
areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before an
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and define
the performance of the laboratory for
each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
where R and s are calculated as in
Section 8.2.3.
The UCL and LCL can be used to
construct control charts!?) that are
useful in observing trends in
performance. The control limits above
be replaced by method performance
criteria as they become available from
the U.S. Environmental Protection
Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined as
R ± s.The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcula-
tion of R and s. Alternately, the analyst
may use four wastewater data points
gathered through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly!7).
8.4. The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample analysis
must be at least 10% of all samples or
one sample per month, whichever is
greater. One aliquot of the sample must
be spiked and analyzed as described in
Section 8.2. If the recovery for a
particular parameter does not fall
within the control limits for method
performance, the results reported for
that parameter in all samples processed
as part of the same set must be quali-
fied as described in Section 13.3. The
laboratory should monitor the frequency
of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate through
the analysis of a one-liter aliquot of
reagent water, that all glassware and
reagent interferences are under control.
Each time a set of samples is extracted
or there is a change in reagents, a
laboratory reagent blank should be
processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. When doubt
exists over the identification of a peak
on the chromatogram, confirmatory
techniques such as gas chromatography
with a dissimilar column, specific
element detector, or mass spectrometer
must be used. Whenever possible, the
laboratory should perform analysis of
standard reference materials and parti-
cipate in relevant performance
evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices^) should be
followed, except that the bottle must
not be prerinsed 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.
9.2 The samples must be iced or
refrigerated at 4 °C from the time of
collection until extraction.
609-4
July 1982
-------
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction'2).
10. Sample Extraction
10.1 Mark the water meniscus on the
side of the sample bottle for later deter-
mination of sample volume. Pour the
entire sample into a two-liter separatory
funnel. Check the pH of the sample
with wide-range pH papter and adjust to
within the range of 5 to 9 with diluted
sodium hydroxide or sulfuric acid.
10.2 Add 60 ml methylene chloride
to the sample bottle, seal, and shake
for 30 seconds to rinse the inner
surface. Transfer the solvent to the
separatory funnel and extract the
sample by shaking the funnel for two
minutes with periodic venting to
release excess pressure. Allow the
organic layer to separate from the
water phase for a minimum of 10
minutes. 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 yvool,
centrifugation, or other physical
methods. Collect the methylene chloride
extract in a 250-mL Erlehmeyer flask.
10.3 Add a second 60rmL 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.
10.4 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 if the requirements of
Section 8.2 are met.
10.5 Pour the combined extract
through a drying column containing
about 10 cm of anhydrous sodium
sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer
flask and column with 20 to 30 mL of
methylene chloride to complete the
quantitative transfer.
10.6 Sections 10.7 and 10.8
describe a procedure for exchanging
the methylene chloride solvent to
hexane while concentrating the extract
volume to 1.0 mL. When it is not'
necessary to achieve the MDL in Table
2, the solvent exchange may be made
by the addition of 50 mL of hexane and
concentration to 10 mL as described in
method 606, Section 10.7.
10.7 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 1 mL 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 1 5 to 20 minutes.
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 1 mL, remove
the K-D apparatus and allow it to drain
and cool for at least 10 minutes.
Remove the Snyder column and rinse
the flask and its lower joint into the
concentrator tube with 1 to 2 mL of
methylene chloride. A 5-mL syringe is
recommended for this operation.
10.8 Add 1 to 2 mL of hexane to the
concentrator tube, and a clean boiling
chip. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder
column by adding about 0.5 mL of
hexane to the top. Place this micro K-D
apparatus on a water bath (60 to
65 °C) so that the concentrator tube is
partially immersed in the hot water.
Adjust the vertical position of the
apparatus and water temperature as
required to complete the concentration
in 5 to 10 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 liquid reaches 0.5 mL,
remove the K-D apparatus and allow it
to drain for at least 10 minutes while
cooling. Remove the micro-Snyder
column and rinse its lower joint into the
concentrator tube with a small volume
of hexane. Adjust the final volume to
1.0 mL and stopper the concentrator
tube and store refrigerated if further
processing will not be performed
immediately. Unless the sample is
known to require cleanup, proceed
with gas chromatographic analysis.
10.9 Determine the original sample
volume by refilling the sample bottle to
the mark with water and measuring the
volume in a 1000-mL graduated
cylinder. Record the sample volume to
the nearest 5 mL.
11. Cleanup and Separation
11.1 . Cleanup procedures may not be
necessary for a relatively clean sample
matrix. The cleanup procedure recom-
mended in this method has been used
for the analysis of various clean waters
and industrial effluents. If particular
circumstances demand the use of an
alternative cleanup procedure, the
analyst must demonstrate, that the
recovery of each compound of interest
is no less than 85%.
11.2 Prepare a slurry of 10g of
activated Florisil in methylene chloride
in hexane (1 + 9)(V/V). Use it to pack
a 10-mm ID chromatography column,
gently tapping the column to settle the
Florisil. Add 1 cm of anhydrous sodium
sulfate to the top of the Florisil.
11.2.1 Just prior to exposure of the
sodium sulfate layer to the air transfer
the 1 mL of sample extract onto the
column using an additional 2 mL of
hexane to complete the transfer.
7 7.2.2 Just prior to exposure of the
sodium sulfate layer to the air, add 30
mL of methylene chloride in hexane (1
+ 9)(V/V) and continue the elution of
the column. Elution of the column
should be at a rate of about 2 mL skill
per min. Discard the eluate from this
fraction.
11.2.3 Next elute the column with
30 mL of acetone/methylene chloride
(1 + 9MV/V) into a 500-mL K-D flask
equipped with a 10-mL concentrator
tube. Concentrate the collected
fraction by the K-D technique
prescribed in Sections 10.6, 10.7, and
10.8 including the solvent exchange in
1 mL of hexane. This fraction should
contain the nitroaromatics and
isophorone.
11.2.4 Analyze by gas
chromatography.
12. Gas Chromatography
12.1 Isophorone and nitrobenzene
are analyzed by injection of a portion of
the extract into an FIDGC. The dinitro-
toluenes are analyzed by a separate
injection into an ECGC. Table 1
summarizes some recommended gas
chromatographic column materials and
operating conditions for the
instruments. This Table includes
retention times and MDL obtained
under these conditions. Examples of
the parameter separations achieved by
Column 1 are shown in Figures 1 and
2. Other packed columns, chromato-
graphic conditions, or detectors may
be used if the requirements of Section
8.2 are met. Capillary (open-tubular)
columns may also be used if the
relative standard deviations of
responses for replicate injections are
demonstrated to be less than 6% and
the requirements of Section 8.2 are
met.
609-5
July 1982
-------
12.2 Calibrate the system daily as
described in Section 7.
12.3 If the internal standard
approach is being used, the analyst
must not add the internal standard to
sample the extract until immediately
before injection into the instrument.
Mix thoroughly.
12.4 Inject 2 to 5 [A. of the sample
extract using the solvent-flush
technique'9'. Smaller (1.0 nU volumes
can be Injected if automatic devices are
employed. Record the volume injected
to the nearest 0.05 \iL, and the
resulting peak size in area or peak
height units.
12.5 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.
12.6 If the response exceeds the
working range of the system, dilute the
extract and reanalyze.
12.7 If the measurement of the peak
response is prevented by the presence
of interferences, further cleanup is
required.
13. Calculations
13.1 Determine the concentration of
individual compounds in the sample.
13. 1. 1 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 in Section
7.2.2. The concentration in the sample
can be calculated from equation 2:
(As)(ls)
(v-)(V )
(A)(Vt)
Eq. 2. Concentration,
where:
A - Amount of material injected, in
nanograms.
Vj = Volume of extract injected
(nU.
Vt ** Volume of total extract (\A-).
Vs ** Volume of water extracted
(mL).
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
equation -3.
Eq. 3
Concentration, n = (A.S)(RF)(VO)
where:
As = Response for the parameter to
be measured.
AJS = Response for the internal
standard.
ls = Amount of internal standard
added to each extract (ng).
V0 = Volume of water extracted, in
liters.
13.2 Report results in micrograms
per liter without correction for recovery
data. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
13.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in Section 8.3, data for
the affected parameters must be
labeled as suspect.
14. Method Performance
14.1 Method detection limits—The
method detection limit (MOD is defined
as the minimum concentration of a
substance that can be measured and
reported with 99% confidence that the
value is above zero'11. The MDL
concentrations listed in Table 1 were
obtained using reagent water'10).
Similar results were achieved using
representative wastewaters.
14.2 This method has been tested
for linearity of recovery from reagent
water and has been demonstrated to
be applicable over the concentration
range from 7 x MDL to 1000 x
MDLdO).
14.3 In a single laboratory (Battelle,
Columbus Laboratories), using spiked
wastewater samples, the average
recoveries presented in Table 2 were
obtained<2>. Each spiked sample was
analyzed in triplicate on two separate
days. The standard deviation of the
percent recovery is also included in
Table 2.
14.4 The U.S. Environmental Protec-
tion Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. "Determination of Nitroaromatics
and Isophorone in Industrial and
Municipal Wastewaters." Report for
EPA Contract 68-03-2624 (In
preparation).
3. ASTM Annual Book of Standards,
Part 31, D 3694. "Standard Practice
for Preparation of Sample Containers
and for Preservation," American
Society for Testing and Materials,
Philadelphia, PA, p. 679, 1 980.
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,"
(29CFR1 910), 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. "Handbook of Analytical Quality
Control in Water and Wastewater
Laboratories," EPA-600/4-79-01 9,
U.S. Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268, March 1979.
8. ASTM Annual Book of Standards,
Part 31, D 3370, "Standard Practice
for Sampling Water," American
Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. Burke, J.A., "Gas Chromatography
for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the
Association of Official Analytical
Chemists, 48, 1037 (1965).
10. "Determination of Method
Detection Limit and Analytical Curve
for EPA Methods 609 —Nitroaromatics
and Isophorone," special letter report
for EPA Contract 68-03-2624.
Environmental Monitoring and Support
Laboratory—Cincinnati, Ohio 45268.
609-6
July 1982
-------
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time Method Detection
(mm.) limit-1i in/I \
Parameter
Nitrobenzene
2, 6-Dinitrotoluene
Isophorone
2,4-Dinitrotoluene
Column 1
', 3.31
3.52
4.49
5.35
Column 2
4.31
4.75
5.72
6.54
EC
13.7
0.01
15.7
O.O2
FID
3.6
5.7
Column 1 conditions: Gas-Chrom Q (80/100mesh) coated with 1.95% QF-1/1.5%
OV-17 packed in a pyrex glass column 7.2 m (4 ft) long x 2 mm or 4 mm ID.
Nitrogen carrier gas at a flow rate of 44 mL/min was used when determining
isophorone and nitrobenzene by FID. The Column temperature was isothermal at
85°C. Methane <10%)/Argon (90%) carrier gas at flow rate of 44 mL/min wasused
when determining the dinitrotoluenes by ECGC. The column temperature was
isothermal at 145°C.
Column 2 conditions: Gas-Chrom Q (8O/100 mesh) coated with 3%OV-1 packed in
a pyrex glass column 3.Om (1O ft) long x 2mmor4mmlD. Nitrogen carrier gas at a
flow rate of 44 mL/min was used when determining isophorone and nitrobenzene by
FID. The column temperature was isothermal at 10O°C. Methane (10%)/Argon
(90%) carrier gas flow rate of 44 mL/min was used when determining the
dinitroltoluenes by ECGC. The column temperature was isothermal,, 15O°C.
A 2 mm ID column was used with the FIDGC and a 4 mm ID column was used with
the ECGC.
Table 2. Single Opera tor A ccurac y and Precision
Parameter
2,4-Dinitrotoluene
2, 6-Dinitrotoluene
Isophorone
Nitrobenzene
Average
Percent
Recovery
; 63
66
73
71
Standard
Deviation
%
3.1
3.2
4.6
5.9
Spike
Range
(ng/L)
5-1OO
5-5O
50-60
9O-1OO
Number
of
Analyses
21
24
21
24
Matrix
Types
4
4
4
4
609-7 July 1982
-------
Column: 1.5% OV-J7 +1.95% QF-1
on Gas Chrom Q
Temperature: 85°C.
Detector: Flame ionization
Column: 1.5% OV-17 +1.95% QF-1
on Gas Chrom Q
Temperature: 145°C.
Detector: Electron capture
0)
I
0) S
I I
24 6 8 10 12
Retention time, minutes
Figure 1. Gas chromatogram
of nitrobenzene
and isophorone.
2468
Retention time, minutes
Figure 2. Gas chromatogram
of dinitrotoluenes.
609-8
July 1982
-------
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
S-EPA
Research and Development
Test Method
Polynuclear Aromatic
Hydrocarbons — Method 610
1. Scope and Application
1.1 This method covers the
determination of certain polynuclear
aromatic hydrocarbons (PAH). The
following parameters may be
determined by this method:
Parameter
STORET No.
CAS No.
Acenaphthene
Acenaphthylene
Anthracene
Benzd (a) anthracene
Benzo (a) pyrene
Benzo (b) fluoranthene
Benzo (ghi) perylene
Benzo (k) fluoranthene
Chrysene
Dibenzo (a, h) anthracene
Fluoranthene
Fluorene
Indeno (1, 2, 3-cd) pyrene
Naphthalene
Phenanthrene
Pyrene
34205
34200
34220
34526
34247
34230
34521
34242
34320
34556
34376
34381
34403
34696
34461
34469
83-32-9
208-96-8
120-12-7
56-55-3
50-32-8
205-99-2
191-24-2
207-08-9
218-01-9
53-70-3
206-44-0
86-73-7
193-39-5
91-20-3
85-01-8
1 29-00-0
1.2 This is a chromatographic
method applicable to the
determination of the compounds listed
above in municipal and industrial
discharges as provided under 40 CFR
1 36.1. When this method is used to
analyze unfamiliar samples for any
or all of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results
for many of the parameters listed in
Section 1.1, using the extract
produced by this method.
1.3 This method provides for both
high performance liquid
chromatographic (HPLC) and gas
chromatographic (GC) approaches to
the determination of PAHs. The gas
chromatographic procedure does not
adequately resolve the following four
pairs of compounds: anthracene and
phenanthrene; chrysene and benzo (a)
anthracene; benzo (b) fluoranthene
and benzo (k) fluoranthene; and
dibenzo (a, h) anthracene and indeno
(1, 2, 3-cd) pyrene. Unless the
purpose for the analysis can be served
by reporting the sum of an unresolved
pair, the liquid chromatographic
approach must be used for these
compounds. The liquid
610-1
July 1982
-------
chromatographic method does
resolve all 16 of the PAHs listed.
1.4 The method detection limit
(MDL. defined in Section 15)m for
each parameter is listed in Table 1.
The MDL for a specific wastewater
may differ depending upon the nature
of interferences in the sample matrix.
1.5 The sample extraction and
concentration steps in this method are
essentially the same as in methods
606, 608, 609, 611 and 612.
Therefore, a single sample may be
extracted to measure the parameters
included in the scope of each of these
methods, provided the concentration
is high enough to permit selecting
aliquots of the extract for cleanup,
when required. Selection of the
aliquots must be made prior to the
solvent exchange steps of this
method. The analyst is allowed the
latitude, under Gas Chromatography
(Section 13), to select
chromatographic conditions
appropriate for the simultaneous
measurement of combinations of
these parameters.
1.6 Any modification of this method,
beyond those expressly permitted,
shall be considered as major
modifications subject to application
and approval of alternate test
procedures under 40 CFR 136.4
and 136.5.
1.7 This method is restricted to use
by or under the supervision of
analysts experienced in the use of
HPLC and GC and in the
interpretation of liquid and gas
chromatograms. Each analyst must
demonstrate the ability to generate
acceptable results with this method
using the procedure described in
Section 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is solvent
extracted with methylene chloride
using a separatory funnel. The
methylene chloride extract is dried
and concentrated to a volume of 10
mL or less. The solvent is exchanged
to cyclohexane prior to cleanup.
Following cleanup, when using HPLC
for determination of the PAHs, the
solvent is exchanged to acetonitrile.
Ultraviolet (UV) and fluorescence
detectors are used with HPLC. When
cleanup is not required and when
flame ionization detector GC is
used for determination, the
methylene chloride extract may be
analyzed directly. When cleanup is
required, the cyclohexane exchange
is made. Instrumental conditions are
described which permit the separation
and measurement of the PAH
compounds'21'
2.2 A silica gel column cleanup
procedure is provided to aid in the
elimination of interferences that may
be encountered.
3. Interferences
3.1 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
interferences under the conditions of
the analysis by running laboratory
reagent blanks as described in
Section 8.5.
3.7.7 Glassware must be
scrupulously cleaned'3'. 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. It should then be drained dry,
and heated in a muffle'furnace at
400°C for 15 to 30 minutes. Some
thermally stable materials, such as
PCBs, may not be eliminated by
this treatment. Solvent rinses with
acetone and pesticide quality hexane
may be substituted for the muffle
furnace heating. Volumetric ware
should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and
stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted
or capped with aluminum foil.
3.7.2 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.
3.2 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 industrial complex or
municipality being sampled. The
cleanup procedure in Section 11 can
be used to overcome many of these
interferences, but unique samples
may require additional cleanup
approaches to achieve the MDL listed
in Table 1.
3.3 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 specific PAH
compounds covered by this method,
other PAH compounds may interfere.
4. Safety
4.1 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 awareness file
of OSHA regulations regarding the
safe handling of the chemicals
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. Additional references to
laboratory safety are available and
have been identified'4"6' for the
information of the analyst.
4.2 The following paramenters
covered by this method have been
tentatively classified as known or
suspected, human or mammalian
carcinogens; benzo (a) anthracene,
benzo (a) pyrene and dibenzo (a, h)
anthracene.
5. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling.
5.7.7 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive.
If amber bottles are not available,
protect samples from light. The
container must be washed, rinsed
with acetone or methylene chloride,
and dried before use to minimize
contamination.
5.7.2 Automatic sampler (optional) -
Must incorporate glass sample
containers for the collection of a
minimum of 250 mL. Sample
containers must be kept refrigerated
at 4°C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however,
the compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
610-2
July 1982
-------
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.1 Separatory funnel - 2000-mL,
with Teflon stopcock.
5.2.2 Drying column -
Chromatographic column, 400 mm
long x 19 mm ID with coarse frit.
5.2.3 Concentrator tube, Kuderna-
Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent).
Calibration must be checked at the
volumes employed in the test. Ground,
glass stopper is used to prevent
evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-
Danish - 500-mL (Kontes K-570001 -
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.5 Snyder column, Kuderna-
Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Snyder column, Kuderna-
Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.7 Vials - Amber glass, 10- to
15- mL capacity, with Teflon-lined
screwcap.
5.2.8 Chromatographic column -
250 mm long x 10 mm ID with coarse
fritted disc at bottom and Teflon
stopcock.
5.3 Boiling chips - approximately
10/40 mesh. Heat to 40Q°C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath - Heated, with
concentric ring cover, capable of
temperature control (±2°C). The bath
should be used in a hood.
5.5 Balance — Analytical, capable of
accurately weighing 0.0001 g.
5.6 High performance liquid
Chromatographic apparatus (modular):
5.6.1 Gradient pumping system,
constant flow.
5.6.2 Reverse phase column, 5
micron HC-ODS Sil-X, 250 mm x 2.6
mm ID (Perkin-Elmer No. 089-0716 or
equivalent).
5.6.3 Fluorescence detector, for
excitation at 280 nm and emission
greater than 389 nm cutoff (Corning
3-75 or equivalent). Fluorometers
should have dispersive optics for
excitation and can utilize either filter
or dispersive optics at the emission
detector.
5.6.4 UV detector, 254 nm, coupled
to fluorescence detector.
5.6.5 Strip-chart recorder
compatible with detectors. Use of a
data system for measuring peak areas
and retention times is recommended.
5.7 Gas chromatograph - An
analytical system complete with
temperature programmable gas
chromatograph suitable for on-column
injection or splitless injection and all
required accessories including
syringes, analytical columns, gases,
detector, and strip-chart recorder.
A data system is recommended for
measuring peak areas.
5.7.1 Column - 1.8 m long x 2 mm
ID pyrex glass packed with 3% OV-17
on Chromosorb W-AW-DCMS
(100/120 mesh) or equivalent. This
column was used to develop the
retention time data in Table 2.
Guidelines for the use of alternate
column packings are provided in
Section 13.
5.7.2 Detector - Flame ionization.
This detector has proven effective in
the analysis of wastewaters for the
compounds listed in the scope
excluding the four pairs of unresolved
compounds listed in Section 1.3.
Guidelines for the use of alternate
detectors are provided in Section
12.2.
6. Reagents
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Sodium thiosulfate - (ACS)
Granular.
6.3 Cyclohexane, methanol, acetone,
methylene chloride, and pentane -
Pesticide quality or equivalent.
6.4 Acetonitrile, high purity HPLC
quality, distilled in glass.
6.5 Sodium sulfate - (ACS)
Granular, anhydrous. Purify by
heating at 400°C for four hours
in a shallow tray.
6.6 Silica gel - Grade 923 (100/200
mesh) dessicant (Davison Chemical or
equivalent). Before use, activate for at
least 16 hours at 130°C in a shallow
glass tray, loosely covered with foil.
6.7 Stock standard solutions (1.00
/ug/yuL) - Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.7.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 g of pure material,
Dissolve the material in HPLC quality
acetonitrile, dilute to volume in a 10-
mL volumetric flask. Larger volumes
can be used at the convenience of the
analyst. If compound purity is certified
at 96% or greater, the weight can be
used without correction to calculate
the concentration of the stock
standard. Commercially prepared
stock standards can be used at any
concentration if they are certified by
the manufacturer or by an
independent source.
6.7.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards will be available from the
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio 45268.
6.7.3 Stock standard solutions must
be replaced after six months, or
sooner if comparison with check
standards indicate a problem.
7. Calibration
7.1 Establish liquid or gas
Chromatographic operating
parameters to produce resolution of
the parameters equivalent to that
indicated in Tables 1 or 2. The
Chromatographic system can be
calibrated using the external standard
technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with
acetonitrile. One of the external
standards should be at a
concentration near, but above, the
MDL 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.
7.2.2 Analyze each calibration
standard (5 to 25 //L for HPLC and 2
to 5 fjL for GC), and tabulate peak
height or area responses against the
mass injected. The results may be
used to prepare a calibration curve for
each compound. Alternatively, if the
ratio erf response to amount injected
(calibration factor) is a constant over
the working range {< 10% relative
610-3
July 1982
-------
standard deviation, RSD), linearity
through the origin can be assumed
and the average ratio on calibration
factor can be used in place of a
calibration curve.
7.2.3 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 any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
^
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested
that is applicable to all samples.
7.3.7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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 internal standards, and dilute to
volume with acetonitrile. One of the
standards should be at a concen-
tration near, but above, the MDL
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.
7.3.2 Analyze each calibration
standard (5 to 25 pL for HPLC and 2
to 5 fjL for GC) and tabulate peak
height or area responses against
concentration for each compound
and internal standard, and calculate
response factors (RF) for each
compound using Equation 1.
Eq. 1 RF = (A3Cis)/(A,s Cs)
where:
As =Response for the parameter to
be measured.
Ai, =Response for the internal
standard.
Ci» =Concentration of the internal
standard, (/ug/L).
C» =Concentration of the parameter
to be measured, (/L/g/L).
If the RF value over the working range
is a constant « 10% RSD), the RF can
be assumed to be invariant and the
average RF can be used for calcula-
tions. Alternatively, the results can be
used to plot a calibration curve of
response ratios, AS/A|S, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of
interferences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist
of an initial demonstration of labora-
tory capability and the analysis of
spiked samples as a continuing check
on performance. The laboratory is
required to maintain performance
records to define the quality of data
that is generated. Ongoing perform-
ance checks must be compared with
established performance criteria
to determine if the results of
analyses are within accuracy and
precision limits expected of the
method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method. This ability is
established as described in Section
8.2.
3.7.2 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike
and analyze a minimum of 10% of
all samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform
the following operations.
3.2.7 Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate in acetronitrile 1000
times more concentrated than the
selected concentrations. Quality
control check sample concentrates,
appropriate for use with this method,
will be available from the U.S.
Environmental Protection Agency,
Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
3.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be
used in place of the reagent water,
but one or more additional aliquots
must be analyzed to determine
background levels, and the spike level
must exceed twice the background
level for the test to be valid. Analyze
the aliquots according to the method
beginning in Section 10.
3.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater
background corrections must be made
before R and s calculations are
performed.
3.2.4 Using Table 3, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or |X-R| > 2p, review potential
problem areas and repeat the test.
3.2.5 The U.S. Environmental
Protection Agency plans to estab-
lish performance criteria for R and
s based upon the results of inter-
laboratory testing. When they
become available, these criteria must
be met before any samples may be
analyzed.
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter
being measured.
3.3.7 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R — 3 s
where R and s are calculated as in
Section 8.2.3.
The UCL and LCL can be used to
construct control charts171 that are
useful in observing trends in perfor-
610-4
July 1982
-------
mance. The control limits above must
be replaced by method performance
criteria as they become available from
the U.S. Environmental Protection
Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined
as R ± s. The accuracy ^statement
should be developed bythe analysis
of four aliquots of wastewater as
described in Section 8.2.2, followed
by the calculation of R and s.
Alternately, the analyst;may use four
wastewater data points;gathered
through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly171. '
8.4 . The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample [per month,
whichever is greater. One aliquot of
the sample must be spiked and :
analyzed as described in Section
8.2. If the recovery for a particular
parameter does not fall within the
control limits for method performance,
the results reported for,that parameter
in all samples processed as part of
the same set must be qualified as
described in Section 1 4'.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagents interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as
a safeguard against laboratory
contamination. ,
8.6 It is recommended that the
laboratory adopt additional quality
assurance practices foriuse with this
method. The specific practices that
are most productive depend upon the
needs of the laboratory and the nature
of the samples. Field duplicates may
be analyzed to monitor the precision
of the sampling technique. When
doubt exists over the identification
of a peak on the chromatogram,
confirmatory techniques such as
chromatography with a dissimilar
column or detector must be used. This
may include the use of a mass
spectrometer. Whenever possible, the
laboratory should perform analysis of
standard reference materials and
participate in relevant performance
evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices'8' 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.
9.2 The samples must be iced or
refrigerated at 4°C from the time of
collection until extraction. PAHs are
known to be light sensitive, therefore,
samples, extracts and standards
should be stored in amber or foil
wrapped bottles in order to minimize
photolytic decomposition. Fill the
sample bottle and, if residual chlorine
is present, add 80 mg of sodium
thiosulfate per liter of sample. U.S.
Environmental Protection Agency
methods 330.4 and 330.5 may be
used for measurement of residual
chlorine191. Field test kits are available
for this purpose.
9.3 All samples must be extracted
within 7 days, and analysis completed
within 40 days of extraction121.
10. Sample Extraction
10.1 Mark the water meniscus on
the side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel.
10.2 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner surface.
Transfer the solvent to the separatory
funnel and extract the sample by
shaking the funnel for two minutes
with periodic venting to release
excess pressure. Allow the organic
layer to separate from the water
phase for a minimum of ten minutes.
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 250-mL
Erlenmeyer flask.
10.3 Add a second 60-mL volume of
methylene chloride to the sample
bottle, rinse and repeat the extraction
procedure a second time, combining
the extracts in the Erlenmeyer flask.
Perform a third extraction in the same
manner.
10.4 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 if the requirements
of Section 8.2 are met.
10.5 Pour the combined extract
through a drying column containing
about 10 cm of anhydrous sodium
sulfate, and collect the extract in
the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20
to 30 ml_ of methylene chloride to
complete the quantitative transfer.
10.6 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 1 mL 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 concen-
tration in 15 to 20 minutes. 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 1
mL, remove the K-D apparatus and
allow it to drain and cool for at least
10 minutes. Remove the Snyder
column and rinse the flask and its
lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride.
A 5-mL syringe is recommemded for
this operation. Stopper the
concentrator tube and store
refrigerated if further processing will
not be performed immediately. If the
extracts will be stored longer than
two days, they should be transferred
to Teflon-sealed screw-cap bottles
and protected from light.
10.7 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.
610-5
July 1982
-------
11. Cleanup and Separation
11.1 Cleanup procedures may not
be necessary for a relatively clean
sample matrix. The cleanup
procedures recommended in this
method have been used for the
analysis of various clean waters and
industrial effluents. If particular
circumstances demand the use of an
alternative cleanup procedure, the
analyst must determine the elution
profile and demonstrate that the
recovery of each compound of interest
is no less than 85%.
11.2 Before the silica gel cleanup
technique can be utilized, the extract
solvent must be exchanged to cyclo-
hexane. Add a 1- to 10- mL aliquot
of sample extract (in methylene
chloride) and a boiling chip to a clean
K-D concentrator tube. Add 4 mL
cyclohexane and attach a micro-
Snyder column. Prewet the micro-
Snyder column by adding 0.5 mL
methylene chloride to the top. Place
the micro-K-D apparatus on a boiling
(1 OO'C) 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
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 0.5 mL, remove
the K-D apparatus and allow it to
drain for at least 10 minutes while
cooling. Remove the micro-Snyder
column and rinse its lower joint
into the concentrator tube with a
minimum of cyclohexane. Adjust the
extract volume to about 2 mL.
11.3 Silica gel column cleanup for
PAHs.
/1.3.1 Prepare a slurry of 10g
activated silica gel in methylene
chloride and place this in a 10-mm ID
chromatography column. Gently tap
the column to settle the silica gel and
elute the methylene chloride. Add 1 to
2 cm of anhydrous sodium sulfate to
the top of the silica gel.
71.3.2 Preelute the column with 40
mL of pentane. Discard the eluate and
just prior to exposure of the sodium
sulfate layer to the air, transfer the 2
mL of cyclohexane sample extract
onto the column, using an additional
2 mL of cyclohexane to complete the
transfer.
11.3.3 Just prior to exposure of the
sodium sulfate layer to the air, add 25
mL pentane and continue elution of
the column. Discard the'pentane
eluate.
11.3.4 Elute the column with 25 mL
of methylene chloride/pentane (4 + 6)
(V/V) and collect the eluate in a 500-
mL K-D flask equipped with a 10-mL
concentrator tube. Elution of the
column should be at a rate of about 2
mL/min.
11.3.5 Concentrate the collected
fraction to less than 10 mL by K-D
techniques as in Section 10.6, using
pentane to rinse the walls of the glass-
ware. Proceed with HPLC or GC
analysis.
12. High Performance Liquid
Chromatography (HPLC)
12.1 To the extract in the
concentrator tube, add 4 mL of
acetonitrile and a new boiling chip,
then attach a micro-Snyder column.
Increase the temperature of the hot
water bath to 95 to 100°C.
Concentrate the solvent as in Section
10. After cooling, remove the micro-
Snyder column and rinse its lower
joint into the concentrator tube with
about 0.2 mL acetonitrile. Adjust the
extract volume to 1.0 mL.
12.2 Table 1 summarizes the
recommended HPLC column materials
and operating conditions for the
instrument. This table includes
retention times, capacity factors, and
MDL that were obtained under these
conditions. The UV detector is
recommended for the determination
of naphthalene, acenaphthylene,
acenapthene, and fluorene, and
the fluorescence detector is
recommended for the remaining
PAHs. Examples of the parameter
separations achieved by this HPLC
column are shown in Figures 1
and 2. Other HPLC columns,
chromatrograpic conditions or
detectors may be used if the
requirements of Section 8.2 are met.
12.3 Calibrate the system daily as
described in Section 7.
12.4 If the internal standard
approach is being used, the internal
standard must be added to sample
extract and mixed thoroughly,
immediately, before injection into the
instrument.
12.5 Inject 5 to 25 /jL of the sample
extract using a high pressure syringe
or a constant volume sample injection
loop. Record the volume injected to
the nearest 0.1 /uL, and the resulting
peak size in height or area units. Re-
equilibrate the liquid chromato-
graphic column at the initial
gradient conditions for at least 10
minutes between injections.
12.6 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.
12.7 If the peak height or area
exceeds the linear range of the
system, dilute the extract with
acetonitrile and reanalyze.
12.8 If the peak area measurement
is prevented by the presence of
interferences, further cleanup is
required.
13. Gas Chromatography
13.1 The packed column GC
procedure will not resolve certain
isomeric pairs as indicated in
Section 1.3 and Table 2. The liquid
chromatographic procedure (Section
12) must be used for these materials.
Capillary (open-tubular) columns
may be used if the relative standard
deviations of responses for replicate
injections are demonstrated to be
less than 6% and the requirements
of Section 8.2 are met.
13.2 To achieve maximum
sensitivity with this method, the
extract must be concentrated to 1.0
mL. Add a clean boiling chip to the
methylene chloride extract in the
concentrator tube. Attach a two-ball
micro-Snyder column. Prewet the
micro-Snyder column by adding about
0.5 mL of methylene chloride to the
top. Place the micro K-D apparatus on
a hot water bath (60 to 65°C) 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 the concentration in 5 to
10 minutes. At the proper rate of
distillation the balls will actively
chatter but the chambers will not
flood. When the apparent volume of
liquid reaches 0.5 mL, remove the
K-D apparatus. Drain and cool for at
least 10 minutes. Remove the micro-
Snyder column and rinse its lower
joint into the concentrator tube with a
small volume of methylene chloride.
Adjust the final volume to 1.0 mL and
stopper the concentrator tube.
13.3 Table 2 describes the
recommended GC column and
operating conditions for the
instrument. This table includes
61O-6
July 1982
-------
retention times that were obtained
under these conditions. An example
of the parameter separations achieved
by this column is shown in Figure 3.
Other packed columns, bhromato-
graphic conditions, or detectors
may be used if the requirements
of Section 8.2 are met. Capillary
(open-tubular) columns ;may also be
used if the relative standard *
deviations of responses for replicate
injections are demonstrated to be less
than 6% and the requirements of
Section 8.2 are met.
13.4 Calibrate the GC
system daily as described in
Section 7.
13-5 If the internal standard
approach is being used,;add the
internal standard to sample extract
and mix thoroughly, immediately,
before injection into the instrument.
13.6 Inject 2 to 5//L of the
sample extract using the solvent-
flush technique1101. Smaller
(1.0 /jL) volumes may be injected if
automatic devices are erriployed.
Record the volume injecjed to the
nearest 0.05 fjL, and the resulting
peak size in area or peak height units.
13.7 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, tjie experience
of the analyst should wdigh heavily in
the interpretation of chromatograms.
13.8 If the response for the peak
exceeds the working range of the
system, dilute the extract and
reanalyze. .
13.9 If the measurement of the
peak response is prevented by the
presence of interferences, further
cleanup is required.
14. Calculations
14.1 Determine the concentration of
individual parameters in the sample.
14.1.1 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 in Section 7.2.2. The
concentration in the sample can be
calculated from Equation 2:
Eq. 2. Concentration,/ug/L = (Vi)(Vs)—
where:
A = Amount of material injected, in
nanograms.
Vi = Volume of extract injected (fjL),
Vt = Volume of total extract (//L).
Vs = Volume of water extracted (ml_).
14.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
Equation 3.
Eq. 3. Concentration,//g/L = (Ais)(RF)(Vo)
where:
As = Response for the parameter to
be measured.
Ais = Response for the internal
standard.
U = Amount of internal standard
added to each extract tug).
V0 = Volume of water extracted,
in liters.
14.2 Report results in micrograms
per'liter without correction for
recovery data. When duplicate and
spiked samples are analyzed, report
all data obtained with the sample
results.
14.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits established in Section
8.4, data for the affected parameters
must be labeled as suspect.
15. Method Performance
15.1 Method detection limits -
The method detection limit (MDL) is
defined as the minimum concen-
tration of a substance that can be
measured and reported with 99%
confidence that the value is above
zero'11. The MDL concentrations listed
in Table l.were obtained using
reagent water11-1'. Similar results
were achieved using representative
wastewaters. MDL for the GC
approach were not determined.
15.2 This method has been tested
for linearity of recovery from spiked
reagent water and has been
demonstrated to be applicable over
the concentration range from 8 x MDL
to 800 x MDL(11>, with the following
exception: benzo(ghi)perylene recovery
at 80 x and 800 x MDL were low
(35% and 45% respectively).
15.3 In a single laboratory (Battelle
Columbus Laboratories), using spiked
wastewater samples, the average
recoveries presented in Table 3 were
obtained . Each spiked sample was
analyzed in triplicate on two separate
days. The standard deviation of the
precent recovery is also included in
Table 3.
15.4 The U.S. Environmental
Protection Agency is in the process
of conducting an interlaboratory
method study to fully define the
performance of this method.
References
1. See Appendix A.
2. Determination of Polynuclear
Aromatic Hydrocarbons in
Industrial and Municipal
Wastewaters," Report for EPA
Contract 68-03-2624
(In preparation).
3. ASTM Annual Book of
Standards, Part 31, D 3694.
"Standard Practice for
Preparation of Sample
Containers and for Preserva-
tion," American Society for
Testing and Materials,
Philadelphia, PA, p. 679, 1 980.
4. "Carcinogens - Working With
Carcinogens," Department of
Health, Education, and Welfare,
Public Health Service, Center for
Disease Control, National Insti-
tute for Occupational Safety and
Health, Publication No. 77-206,
Aug. 1977.
5. "OSHA Safety and Health Stan-
dards, General Industry,"
(29CFR-1910), Occupational
Safety and Health Adminis-
tration, OSHA 2206, (Revised,
January 1 976).
6. "Safety in Academic Chemistry
Laboratories," American Chem-
ical Society Publication, Com-.
mittee on Chemical Safety,
3rd Edition, 1979. , -
7. "Handbook of Analytical Quality
Control in Water and Waste-
water Laboratories," EPA-600/
4-79-019, U.S. Environmental
Protection Agency Environ-
mental Monitoring and Support
Laboratory, Cincinnati, Ohio
45268, March 1979.
8. ASTM Annual Book of
Standards, Part 31, D 3370,
"Standard Practice for Sampling
Water," American Society
for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
610-7
July 1982
-------
9. "Methods 330.4 (Titrimetric,
DPD-FAS) and 330.5 (Spectre-
photometric, DPD) for Chlorine,
Total Residual/' Methods for
Chemical Analysis of Water and
Wastes, EPA 600-4/79-020,
U.S. Environmental Protection
Agency, Environmental Moni-
toring and Support Labora-
tory, Cincinnati, Ohio 45268,
March 1979.
10. Burke, J. A., "Gas Chromatog-
raphy for Pesticide Residue
Analysis; Some Practical
Aspects," Journal of the Asso-
ciation of Official Analytical
Chemists, 45,1037 (1965).
11. Cole, T., Riggins, R., and Glaser,
J., "Evaluation of Method Detec-
tion Limits and Analytical Curve
for EPA Method 610 - PNAs,"
International Symposium on
Polynuclear Aromatic Hydro-
carbons, 5th, Battelle Columbus
Laboratory, Columbus, Ohio
(1980).
Table 1. High Performance Liquid Chromatography Conditions and Method
Detection Limits
Parameter
Naphthalene
Acenaphthylene
Acenaphthene
Fluor en e
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzofajan thra cene
Chrysene
Benzo(b)fluoranthene
Benzofkjflouranthene
Benzof ajpyrene
Dibenzo(a,h)anthracene
Benzo(ghi)perylene
lndeno(1 ,2,3-cdjpyrene
Retention Time
(min)
16.6
18.5
20.5
21.2
22.1
23.4
24.5
25.4
28.5
29.3
31.6
32.9
33.9
35.7
36.3
37.4
Capacity
Factor
(k')
12.2
13.7
15.2
15.8
16.6
17.6
18.5
19.1
21.6
22.2
24.0
25.1
25.9
27.4
27.8
28.7
Method
Detection Limit
(W/Lf
1.8
2.3
1.8
0.21
0.64
O.66
0.21
0.27
0.013
0.15
0.018
0.017
O.O23
0.030
0.076
0.043
HPLC conditions: Reverse phase HC-ODS Sil-X 2.6 mm x 250 mm Perkin-Elmer
column; isocratic elution for 5 min using acetonitrile/water (4 + 6), then linear
gradient elution to 1OO% acetonitrile over 25 minutes; flow rate is 0.5 mL/min.
If columns having other internal diameters are used, the flow rate should be
adjusted to maintain a linear velocity of 2 mm/sec.
aThe method detect/on limit for naphthalene, acenaphthylene, acenaphthene,
and fluorene were determined using a UV detector. All others were
determined using a fluorescence detector.
Table 2. Gas Chromatographic
Operating Conditions
and Retention Times
Parameter Retention Time
(min)
Naphthalene 4.5
A cenaphth ylene 10.4
Acenaphthene 10.8
Fluorene 12.6
Phenanthrene 15.9
Anthracene 15.9
Fluoranthene 19.8
Pyrene 20.6
Benzo(a)anthracene 20.6
Chrysene 24.7
Benzo(blfluoranthene 28.0
Benzo(k)fluoranthene 28.0
Benzo(a)pyrene 29.4
Dibenzo(a,hjanthracene 36.2
Indenod',2,3-cdjpyrene 36.2
Benzo(ghi)perylene 35.6
GC conditions: Chromosorb W-AW-
DCMS (100/120 mesh) coated with
3% OV-17, packed in a 1.8 m long x
2 mm ID glass column, with nitrogen
carrier gas at a flow rate of 40 mL/
min. Column temperature was held
at 100°C for 4 min, then pro-
grammed at 8°/minute to a final
hold at 280°C.
610-8
July 1982
-------
Table 3 Single Operator Accuracy and Precision
Average
Parameter Percent
Recovery
Acenaphthene
A cenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b)fluoranthene
Benzo(ghi)perylene
Benzo(k)fluoranthene
Chrysene
Dibenzo(a,h)anthracene
Fluoranthene
Fluoren e '.
lndeno(1 ,2,3-cd)pyrene ,
Naphthalene ,
Phenanthrene
Pyrene
88
93
93
89
94
97
86
94
88
87
116
90
94
78
98
96
Standard
Deviation
%
5.7
6.4
6.3
6.9
7.4
12.9
7.3
9.5
9.0
5.8
9.7
7.9
6.4
8.3
8.4
8.5
Spike Number
Range of
f/jg/L) Analyses
11.6-25
250-450
7.9-11.3
0.64-0.66
O.21-O.3O
O.24-O.30
0.42-3.4
0.14-6.2
2.0-6.8
0.4-1.7
0.3-2.2
6.1-23
0.96-1.4
20-70
3.8-5.0
2.3-6.9
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
Matrix
Types
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Column: HC-ODS SIL-X
Mobile phase: 40% to, 100% Acetonitrile in water
Detector: Ultra violet at 254nm
12 16 20 24 28
Retention time, minutes
32 36
Figure 1 . Liquid chromatogram of polynuclear
aromatic hydrocarbons.
610-9
July 1982
-------
Column: HC-ODS SIL-X
Mobile phase: 40% to 100% Acetonitrile
in water
Detector: Fluorescence
8 12 16 20 24 28
Retention time, minutes
32 36
Figure 2. Liquid chromatogram of polynuclear
aromatic hydrocarbons.
Column: 3% OV-17 on Chromosorb W-AW-DCMS
Program: 100°C. 4 min.,8° per min. to 280°C.
Detector: Flame ionization
4 8
12 16 20 24 28
Retention time, minutes
32 36
610-10
Figure 3. Gas chromatogram of polynuclear
aromatic hydrocarbons.
July 1982
-------
vvEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Haloethers —
Method 611
1. Scope and Application
1.1 This method covers the
determination of certain haloethers.
The following parameters can be
determined by this method:
Parameter
Bis(2-chloroethyl) ether
Bis(2-chloroethoxy) methane
Bis(2-chloroisopropyl) ether
4-Bromophenyl phenyl ether
4-Chlorophenyl phenyl ether
STORET No.
34273
34278
34283
34636
34641
CAS No.
1 1 1 -44-4
111-91-1
108-60-1
101-55-3
7005-72-3
1.2 This is a gas chromatographic
(GC) method applicable to the
determination of the compounds listed
above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any
or all of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. This method
describes analytical conditions for a
second GC column that can be used
to confirm measurements made
with the primary column. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of
results for all of the parameters
listed above, using the extract
from this method.
1.3 The method detection limit
{MDL, defined in Section 14.1)11' for
each parameter is listed in Table 1.
The MDL for a specific wastewater
may differ from that listed, depending
upon the nature of interferences in
the sample matrix.
1.4 The sample extraction and
concentration steps in this method are
essentially the same as in methods
606, 608, 609, and 612. Thus, a
single sample may be extracted to
measure the parameters included in
the scope of each of these methods.
When cleanup is required, the
concentration levels must be high
enough to permit selecting aliquots,
as necessary, to apply appropriate
cleanup procedures. The analyst is.
allowed the latitude, under Gas
Chromatography (Section 12), to
select chromatographic conditions
appropriate for the simultaneous
measurement of combinations of
these parameters.
1.5 Any modification of this method,
beyond those expressly permitted,
shall be considered as major
modifications subject to application
and approval of alternate test
procedures under 40 CFR 136.4
and 136.5. •>
1.6 This method is restricted to
use by or under the supervision of
analysts experienced in the use of
611-1
July 1982
-------
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 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is solvent
extracted with methylene chloride
using a separatory funnel. The
methylene chloride extract is dried
and exchanged to hexane during
concentration to a volume of 10 mL or
less. GC conditions are described
which permit the separation and
measurement of the compounds in
the extract using a halide
specific detector21.
2.2 The method provides a Florisil
column cleanup procedure to aid in
the elimination of interferences that
may be encountered.
3. Interferences
3.1 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 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 as described in
Section 8.5.
3.1.1 Glassware must be
scrupulously cleaned.131 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 reagent
water. It should then be drained dry,
and heated in a muffle furnace at
400°C for 15 to 30 minutes. Some
thermally stable materials, such as
PCBs, may not be eliminated by this
treatment. Solvent rinses with
acetone and pesticide quality hexane
may be substituted for the muffle
furnace heating. Volumetric ware
should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and
stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted or
capped with aluminum foil.
3.1.2 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.
3.2 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 industrial complex
or municipality being sampled. The
cleanup procedures in Section 11 can
be used to overcome many of these
interferences, but unique samples
may require additional cleanup
approaches to achieve the MDL listed
in Table 1.
3.3 Dichlorobenzenes are known to
coelute with haloethers under some
gas chromatographic conditions. If
these materials are present together
in a sample, it may be necessary to
analyze the extract with two different
column packings to completely resolve
all of the compounds.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals specified inithis method.
A reference file of material data
handling 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. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling.
5.1.1 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive.
If amber bottles are not available,
protect samples from light. The
container must be washed, rinsed
with acetone or methylene chloride,
and dried before use to minimize
contamination.
5.7.2 Automatic sampler (optional) -
Must incorporate glass sample
containers for the collection of a
minimum of 250 mL Sample
containers must be kept refrigerated
at 4°C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however,
the compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.1 Separatory funnel - 2000-mL
with Teflon stopcock.
5.2.2 Drying column -
Chromatographic column 400 mm
long x 19 mm ID, with coarse frit.
5.2.3 Chromatographic column -
400-mm long x 19 mm ID glass with
coarse fritted plate on bottom and
Teflon stopcock (Kontes K-420540-
0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-
Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent).
Calibration must be checked at the
volumes employed in the test. Ground
glass stopper is used to prevent
evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-
Danish - 500-mL (Kontes K-570001-
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.6 Snyder column, Kuderna-
Danish - Three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Vials - Amber glass, 10- to
15r mL capacity, with Teflon-lined
screwcap.
5.3 Boiling chips - Approximately
10/40 mesh. Heat to 400°C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath - Heated, with
concentric ring cover, capable of
temperature control (±2°C). The bath
should be used in a hood.
5.5 Balance - Analytical, capable of
accurately weighing 0.0001 g.
5.6 Gas chromatograph - An
analytical system complete with
temperature programmable gas
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical columns,
gases, detector, and strip-chart
recorder. A data system is recom-
mended for measuring peak areas.
5.6.1 Column 1 - 1.8 m long x 2
mm ID pyrex glass, packed with
Supelcoport, (100/120 mesh) coated
611-2
July 1982
-------
with 3% SP-1000 or equivalent. This
column was used to develop the
method performance statements in
Section 14. Guidelines for the use of
alternate column packings are
provided in Section 12.1.
5.6.2 Column 2 - 1.8 m long x 2
mm ID pyrex glass, packed with
Tenax-GC (60/80 mesh) or
equivalent.
5.6.3 Detector - Halide specific:
electrolytic conductivity or
microcoulometric. These detectors
have proven effective in the analysis
of wastewaters for the parameters
listed in the scope of this method. The
Hall conductivity detector was used to
develop the method performance
statements in Section 14. Guidelines
for the use of alternate detectors are
provided in Section 12.1. Although
less selective, an electron capture
detector is an acceptable alternative.
6. Reagents
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Sodium thiosulfate - (ACS)
Granular.
6.3 Acetone, methanol, methylene
chloride, hexane, and petroleum ether
(boiling range 30 to 60°C) - Pesticide
quality or equivalent.
6.4 Sodium sulfate - (ACS)
Granular, anhydrous. P;urify by
heating at 400°C for four hours in
a shallow tray.
6.5 Florisil - PR Grade (60/100
mesh); purchase activated at 1250°F
and store in the dark in glass
container with glass stoppers or foil-
lined screw caps. Before use, activate
each batch overnight at 130°C in a
foil-covered glass container.
6.6 Ethyl ether - Nanograde,
redistilled in glass, if necessary.
6.6.1 Must be free of peroxides as
indicated by EM Laboratories Quant
test strips. (Available from Scientific
Products Co., Cat. No. P1126-8, and
other suppliers.)
6.6.2 Procedures recommended for
removal of peroxides are provided
with the test strips. After cleanup 20
mL ethyl alcohol preservative must be
added to each liter of ether.
6.7 Stock standard solutions (1.00
fig/fjL) - Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.7.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 g of pure material.
Dissolve the material in pesticide
quality acetone and dilute to volume
in a 10-mL volumetric flask. Larger
volumes can be used at the
convenience of the analyst. If
compound purity is certified at 96%
or greater, the weight can be used
without correction to calculate the
concentration of the stock standard.
Commercially prepared stock
standards can be used at any
concentration if they are certified
by the manufacturer or by an
independent source.
6.7.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards will be available for the
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio 45268.
6.7.3 Stock standard solutions must
be replaced after six months, or
sooner if comparison with check
standards indicate a problem.
7. Calibration
7.1 Establish gas chromatographic
operating parameters to produce
retention times equivalent to those
listed in Table 1. The GC chromato-
graphic system may be calibrated
using the external standard technique
(Section 7.2) or the internal standard
technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with hexane.
One of the external standards should
be at a concentration near, but above,
the MDL and the other concentra-
tions should correspond to the
expected range of concentrations
found in real samples or should
define the working range of the
detector.
7.2.2 Using injections of 2 to 5 /jL of
each calibration standard, 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
calibration curve.
7.2.3 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 any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested
that is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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 internal standards, and dilute to
volume with hexane. One of the
standards should be at a concentra-
tion near, but above, the MDL 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.
7.3.2 Using injections of 2 to 5 //L
of each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1 RF = (AsC,s)/(AisCs)
where:
As = Response for the parameter to
be measured.
Als = Response for the internal
standard.
Cis = Concentration of the internal
standard, (//g/L).
Cs = Concentration of the parameter
to be measured, (/ug/L).
611-3
July 1982
-------
If the RF value over the working range
Is a constant « 10% RSD), the RF can
be assumed to be invariant and the
average RF can be used for calcula-
tions. Alternatively, the results can
be used to plot a calibration curve of
response ratios, As/AiS, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of
interferences from the reagents.
7.5 The cleanup procedure in
Section 11 utilizes Florisil column
chromatography. Florisil from different
batches or sources may vary in
adsorption capacity. To standardize
the amount of Florisil which is used,
the use of lauric acid value171 is
suggested. The referenced procedure
determines the adsorption from
hexane solution of lauric acid (mg) per
gram Florisil. The amount of Florisil to
be used for each column is calculated
by dividing 110 by this ratio and
multiplying by 20 g.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist
of an initial demonstration of
laboratory capability and the analysis
of spiked samples as a continuing
check on performance. The laboratory
is required to maintain performance
records to define the quality of data
that is generated. Ongoing
performance checks must be
compared with established
performance criteria to determine if
the results of analyses are within
accuracy and precision limits expected
of the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method. This ability is
established as described in Section
8.2
8.1.2 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform
the following operations!.
8.2.1 Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be
used in place of the reagent water,
but one or more additional aliquots
must be analyzed to determine
background levels, and the spike level
must exceed twice the background
level for the test to be valid. Analyze
the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater back-
ground corrections must be made
before R and s calculations are
performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or |X-R| > 2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental
Protection Agency plans to
establish performance criteria for
R and s based upon the results of
interlaboratory testing. When they
become available, these criteria must
be met before any samples may be
analyzed.
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter being
measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R — 3 s
where R and s are calculated as in
Section 8.2.3. The UCL and LCL can
be used to construct control charts
that are useful in observing trends in
performance. The control limits above
must be replaced by method per-
formance criteria as they become
available from the U.S. Environmental
Protection Agency.
3.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined
as R ± s. The accuracy statement
should be developed by the analysis of
four aliquots of wastewater as
described in Section 8.2.2, followed
by the calculation of R and s.
Alternately, the analyst may use four
wastewater data points gathered
through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly'8'
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of
the sample must be spiked and
analyzed as described in Section 8.2
If the recovery for a particular
parameter does not fall within the
control limits for method performance,
the results reported for that parameter
in all samples processed as part of
the same set must be qualified as
described in Section 13.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagent interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as
a safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
611-4
July 1982
-------
assurance practices forl use with this
method. The specific practices that
are most productive depend upon the
needs of the laboratory and the nature
of the samples. Field duplicates may
be analyzed to monitor the precision!
of the sampling technique. When
doubt exists over the identification of
a peak on the chromatogram,
confirmatory techniques such as GC
with a dissimilar column, specific
element detector, or mass spec-
trometer must be used. Whenever
possible, the laboratory should
perform analysis of standard
reference materials and participate
in relevant performance evaluation
studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices191 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 a free as possible of Tygon and
other potential sources of
contamination.
9.2 The samples must be iced or
refrigerated at 4°C from the time of
collection until extraction. Fill the
sample bottles and, if residual
chlorine is present, add 80 mg of
sodium thiosulfate per each liter of
water. U.S. Environmental Protection
Agency methods 330.4 and 330.5
may be used to measure the residual
chlorine1101. Field test kits are available
for this purpose.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction .
10. Sample Extraction
10.1 Mark the water meniscus on
the side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel.
10.2 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner walls.
Transfer the solvent to the separatory
funnel and extract the sample by
shaking the funnel for two minutes
with periodic venting .to release
excess pressure. Allow the organic
layer to separate from the water
phase for a minimum of 10 minutes.
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 250-mL
Erlenmeyer flask.
10.3 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.
10.4 Assemble a Kuderna-Danish
(K-D) concentrator by attaching a 10-
ml_ concentrator tube to a 500-mL
evaporative flask. Other concen-
tration devices or techniques may
be used in place of the K-D if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract
through a drying column containing
about 10 cm of anhydrous sodium
sulfate, and collect the extract in the
K-D concentrator. Rinse the
Erlenmeyer flask and column with 20
to 30 mL of methylene chloride to
complete the quantitative transfer.
10.6 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 1 mL 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 15 to 20 minutes. 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 1
mL, remove the K-D apparatus and
allow it to drain and cool for at least
10 minutes.
NOTE: Some of the haloethers are
very volatile and significant losses will
occur in concentration steps if care is
not exercised. It is important to
maintain a constant gentle
evaporation rate and not to allow the
liquid volume to fall below 1 to 2 mL
before removing the K-D from the hot
water ba.th.
10.7 Momentarily remove the
Snyder column, add 50 mL of hexane
and a new boiling chip and replace
the column. Raise the temperature
of the water bath to 85 to 90°C.
Concentrate the extract as in Section
10.6 except use hexane to prewet the
column. When the apparent volume of
liquid reaches 1 to 2 mL, remove the
K-D and allow it to drain and cool at
least 10 minutes. Remove the Snyder
column and rinse the flask and its
lower joint into the concentrator tube
with 1 to 2 mL of hexane. A 5-mL
syringe is recommended for this
operation. Stopper the concentrator
tube and store refrigerated if further
processing will not be performed
immediately. If the extracts will be
stored longer than two days, they
should be transferred to Teflon-sealed
screw-cap bottles.
10.8 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. Cleanup and Separation
11.1 Cleanup procedures may not
be necessary for a relatively clean
sample matrix. The cleanup procedure
recommended in this method has been
used for the analysis of various clean
waters and industrial effluents. If
particular circumstances demand the
use of an alternative cleanup pro-
cedure, the analyst must determine
the elution profile and demonstrate
that the recovery of each compound
of interest is no less than 85%.
11.2 Florisil column cleanup for
haloethers:
71.2.1 Adjust the sample extract
volume to 10 mL.
11.2.2 Place a charge (nominally
20 g, actual amount determined as in
Section 7.5) of activated Florisil in a
19-mm ID chromatographic column.
After settling the Florisil by tapping
the column, add about one-half inch
layer of anhydrous granular sodium
sulfate to the top. Allow the Florisil
to cool.
7 7.2.3 Pre-elute the column with
50 to 60 mL of petroleum ether.
Discard the eluate and just prior to
exposure of the sulfate layer to air,
quantitatively transfer the sample
extract into the column by decantation
and subsequent petroleum ether
washings. Discard the eluate. Just
prior to exposure of the sodium
sulfate layer to the air,, begin eluting
the column with 300 mL of ethyl
ether/petroleum ether (6 + 94) (V/V).
Adjust the elution rate to approx-
611-5
July 1982
-------
innately 5 mL/min and collect the
eluate in a 500-mL K-D flask
equipped with a 10-mL concentrator
tube. This fraction should contain all
of the haloethers.
11.2,4 Concentrate the fraction by
K-D as in Section 10.6 except prewet
the Snyder column with hexane.
When the apparatus is cool, remove
the column and rinse the flask and
its lower joint into the concentrator
tube with hexane. Adjust the volume
to 10 ml. Analyze by GC (Section 12.)
12. Gas Chromatography
12.1 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. This table
includes retention times and MDL
that were obtained under these
conditions. Examples of the
parameter separations achieved
by these columns are shown in
Figures 1 and 2. Other packed
columns, chromatographic conditions,
or detectors may be used if the
requirements of Section 8.2 are met.
Capillary (open-tubular) columns may
also be used if the relative standard
deviations of responses for replicate
injections are demonstrated to be less
than 6% and the requirements of
Section 8.2 are met.
12.2 Calibrate the system daily as
described in Section 7.
12.3 If the internal standard
approach is being used, the analyst
must not add the internal standard to
sample extracts until immediately
before injection into the instrument.
Mix thoroughly.
12.4 Inject 2 to 5/uL of the sample
extract using the solvent-flush
technique". Smaller (LOjuL) volumes
can be injected if automatic devices
are employed. Record the extract
volume to the nearest 0.1 mL and the
volume injected to the nearest 0.05
(A., and the resulting peak size in area
or peak height units.
12.5 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.
12.6 If the response for the peak
exceeds the working range of the
system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak
response is prevented by the presence
of interferences, further cleanup is
required.
13. Calculations
13.1 Determine the concentration of
individual compounds in the sample.
13. 1. 1 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 in Section 7.2.2. The
concentration in the sample can be
calculated from equation 2:
Eq. 2. Concentration, Jug/L= (Vi)(Vs)
where:
A = Amount of material injected, in
nanograms.
Vi = Volume of extract injected (yuL).
Vt = Volume of total extract (jjL).
Vs = Volume of water extracted (mL).
13.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
equation 3.
„ (As)ds)
Eq. 3. Concentration,//g/L=(A.s)(RF)(v0)
where:
As = Response for the parameter to
be measured.
Ais = Response for the internal
standard.
ls = Amount of internal standard
added to each extract (/ug).
V0 = Volume of water extracted, in
liters.
73.2 Report results in micrograms
per liter without correction for
recovery data. When duplicate and
spiked samples are analyzed, report
all data obtained with the sample
results.
13.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in Section 8.4, data tor
the affected parameters must be
labeled as suspect.
14. Method Performance
14.1 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
zero111. The MDL concentrations listed
in Table 1 were obtained using
reagent water1121. Similar results were
achieved using representative
wastewaters.
14.2 This method has been tested
for linearity of recovery from spiked
reagent water and has been
demonstrated to be applicable for the
concentration range from 4X MDL to
1000 x MDL'121.
14.3 In a single laboratory
(Monsanto Research Center), using
spiked wastewater samples, the
average recoveries presented in Table
2 were obtained'2'. Each spiked
sample was analyzed in triplicate on
three separate occasions. The
standard deviation of the percent
recovery is also included in Table 2.
14.4 The U.S. Environmental
Protection Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. "Determination of Haloethers
in Industrial and Municipal
Wastewaters." Report for EPA
Contract 68-03-2633 (In
preparation).
3. ASTM Annual Book of
Standards, Part 31, D 3694.
"Standard Practice for
Preparation of Sample
Containers and for
Preservation," American
Society for Testing and
Materials, Philadelphia, PA,
p. 679, 1980.
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,"
(29CFR1910), 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.
611-6
July 1982
-------
7. Mills, P.A., "Variation of
Florisil Activity: Simple Method
for Measuring Absorbent
Capacity and It's Use in
Standardizing Florisil
Columns," Journal of the
Association of Official
Analytical Chemists, 51, 29
(1968).
8. "Handbook of Analytical
Quality Control in Water and
Wastewater Laboratories,"
EPA-600/4-79r019, U.S.
Environmental Protection
Agency, Environmental
Monitoring and Support
Laboratory, Cincinnati, Ohio
45268, March 1979.
9. ASTM Annual Book of
Standards, Part 31, D 3370,
"Standard Practice for
Sampling Water," American
Society for Testing and
Materials, Philadelphia, PA,
p. 76, 1980.
10. "Methods 330J4 (Titrimetric,
DPD-FAS)and;330.5
(Spectrophotometric, DPD) for
Chlorine, Total Residual,"
Methods for Chemical Analysis
of Water and Wastes, EPA
600-4/79-020; U.S.
Environmental -Protection
Agency, Environmental
Monitoring and Support
Laboratory, Cincinnati, Ohio
45268, March 1979.
i
11. Burke, J. A., "Gas
Chromatography for Pesticide
Residue Analysis; Some
Practical Aspects," Journal of
the Association of Official
Analytical Chemists, 48,
1037(1965). '
12. "EPA Method Validation Study
21 Method 611 (Haloethers),"
Report for EPA Contract 68-
03-2633 (In Preparation).
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
Bis(2-chloroisopropyl) ether
Bis(2-chloroethyl) ether
Bis(2-chloroethoxy) methane
4-Chlorophenyl phenyl ether
4-Bromophenyl phenyl ether
Retention Time
(min.)
Column 1 Column 2
8.4 9.7
9.3 9.1
13.1 10.0
19.4 15.0
21.2 16.2
Method
Detection Limit
ffjg/L)
0.8
0.3
0.5
3.9
2.3
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-1000
packed in 1.8 m long x 2 mm ID glass column with helium carrier gas at a flow
rate of 40 mL/min. Column temperature: 60°C for 2 min after injection then
program at 8°C/min to 230°C and hold for 4 min. Under these conditions
the retention time for Aldrin is 22.6 min.
Column 2 conditions: Tenax-GC (60/80 mesh) packed in a 1.8 m long/x 2mm
ID glass column with helium carrier gas at 40 mL/min flow rate. Column
temperature: 150°C for 4 min after injection then program at 16°C/min to
310°C. Under these conditions the retention time for Aldrin is 18.4 min.
Table 2. Single Operator Accuracy and Precision
Parameter
Average
Percent
Recovery
Standard
Deviation
Spike Number
Range of Matrix
(jjg/L) Analyses Types
Bis(2-chloroethyl)ether 59 4.5 97 27 3
Bis(2-chloroethoxy)methane 62 5.3 138 27 3
Bis(2-chloroisopropyl)ether 67 4.O 54 27 3
4-Bromophenyl phenyl ether 78 3.5 14 27 3
4-Chlorophenyl phenyl ether 73 4.5 30 27 3
611-7
July 1982
-------
Column: 3% SP-1000 on Supelcoport
Program: 60°C.-2 minutes 8°/minute to 230°C.
Detector: Hall electrolytic conductivity
24 6 8 10 12 14 16 18 2O 22 24
Retention time, minutes
Figure 1. Gas chromatogram of haloethers.
Column: Tenax GC
Program: 150°C.-4 minutes 16°/minute to 310°C.
Detector: Hall electrolytic conductivity
_L
_L
611-8
O 4 8 12 16 2O
Retention time, minutes
Figure 2. Gas chromatogram of haloethers.
July 1982
24
-------
&EPA
United States
Environmental Protection
Agency
Environmental Monitormg and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Chlorinated Hydrocarbons
Method 61 2
1. Scope and Application
1.1 This method covers the
determination of certain chlorinated
hydrocarbons. The following
parameters can be determined by this
method:
Parameter
STORET No.
CAS No.
2-Chloronaphthalene
1 ,2-Dichlorobenzene
1 ,3-Dichlorobenzene
1 ,4-Dichlorobenzene
Hexachlorobenzene
Hexachlorobutadiene
Hexachlorocyclopentadiene
Hexachloroethane
1 ,2,4-Trichlorobenzene
34581
34536
34566
34571
39700
34391
34386
34396
34551
91-58-7
95-50-1
541-73-1
106-46-7
118-74-1
87-68-3
77-47-4
67-72-1
120-82-1
1.2 This is a gas chromatographic
(GC) method applicable to the
determination of the compounds listed
above in municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any or
all of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions
appropriate for the qualitative and
quantitative confirmation of results
for all of the parameters listed above,
using the extract produced by this
method.
1.3 The method detection limit
(MDL defined in Section 14.1)m for
each parameter is listed in Table 1.
The MDL for a specific wastewater
may differ from that listed, depending
upon the nature of interferences in
the sample matrix.
1.4 The sample extraction and
concentration steps in this method
are essentially the same as in
methods 606, 608, 609, and 611.
Thus, a single sample may be
extracted to measure the parameters
included in the scope of each of these
methods. When cleanup is required,
the concentration levels must be high
enough to permit selecting aliquots,
as necessary, to apply appropriate
cleanup procedures. The analyst is
allowed the latitude, under Gas
Chromatography (Section 12), to
select chromatographic conditions
appropriate for the simultaneous
measurement of combinations of
these parameters, provided that the
requirements of Section 8.2 are met.
1.5 Any modification of this method,
beyond those expressly permitted,
shall be considered as major
modifications subject to application
and approval of alternate test
procedures under 40 CFR 136.4
and 136.5.
612-1
July 1982
-------
1.6 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 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is solvent
extracted with methylene chloride
using a separatory funnel. The
methylene chloride extract is dried
and solvent exchanged to hexane
during concentration to a volume of
10 mL or less. GC conditions are
described which permit the
separation and measurement of
the compounds in the extract using
an ECD'5".
2.2 The method provides a Florisil
column cleanup procedure to aid in
the elimination of interferences that
may be encountered.
3. Interferences
3.1 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 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 as described in
Section 8.5.
3.1.1 Glassware must be
scrupulously cleaned'31. 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. It should then be
drained dry, and heated in a muffle
furnace at 400°C for 15 to 30
minutes. Some thermally stable
materials, such as PCBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Volu-
metric ware should not be heated
in a muffle furnace. After drying and
cooling, glassware should be sealed
and stored in a clean environment to
prevent any accumulation of dust or
other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 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.
3.2 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 industrial complex
or municipality being sampled. The
cleanup procedure in Section 11 can
be used to overcome many of these
interferences, but unique samples
may require additional cleanup
approaches to achieve the detection
limits listed in Table 1.
4. Safety
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 awareness file
of OSHA regulations regarding the
safe handling of the chemicals
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. Additional references to
laboratory safety are available and
have been identified14"61 for the
information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling.
5.1.1 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive.
If amber bottles are not available,
protect samples from light. The
container must be washed, rinsed
with acetone or methylene chloride,
and dried before use to; minimize
contamination.
5.1.2 Automatic sampler (optional) -
Must incorporate glass sample
containers for the collection of a
minimum of 250 mL. Sample
containers must be kept refrigerated
at 4°C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber may be
used. Before use, however, the
compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter
is required to collect flow
proportional composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only). ,
5.2.1 Separatory funnel - 2000-mL,
with Teflon stopcock.
5.2.2 Drying column -
Chromatographic column 400 mm
long x 19 mm ID with coarse frit.
5.2.3 Concentrator tube, Kuderna-
Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent).
Calibration must be checked at the
volumes employed in-the test. Ground
glass stopper is used to prevent
evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-
Danish - 500-mL (Kontes K-570001-
0500 or equivalent). Attach to
concentrator tube with springs.
5.2.5 Snyder column, Kuderna-
Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Snyder column, Kuderna-
Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.7 Vials - Amber glass, 10- to
15- mL capacity, with Teflon- fined
screwcap.
5.2.8 Chromatography column -
300 mm long x 10 mm ID with coarse
fritted disc at bottom and Teflon
stopcock.
5.3 Boiling chips - approximately
10/40 mesh. Heat to 400°C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath - Heated, with
concentric ring qover, capable of
temperature control (± 2°C). The bath
should be used in a hood.
5.5 Balance - Analytical, capable of
accurately weighing 0.0001 g.
5.6 Gas chromatograph - An
analytical system complete with gas
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical
columns, gases, ECD, and strip-chart
recorder. A data system is
recommended for measuring
peak areas.
5.6.1 Column - 1.8 m long x 2 mm
ID pyrex glass, packed with 1.5% OV-1
+ 2.4% OV-225 on Supelcoport (80/
100 mesh) or equivalent. This column
was used to develop the method
performance statements in Section
5/2-2
July 1982
-------
14. Guidelines'for the use of alternate
column packings are provided in
Section 12.1
5.6.2 Detector - Electron capture.
This detector has proven effective in
the analysis of wastewaters for the
parameters listed in the scope, and
was used to develop the accuracy and
precision statements in Section 14.
Guidelines for the use of'alternate
detectors are provided in Section
12.1.
6. Reagents
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Acetone, methanol, methylene
chloride, hexane, petroleum ether
(Boiling range 30 to 60°C) - Pesticide
quality or equivalent.
6.3 Sodium sulfate - (ACS)
Granular, anhydrous. Purify by
heating at 400°C for four hours in
a shallow tray.
6.4 Florisil - PR grade (60/100
mesh); purchase activated at 1250°F
and store in the dark in glass
containers with glass stoppers or foil-
lined screw caps. Before use, activate
each batch at, 130°C in foil-covered
glass containers.
6.5 Stock standard solutions (1.00
yug/juL) - Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.5.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 g of pure material.
Dissolve the material in pesticide
quality isooctane, dilute to volume
in a 10-mL volumetric flask. Larger
volumes can be used at the
convenience of the analyst. If
compound purity is certified at 96%
or greater, the weight can be used
without correction to calculate the
concentration of the stock standard.
Commercially prepared stock
standards can be used at'any
concentration if they are Certified
by the manufacturer or by an
independent source.
6.5.2 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 frorn them.
Quality control check standards that
can be used to determine the
accuracy of calibration standards
will be available from the U.S.
Environmental Protection Agency,
Environmental Monitoring and
Support Laboratory, Cincinnati,
Ohio, 45268.
6.5.3 Stock standard solutions must
be replaced after six months, or
sooner if comparison with check
standards indicate a problem.
7. Calibration
7.1 Establish GC operating
conditions to produce resolution
of the parameters equivalent to
those indicated in Table 1. The GC"
system may be calibrated using
external standard technique
(Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with isooctane.
One of the external standards should
be at a concentration near, but above,
the MDL and the other concen-
trations should correspond to
the expected range of concen-
trations found in real samples
or should define the working range
of the detector.
7.2.2 Using injections of 2 to 5 fjL
of each calibration standard, 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
calibration curve.
7.2.3 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 any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration
curve or calibration factor must be
prepared for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested
that is applicable to all samples.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter 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 internal standards, and dilute to
volume with isooctane. One of the
standards should be at a concen-
tration near, but above, the MDL
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.
7.3.2 Using injections of 2 to 5 fjL
of each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1 RF = (AsCis)/(AisCs)
where:
As = Response for the parameter to
be measured.
Ais = Response for the internal
standard.
ds = Concentration of the internal
standard, (/ug/L).
Cs = Concentration of the parameter
to be measured, (/ug/L).
If the RF value over the working range
is a constant « 10% RSD), the RF
can be assumed to be invariant
and 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. RF.
7.3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
7.4 Before using any cleanup
procedure, the analyst must process a
•series of calibration standards through
the procedure to validate elution
patterns and the absence of
interferences from the reagents.
612-3
July 1982
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8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist
of an initial demonstration of
laboratory capability and the analysis
of spiked samples as a continuing
check on performance. The laboratory
is required to maintain performance
records to define the quality of data
that is generated. Ongoing perform-
ance checks must be compared
with established performance
criteria to determine if the results
of analyses are within accuracy
and precision limits expected of
the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method. This ability is
established as described in Section
8.2.
8.1.2 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
5.7.5 The laboratory must spike and
analyze a minimum of 10% of all
samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
8,2.2 Using a pipet, add 1.00 mL
of the check sample concentrate to
each of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be.
used in place of the reagent water but
one or more additional aliquots must
be analyzed to determine background
levels, and the spike level must
exceed twice the background level
for the test to be valid, Analyze the
aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater back-
ground corrections must be made
before R and s calculations are
performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or |X-R| > 2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental
Protection Agency plans to
establish performance criteria for
R and s based upon the results of
interlaboratory testing. When they
become available, these criteria must
be met before any samples may be
analyzed.
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter being
measured.
8.3.1 Calculate upper and lower
control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in
Section 8.2.3.
The UCL and LCL can be used to
construct control charts'71 that are
useful in observing trends in perfor-
mance. The control limits above must
be replaced by method performance
criteria as they become available
from the U.S. Environmental Protection
Agency.
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined
as R ± s. The accuracy statement
should be developed by the analysis
of four aliquots of wastewater as
described in Section 8.2.2, followed
by the calculation of R and s.
Alternately, the analyst may use four
wastewater data points gathered
through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly171,
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all,
samples or one sample per month,
whichever is greater. One aliquot of
the sample must be spiked and
analyzed as described in Section 8.2. If
the recovery for a particular parameter
does not fall within the control limits
for method performance, the results
reported for that parameter in all
samples processed as part of the
same set must be qualified as
described in Section 13.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of one-liter
aliquot of reagent water, that all
glassware and reagents interferences
are under control. Each time a set
of samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed
as a safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may
be analyzed to monitor the precision
of the sampling technique. When
doubt exists over the identification of
a peak on the chromatogram,
confimatory techniques such as gas
chromatography with a dissimilar
column, specific element detector,
or mass spectrometer must be used.
Whenever possible, the laboratory
should perform analysis of standard
reference materials and participate in
relevant performance evaluation
studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices181 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 and
other potential sources of
contamination.
9.2 The samples must be iced or
refrigerated at 4°C from the time of
collection until extraction.
612-4
July 1982
-------
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction'21.
10. Sample Extraction
10.1 Mark the water rneniscus on
the side of the sample bottle for
later determination of sample volume.
Pour the entire sample irito a two-
liter separatory funnel. :
10.2 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner
surface. Transfer the solvent to the
separatory funnel and extract the
sample by shaking the funnel for two
minutes with periodic venting to
release excess pressure. Allow the
organic layer to separate'from the
water phase for a minimum of 10
minutes. 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 tjlass wool,
centrifugation, or other physical
methods. Collect the methylene
chloride extract in a 250-mL
Erlenmeyer flask.
10.3 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.
10.4 Assemble a Kuder.na-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 Kuderna-Danish if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract
through a drying column containing
about 10 cm of anhydrous sodium
sulfate, and collect the extract in the
K-D concentrator. Rinse the
Erlenmeyer-flask and column with 20
to 30 mL of methylene chloride to
complete the quantitative transfer.
10.6 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 1 mL 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 concen-
tration in 15 to 20 minutes. 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 1 to
2 mL, remove the K-D apparatus from
the water bath and allow it to drain
for at least 10 minutes while cooling.
NOTE: The dichlorobenzenes have a
sufficiently high volatility that signif-
icant losses may occur in concentra-
tion steps if care is not exercised. It is
important to maintain a constant
gentle evaporation rate and not to
allow the liquid volume to fall below 1
to 2 mL before removing the K-D from
the hot water bath.
10.7 Momentarily remove the
Snyder column, add 50 mL hexane
and a new boiling chip and replace
the column. Raise the temperature of
the water bath to 85 to 90°C.
Concentrate the extract as in Section
10.6, except using hexane to prewet
the column. Remove the Snyder
column and rinse the flask and its
lower joint into the concentrator tube
with 1 to 2 mL of hexane. A 5-mL
syringe is recommended for this
operation. Stopper the concentrator
tube and store refrigerated if further
processing will not be performed
immediately.
10.8 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.
10.9 Unless the sample is known to
require cleanup, proceed to analysis
by gas chromatography.
11. Cleanup and Separation
11.1 Cleanup procedures may not
be necessary for a relatively clean
sample matrix. The cleanup
procedures recommended in this
method have been used for the
analysis of various clean waters and
industrial effluents. If particular
circumstances demand the use of an
alternative cleanup procedure, the
analyst must determine the elution
profile and demonstrate that the
recovery of each compound of interest
is no less than 85%.
11.2 Florisil column cleanup for
chlorinated hydrocarbons.
71.2.1 Adjust the sample extract to
10 mLwith hexane.
11.2.2 Place a 12-g charge of
activated Florisil in a 10-mm ID
chromatography column. After settling
the Florisil by tapping the column,
add a 1 to 2 cm layer of anhydrous
granular sodium sulfate to the top.
Allow to cool, then pre-elute the
column, with 100 mL of petroleum
ether. Discard the eluate and just
prior to exposure of the sodium
sulfate layer to air, quantitatively
transfer the sample extract into
the column by decantation and
subsequent petroleum ether.wash-
ings. Discard the eluate. Just prior to
exposure of the sodium sulfate layer
to the air, begin eluting the column
with 200 mL petroleum ether and
collect the eluate in a 500-mL K-D
flask equipped with a 10-mL concen-
trator tube. This fraction should
contain all of the chlorinated
hydrocarbons.
11.2.3 Concentrate the fraction by
K-D as in section 10.6 except prewet
the column with hexane. When the
apparatus is cool, remove the Snyder
column and rinse the flask and its
lower joint into the concentrator tube
with 1 to 2 mL of hexane. Analyze
by GC.
12. Gas Chromatography
12.1 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. This table
includes retention times and MDL
that were obtained under these
conditions. Examples of the param-
eter separations achieved by this
column are shown in Figures 1
and 2. Other packed columns, chrom-
atographic conditions, or detectors
may be used if the requirements of
Section 8.2 are met. Capillary (open-
tubular) columns may also-be used if
the relative standard deviations of
responses for replicate injections are
demonstrated to be less than 6% and
the requirements of Section 8.2
are met.
12.2 Calibrate the system daily as
described in Section 7.
12.3 If the internal standard
approach is being used, the internal
standard must be added to the sample
extract and mixed thoroughly,
immediately before injection into the
instrument.
12.4 Inject 2 to 5 /uL of the sample
extract using the solvent-flush
technique191. Smaller (LOyuL) volumes
can be injected if automatic devices
are employed. Record the volume of
the extract to the nearest 0.1 mL,
the volume injected to the nearest
0.05 fjL, and the resulting peak size
in area or peak height units.
612-5
July 1982
-------
12.5 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.
12.6 If the response for the peak
exceeds the working range of the •
system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak
response is prevented by the presence
of interferences, further cleanup is
required.
13. Calculations
13.1 Determine the concentration of
individual compounds in the sample.
13.1.1 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 in Section 7.2.2. The concentra-
tion in the sample can be calculated
from Equation 2:
Eq. 2. Concentration,
where:
(Vi){Vs)
A = Amount of material injected, in
nanograms.
Vi = Volume of extract injected (fA.).
Vi = Volume of total extract (/uL).
Va = Volume of water extracted (mL).
13.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
Equation 3.
(As)(ls)
Eq. 3. Concentration, /L/g/L = (Als)(RF)(V0)
where:
As = Response for the parameter
to be measured.
Aia = Response for the internal
standard.
la = Amount of internal standard
added to each extract (/L/g).
V0 = Volume of water extracted,
in liters.
13.2 Report results in micrograms
per liter without correction for
recovery data. When duplicate and
spiked samples are analyzed, report
all data obtained with the sample
results.
13.3 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in section 8.4, data for
the affected parameters must be
labeled as suspect.
14. Method Performance
14.1 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
zero111. The MDL concentrations listed
in Table 1 were obtained using
reagent water1101. Similar results
were achieved using representative
wastewaters.
14.2 This method has been tested
for linearity of spike recovery from
reagent water and has been
demonstrated to be applicable over the
concentration range from the 4 x MDL
up to 1000 x MDL'101.
14.3 In a single laboratory (EMSL-
Cincinnati), using three wastewaters
spiked at six concentration levels, the
average recoveries presented in Table
2 were obtained. The standard
deviation of the percent recovery is
also included in Table 2.
14.4 The U.S. Environmental Pro-
tection Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. "Determination of Chlorinated
Hydrocarbons In Industrial and
Municipal Wastewaters."
Report for EPA Contract 68-
03-2625 (In preparation).
3. ASTM Annual Book of
Standards, Part 31, D 3694.
"Standard Practice for
Preparation of Sample
Containers and for
Preservation," American
Society for Testing and
Materials, Philadelphia, PA,
p. 679, 1980.
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,"
(29CFR1910), 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. "Handbook of 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.
8. ASTM Annual Book of
Standards, Part 31, D 3370,
''Standard Practice for
Sampling Water," American
Society for Testing and
Materials, Philadelphia, PA,
p. 76, 1980.
9. Burke, J. A. "Gas Chroma-
tography for Pesticide Residue
Analysis; Some Practical
Aspects," Journal of the
Association of Official
Analytical Chemists, 48.
1037(1965).
10. "Development of Detection
Limits, EPA Method 612,
Chlorinated Hydrocarbons,"
Special letter report for EPA
Contract 68-03-2625,
Environmental Monitoring
and Support Laboratory -
Cincinnati, Ohio 45268.
612-6
July 1982
-------
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
1,3-Dichlorobenzene
1,4-Dichlorobenzene
Hexachloroethane
1,2-Dichlorobenzene
Hexachlorobutadiene '.
1,2,4- Trichlorobenzene
2-Chloronaphthalene
Hexachlorobenzene
Retention Time
(min.)
6.8
7.6
8.3
9.3
20.0
22.3
3.6*
10.1a
Method
Detection Limit
(fJff/L)
1.19
1.34
O.O3
1.14
0.34
0.05
0.94
0.05
Column conditions: Supelcoport (80/100 mesh) coated with 1.5% OV-1/2.4%
OV-225 packed in a 1.8 m x 2 mm ID g/ass column with 5% Methane/95%
Argon carrier gas at a flow rate of 25 mL/min. Column temperature, isothermal
at 75°C, except as other wise indicated
a - Column temperature 165°C.
Table 2. Single Operator Accuracy and Precision
Parameter
2-Chloronaphthalene
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
Hexachlorobenzene
Hexa chlorobutadiene
Hexachloroethane
A verage
Percent
Recovery
76
82
86
89
95
96
99
Standard Spike Number
Deviation Range of Matrix
% fag/L) Analyses Types
25
10
18
20
12
1O
12
19.1-268
29.8-356
20.4-238
23.0-324
1.29-14.9
3.12-36.8
1.02-14.8
18
18
18
18
18
18
18
3
3
3
3
3
3
3
1,2,4-Trichlorobenzene 96 16 15.1-216 18
612-7 July 1982
-------
Column: 1.5% OV-1 +2.4% OV-225 on
Supelcoport 80/100
Temperature: 75°C.
Detector: Electron capture
8 12 16 20 24
Retention time, minutes
Figure 1. Gas chromatogram of chlorinated
hydrocarbons.
Column: 1.5% OV-1 +2.4%
OV-225 on
Supelcoport 80/100
Temperature: 165°C.
Detector: Electron capture
I
i
8
0 4 8 12
Retention time, minutes
figure 2. Gas chromatogram of
chlorinated hydrocarbons.
612-8
July 1982
-------
vvEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
2,3,7,8-Tetrachlorodibenzo-p
Dioxin — Method 613
1. Scope and Application
1.1 This method covers the determi-
nation of 2,3,7,8-tetrachlorodibenzo-
p-dioxin (2,3,7,8-TCDD). The following
parameter may be determined by this
method:
Parameter
STORET No.
CAS No.
2,3,7,8-TCDD 34675
1.2 This is a gas chromatographic/
mass spectrometer (GC/MS) method
applicable to the determination of
2,3,7,8-TCDD in municipal and
industrial discharges as provided under
40 CFR 1 36.1. Method 625 may be
used to screen samples for
2,3,7,8-TCDD. When the screening
test is positive, the final qualitative
confirmation and quantification must
be made using method 613.
1.3 The method detection limit (MDL,
defined in Section 14.1)(1)for
2,3,7,8-TCDD is listed in Table 1. The
MDL for a specific wastewater may be
different depending upon the nature of
interferences in the sample matrix.
1.4 Because of the extreme toxicity
of this compound, the analyst must
prevent exposure to himself, or to
others, by materials known or believed
to contain 2,3,7,8-TCDD. Section 4 of
this method contains guidelines and
protocols that serve as minimum safe-
handling standards in a limited access
laboratory.
1.5 Any modification of this method,
beyond those expressly permitted,
shall be considered as major modifica-
tions subject to application and
approval of alternate test procedures
under 40 CFR 1 36.4 and 136.5.
1746-01-6
1.6 This method is restricted to use
only by or under the supervision of
analysts experienced in the use of gas
chromatograph/mass spectrometers
and skilled in the interpretation of mass
spectra. Each analyst must demonstrate
the ability to generate acceptable
results with this method using the
procedure described in Section 8.2.
2. Summary of Method
2.1 A one-liter sample of wastewater
is spiked with an internal standard of
labeled 2,3,7,8-TCDD. The spiked
sample is then extracted with methy-
lene chloride using separately funnel
techniques. The extract is concen-
trated and exchanged to hexane while
being concentrated to a volume of 1.0
mL or less. Capillary column GC/MS
conditions are described which allow
for the separation and measurement of
2,3,7,8-TCDD in the extract<2,3).
2.2 The method provides selected
column chromatographic cleanup
procedures to aid in the elimination of
interferences that may be encountered.
3. Interferences
3.1 • Method interferences may be
caused by contaminants in solvents,
reagents, glassware, and other sample
613-1
July 1982
-------
processing hardware that lead to
discrete artifacts and/or elevated
backgrounds at the ions monitored. 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 8.5.
3.1.1 Glassware must be scrupulously
cleaned.Ml 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
muffle furnace at 400 °C for 1 5 to 30
minutes. Some thermally stable
materials, such as PCBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Volumetric
ware should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and stored
in a clean environment to prevent any
accumulation of dust or other
contaminants. Store it inverted or
capped with aluminum foil.
3.1.2 The use of high purity reagents
and solvents helps to minimize inter-
ference problems. Purification of
solvents by distillation in all-glass
systems may be required.
3.2 Matrix interferences may be
caused by contaminants that are co-
extracted from the sample. The extent
of matrix interferences will vary
considerably from source to source,
depending upon the nature and diver-
sity of the industrial complex or munici-
pality being sampled. 2,3,7,8-TCDD is
often associated with other interfering
chlorinated compounds which are at
concentrations several magnitudes
higher than that of 2,3,7,8-TCDD. The
cleanup procedures in Section 11 can
be used to overcome many of these
interferences, but unique samples may
require additional cleanup
approaches'1'5"7I to eliminate false
positives and achieve the method
detection limit listed in Table 1.
3.3 The primary column, SILAR-1OC,
resolves 2,3,7,8-TCDD from the other
21 isomersi3). Positive results obtained
using any other gas chrorhatographic
column must be confirmed using this
column.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are identified!8-1 °).
Benzene and 2,3,7,8-TCDD have been
identified as suspected human or
mammalian carcinogens.
4.2 Each laboratory must develop a
strict safety program for handling of
2,3,7,8-TCDD. The following labora-
tory practices are recommended:
4.2.1 Contamination of the labora-
tory will be minimized by conducting all
manipulations in a hood.
4.2.2 The effluents of sample
splitters for the gas chfomatograph and
roughing pumps on the GC/MS should
pass through either a column of
activated charcoal or be bubbled
through a trap containing oil or high-
boiling alcohols. ;
4.2.3 Liquid waste should be
dissolved in methanol or ethanol and
irradiated with ultraviolet light with
wavelength greater than 290 nm for
several days. (Use F 40 BL lamps or
equivalent.) Analyze liquid wastes and
dispose of the solutions when
2,3,7,8-TCDD can no longer be
detected.
4.3 Dow Chemical U.S.A. has issued
the following precautions (revised
11/78) for safe handling of
2,3,7,8-TCDD in the laboratory:
4.3.1 The following statements on
safe handling are as complete as
possible on the basis of available
toxicological information. The
precautions for safe handling and use
are necessarily general in nature since
detailed, specific recommendations can
be made only for the particular exposure
and circumstances of each individual
use. Inquiries about specific operations
or uses may be addressed to the Dow
Chemical Company. Assistance in
evaluating the health hazards of
particular plant conditions may be
obtained from certain consulting
laboratories and from State Depart-
ments of Health or of Labor, many of
which have an industrial health service.
2,3,7,8-TCDD is extremely toxic to
laboratory animals. However, it has
been handled for years without injury in
analytical and biological laboratories.
Techniques used in handling radio-
active and infectious materials are
applicable to 2,3,7,8-TCDD.
4.3.1.1 Protective Equipment:
Throw-away plastic gloves, apron or
lab coat, safety glasses and lab hood
adequate for radioactive work.
4.3.1.2 Training: Workers must be
trained in the proper method of
removing of contaminated gloves and
clothing without contacting the
exterior surfaces.
4.3.1.3 Personal Hygiene: Thorough
washing of hands and forearms after
each manipulation and before breaks
(coffee, lunch, and shift).
4.3.1.4 Confinement: Isolated work
area, posted with signs, segregated
glassware and tools, plastic-backed
absorbent paper on benchtops.
4.3.1.5 Waste: Good technique
includes minimizing contaminated
waste. Plastic bag liners should be
used in waste cans. Janitors must be
trained in safe handling of waste.
4.3.1.6 Disposal of Wastes:
2,3,7,8-TCDD decomposes above
800 °C. Low-level waste such as the
absorbent paper, tissues, animal
remains and plastic glvoes may be
burned in a good incinerator. Gross
quantities (milligrams) should be
packaged securely and disposed
through commercial or governmental
channels which are capable of handling
high-level radioactive wastes or
extremely toxic wastes. Liquids should
be allowed to evaporate in a good hood
and in a disposable container. Residues
may then be handled as above.
4.3.1. 7 Decontamination: Personal—
any mild soap with plenty of scrubbing
action: Glassware, Tools, and
Surfaces—Chlorothene NU Solvent
(Trademark of the Dow Chemical
Company) is the least toxic solvent
shown to be effective. Satisfactory
cleaning may be accomplished by
rinsing with Chlorothene, then washing
with any detergent and water. Dish
water may be disposed to the sewer. It
is prudent to minimize solvent wastes
because they may require special
disposal through commercial sources
which are expensive.
4.3.1.8 Laundry: Clothing known to
be contaminated should be disposed
with the precautions described under
"Disposal of Wastes." Lab coats or
other clothing worn in 2,3,7,8-TCDD
6/3-2
July 1982
-------
work area may be laundered. Clothing
should be collected in plastic bags.
Persons who convey the bags and
launder the clothing should be advised
of the hazard and trained in proper
handling. The clothing may be put into
a washer without contact if the
launderer knows the problem. The
washer should be run through a cycle
before being used again for other
clothing.
4.3.1.9 Wipe Tests: A useful method
of determining cleanliness of work
surfaces and tool is to wipe the surface
with a piece of filter paper. Extraction
and analysis by gas chromatography
can achieve a limit of sensitivity of 0.1
pig per wipe. Less than 1 'pig
2,3,7,8-TCDD per sample indicates
acceptable cleanliness; anything higher
warrants further cleaning. More than
10 ptg on a wipe sample indicates an
acute hazard and requires prompt
cleaning before further use of the
equipment or work space and indicates
further that unacceptable work
practices have been employed in the
past.
4.3.1.1O Inhalation: Any procedure
that may produce airborne contamina-
tion must be done with good ventilation.
Gross losses to a ventilation system
must not be allowed. Handling of the
dilute solutions normally used in
analytical and animal work presents no
inhalation hazards except in case of an
accident.
4.3.1.11 Accidents: Remove
contaminated clothing immediately,
taking precautions not to contaminate
skin or other articles. Wash exposed
skin vigorously and repeatedly until
medical attention is obtained.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
or composite sampling. .
5.1.1 Grab sample bottle—Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If
amber bottles are not available, protect
samples from light. The container must
be washed, rinsed with acetone or
methylene chloride, and dried before
use to minimize contamination.
5.1.2 Automatic sampler (optional) —
Must incorporate glass sample
containers for the collection of a
minimum of 25O mL. Sample
containers must be kept refrigerated at
4 °C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however, the
compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the potential
for contamination of the sample. An
integrating flow meter is required to
collect flow proportional composites.
5.1.3 Clearly label all samples as
"POISON" and ship according to
U.S.D.O.T. requirements.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.7 Separatory funnel—2000-mL
and 125-mL, with Teflon stopcock.
5.2.2 Concentrator tube, Kuderna-
Danish— 10-mL, graduated (Kontes
K-570050-1 025 or equivalent).
Calibration must be checked at the
volumes employed in the test. Ground
glass stopper is used to prevent
evaporation of extracts.
5.2.3 Evaporative flask, Kuderna-
Danish—500-mL (Kontes
K-570001 -0500 or equivalent).
Attach to concentrator tube with
springs.
5.2.4 Snyder column, Kuderna-
Danish—three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.5 Snyder column, Kuderna-
Danish—two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.6 Vials—Amber glass, 10- to
1 5-mL capacity, with Teflon-lined
screw cap.
5.2.7 Chromatography column —300
mm long x 10 mm ID with coarse
fritted disc at bottom and Teflon
stopcock.
5.2.8 Chromatography column—40O
mm long x 11 mm ID with coarse
fritted disc at bottom and Teflon
stopcock.
5.3 Boiling chips—approximately
10/40 mesh. Heat to 400 °C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath—Heated, with
concentric ring cover, capable of
temperature control (± 2 °C). The bath
should be used in a hood.
5.5 GC/MS system.
5.5.1 Gas chromatograph—An
analytical system complete with all
required accessories including syringes,
analytical columns, and gases. The
injection port must be designed for
capillary columns. Either split, splitless,
or on-column injection techniques may
be employed, as long as the require-
ments of Section 7.1.1 are achieved.
5.5.2 Primary column—50 m long x
0.25 mm ID glass, coated with SILAR-
10C (or equivalent). An equivalent
column must resolve 2,3,7,8-TCDD
from the other 21 TCDD isomers.
Guidelines for the use of alternate
columns are provided in Section 1 2.1.
5.5.3 Mass Spectrometer—Either
low resolution mass spectrometers
(LRMS) or high resolution mass spec-
trometers (HRMS) may be used. The
mass spectrometer must be equipped
with a 70 volt (nominal) ion source and
be capable of acquiring ion abundance
data in real time Selected Ion
Monitoring (SIM) for groups of four or
more ions.
5.5.4 GC/MS interface—Any gas
chromatograph to mass spectrometer
interface can be used that achieves the
requirements of Section 7.1.1
constructed of glass or glass-lined
materials .are recommended. Glass
surfaces can be deactivated by
silanizing with dichlorodimethylsilane.
To achieve maximum sensitivity, the
exit end of the capillary column should
be placed in the ion source. A short
piece of fused silica capillary can be
used as the interface to overcome
problems associated with straightening
the exit end of glass capillary columns.
5.5.5 The SIM data acquired during
the chromatographic program is
defined as the Selected Ion Current
Profile (SICP). The SICP can be
acquired under computer control or as
real time analog output. If computer
control is used, there must be software
available to plot the SICP and report
peak height or area data for any ion in
the SICP between specified time or
scan number limits.
5.6 Balance—Analytical, capable of
accurately weighing 0.0001 g.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an inter-
ferent is not observed at the MDL of
2,3,7,8-TCDD.
6.2 Sodium hydroxide solution—
(ACS) Dissolve 400 g NaOH in reagent
water and dilute to one liter. Wash the
solution with methylene chloride and
with hexane before use.
6.3 Sodium thiosulf ate—(ACS)
Granular.
6.4 Sulfuric acid (Cone.)—(ACS) sp.
gr. 1.84.
613-3
July 1982
-------
6.5 Methylene chloride, hexane,
benzene, tetradecane—Pesticide
quality or equivalent.
6.6 Sodium sulfate—(ACS) Granular,
anhydrous (purified by heating at
400 °C for four hours in a shallow
tray).
6.7 Alumina—neutral, 80/200 mesh
(Fisher Scientific Co., No. A-540 or
equivalent). Before use, activate for 24
hours at 130 °C in a foil covered glass
container.
6.8 Silica gel—high purity grade,
100/120 mesh, (Fisher Scientific Co.,
No. S-679 or equivalent).
6.9 Stock standard solutions (1.00
f
-------
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL aliquots
of reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recoyery (s), for the
results. Wastewater background
corrections must be made before R and
s calculations are performed.
8.2.4 Using Table 2, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s > 2p
or |X —R| > 2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and s based
upon the result of interlaboratory
testing. When they becopne available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria and define
the performance of the laboratory for
each spike concentratio^and
parameter being measured.
8.3.1 Calculate upper and lower
control limits for method'performance:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
where R and s are calculated as in
Section 8.2.3. The UCL and LCL can
be used to construct control charts!11)
that are useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they become avail-
able from the U.S. Environmental
Protection Agency. '
8.3.2 The laboratory must develop
and maintain separate accuracy state-
ments of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcula-
tion of R and s. Alternately, the analyst
may use four wastewater data points
gathered through the requirement for
continuing quality'control in Section
8.4. The accuracy statements should
be updated regularly do).
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of the
sample must be spiked and analyzed as
described in Section 8.2. If the
recovery for 2,3,7,8-TCDD does not
fall within the control limits for method
performance, the results reported for
2,3,7,8-TCDD in all samples processed
as part of the same set must be
qualified as described in Section 13.4.
The laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagent interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as a
safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. Whenever
possible, the laboratory should perform
analysis of standard reference
materials and participate in relevant
performance evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices!12) 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 and
other potential sources of
contamination.
9.2 The samples must be iced or
refrigerated at 4 °C and protected from
light from the time of collection until
extraction. If the sample contains
residual chlorine, add 80 mg of sodium
thiosulfate per each liter of sample.
U.S. Environmental Protection Agency
methods 330.4 and 330.5 may be
used for measurement of residual
chlorinedS). Field test kits are available
for this purpose.
9.3 Label all samples and containers
POISON and ship according to
applicable U.S. Department of
Transportation regulations.
9.4 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction'2).
10. Sample Extraction
CAUTION: When using this method to
analyze for 2,3,7,8-TCDD, all of the
following operations must be
performed in a limited access
laboratory with the analyst wearing full
protective covering for all exposed skin
surfaces. See Section 4.2.
10.1 Mark the water meniscus on the
side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel.
70.2 Add 1.00 mL of internal
standard spiking solution to the sample
in the separatory funnel. If the final
extract will be concentrated to a fixed
volume below 1.00 mL (Section 1 2.3),
only that volume of spiking solution
should be added to the sample so that
the final extract will represent 25
ng/mL at the time of analysis.
70.3 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
30 seconds to rinse the inner surface.
Transfer the solvent to the separatory
funnel and extract the sample by
shaking the funnel for two minutes
with periodic venting to release excess
pressure. Allow the organic layer to
separate from the water phase for a
minimum of 10 minutes. If the emul-
sion 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 tech-
nique depends upon the sample, but
may include stirring, filtration of the
emulsion through glass wool, centrifu-
gation, or other physical methods.
Collect the methylene chloride extract
in a 250-mL Erlenmeyer flask.
10.4 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.
10.5 Assemble a Kuderna-Danish
(rvD) concentrator by attaching a
613-5
July 1982
-------
10-mL concentrator tube to a 500-mL
evaporativs flask. Other concentration
devices or techniques may be used in
place of the K-D if the requirements of
Section 8.2 are met.
10.6 Pour the combined extract into
the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20
to 30 mL of methylene chloride to
complete the quantitative transfer.
10.7 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 1 mL 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 1 5 to 20 minutes.
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 1 mL, remove
the K-D apparatus from the water bath
and allow it to drain and cool for at
least 10 minutes.
10.8 Momentarily remove the Snyder
column, add 50 mil hexane and a new
boiling chip and replace the column.
Raise the temperature of the water
bath to 85 to 90 °C. Concentrate the
extract as in Section 10.7, except
using hexane to prewet the column.
Remove the Snyder column and rinse
the flask and its lower joint into the
concentrator tube with 1 to 2 mL of
hexane. A 5-mL syringe is
recommended for this operation. Set
aside a K-D glasswa/e for reuse in
Section 10.14:
10.9 Pour the hexane from the
concentrator tube into a 12 5-mL
separatory funnel. Rinse the
concentrator tube four times with
10-mL aliquots of hexane. Combine all
rinses in the 125-mL separatory
funnel.
10.10 Add 50 mL of 10N sodium
hydroxide solution to the funnel and
shake for 30 to 60 seconds. Discard
the aqueous phase.
10.11 Perform a second wash of the
organic layer with 50 mL of reagent
water. Discard the aqueous phase.
10.12 Wash the hexane layer with at
least two 50-mL aliquots of concen-
trated sulfuric acid. Continue washing
the hexane layer with 50-mL aliquots
of concentrated sulfuric acid until the
acid layer remains colorless. Discard all
acid fractions.
10.13 Wash the hexane layer with
two 50-mL aliquots of reagent water.
Discard the aqueous phases.
10.14 Transfer the hexane layer into
a 125-mL Erlenmeyer flask containing
1 to 2 g anhydrous sodium sulfate.
Swirl the flask for 30 seconds and
decant the hexane into the
reassembled K-D apparatus. Complete
the quantitative transfer with two
10-mL hexane rinses of the Erlenmeyer
flask.
10.15 Replace the one or two clean
boiling chips and concentrate the
extract to 6 to 10 mL as described in
Section 10.8.
10.16 Add a clean boiling chip and
attach a micro-Snyder column. Prewet
the column by adding about 1 mL
hexane to the top. Place the K-D
apparatus on the 80 °C 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 the concentration in 5 to 10
minutes. At the proper rate of distilla-
tion the balls of the column will
actively chatter but the chambers will
not flood. When the apparent volume
of liquid reaches about 0.5 mL, remove
the K-D apparatus and allow it to drain
for at least 10 minutes while cooling.
Remove the micro Snyder column and
rinse its lower joint into the concen-
trator tube with 0.2 mL hexane. Adjust
the extract volume to 1.0 mL with
hexane. Stopper the concentrator tube
and store refrigerated and protected
from light if GC/MS analysis or cleanup
will not be performed immediately.
10.17 Determine the original sample
volume by refilling the sample bottle to
the mark with water and measuring the
volume in a 1000-mL graduated
cylinder. Record the sample volume to
the nearest 5 mL.
11. Cleanup and Separation
11.1 Cleanup procedures may not be
necessary for a relatively clean sample
matrix. The cleanup procedures
recommended in this method have
been used for the analysis of various
clean waters and industrial effluents.
The single operator precision and
accuracy data in Table 2 was gathered
using the recommended cleanup
procedures. If particular circumstances
demand the use of an alternative
cleanup procedure, the analyst must
determine the elution profile and
demonstrate that the recovery of
2,3,7,8-TCDD is reproducible and
equivalent to the recovery of the
internal standard. Two cleanup column
options are offered to the analyst in
this section. The alumina column
should be used first to overcome
interferences. If background problems
are still encountered, the silica gel
column may be helpful. Other cleanup
procedures have been described to
overcome special interference
problems
-------
500-mL K-D flask equipped with a
10-mL concentrator tube.
11.3.4 Evaporate the fraction to 1.0
mL by standard K-D techniques and
analyze by GC/MS.
12. GC/MS Analysis
12.1 Table 1 summarizes the
recommended gas chromatographic
capillary column and operating condi-
tions for the instrument. Included in
this table are the estimated retention
time and MDL that can be achieved by
this method. Other capillary columns or
chromatographic conditions may be
used to screen samples if the
requirements of Section 8.2 are met.
Confirmation of 2,3,7,8-TCDD must
be accomplished using a GC column
that separates 2,3,7,8-TCDD from all
other TCDD isomers.
12.2 Analyze standards and samples
with the mass spectrometer operating
in the SIM mode using a dwell time to
give at least seven points per peak. For
LRMS, use ions at m/e 320, 322, and
257 for 2,3,7,8-TCDD arid either the
ion at m/e 328 for 37QI 2,3,7,8-TCDD
or m/e 332 for 13C 2,3,7,8-TCDD. For
HRMS, use ions at m/e 319.8965 and
321.8936 for 2,3,7,8-TCDD and
either the ion at m/e 327:8847 for
37CI 2,3,7,8-TCDD or m/e 331.9367
for 13C 2,3,7,8-TCDD. ;
12.3 If lower detection limits are
required, the extract may'be carefully
evaporated to dryness under a gentle
stream of nitrogen with the concen-
trator tube in a water bath at about
40 °C. Do this immediately before
GC/MS analysis. Redissoive the extract
in the desired final volume of ortho-
xylene or tetradecane. The method
performance data reported in Section
14 was gathered using a final extract
volume of 0.2 mL.
12.4 Calibrate the system daily as
described in Section 7.1 .3* The
volume of calibration standard injected
must be measured, or be the same as
all sample injection volumes. '.
12.5 Inject a 2 to 5 pL aliquot of the
sample extract.
12.6 The presence of 2',3,7,8-TCDD
is qualitatively confirmed if all of the
following criteria are achieved.
12.6.1 The gas chromatographic
column must resolve 2,3;7,8-TCDD
from the other 21 TCDD isomers.
12.6.2 The ions for native 2,3,7,8-
TCDD (LRMS-m/e 320, 322, and 257
and HRMS-m/e 320 and 322} and
labeled 2,3,7,8-TCDD (m/e 328 or
332) must exhibit a simultaneous
maximum at a retention time that
matches that of native 2,3,7,8-TCDD
in the calibration standard, within the
performance specifications of the
analytical system.
12.6.3 The chlorine isotope ratio at
m/e 320 and m/e 322 must agree to
within ± 10% of that in the calibration
standard.
12.6.4 The signal of all peaks must
be greater than 2.5 times the noise
level.
12.7 For quantitation, measure the
response of the m/e 320 peak for
2,3,7,8-TCDD and the m/e 332 peak
for 13d 2 2,3,7,8-TCDD or the m/e
328 peak for 37CI4 2,3,7,8-TCDD.
12.8 Co-eluting impurities are
suspected if all criteria are achieved
except those in Section 1 2.6.3. In this
case, another SIM analysis using ions
at m/e 257, 259, 320 and either m/e
328 or m/e 322 can be performed.
The ions at m/e 257 and m/e 259 are
indicative of the loss of one chlorine
and one carbonyl group from 2,3,7,8-
TCDD. If the ions m/e 257 and m/e
259 give a chlorine isotope ratio that
agrees to within ± 10% of the same
cluster in the calibration standards,
than the presence of TCDD can be
confirmed. Co-eluting ODD, DDE, and
PCS residues can be confirmed, but
will require another injection using the
appropriate SIM ions or full repetitive
mass scans. If the response for 37CI
2,3,7,8-TCDD at m/e 328 is too large,
PCB contamination is suspected and
can be confirmed by examining the
response at both m/e 326 and m/e
328. The 37CI 2,3,7,8-TCDD internal
standard gives negligible response at
m/e 326. These pesticide residues can
be removed using the alumina column
cleanup.
12.9 If broad background interfer-
ence restricts the sensitivity of the
GC/MS analysis, the analyst should
employ additional cleanup procedures
and reanalyze by GC/MS.
12.10 In those circumstances where
these procedures do not yield a
definitive conclusion, then the use of
high resolution mass spectrometry is
suggested.!5)
13. Calculations
13.1 Calculate the concentration of
2,3,7,8-TCDD in the sample using the
response factor (RF) determined in
7.1.2 and equation 2.
(Ai.) (I.)
Eq. 2
Concentration,
(Ais)(RF)(V0)
where:
As = SIM response for 2,3,7,8-
TCDD ion at m/e 320.
Ais = SIM response for the internal
standard ion at m/e 328 or
332.
ls = Amount of internal standard
added to each extract (pig).
V0 = Volume of water extracted, in
liters.
13.2 For each sample, calculate the
percent recovery of the internal
standard by comparing the area of the
ion peak measured in the sample to the
area of the same peak in the calibration
standard. If the recovery is below
50%, the results of the sample
analysis must be qualified as described
in Section 13.4.
13.3 Report results in micrograms
per liter. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
13.4 For samples processed as part
of a set where the laboratory spiked
S9mple recovery falls outside of the
control limits in Section 8.3 or the
internal standard recovery is below
50%, the data for 2,3,7,8-TCDD must
be labeled as suspect.
14. Method Performance
14.1 Method detection limit—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 zero<1>. The MDL
concentration listed in Table 1 was
obtained using reagent water!14).
14.2 In a single laboratory (Monsanto
Research Corporation), using spiked
samples, the average recovery
presented in Table 2 was obtained.!14)
The average standard deviation of the
percent recovery is also included in
Table 2. The results for quality control
checks for this study are presented in
Table 3.
14.3 The U.S. Environmental Protec-
tion Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
613-7
July 1982
-------
2. "Determination of 2,3,7,8-TCDD in
Industrial and Municipal Wastewaters."
Report for EPA Control 68-03-2635
(In preparation).
3. Buser, H.R., and Rappe, C. "High
Resolution Gas Chromatography of the
22 Tetrachlorodibenzo-p-dioxin
Isomers," Analytical Chemistry, 52,
2257, (1980).
4. ASTM Annual Book of Standards,
Part 31, D 3694. "Standard Practice
for Preparation of Sample Containers
and for Preservation," American
Society for Test and Materials,
Philadelphia, PA, p. 679, (1980).
5. Harless, R.L., E.O. Oswald, and
M.K. Wilkinson, "Sample Preparation
and Gas Chromatography/Mass Spec-
trometry Determination of 2,3,7,8-
Tetrachlorodibenzo-p-dioxin."/1/73/Xif/-
cal Chemistry, 52, 1239 (1980).
6. Lamparski, L.L. and T.J. Nestrick.
" Determination of Tetra-, Hepta-, and
Octachlorodibenzo-p-dioxin Isomers in
Paniculate Samples at Parts per Trillion
Levels," Analytical Chemistry, 52,
2045(1980).
7. Longhorst, M.L. and L.A. Shadoff,
"Determination of Parts-per-Trillion
Concentrations of Tetra-, Hexa-, and
Octachlorodibenzo-p-dioxins in Human
Milk," Analytical Chemistry, 52,
2037, (1980).
8. "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.
9. "OSHA Safety and Health
Standards, General Industry,"
(29CFR1910), 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. "Handbook of 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.
12. ASTM Annual Book of Standards,
Part 31, D 3370, "Standard Practice
for Sampling Water," American
Society for Testing and Materials,
Philadelphia, PA 1980.
13. "Methods 330.4 (Titrimetric,
DPD-FAS) and 330.5 (Spectrophoto-
metric DPD) for Chlorine, Total
Residual," Methods for Chemical
Analysis of Water and Wastes, EPA
600/4-79-020, U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1979.
14. "Determination of Method Detec-
tion Limits for EPA Method 613,"
Special letter report for EPA Contract
68-03-2863, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
Table 1.
Parameter
Chromatographic Conditions and Method
Detection Limit
Retention Time
(min.J
Detection
Limit (g/L)
2,3,7,8-TCDD
34.5
0.002
Column conditions: SILAR-10C coated onaSOm long O.25
mm ID glass column with helium carrier gas at 3O cm/sec
linear velocity, split/ess injector. Column temperature pro-
grammed: isothermal, 100°C for 3 minutes, then pro-
grammed at 2O°C/min to 18O°C, and 2 °C/min to 250°C.
Table 2. Single Operator Accuracy and Precision
Parameter
2,3,7,8-TCDD
2,3,7,8-TCDD
2,3,7,8-TCDD
Matrix
Reagent water
Industrial waste
Municipal waste
Spike
vafl-
0.005
0.005
O.025
Average
Percent
Recovery
95.4
85.8
92.4
Standard
Deviation
%
10.2
6.6
18.7
Table 3. Quality Control Results (14)
Matrix
Reagent water
Industrial waste
Municipal waste
320/322
Isotope Ratios
O.79 ± O.O4
0.81 ± 0.01
i O.83 ± O.O2
Internal
Standard
Recovery
87%
104%
14%
613-8
July 1982
-------
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
v>EPA
Research and Development
Test Method
Purgeables -
Method 624
1. Scope and Application
1.1 This method covers the determi-
nation of a number of purgeable
organics. The following parameters
may be determined by this method:
Parameter
STORET No.
CAS No.
Benzene
Bromodichloromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chloroethane
2-Chloroethylvinyl ether
Chloroform
Chloromethane
Dibromochloromethane
1 ,2-Dichlorobenzene
1 ,3-Dichlorobenzene
1 ,4-Dichlorobenzene
1 , 1 -Dichloroethane
1 ,2-DichIoroethane
1 , 1 -Dichloroethene
trans-1 ,2-Dichloroethene
1 ,2-Dichloropropane
cis-1 ,3-Dichloropropene
trans-1 ,3-Dichloropropene
Ethyl benzene
Methylene chloride
1 , 1 ,2,2-Tetrachloroethane
Tetrachloroethene
Toluene
1,1,1 -Trichloroethane
1 ,1 ,2-Trichloroethane
Trichloroethene
Trichlorofluoromethane
Vinyl chloride
34030
32101
32104
34413
32102
34301
34311
34576
32106
34418
32105
34536
34566
34571
34496
34531
34501
34546
34541
34704
34699
34371
34423
34516
34475
34010
34506
34511
39180
34488
39175
71-43-2
75-27-4
75-25-2
74-83-9
56-23-5
108-90-7
75-00-3
110-75-8
67-66-3
74-87-3
124-48-1
95-50-1
541-73-1
106-46-7
75-34-3
107-06-2
75-35-4
156-60-5
78-87-5
10061-01-5
10061-02-6
100-41-4
75-09-2
79-34-5
127-18-4
108-88-3
71-55-6
79-00-5
79-01-6
75-69-4
75-01-4
1.2 The method may be extended to
screen samples for acrolein (STORET
No. 34210, CAS No. 107-02-8) and
acrylonitrile (STORET 3421 5, CAS No.
107-13-1), however, the preferred
method for these two compounds is
method 603.
1.3 This is a purge and trap gas
chromatographic/mass spectrometer
624-1
July 1982
-------
(GC/MS) method applicable to the
determination of the compounds listed
above in municipal and industrial
discharges as provided under 40 CFR
136.1.
1.4 The method detection limit (MDL,
defined in Section 14.1) d > for each
parameter is listed in Table 1. The MDL
for a specific wastewater differ from
those listed, depending upon the
nature of interferences in the sample
matrix.
1.5 Until the U.S. Environmental Pro-
tection Agency establishes perfor-
mance criteria based upon the results
of interlaboratory testing, any
alternative GC/MS method which
meets the performance criteria
described in Section 8.2 will be
permitted. Performance must be
verified for such modification by
analyzing wastewater as described in
Section 8.2.2. In addition, the
laboratory must successfully partici-
pate in the applicable performance
evaluation studies.
1,6 This method is restricted to use
by or under the supervision of analysts
experienced in the use of purge and
trap systems and gas chromatograph/
mass spectrometers and skilled in the
interpretation of mass spectra. Each
analyst must demonstrate the ability to
generate acceptable results with this
method using the procedure described
in Section 8.2.
2. Summary of Method
2.1 An inert gas is bubbled through a
5-mL sample contained in a specially-
designed purging chamber at ambient
temperature. The purgeables are
efficiently transferred from the
aqueous phase to the vapor phase. The
vapor is swept through a sorbent
column where the purgeables are
trapped. After purging is completed,
the sorbent column is heated and
backflushed with the inert gas to
desorb the purgeables onto a gas
chromatographic column. The gas
chromatograph is temperature
programmed to separate the
purgeables which are then detected
with a mass spectrometer(2,3).
3. Interferences
3.1 Impurities in the purge gas,
organic compounds out-gassing from
the plumbing ahead of the trap and
solvent vapors in the laboratory
account for the majority of contamina-
tion problems. The analytical system
must be demonstrated to be free from
contamination under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5. The use of non-TFE plastic tubing,
non-TFE thread sealants, or flow
controllers with rubber components in
the purging device should be avoided.
3.2 Samples can be contaminated by
diffusion of volatile organics (particu-
larly fluorocarbons and methylene
chloride) through the septum seal into
the sample during shipment and
storage. A field reagent blank prepared
from reagent water and carried through
the sampling and handling protocol can
serve as a check on such
contamination.
3.3 Contamination by carry over can
occur whenever high level and low
level samples are sequentially
analyzed. To reduce carry over, the
purging device and sample syringe
must be rinsed with reagent water
between sample analyses. Whenever
an unusually concentrated sample is
encountered, it should be followed by
an analysis of reagent water to check
for cross contamination. For samples
containing large amounts of water-
soluble materials, suspended solids,
high boiling compounds or high purge-
able levels, it may be necessary to
wash out the purging device with a
detergent solution, rinse it with distilled
water, and then dry it in a 105 °C oven
between analyses. The trap and other
parts of the system are also subject to
contamination; therefore, frequent
bakeout and purging of the entire
system may be required.
4. Safety
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified!5-7' for the
information of the analyst.
4.2 The following parameters
covered by this method have been
tentatively classified as known or
suspected, human or mammalian
carcinogens: benzene, carbon
tetrachloride, chloroform,
1,4-dichlorobenzene, and vinyl
chloride. Primary standards of these
toxic compounds should be prepared in
a hood. A NIOSH/MESA approved
toxic gas respirator should be worn
when the analyst handles high
concentrations of these toxic
compounds.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
sampling.
5.1.1 Vial—25-mL capacity or larger,
equipped with a* screw cap with hole in
center (Pierce #1 3075 or equivalent).
Detergent wash, rinse with tap and
distilled water, and dry at 105 °C
before use.
5.1.2 Septum—Teflon-faced silicone
(Pierce #12722 or equivalent).
Detergent wash, rinse with tap and
distilled water, and dry at 105 °C for
one hour before use.
5.2 Purge and trap device—The
purge and trap device consists of three
separate pieces of equipment: the
sample purger, trap, and the desorber.
Several complete devices are now
commercially available.
5.2.1 The sample purger must be
designed to accept 5-mL samples with
a water column at least 3 cm deep.
The gaseous head space between the
water column and the trap must have a
total volume of less than 1 5-mL. The
purge gas must pass through the water
column as finely divided bubbles with a
diameter of less than 3 mm at the
origin. The purge gas must be intro-
duced no more than 5 mm from the
base of the water column. The sample
purger, illustrated in Figure 1, meets
these design criteria.
5.2.2 The trap must be at least 25
cm long and have an inside diameter of
at least 0.105 inch. The trap must be
packed to contain the following mini-
mum lengths of adsorbents: 1.0 cm of
methyl silicone coated packing (Sec-
tion 6.3.2), 1 5 cm of 2,6-diphenylene
oxide polymer (Section 6.3.1), and 8
cm of silica gel, (Section 6.3.3). The
minimum specifications for the trap are
illustrated in Figure 2.
5.2.3 The desorber should be
capable of rapidly heating the trap to
180 °C. The polymer section of the
trap should not be heated higher than
180 °C and the remaining sections
should not exceed 220 °C. The
desorber design, illustrated in Figure 2,
meets these criteria.
624-2
July 1982
-------
5.2.4 The purge and trap device may
be assembled as a separate unit'br be
coupled to a gas chromatograph-as
illustrated in Figures 3 and 4.
5.3 GC/MS system.
5.3. 7 Gas chromatograph—An ana-
lytical system complete with a temper-
ature programmable gas chromato-
graph suitable for on-column injection
and all required accessories including
syringes, analytical columns, and
gases.
5.3.2 Column—6 ft long x 0.1 in ID
stainless steel or glass, packed with
1 % SP-1000 on Carbopack B (60/80
mesh) or equivalent. This column was
used to develop the method perfor-
mance statements in Section 14.
Guidelines for the use of alternate
column packings are provided in
Section 11.1.
5.3.3 Mass spectrometer—Capable
of scanning from 20 to 260 amu every
seven seconds or less, utilizing 70
volts (nominal) electron energy in the
electron impact ionizatiop mode and
producing a mass spectrum which
meets all the criteria in Table 2 when
50 ng of 4-bromofluorobenzene (BFB)
is injected through the gas chromato-
graph inlet. ;
5.3.4 GC/MS interface—Any gas
chromatograph to mass spectrometer
interface that gives acceptable
calibration points at 50 ng or less per
•injection for each of the parameters of
interest and achieves all acceptable
performance criteria (see Section 10)
may be used. Gas chromatograph to
mass spectrometer interfaces con-
structed of all-glass or glass-lined
materials are recommended. Glass can
be deactivated by silanizing with
dichloro-dimethylsilane. '
5.3.5 Data system—A computer
system must be interfaced to the mass
spectrometer that allows the
continuous acquisition and storage on
machine readable media iof all mass
spectra obtained throughout the
duration of the chromatographic
program. The computer must have
software that allows searching any
GC/MS data file for ions 'of a specified
mass and plotting such ion abundances
versus time or scan number. This type
of plot is defined as an Extracted Ion
Current Profile (EICP). Software must
also be available that allows integrating
the abundance in any EICP between
specified time or scan number limits.
5.4 Syringes— 5-mL glass hypoder-
mic with Luerlok tip (two each), if
applicable to the purging device.
5.5 Micro syringes—25-mL, 0.006
Inch ID needle.
5.6 Syringe valve—two-way, with
Luer ends (three each), if applicable to
the purging device.
5.7 Syringe—5-mL, gas-tight with
shut-off valve.
5.8 Bottle— 1 5-mL, screw-cap, with
Teflon cap liner.
5.9 Balance—Analytical, capable of
accurately weighing 0.0001 g.
6. Reagents
6.1 Reagent water— Reagent water is
defined as a water in which an inter-
ferent is not observed at the MDL of
the parameters of interest.
6.1.1 Reagent water may be gener-
ated by passing tap water through a
carbon filter bed containing about 453
g of activated carbon (Calgon Corp.,
Filtrasorb-300 or equivalent).
6.1.2 A water purification system
(Millipore Super-Q or equivalent) may
be used to generate reagent water.
6.1.3 Reagent water may also be
prepared by boiling water for 1 5
minutes. Subsequently, wh'ile maintain-
ing the temperature at 90 °C, bubble a
contaminant-free inert gas through the
water for one hour. While still hot,
transfer the water to a narrow-mouth
screw-cap bottle and seal with a
Teflon-lined septum and cap.
6.2 Sodium thiosulfate—(ACS)
Granular.
6.3 Trap materials
6.3. 7 2,6-Diphenyiene oxide
polymer—Tenax (60/8O mesh),
chromatographic grade or equivalent.
6.3.2 Methyl silicone packing—3%
OV-1 on Chromosorb-W (60/80 mesh)
or equivalent.
6.3.3 Silica gel, Davison Chemical,
(35/60 mesh), grade-1 5 or equivalent.
6.4 Methanol—Pesticide quality or
equivalent.
6.5 Stock standard solutions—Stock
standard solutions may be prepared
from pure standard materials or
purchased as certified solutions.
Prepare stock standard solutions in
methanol using assayed liquids or
gases as appropriate. Because of the
toxicity of some of the ocganohalides,
primary dilutions of these materials
should be prepared in a hood. A
NIOSH/MESA approved toxic gas
respirator should be used when the
analyst handles high concentrations of
such materials.
6.5.7 Place about 9.8 mL of
methanol into a 10-mL ground glass
stoppered volumetric flask. Allow the
flask to stand, unstoppered, for about
10 minutes or until all alcohol wetted
surfaces have dried. Weigh the flask to
the nearest 0.1 mg.
6.5.2 Add the assayed reference
material as described below:
6.5.2.1 Liquids—Using a 100-fA.
syringe, immediately add two or more
drops of assayed reference material to
the flask, then reweigh. The liquid
must fall directly into the alcohol
without contacting the neck of the
flask.
6.5.2.2 Gases—To prepare standards
for any of the four halocarbons that
boil below 30 °C (bromomethane,
chloroethane, chloromethane, and vinyl
chloride), fill a 5-mL valved gas-tight
syringe with the reference standard to
the 5.0-mL mark. Lower the needle to
5 mm above the methanol meniscus.
Slowly introduce the reference stan-
dard above the surface of the liquid.
The heavy gas rapidly dissolves in the
methanol.
6.5.3 Reweigh, dilute to volume,
stopper, then mix by inverting the flask
several times. Calculate the concentra-
tion in micrograms per microliter from
the net gain in weight. When
compound purity js assayed to be 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.
6.5.4 Transfer the stock standard
solution into a Teflon-sealed screw-cap
bottle. Store, with minimal headspace,
at -10 ° to - 20 °C and protect from
light.
6.5.5 Prepare fresh standards weekly
for the four gases and 2-chloroethyl-
vinyl ether. All other standards must be
replaced after one month, or sooner if
comparison with check standards indi-
cate a problem.
6.6 Secondary dilution standards-
Using stock standard solutions, prepare
secondary dilution standards in
methanol that contain the compounds
of interest, either singly or mixed
together. The secondary dilution
standards should be prepared at
concentrations such that the aqueous
calibration standards prepared in
Section 7.3.1 or 7.4.1 will bracket the
624-3
July 1982
-------
working range of the analytical system.
Secondary dilution standards should be
stored with minimal headspace and
should be checked frequently for signs
of degradation or evaporation, espe-
cially just prior to preparing calibration
standards from them. Quality control
check standards that can used to
determine the accuracy of calibration
standards, will be available from the
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
6.7 Surrogate standard spiking
solution—Select a minimum of three
surrogate compounds from Table 3.
Prepare stock standard solutions for
each surrogate standard in methanol as
described in Section 6.5. Prepare a
surrogate standard spiking solution
from these stock standards at a con-
centration of 150 ng/10 mL in water.
Store the spiking solution at 4 °C in
Teflon sealed glass containers with a
minimum of headspace. The solutions
should checked frequently for stability.
They should be replaced after six
months. The addition of 10 ^L of this
solution to 5 mL of sample or standard
is equivalent to a concentration of 30
ftg/L of each surrogate standard.
Surrogate standard spiking solutions,
appropriate for use with this method,
will be available from the U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
6.8 BFB Standard—Prepare a 25
^g/frt. solution of BFB in methanol.
7. Calibration
7.1 Assemble a purge and trap
device that meets the specifications in
Section 5.2. Condition the trap over-
night at 180 °C by back flushing with
an inert gas flow of at least 20
mL/min. Prior to use, daily condition
traps 10 minutes while backf lushing at
180°C.
7.2 Connect the purge and trap
device to a gas chromatograph. The
gas chromatograph must be operated
using temperature and flow rate
parameters equivalent to those in Table
1. Calibrate the purge and trap-GC/MS
system using either the external stan-
dard technique (Section 7.3) or the
internal standard technique (Section
7.4).
7.3 External standard calibration
procedure:
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter by carefully
adding 20.0 jd. of one or more secon-
dary dilution standards to 50, 250, or
500 mL of reagent water. A 25-piL
syringe with a 0.006 inch ID needle
should be used for this operation. One
of the external standards should be at a
concentration near, but above, the
MDL (See Table 1) and the other
concentrations should correspond to
the expected range of concentrations
found in real samples or should define
the working range of the GC/MS
system. Aqueous standards may be
stored up to 24 hours, if held in sealed
vials with zero headspace as described
in Section 9.2. If not so' stored, they
must be discarded after one hour.
7.3.2 Analyze each calibration
standard according to Section 11, and
tabulate the area response of the
primary characteristic ion (See Table 4)
against 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 «10% relative standard devia-
tion, RSD), linearity through the origin
can be assumed and the average ratio
or calibration factor can be used in
place of a calibration curve.
7.3.3 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 any parameter varies
from the predicted response by more
than ± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that parameter.
7.4 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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.
Because of these limitations, no
internal standard can be suggested that
is applicable to all samples. Due to their
generally unique retention times,
bromochloromethane, 2-bromo-1 -
chloropropane, and 1,4"-dichlorobutane
have been used successfully as internal
standards.
7.4.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest as
described in Section 7.3.1.
7.4.2 Prepare a spiking solution
containing each of the internal
standards using the procedures
described in Sections 6.5 and 6.6. It is
recommended that the secondary dilu-
tion standard be prepared at a concen-
tration of 1 5 ng/mL of each internal
standard compound. The addition of
1 0 fA. of this standard to 5.0 mL of
sample or calibration standard would
be equivalent to 30
7.4.3 Analyze each calibration
standard, according to Section 1 1 ,
adding 1 0 piL of internal standard
spiking solution directly to the syringe
(Section 1 1 .4). Tabulate the area
response of the characteristic ions
against concentration for each
compound and internal standard and
calculate response factors (RF) for
each compound using equation 1 .
Eq. 1 RF = (AsCis)/(AisCs)
where:
As = Area of the characteristic ion
for the parameter to be
measured.
Ajs = Area of the characteristic ion
for the internal standard.
Cis = Concentration of the internal
standard.
Cs = Concentration of the
parameter to be measured.
If the RF value over the working range
is a constant «1 0% RSD), the RF can
be assumed to be invariant and the
average RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
or response ratios, As/Ajs, vs. RF.
7.4.4 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 1 0%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared for that compound.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with
established performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
624-4
July 1982
-------
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision with
this method. This ability is established
as described in Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted to
certain options to improve the separa-
tions or lower the cost of measure-
ments. Each time such modifications
are made to the method, the analyst is
required to repeat the procedure in
Section 8.2.
8.1.3 The laboratory must spike all
samples with surrogate standards to
monitor continuing laboratory
performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each parameter to be
measured. Using stock standards,
prepare a quality controlcheck sample
concentrate in methanol 500 times
more concentrated than ithe selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268. ;
8.2.2 Using a syringe,!add 10 pL of
the check sample concentrate and 10
\A- of the surrogate standard dosing
solution (Section 6.7) to each of a
minimum of four 5-mL aliquots of
reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed jtwice the
background level for the|test to be
valid. Analyze the aliquots according to
the method beginning in-Section 11.
j
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for all
parameters and surrogate standards.
Wastewater background corrections
must be made before R and s calcu-
lations are performed. ,
8.2.4 Using Table 5, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s > p or
|X —R| > p, review potential problem
areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and s based
upon the results of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria for each
of the surrogate standards.
8.3.1 Calculate upper and lower
control limits for method performance
for each surrogate standard, using the
values for R and s calculated in Section
8.2.3:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
The UCL and LCL can be used to
construct control charts<8) that are
useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they become avail-
able from the U.S. Environmental
Protection Agency.
8.3.2 For each surrogate standard,
the laboratory must develop and main-
tain separate accuracy statements of
laboratory performance for wastewater
samples. An accuracy statement for
the method is defined as R ± s. The
accuracy statement should be
developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcu-
lation of R and s. Alternately, the
analyst may use four wastewater data
points gathered through the require-
ment for continuing quality control in
Section 8.4. The accuracy statements
should be updated regularly(8).
8.4 The laboratory is required to
spike all of their samples with the
surrogate standard spiking solution to
monitor spike recoveries. If the
recovery for any surrogate standard
does not fall within the control limits
for method performance, the results
reported for that sample must be
qualified as described in Section 13.3.
The laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Each day, the analyst must
demonstrate, through the analysis of
reagent water, that interferences from
the analytical system are under control.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. Whenever
possible, the laboratory should perform
analysis of standard reference
materials and participate in relevant
performance evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 All samples must be iced or
refrigerated from the time of collection
until extraction. If the sample contains
residual chlorine, add sodium
thiosulfate preservative (10 mg/40 mL
is sufficient for up to 5 ppm CI2) to the
empty sample bottles just prior to
shipping to the sampling site. U.S.
Environmental Protection Agency
methods 330.4 and 330.5 may be
used for measurement of residual
chlorine!9*. Field test kits are available
for this purpose.
9.2 Grab samples must be collected
in glass containers having a total
volume of at least 25 mL. Fill the
sample bottle just to overflowing in
such a manner that no air bubbles pass
through the sample as the bottle is
being filled. Seal the bottle so that no
air bubbles are entrapped in it. If
preservative has been added, shake
vigorously for one minute. Maintain the
hermetic seal on the sample bottle until
time of analysis.
9.3 Experimental evidence indicates
that some aromatic compounds,
notably benzene, toluene, and ethyl
benzene are susceptible to rapid
biological degradation under certain
environmental conditions^).
Refrigeration along may not be
adequate to preserve these compounds
in wastewaters for more than seven
days. For this reason, a separate
sample should be collected, acidified,
and analyzed when these aromatics are
to be determined. Collect about 500
mL of sample in a clean container.
Adjust the pH of the sample to about 2
by adding HCI (1 + 1) while stirring.
Check pH with narrow "range (1.4 to
2.8) pH paper. Fill a sample container
as described in Section 9.2. If chlorine
residual is present, add sodium thio-
sulfate to another sample container
and fill as in Section 9.2 and mix
thoroughly.
9.4 All samples must be analyze
within 14 days of collection.
10. Daily GC/MS Performance
Tests
10.1 At the beginning of each day
that analyses are to be performed, the
624-5
July 1982
-------
GC/MS system must be checked to see
if acceptable performance criteria are
achieved for BFBCio). The performance
test must be passed before any
samples, blanks, or standards are
analyzed, unless the instrument has
met the DFTPP test described in
method 625 earlier in the dayd D.
10.2 These performance tests
require the following instrumental
parameters.
Electron Energy: 70 Volts (nominal)
Mass Range: 20 to 260
Scan Time: to give at least 5
scans per peak but
not to exceed 7
seconds per scan.
10.3 At the beginning of each day,
inject 2 {A. of BFB solution directly on
column. Alternately, add 2 f/L of BFB
solution to 5.0 mL of reagent water or
standard solution and analyze
according to Section 11. Obtain a
background corrected mass spectrum
of BFB and check that all the key ion
criteria in Table 2 are achieved. If all
the criteria are not achieved, the
analyst must retune the mass
spectrometer and repeat the test until
all criteria are achieved.
11. Sample Extraction and
Gas Chromatography
11.1 Table 1 summarizes the
recommended operating conditions for
the gas chromatograph. This table
includes retention times and method
detection limits that were achieved
under these conditions. An example of
the parameter separations achieved by
Column 1 is shown in Figure 5. Other
packed columns or chromatographic
conditions may be used if the
requirements of Section 8.2 are met.
11,2 After achieving the key ion
abundance criteria in Section 10,
calibrate the system daily as described
in Section 7.
11.3 Adjust the purge gas (helium)
flow rate to 40 ± 3 mL/min. Attach
the trap inlet to the purging device, and
set the device to purge. Open the
syringe valve located on the purging
device sample introduction needle.
11.4 Remove the plunger from a
5-mL syringe and attach a closed
syringe valve. Open the sample or
standard bottle which has been
allowed to come to ambient
temperature, and carefully pour the
sample into the syringe barrel to just
short of overflowing. Replace the
syringe plunger and compress the
sample. Open the syringe valve and
vent any residual air while adjusting the
sample volume to 5.0 mL. Since this
process of taking an aliquot destroys
the validity of the sample for future
analysis, the analyst should fill a
second syringe at this time to protect
against possible loss of data. Add 10.0
nL of the surrogate spiking solution
(Section 6.7) and, if applicable, 10.0
fA. of the internal standard spiking
solution (Section 7.4.2) through the
valve bore, then close the valve. The
surrogate and internal standards may
be mixed and added as a single spiking
solution.
11.5 Attach the syringe-syringe
valve assembly to the syringe valve on
the purging device. Open the syringe
valves and inject the sample into the
purging chamber.
11.6 Close both valves and purge the
sample for 11.0 ±0.1 minutes at
ambient temperature. [
11.7 At the conclusion of the purge
time, attach the trap to the
chromatograph, adjust the device to
the desorb mode, and begin the gas
chromatographic temperature program.
Concurrently, introduce the trapped
materials to the gas chromatographic
column by rapidly heating the trap to
1 80 °C while backflushing the trap
with an inert gas between 20 and 60
mL/min for four minutes. If this rapid
heating requirement cannot be met, the
gas chromatographic column must be
used as a secondary trap by cooling it
to 30 °C (or subambient, if problems
persist) instead of the recommended
initial temperature of 45 <°C.
11.8 While the trap is being desorbed
into the gas chromatograph, empty the
purging chamber using the sample
introduction syringe. Wash the
chamber with two 5-mL flushes of
reagent water.
11.9 After desorbing the sample for
four minutes, recondition the trap by
returning the purge and trap device to
the purge mode. Wait 1 5 seconds then
close the syringe valve on the purging
device to begin gas flow through the
trap. The trap temperature should be
maintained at 1 80 °C. Trap
temperatures up to 230 PC may be
employed, however, the higher
temperature will shorten the useful life
of the trap. After approximately seven
minutes turn off the trap heater and
open the syringe valve to stop the gas
flow through the trap. When cool, the
trap is ready for the next sample.
11.10 If the response for any ion
exceeds the working range of the
system, dilute the sample aliquot in the
second syringe wjth reagent water and
reanalyze.
12. Qualitative Identification
12.1 Obtain EICPs for the primary ion
(Table 4) and at least two secondary
ions for each parameter of interest. The
following criteria must be met to make
a quantitative identification.
12.1.1 The characteristic ions of
each parameter of interest must
maximize in the same or within one
scan of each other.
12.1.2 The retention time must fall
within ±30 seconds of the retention
time of the authentic compound.
12.1.3 The relative peak heights of
the three characteristic ions in the
EICPs must fall within ±20% of the
relative intensities of these ions in a
reference mass spectrum. The
reference mass spectrum can be
obtained from a standard analyzed in
the GC/MS system or from a reference
library.
12.2 Structural isomers that have
very similar mass spectra and less than
30 seconds difference in retention
time, can be explicitly identified only if
the resolution between authentic
isomers in a standard mix is
acceptable. Acceptable resolution is
achieved if the baseline to valley height
between the isomers is less than 25%
of the sum of the two peak heights.
Otherwise, structural isomers are
identified as isomeric pairs.
13. Calculations
13.1 When a parameter has been
identified, the quantitation of that
parameter should be based on the
integrated abundance from the EICP of
the first listed characteristic ion given
in Table 4. If the sample produces an
interference for the primary ion, use a
secondary characteristic ion to
quantitate. Quantitation may be
performed using the external or internal
standard techniques.
13.1.1 If the external standard
calibration procedure is used, calculate
the concentration of the parameter
being measured from the area of the
characteristic ion using the calibration
curve or calibration factor in Section
7.3.2.
13.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.4.3 and
equation 2.
Eq. 2.
Concentration ng/L = (AsCis)/(Ais)(RF)
where:
624-6
July 1982
-------
As = Area of the characteristic ion
for the parameter or surrogate
standard to be measured.
Ais = Area of the characteristic ion
for the internal standard.
Cis = Concentration of the internal
standard.
13.2 Report results in micrograms
per liter. The results for'cis- and
trans-1,3 dichloropropene should be
reported as total 1,3-dichloropropene
(STORE! No. 34561, CAS No.
542-75-6). When duplicate and spiked
samples are analyzed/report all data
obtained with the sample results.
13.3 If any of the surrogate standard
recoveries fall outside the control limits
which were established;as directed in
Section 8.4, data for all parameters
determined by this method in that
sample must be labeled as suspect.
14. Method Performance
14.1 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
zerod). The MDL concentrations listed
in Table 1 were obtained using reagent
water! 12). Similar results were
achieved using representative
waste waters. '
i
14.2 The average recoveries and the
average standard deviations of the
percent recoveries, presented in Table
5, were the result of a study of the
accuracy and precision of this method
by several laboratories. The values
listed represent the results from 2 to 4
laboratories!13'.
14.3 The U.S. Environmental Protec-
tion Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
References ;
1. See Appendix A.
2. Bellar, T.A., and J.J. Lichtenberg,
Journal American Water.Works
Association, 66, p. 739, (1974).
3. Bellar, T.A., and J.J.:Lichtenberg,
"Semi-Automated Headspace Analysis
of Drinking Waters and Industrial
Waters for Purgeable Volatile Organic
Compounds," Measurement of Organic
Pollutants in Water and Wastewater,
C.E. Van Hall, editor, American Society
for Testing and Materials, Philadelphia,
PA. Special Technical Publication 686,
1978.
4. "Sampling and Analysis Procedures
for Screening of Industrial Effluents for
Priority Pollutants." U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, OH 45268,
March 1977, Revised April 1977.
Effluent Guidelines Division,
Washington, DC 10460.
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, Aug. 1977.
6. "OSHA Safety and Health
Standards, General Industry,"
(29CFR1910), 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. "Handbook of Analytical Quality
Control in Water and Wastewater
Laboratories," EPA-600/4-79-01 9,
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268,
March 1979.
9. "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual/'
Methods for Chemical Analysis of
Water and Wastes, EPA
6OO/4-79-020, U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1 979.
10. Budde, W.L. and Eichelberger,
J.W., "Performance Tests for the
Evaluation of Computerized Gas
Chromatography/Mass Spectrometry
Equipment and Laboratories,"
EPA-600/4-80-025, U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, OH 45268, p.
16, April 1980.
11. Eichelberg, J.W. Harris, L.E., and
Budde, W.L., "Reference Compound to
Calibrate Ion Abundance Measurement
in Gas Chromatography—Mass
Spectrometry Systems,", Analytical
Chemistry, 47, 995-1000 (1979).
1 2. "Method Detection Limit for
Methods 624 and 625," Olynyk, P.,
Budde, W.L. Eichelberger, J.W.,
unpublished report, October 1980.
13. Kleopfer, R.D., "POTW Toxic
Study, Analytical Quality Assurance
Final Report," U.S. Environmental
Protection Agency, Region VII, Kansas
City, Kansas 6611 5, 1981.
624-7
July 1982
-------
Table 7. Chromatographic Conditions and Method Detection Limits
Parameter
Chloromethane
Bromomethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorofluoromethane
1, 1 -Dichloroethene
1, 1 -Dichloroethane
trans- 1 ,2-Dlchloroethene
Chloroform
1, 2-Dichloroethane
1, 1, 1 -Trichloroethane
Carbon tetrachloride
Bromodichloromethane
1, 2-Dichloropropane
trans- 1 ,3-Dichloropropene
Trlchloroethene
Benzene
Dibromochloromethane
1, 1 ,2-Trichloroethane
cis- 1 ,3-Dichloropropene
2-Chloroethylvinyl ether
Bromoform
1, 1 ,2,2-Tetrachloroethane
Tetrachloroethene
Toluene
Chlorobenzene
Ethyl benzene
1, 3-Dichlorobenzene
1, 2-Dichlorobenzene
1 f4-Dichlorobenzene
Retention Time
(min.)
Column 1
2.3
3.1
3.8
4.6
6.4
8.3
9.0
10.1
10.8
11.4
12.1
13.4
13.7
14.3
15.7
15.9
16.5
17.0
17.1
17.2
17.2
18.6
19.8
22.1
22.2
23.5
24.6
26.4
33.9
35.0
35.4
Method
Detection
Limit (\ig/L)
nd
nd
nd
nd
2.8
nd
2.8
4.7
1.6
1.6
2.8
3.8
2.8
2.2
6.0
5.0
1.9
4.4
3.1
5.0
nd
nd
i . _
; 4.7
6.9
4. 1
' 6.0
6.0
7.2
nd
nd
nd
nd » not determined
Column conditions: CarbopakB (60/80 mesh) coated with 1 % SP- WOO packed in a
6 ft by 2 mm ID glass column with helium carrier gas at a flow rate, of 30 mL/min.
Column temperature is isothermal at 45° C for 3 min, then programmed at 8 °Cper
minute to 220° C and held for 15 min.
Table 2. BFB Key Ion Abundance Criteria
Mass Ion Abundance Criteria
50 15 to 40% of mass 95
75 30 to 60% of mass 95
95 Base Peak, 100% Relative Abundance
96 5 to 9% of mass 95
173 <2%of mass 174
174 >50% of mass 95
175 5 to 9% of mass 174
176 >95% but < 101% of mass 174
177 5 to 9% of mass 176
624-8 July 1982
-------
Table 3. Suggested Surrogate and Internal Standards
Compound
Surrogate Standards ;
Benzene d-6 ,
4-Bromofluorobenzene
1 ,2-Dichloroethane d-4
1 ,4-Difluorobenzene
Ethylbenzene d-5
Ethylbenzene d-1O
Fluorobenzene
Pentafluorobenzene
Internal Standards
Bromochloromethane ;
2-Bromo- 1 -chloropropane
1 ,4-Dichlorobutane
Retention Time
(min.)a
17.0
28.3
12.1
19.6
26.4
26.4
18.4
23.5
9.3
19.2
25.8
Primary
Ion
84
95
1O2
114
111
98
96
168
128
77
55
Secondary
Ions
174, 176
63, 88
7O
—
49, 130,51
79, 156
90,92
aFor chromatographic conditions, see Table 1.
Table 4. Characteristic Ions for Purgeable Organics
Parameter
Chloromethane
Bromomethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorofluoromethane
7, 1 ' -Dichloroethene
1, 1 -Dichloroethane
trans- 1 ,2-Dichloroethene
Chloroform
1 ,2-Dichloroethane
1, 1, 1 -Trichloroethane '
Carbon tetrachloride
Bromodichloromethane
1 ,2-Dichloropropane
trans- 1 ,3-Dichloropropene
Trichloroethene ',
Benzene :
Dibromochloromethane
1, 1 ,2-Trichloroethane
cis- 1 ,3-Dichrloropropene
2-Chloroethylvinyl ether ,
Bromoform ,
1, 1 ,2,2-Tetrachloroethane
Tetrachloroethene
Toluene
Chlorobenzene \
Ethyl benzene
1 ,3-Dichlorobenzene
1, 2-Dichlorobenzene
1 ,4-Dichlorobenzene
Primary
Ion
5O
94
62
64
84
1O1
96
63
96
83
98
97
117
127
112
75
13O
78
127
97
75
106
173
168
164
92
112
106
146
146
146
Secondary Ions
52
96
64
66
49, 51, 86
1O3
61,98
65, 83, 85, 98, WO
61, 98
85
62, 64, WO
99, 117, 119
119, 121
83, 85, 129
63, 65, 1 14
77
95, 97, 132
129, 2O8, 206
83, 85, 99, 132, 134
77
63, 65
171, 1 75, 250, 252, 254, 256
83, 85, 131, 133, 166
129, 131, 166
91
114
91
148, 113
148, 1 13
148, 1 13
624-9 July 1982
-------
Table 5. Accuracy and Precision for Purgeable Organics
Reagent Water
Wastewater
Parameter
Benzene
Bromodichtoromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chloroethane
2-Chloroethylvlnyl ether
Chloroform
Chloromethane
Dibromochloromethane
1, 1 -Dichloroethane
1, 2-Dichloroethane
1, 1-D!chloroethene
trans- 1 ,2-Dichloroethene
1, 2-Dichloropropane
cis- 1 ,3-Dichloropropene
trans- 1 ,3-Dichloropropene
Ethyl benzene
Methylene chloride
1, 1,2,2- Tetrachloroethane
Tetrachloroethene
Toluene
1, 1, 1 -Trichloroethane
1, 1 ,2-Trichloroethane
Trichloroethene
Trichlorofluoromethane
Vinyl chloride
Samples were spiked between
Average Standard
Percent Deviation
Recovery (%)
99
102
104
100
1O2
1OO
97
1O1
101
99
103
101
1OO
102
99
1O2
105
104
100
96
102
1O1
101
101
1O1
101
103
1OO
10 and
9
12
14
20
16
7
22
13
10
19
1
/
10
8
17
12
8
15
1
1
8
15
1
9
9
9
1
10
9
11
13
A verage
Percent
Recovery
98 \
103
105
88
104
102
103
95
101
99
104
104
1O2
99
101
1O3
102
100
1O3
89
104
100
98
102
104
100
107
98
Standard
Deviation
10
10
16
23
15
9
31
17
12
24
14
15
1O
15
10
12
19
18
10
28
14
11
14
16
15
12
19
25
1000 jxg/L.
'
Optional
Foam x-
Trap (
I
V
p.^_Exit 1/4 in.
"^T PJD.
L«_ 14mm
* ) O.D.
/K Inlet V* in-
U-— O.D.
1/4 in.
O.D. exit
Inlet
way Syringe valve
20 gauge syringe needle
O.D. Rubber Septum
10mm O.D. V,t in. O.D.
-Inlet ^/Stainless Steel
Vt in. O.D.
13X molecular
sieve purge
gas filter
Purge gas
flow control
10mm glass frit
medium porosity
624-10
Figure 1 . Purging device
July 1982
-------
Packing procedure
Construction
Glass
wool 5mm
Grade 15
Silica gel 8cm
Tenax 15cm
3% OV-1 1cm\
Glass 5mm
wool Trap inlet
Compression fitting
•nut and ferrules
14ft 7-^ foot resistance
wire wrapped solid
Thermocouple/controller
•sensor
Tubing 25 cm.
0.105 in. I.D.
0.125 in. O.D.
stainless steel
Figure 2. Trap packings and construction to include desorb capability
Carrier gas flow control
Pressure regulator
Purge gas
flow control \
13X molecular
sieve -filter
Liquid injection ports
Column oven
— Confirmatory column
To detector
Analytical column
optional 4-port column
6-port selection valve
valve / Trap inlet
/ x Resistance wire
^-Heater control
Purging
device
Note:
All lines between
trap and GC
should be heated
to 80°C
Figure 3. Schematic of purge and trap device — purge mode
624-11
July 1982
-------
Carrier gas flow control
~
Pressure regulator
. Liquid injection ports Co,umn
oven
Purge gas . .
flow control v ,
13X molecular
sieve filter
L._— Confirmatory column
\ >> To detector
~—Analytical column
optional 4-port column,
selection valve !
6-port jrap in/et
valve I Resistance wire
Purging
device
Heater control
Note:
All lines between
trap and GC
should be heated
to 95°C
Figure 4. Schematic of purge and trap device — desorb mode
Column: 1% SP-1000 on Supelcoport
Program: 45°C. 3 min., 8° per min. to 220°C.
Detector: Mass spectrometer
8
10 12 14 16 18 20
Retention time, minutes
26 28
Figure 5. Gas chromatogram of volatile organics by purge and trap.
624-12 July 1982
-------
SEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
Test Method
Base/Neutrals and Acids —
Method 625
1. Scope and Application
1.1 This method covers the determi-
nation of a number of organic
compounds that are partitioned into an
organic solvent and are amenable to
gas chromatography. The parameters
listed in Tables 1 and 2 may be
qualitatively and quantitatively
determined using this method.
1.2 The method may be extended to
include the parameters listed in Table 3.
Benzidine can be subject to oxidative
losses during solvent concentration.
a-BHC, y-BHC, endosulfan I and II, and
endrin are subject to decomposition
under the alkaline conditions of the
extraction step. Hexachlorocyclopenta-
diene is subject to thermal decomposi-
tion in the inlet of the gas chromato-
graph, chemical reaction in acetone
solution and photochemical decompo-
sition. N-nitrosodimethylamine is
difficult to separate from the solvent
under the chromatographic conditions
described. N-nitrosodiphenylamine
decomposes in the gas chromator
graphic inlet and cannot be separated
from diphenylamine. The preferred
method for each of these parameters is
listed in Table 3.
1.3 This is a gas chromatography/
mass spectrometry (GC/MS) method
applicable to the determination of the
compounds listed in Tables 1, 2, and 3
in municipal and industrial discharges
as provided under 40 CFR 1 36.1. Until
the U.S. Environmental Protection
Agency establishes performance cri-
teria based upon the results of inter-
laboratory testing, any alternative
GC/MS method which meets the per-
formance criteria described in Section
8.2 will be permitted. Performance
must be verified for such modification
by analyzing wastewater as described
in Section 8.2.2. In addition, the
laboratory must successfully partici-
pate in the applicable performance
evaluation studies.
1.4 The method detection limit (MDL,
defined in Section 16)<1) for each
parameter is listed in Tables 4 and 5.
The MDL for a specific wastewater
differ from those listed, depending
upon the nature of interferences in the
sample matrix.
1.5 This method is restricted to use
by or under the supervision of analysts
experienced in the operation of gas
chromatograph/mass spectrometers
and skilled in the interpretation of mass
spectra. Each analyst must demon-
strate the ability to generate accept-
able results with this method using the
procedure described in Section 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is serially
extracted with methylene chloride at a
pH greater than 11 and again at pH
less than 2 using a separatory funnel or
a continuous extractor. The methylene
chloride extract is dried and
concentrated to a volume of 1 mL.
Chromatographic conditions are
described which permit the separation
and measurement of the parameters in
the extract. Qualitative identification is
performed using the retention time and
the relative abundance of three
characteristic ions. Quantitative
analysis is performed using either
625-1
July 1982
-------
external or internal standard techniques
with a single characteristic ion.
3. Interferences
3.1 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 total ion current
profiles. All of these materials must be
routinely demonstrated to be free from
interferences under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5.
3.1.1 Glassware must be scrupulously
cleaned<3>. 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 reagent water. It should then be
drained dry, and heated in a muffle
furnace at 400 °C for 15 to 30
minutes. Some thermally stable
materials, such as PCBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Volumetric
ware should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and stored
in a clean environment to prevent any
accumulation of dust or other
contaminants. Store it inverted or
capped with aluminum foil.
3.7.2 The use of high purity reagents
and solvents helps to minimize inter-
ference problems. Purification of
solvents by distillation in all-glass
systems may be required.
3.2 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 diver-
sity of the industrial complex or munici-
pality being sampled.
3.3 The base-neutral extraction may
cause significantly reduced recovery of
phenol, 2-methylphenol, and
2,4-dimethylphenol. The analyst must
recognize that results obtained under
these conditions are minimum
concentrations.
3.4 The packed gas chromatographic
columns recommended for the basic
fraction may not exhibit sufficient
resolution for certain isomeric pairs.
These include anthracene and phenan-
threne; chrysene and benzo(a)anthra-
cene; and benzo(b)fluoranthene and
benzo(k)fluoranthene. The gas
chromatograph retention time and
mass spectra are not sufficiently
different to make an unambiguous
distinction between these compounds.
Alternative techniques should be used
to identify and quantify these specific
compounds. See method 610.
3.5 In samples that contain an
inordinate number of interferences, the
use of chemical ionization (CD mass
spectrometry may make identification
easier. Tables 6 and 7 give
characteristic Cl ions for most of the
compounds covered by this method.
The use of Cl mass spectrometry to
support electron ionization (El) mass
spectrometry is encouraged but not
required. j
4. Safety '•
4.1 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
awareness file of OSHA regulations
regarding the safe handling of the
chemicals 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. Additional references to
laboratory safety are available and
have been identified for the information
of the analyst<4-6).
4.2 The following parameters
covered by this method have been
tentatively classified as known or
suspected, human or mammalian
carcinogens; benzo(a)anthracene,
benzidine, 3,3'-dichlorobenzidine,
benzo(a)pyrene, a-BHC, /5-BHC, cJ-BHC,
y-BHC, dibenzo(a,h) anthracene, Ni-
nitrosodimethylamine, 4,4'-DDT and
polychlorinated biphenyis.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
or composite sampling.
5.1.1 Grab sample bottle—Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If
amber bottles are not available, protect
samples from light. The container must
be washed, rinsed with acetone or
methylene chloride, and dried before
use to minimize contamination.
5.7.2 Automatic sampler (optional) —
Must incorporate glass sample
containers for the collection of a
minimum of 250 mL. Sample
containers must be kept refrigerated at
4 °C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however, the
compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the potential
for contamination of the sample. An
integrating flow meter is required to
collect flow proportional composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.7 Separatory funnel — 2000-mL,
with Teflon stopcock.
5.2.2 Drying column— 1 9 mm ID
chromatographic column with coarse
frit.
5.2.3 Concentrator tube, Kuderna-
Danish— 10-mL, graduated (Kontes
K-570050-1025 or equivalent).
Calibration must be checked at the
volumes employed in the test. Ground
glass stopper is used to prevent
evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-
Danish—500-mL (Kontes
K-570001-0500 or equivalent).
Attach to concentrator tube with
springs.
5.2.5 Snyder column, Kuderna-
Danish—Three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Snyder column, Kuderna-
Danish—Two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.7 Vials—Amber glass, 10-to
15-mL capacity, with Teflon-lined
screw cap.
5.2.8 Continuous liquid-liquid
extractors—Equipped with Teflon or
glass connecting joints and stopcocks
requiring no lubrication. (Hershberg-
Wolf Extractor-Ace Glass Company,
Vineland, N.J. P/N 6841-10 or
equivalent.)
5.3 Boiling chips—approximately
10/40 mesh. Heat to 400 °C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath—Heated, with
concentric ring cover, capable of
temperature control (±2 °C). The bath
should be used in a hood.
625-2
July 1982
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5.5 Balance—Analytical, capable of
accurately weighing 0.0001 g.
5.6 GC/MS system.
5.6.1 Gas chromatograph—An
analytical system complete with a
temperature programmable gas
chromatograph and all required
accessories including syringes,
analytical columns, and gases. The
injection port must be designed for on-
column injection when using packed
columns and for splitless injection
when using capillary columns.
5.6.2 Column for Base Neutrals—1.8
m long x 2 mm ID glass, packed with
Supelcoport (100/1 20imesh) coated
with 3% SP-2250 or equivalent. This
column was used to develop the
accuracy and precision statements in
Table 6 and the MDL data in Table 4.
Guidelines for the use of alternate
column packings are provided in
Section 13.1.
5.6.3 Column for Acids— 1.8m long
x 2 mm ID glass, packed with
Supelcoport (100/1 20 'mesh) coated
with 1 % SP-1 240 DA or equivalent.
This column was used to develop the
accuracy and precision statements in
Table 7, and the MDL data in Table 5.
Guidelines for the use of alternate
column packings are given in Section
13.1.
5,6.4 Mass Spectrometer—Capable
of scanning from 35 to'450 amu every
seven seconds or less utilizing a 70
volt (nominal) electron energy in the
electron impact ionization mode and
producing a mass spectrum which
meets all the criteria in Table 9 when
50 ng of decafluorotriphenyl phosphine
(DFTPP; bis(perfluorophenyl) phenyl
phosphine) is injected through the gas
chromatographic inlet. Any gas
chromatograph to mass spectrometer
interface that gives acceptable
calibration points at 50 ng per injection
for each compound of interest in
Tables 1 through 3 and'achieves all
acceptable performance criteria
(Section 1 2) may be used. Gas
chromatograph to mass spectrometer
interfaces constructed of all glass or
glass lined materials areFrecommended.
Glass can be deactivated by silanizing
with dichlorodimethylsilane.
5.6.5 A computer system must be
interfaced to the mass spectrometer
that allows the continuous acquisition
and storage on machine readable media
of all mass spectra obtained
throughout the duration.of the
chromatographic program. The
computer must have software that
allows searching any GC/MS data file
for ions of a specific mass and plotting
such ion abundances versus time or
scan number. This type of plot is
defined as an Extracted Ion Current
Profile (EICP). Software must also be
available that allows integrating the
abundance in any EICP between
specified time or scan number limits.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an inter-
ferent is not observed at the method
detection limit of each parameter of
interest.
6.2 Sodium hydroxide solution (10
N)—Dissolve 40g NaOH in reagent
water and dilute to 100 mL.
6.3 Sodium thiosulfate—(ACS)
Granular.
6.4 Sulfuric acid solution
(1 + 1 )-Slowly add 50 mL of H2SO4
(sp. gr. 1.84) to 50 mL of reagent
water.
6.5 Acetone, methanol, methylene
chloride—Pesticide quality or
equivalent.
6.6 Sodium sulfate-(ACS) Granular,
anhydrous. Purify by heating at 400 °C
for four hours in a shallow fray.
6.7 Stock standard solutions (1.00
HQlpL) — Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.7.1 Prepare stock standard
solutions by accurately weighing about
0.0100 g of pure material. Dissolve
the material in pesticide quality
acetone or other suitable solvent 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 assayed 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.
6.7.2 Transfer the stock standard
solutions into Teflon-sealed screw-cap
bottles. Store in 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 cali-
bration standards from them. Quality
control check samples, that can be
used to determine the accuracy of
calibration standards will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
6.7.3 Stock standard solutions must
be replaced after six months or sooner
if comparison with quality control
check samples indicate a problem.
6.8 Surrogate standard spiking
solutions—select a minimum of three
surrogate compounds from Table 8.
Prepare a surrogate standard spiking
solution at a concentration of 100
Mg/1.00 ML in acetone. Addition of
1.00 mL of this solution to 1000-mL
of sample is equivalent to a concentra-
tion of 100 i*g/L of each surrogate
standard. Store the spiking solutions at
4 °C in Teflon-sealed containers. The
solutions should be checked frequently
for stability. These solutions must be
replaced after six months, or sooner if
comparison with quality control check
samples indicate a problem. Surrogate
standard spiking solutions, appropriate
for use with this method will be
available from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
6.9 DFTPP standard —Prepare a 25
ng/f*L solution of DFTPP in acetone.
7. Calibration
7.1 Establish gas chromatographic
operating parameters equivalent to
those indicated in Tables 4 or 5. The
GC/MS system can be calibrated using
the external standard technique
(Section 7.2) or the internal standard
technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding volumes of one or more stock
standards to a volumetric flask and
diluting to volume with acetone. One
of the external standards should be at a
concentration near, but above, the
MDL and the other concentrations
should correspond to the expected
range of concentrations found in real
samples or should define the working
range of the GC/MS system.
7.2.2 Analyze 2 to 5 ^L of each
calibration standard and tabulate the
area responses of the primary
characteristic ion of each standard
(Tables 4 and 5) against the mass
injected. The results may 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
625-3
July 1982
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the working range «10% relative
standard deviation, RSD), linearity
through the origin may be assumed and
the average ratio or calibration factor
may be used in place of a calibration
curve.
7.2.3 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 any parameter varies
from the predicted response by more
than ± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar 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. Table
8 lists some recommended internal
standards. Phenanthrene-d10 has been
used for this purpose. Use the base
peak ion as the primary ion for
quantification of the standards. If
interferences are noted, use the next
two most intense ions as the
secondary ions.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding appropriate volumes of one or
more stock standards to a volumetric
flask. To each calibration standard or
standard mixture, add a known
constant amount of one or more
internal standards, and dilute to volume •
with acetone. One of the calibration
standards should be at a concentration
near, but above, the MDL and the other
concentrations should correspond to
the expected range of concentrations
found in real samples or should define
the working range of the GC/MS
system.
7.3.2 Analyze 2 to 5 ftL of each
calibration standard and tabulate the
area of the primary characteristic ion
(Tables 4 and 5) against concentration
for each compound and internal
standard, and calculate response
factors {RF) for each compound using
equation 1.
Eq. 1 RF - (AsCis)/(AisCs)
where:
As = Area of the characteristic ion
for the parameter to be
measured.
AJS = Area of the ch.aracteristic ion
for the internal standard.
Cis = Concentration of the internal
standard, (^g/L).
Cs = Concentration of the
parameter to be measured,
(ng/L).
If the RF value over the working range
is a constant «10% RSD), the RF can
be assumed to be invariant and the
average RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
of response ratios, As/A|g, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration o;f laboratory
capability and the analyses of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing perfprmance
checks must be compared with estab-
lished performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision with
this method. This ability is established
as described in Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the
separations or lower the cost of
measurements. Each time such
modifications are made to the method,
• the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike all
samples with surrogate standards to
monitor continuing laboratory
performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform the
following operations.
8.2.1 Select a representative spike
concentration for each parameter to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1 000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate and 1.0
mL of the surrogate standard dosing
solution (Section 6.8) to each of a
minimum of four 1000-mL aliquots of
reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for all
parameters and surrogate standards.
Wastewater background corrections
must be made before R and s
calculations are performed.
8.2.4 Using Table 6 or 7, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s > p or
|X-R| > p, review potential problem
areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and s based
upon the result of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria for each
of the surrogate standards.
8.3.1 Calculate upper and lower
control limits for method performance
for each surrogate standard, using the
values for R and s calculated in Section
8.2.3:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
The UCL and LCL can be used to
construct control charts!7) that are
useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they become avail-
625-4
July 1982
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able from the U.S. Environmental
Protection Agency.
8.3.2 For each surrogate standard,
the laboratory must develop and
maintain separate accuracy statements
of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcula-
tion of R and s. Alternately, the analyst
may use four wastewater data points
gathered through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly.(7) •
8.4 The laboratory is required to
spike all samples with the surrogate
standard spiking solution to monitor
spike recoveries. If the recovery for any
surrogate standard does not fall within
the control limits for method
performance, the results reported for
that sample must be qualified as
described in Section 1 5.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water,,that all
glassware and reagent interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as a
safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance 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. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. Whenever
possible, the laboratory should perform
analysis of standard reference
materials and participate in relevant
performance evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices!8) 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 and
other potential sources of
contamination.
9.2 The samples must be iced or
refrigerated at 4 °C from the time of
collection until extraction. Fill the
sample bottles and, if residual chlorine
is present, add 80 mg of sodium
thiosulfate per each liter of water. U.S.
Environmental Protection Agency
methods 330.4 and 330.5 may be
used to measure the residual
chlorine'9). Field test kits are available
for this purpose.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction.
10. Separatory Funnel
Extraction
10.1 Samples are usually extracted
using separatory funnel techniques. If
emulsions will prevent achieving
acceptable solvent recovery with
separatory funnel extractions,
continuous extraction (Section 1 1)
may be used. The separatory funnel
extraction scheme described below
assumes a sample volume of one-liter.
When sample volumes of two liters are
to be extracted, use 250-, 100-, and
100-mL volumes of methylene chloride
for the serial extraction of the base/
neutrals and 20O-, 1OO-, and 1OO-mL
volumes of methylene chloride for the
acids.
10.2 Mark the water meniscus on the
side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel. Pipet 1.00 mL
surrogate standard spiking solution into
the separatory funnel and mix well.
Check the pH of the sample with wide-
range pH paper and adjust to pH > 11
with 10 N sodium hydroxide.
10.3 Add 60 mL methylene chloride
to the sample bottle, seal, and shake
for 30 seconds to rinse the inner
surface. Transfer the solvent to the
separatory funnel and extract the
sample by shaking the funnel for two
minutes with periodic venting to
release excess pressure. Allow the
organic layer to separate from the
water phase for a minimum of 10
minutes. 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, centrifu-
gation, or other physical methods.
Collect the methylene chloride extract
in a 250-ml Erlenmeyer flask. If the
emulsion cannot be broken (recovery of
less than 80% of the methylene
chloride, corrected for the water
solubility of methylene chloride),
transfer the sample, solvent, and
emulsion into the extraction chamber
of a continuous extractor and proceed
as described in Section 11.3.
10.4 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. Label
the combined extract as base/neutral
fraction.
10.5 Adjust the pH of the aqueous
phase to less than 2 using sulf uric acid
(1 +1). Serially extract three times
with 60-mL aliquots of methylene
chloride. Collect and combine the
-extracts in a 250-mL Erlenmeyer flask
and label the combined extract as the
acid fraction.
10.6 For each fraction, 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 if the
requirements of Section 8.2 are met.
10.7 For each fraction, pour the
combined extract through a drying
column containing about 10 cm of
anhydrous sodium sulfate, and collect
the extract in the K-D concentrator.
Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene
chloride to complete the quantitative
transfer.
10.8 To the evaporative flask for
each fraction, add one or two clean
boiling chips and attach a three-ball
Snyder column. Prewet the Snyder
column by adding about 1 mL
methylene chloride to the top of the
column. 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 1 5 to
20 minutes. 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 1 mL, remove the K-D
apparatus from the water bath and
625-5
July 1982
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allow it to drain and cool for at least 10
minutes. Remove the Snyder column
and rinse the flask and its lower joint
into the concentrator tube with 1 to 2
mL of methylene chloride. A 5-mL
syringe is recommended for this
operation.
10.9 Add another one or two clean
boiling chips to the concentrator tube
and attach a two-ball micro Snyder
column. Prewet the Snyder column by
adding about 0.5 mL of methylene
chloride to the top of the column. 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. Adjust the vertical
position of the apparatus and the water
temperature as required to complete
the concentration in 5 to 10 minutes.
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 about 0.5 mL,
remove the K-D apparatus from the
water bath and allow it to drain for at
least 10 minutes while cooling.
Remove the Snyder column and rinse
its the flask and its lower joint into the
concentrator tube with 0.2 mL of
acetone or methylene chloride. Adjust
the final volume to 1.0 mL with the
solvent. Stopper the concentrator tube
and store refrigerated if GC/MS
analysis will not be performed
immediately. If the extracts will be
stored longer than two days, they
should be transferred to Teflon-sealed
screw-cap bottles and labeled
base/neutral or acid fraction as
appropriate.
10.10 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. Continuous Extraction
11.1 When experience with a sample
from a given source indicates that a
serious emulsion problem will result or
an emulsion is encountered in Section
10.3, using a separatory funnel, a
coritinuous extractor should be used.
11.2 Mark the water meniscus on the
side of the sample bottle for later
measurement of the sample volume.
Check the pH of the sample with wide-
range pH paper and adjust to pH 11
with 10 N sodium hydroxide. Transfer
the sample to the continuous extractor
and using a pipet, add 1.00 mL of
surrogate standard spiking solution and
mix well. Add 60 mL of methylene
chloride to the sample bottle, seal and
shake for 30 seconds to rinse the inner
surface. Transfer the solvent to the
extractor.
11.3 Repeat the sample bottle rinse
with an additional 50- to 100-mL
portion of methylene chloride and add
the rinse to the extractor.
11.4 Add 200 to 500 mL of
methylene chloride to the distilling
flask, add sufficient reagent water to
ensure proper operation,'and extract
for 24 hours. Allow to cool, then
detach the boiling flask, and dry,
concentrate and seal the extract as in
Section 10.6 through 10.9. Hold the
aqueous phase for acid extraction (See
Section 11.5).
11.5 Charge a clean distilling flask
with 500 mL of methylene chloride and
attach it to the continuous extractor.
Carefully, adjust the pH 6f the aqueous
phase to less than 2 using sulfuric acid
(1 +1). Extract for 24 hours. Dry,
concentrate and label and seal the
extract as described in Sections 10.6
through 10.9. •
12. Daily GC/MS Performance
Tests
12.1 At the beginning ;of each day
that analyses are to be performed, the
GC/MS system must be checked to see
that acceptable performance criteria
are achieved for DFTPP. Each day that
benzidine is to be determined, the
tailing factor criterion described in
Section 1 2.4 must be achieved. Each
day the acids are to be determined, the
tailing factor criterion infection 12.5
must be achieved. ,
12.2 These DFTPP performance test
require the following instrumental
parameters.
Electron Energy 70 volts (nominal)
Mass Range 35to450amu
Scan Time to give at least 5
scans per peak but
not to exceed 7
seconds per scan.
12.3 DFTPP performance test< 10)-
At the beginning of each day, inject
2fA. (50 ng) of DFTPP standard
solution. Obtain a background cor-
rected mass spectra of DFTPP and
check that all the key ion criteria in
Table 9 are achieved. If all the criteria
are not achieved, the analyst must
retune the mass spectrometer and
repeat the test until all criteria are
achieved. The performance criteria
must be achieved before any samples,
blanks, or standards are analyzed. The
tailing factor tests in Section 1 2.4 and
1 2.5 may be performed simultaneously
with the test.
12.4 Column performance test for
base/neutrals—At the beginning of
each day that the base-neutral fraction
is to be analyzed for benzidine, the
benzidine tailing factor must be
calculated. Inject 100 ng of benzidine
either separately or as a part of a stan-
dard mixture that may contain DFTPP
and calculate the tailing factor. The
benzidine tailing factor must be less
than 3.0. Calculation of the tailing
factor is illustrated in Figure 13.<1D
Replace the column packing if the
tailing factor criterion cannot be
achieved.
12.5 Column performance for
acids—At the beginning of each day
that the acids are to be determined,
inject 50 ng of pentachlorophenol
either separately or as a part of a
standard mix that may contain DFTPP.
The tailing factor for pentachlorophenol
must be less than five. Calculation of
the tailing factor is illustrated in Figure
13<1 D. Replace the column packing if
the tailing factor criterion cannot be
achieved.
13. Gas Chrornatography/
Mass Spectrometry
13.1 Table 4 summarizes the
recommended gas chromatographic
operating conditions for the
base/neutral fraction. Table 5
summarizes the recommended gas
chromatographic operating conditions
for determination of the acid fraction.
These tables include retention times
and MDL that were achieved under
these conditions. Examples of the
parameter separations achieved by
these columns are shown in Figures 1
through 1 2. Other packed columns or
chromatographic conditions may be
used if the requirements of Section 8.2
and Section 1 2 are met. Capillary
(open-tubular) columns may also be
used if the relative standard deviations
of responses for replicate injections are
demonstrated to be less than 6% and
the requirements of Section 8.2. and
Section 12 are met.
13.2 After the GC/MS performance
requirements of Section 1 2, calibrate
the system daily as described in
Section 7.
13.3 If the internal standard
approach is being used, the internal
standard must be added to sample
extract and mixed thoroughly, imme-
diately, before injection into the
instrument. This minimizes losses due
to adsorption, chemical reaction or
evaporation.
13.4 Inject 2 to 5 pL of the sample
extract using the solvent-flush
625-6
July 1982
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technique'12>. Smaller (1.0 jA.) volumes
may be injected if automatic devices
are employed. Record the volume
injected to the nearest 0.05 yL.
13.5 If the response for any ion
exceeds the working range of the
GC/MS system, dilute the extract and
reanalyze.
13.6 Perform all qualitative and
quantitative measurements as
described in Sections 14 and 1 5.
When the extracts are not being used
for analyses, store them1 at 4 °C pro-
tected from light in screw-cap vials
equipped with unpierced Teflon-lined
septa.
14. Qualitative Identification
14.1 Obtain an EJCP for the primary
ion and the two other ions listed in
Tables 4 and 5. See Section 7.3 for
ions to be used with internal and
surrogate standards. The following
criteria must be met to make a
qualitative identification.
14.1.1 The characteristic ions for
each compound of interest must
maximize in the same or! within one
scan of each other. ;
14.1.2 The retention time must fall
within ± 30 seconds of the retention
time of the authentic compound.
14.1.3 The relative peak heights of
the three characteristic ions in the
EICP's must fall within i 20% of the
• relative intensities of these ions in a
reference mass spectrum. The refer-
ence mass spectrum can be obtained
by a standard analyzed in the GC/MS
system or from a reference library.
14.2 Structural isomers that have
very similar mass spectra and less than
30 seconds difference in retention
time, can be explicitly identified only if
the resolution between authentic
isomers in a standard mix is acceptable.
Acceptable resolution is achieved if the
baseline to valley height between the
isomers is less than 25% of the sum of
the two peak heights. Otherwise,
structural isomers are identified as
isomeric pairs.
15. Calculations
15.1 When a compound has been
identified, the quantitation of that
compound, will be based on the inte-
grated abundance from the EICP of the
primary characteristic ion in Tables 4
and 5. Use the base peak ion for
internal and surrogate standards. If the
sample produces an interference for
the first listed ion, use a secondary ion
to quantitate. Quantitation will be per-
formed using external or internal
standard techniques.
15. 1. 1 '" If the external standard
calibration procedure is used, calculate
the amount of material injected from
the area of the characteristic ion using
the calibration curve or calibration
factor in Section 7.2.2. The concentra-
tion in the sample can be calculated
from equation 2:
= (A)(Vt>
where:
A = Amount of material injected,
in nanograms.
V| = Volume of extract injected
Eq. 2. Concentration,
Vt = Volume of total extract (^L).
Vs = Volume of water extracted
(mL).
15.1.2 If the internal standard cali-
bration procedure was used, calculate
the concentration in the sample using
the response factor (RF) determined in
Section 7.3.2 and equation 3.
Eq. 3
Concentration, ug/L = R s
(Ais)(RF)(V0)
where:
As = Area of the characteristic ion
for the parameter to be
measured.
AJS = Area of the characteristic ion
for the internal standard.
ls = Amount of internal standard
added to each extract (jig).
V0 = Volume of water extracted
(liters).
15.2 Report results in micrograms
per liter without correction for recovery
data. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
15.3 If the surrogate standard
recovery falls outside the control limits
in Section 8.3, data for all parameters
in that fraction of the sample must be
labeled as suspect.
16. Method Performance
16.1 The method detection limit
(MDL) is defined as the minimum
concentration of a substance that can
be measured and reported with a 99%
confidence that the value is above
zero'1). The MDL concentrations listed
in Tables 4 and 5 were obtained using
reagent water'13>.
16.2 The average recoveries and the
average standard deviations of the
percent recoveries, presented in Table
5, were the result of a study of the
accuracy and precision of this method
by several laboratories. The values
listed represent the results from two to
four laboratories'14'.
16.3 The U.S. Environmental
Protection Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
17. Screening Procedure for
2,3,7,8-TCDD
17.1 If the sample must be screened
for the presence of 2,3,7,8-TCDD, it is
recommended that the reference mate-
rial not be handled in the laboratory
unless extensive safety precautions are
employed. It is sufficient to analyze the
base/neutral extract by selected ion
monitoring (SIM) GC/MS techniques,
as follows:
17.1.1 Concentrate the base/neutral
extract to a final volume of 0.2 mL.
17.1.2 Adjust the temperature of the
base/neutral column (Section 5.6.2) to
220°C.
17.1.3 Operate the mass spec-
trometer to acquire data in the SIM
mode using the ions at m/e 257, 320
and 322 and a dwell time no greater
than 333 milliseconds per ion.
17.1.4 Inject 5 to 7 ^L of the base/
neutral extract. Collect SIM data for a
total of 1 0 minutes.
17.1.5 The possible presence of
2,3,7,8-TCDD is indicated if all three
ions exhibit simultaneous peaks at any
point in the selected ion current
profiles.
17.1.6 For each occurrence where
the possible presence of 2,3,7,8-
TCDD is indicated, calculate and retain
the relative abundances of each of the
three ions.
17.2 False positives to this test may
be caused by the presence of single or
coeluting combinations of compounds
whose mass spectra contain all of
these ions.
17.3,. Conclusive results of the
presence and concentration level of
2,3,7,8-TCDD can be obtained only
from a properly equipped laboratory
through the use of method 61 3 of
other approved alternate test
procedures.
625-7
July 1982
-------
References
1. See Appendix A.
2. "Sampling and Analysis Procedures
for Screening of Industrial Effluents for
Priority Pollutants." U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1977,
Revised April 1977. Available from
Effluent Guidelines Division,
Washington, DC 20460.
3. ASTM Annual Book of Standards,
Part 31, D 3694. "Standard Practice
for Preparation of Sample Containers
and for Preservation," American
Society for Testing and Materials,
Philadelphia, PA, p. 679, (1980).
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,"
(29CFR1910), Occupational Safety
and Health Administration, OSHA
2206, (Revised, January 1976).
6. "Safety in Academic Chemistry
Laboraties," American Chemical
Society Publication, Committee on
Chemical Safety, 3rd Edition, 1979.
7. "Handbook of 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.
8. ASTM Annual Book of Standards,
Part 31, D 3370, "Standard Practice
for Sampling Water," American
Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual,"
Methods for Chemical Analysis of
Water and Wastes, EPA 600-4/79-020,
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268,
March 1979.
10. Eichelberger, J.W., Harris, L.E.,
and Budde, W.L., "Reference Com-
pound to Calibrate Ion Abundance
Measurement in Gas Chromatography-
Mass Spectrometry," Analytical
Chemistry, 47, 995 (1975).
11. McNair, H.M. and Bonelli, E.J.,
"Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52,
1969.
12. Burke, J.A., "Gas Chromatography
for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the
Association of Official Analytical
Chemists, 48, 1037 (1965).
1 3. "Method Detection Limit for
Methods 624 and 625," Olynyk, P.,
Budde, W.L., Eichelberger, J.W.,
unpublished report October, 1 980.
14. Kloepfer, R.D., "POTW Toxic
Study, Analytical Quality Assurance
Final Report," U.S. Environmental
Protection Agency, Region VII, Kansas
City, Kansas 6611 5, 1 981.
625-8
July 1982
-------
Table 1. Base/Neutral Extractables
Parameter
Acenaphthene
Acenaphthylene
Anthracene
Aldrin
Benzo (a) an thracene
Benzo(b)fluoranthene l
Benzo (k)fluoran thene
Benzo(a)pyrene
Benzo(ghi)perylene
Benzyl butyl phthalate
fi-BHC
6-BHC :
Bis(2-chloroethyl)ether
Bis(2-chloroethoxy)methane
Bis(2-ethylhexyl)phthalate
Bis (2-chloroisoprop yl) ether
4-Bromophenyl phenyl ether
Chlordane ,
2-Chloronaphthalene ;
4-Chlprophenyl phenyl ether
Chrysene
4, 4 '-ODD
4,4' -DDE
4,4'-DDT ;
Dibenzo (a, h) anthracene
Di-n-butylphthalate
1, 3-Dichlorobenzene
1 ,2-Dichlorobenzene
1 ,4-Dichlorobenzene
3, 3 '-Dlchlorobenzidine
Dieldrin ,
Diethyl phthalate
Dimethyl phthalate
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Di-n-octylphthalate
Endosulfan sulfate
Endrin aldehyde
Fluoranthene f
'Fluorene
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexachlorobutadiene
Hexachloroethane ]
Indeno (1,2, 3-cd)p yrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-n-propylamine
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB- 1248
PCB-1254
PCB-1260
Phenanthrene
Pyrene
Toxaphene
1 ,2,4-Trichlorobenzene \
STORETNo.
342O5
342OO
34220
39330
34526
34230
34242
34247
34521
34292
39338
34259
34273
34278
39 WO
34283
34636
39350
34581
34641
34320
393 1O
39320
39300
34556
391 1O
34566
34536
34571
34631
39380
34336
34341
34611
34626
34596
34351
34366
34376
34381
39410
39420
397 OO
34391
34396
34403
344O8
34696
34447
34428
34671
39488
39492
39496
395OO
395O4
395O8
34461
34469
39400
34551
CAS No.
83-32-9
208-96-8
12O-12-7
3O9-OO-2
56-55-3
2O5-99-2
207-O8-9
50-32-8
191-24-2
85-68-7
319-85-7
319-86-8
1 1 1-44-4
111-91-1
117-81-7
108-60-1
1 01 -55-3
57-74-9
91-58-7
7005-72-3
218-01-9
72-54-8
72-55-9
5O-29-3
53-70-3
84-74-2
541-73-1
95-50- 1
106-46-7
91-94-1
60-57-1
84-66-2
131-11-3
121-14-2
606-20-2
1 1 7-84-O
1031-07-8
7421-93-4
206-44-O
86-73-7
76-44-8
1O24-57-3
118-74-1
87-68-3
67-72-1
193-39-5
78-59-1
91-20-3
98-95-3
621-64-7
12674-11-2
1 1 104-28-2
11141-16-5
53469-2 1-9
12672-29-6
11097-69-1
1 1O96-82-5
85-O1-8
129-00-0
8001-35-2
12O-82-1
625-9
July 1982
-------
Table 2. Acid Extractables
Parameter
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dlchlorophenol
2,4-Dlmethylphenol
2,4-Dinitrophenol
2-Methyl-4, 6-dlnitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2t 4, 6-Trichlorophenol
STORET No.
34452
34586
34601
34606
34616
.34657
34591
34646
39032
34694
34621
Table 3. Additional Extractable Parameters^
Parameter STORET No.
Benzidine
a-BHC
t-BHC
Endosulfan 1
Endosulfan II
Endrin
Hexachlorocyclopentadiene
N-Nitrosodlmethylamine
N-Nitrosodiphenylamlne
39120
39337
39340
34361
34356
39390
34386
34438
34433
'
CAS No.
92-87-5
319-84-16
58-89-8
959-98-8
33213-65-9
72-2O-8
77-47-4
62-75-9
86-30-6
CAS No.
59-5O-7
95-57-8
120-83-2
1O5-67-9
51-28-5
534-52- 1
88-75-5
100-02-7
87-86-5
108-95-2
88-06-2
Method
605
608
608
608
608
6O8
612
605
6O5
'See Section 1.2 of method
Tebla 4. Chromatographic Conditions, Method Detection Limits and Characteristic Ions for Base/Neutral Extractables
Characteristic Ions
Parameter
1, 3-Dichlorobenzene
1 ,4-Dichlorobenzene
Hexachloroethane
B!s(2-chloroethyl)ether
1, 2-Dichlorobenzene
Bts(2-chtoroisopropyl)ether
N-Nitrosodi-n-propyl amine
Nitrobenzene
Hexachlorobutadiene
1, 2, 4-Trichlorobenzene
Isophorone
Naphthalene
B!s(2-chloroethoxy)rrjethane
Hexachlorocyclopentadiene *
2-Chloronaphthalene
Acenaphthylene
Acenaphthene
Dimethyl phthalate
2, 6-Dinitrotoluene
Fluorene
4-Chlorophenyl phenyl ether
2,4-Dinitrotoluene
Diethylphthalate
N-Nitrosodiphenylamine *
Hexachlorobenzene
a-BHC*
4-Bromophenyl phenyl ether
y-BHC*
Phenanthrene
Anthracene
0-BHC
Heptachlor
6-BHC
Aldrin
Retention
Time
(min.)
7.4
7.8
8.4
8.4
8.4
9.3
11.1
11.4
11.6
11.9
12.1
12.2
13.9
15.9
17.4
17.8
18.3
18.7
19.5
19.5
19.8
20.1
20. 5
21.0
21.1
21.2
22.4
22.8
22.8
23.4
23.4
23.7
24.0
ivietnoa
Detection
Limit (ng/L)
1.9
4.4
1.6
5.7
1.9
5.7
1.9
0.9
1.9
2.2
1.6
5.3
1.9
3.5
1.9
1.6
1.9
1.9
4.2
5.7
22
1.9
1.9
1.9
5.4
1.9
4.2
1.9
3.1
1.9
Electron Impact
Primary
,146
\146
,117
; S3
146
45
130
77
225
180
82
128
93
237
162
152
154
163
165
166
204
165
149
169
284
183
248
183
178
178
' 181
100
\ 183
66
Secondary
148
148
201
63
148
77
42
123
223
182
95
129
95
235
164
151
153
194
89
165
206
63
177
168
142
181
250
181
179
179
183
272
109
263
113
113
199
95
113
79
101
65
227
145
138
127
123
272
127
153
152
164
121
167
141
182
150
167
249
1O9
141
109
176
176
1O9
274
181
220
Chemical lonization
(Methane)
146
146
199
63
146
77
124
223
181
139
129
65
235
163
152
154
151
183
166
183
177
169
284
249
178
178
148
148
201
107
148
135
152
225
183
167
157
107
237
191
153
155
163
21 1
167
21 1
223
170
286
251
179
179
150
150
203
109
15C
137
164
227
209
178
169
137
239
203
181
183
164
223
195
223
251
198
288
277
207
2O7
625-10
July 1982
-------
Table 4. (Continued)
Parameter
Dibutyl phthalate
Heptachlor epoxide
Endosulfan 1*
Fluoranthene
Dieldrin
4,4' -DDE
Pyrene
Endrin *
Endosulfan II*
4, 4' -ODD
Benzidine *
4,4'-DDT
Endosulfan sulfate
Endrin aldehyde
Butyl benzyl phthalate
Bis(2-ethylhexyl) phthalate
Chrysene
Benzo (ajanthracene
3, 3 '-Dichlorobenzidine
Di-n-octylphthalate
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo (a)pyrene
Indenod, 2,3-c,d)pyrene
Dibenzo(a,h)anthracene
Benzo (ghi)perylene
N-Nitrosodimethyl amine*
Chlordane**
Toxaphene *
PCB 1O16*
PCB 1221*
PCB 1232*
PCB 1242*
PCB 1248*
PCB 1254*
PCB 1260**
Retention
Time
(min.)
24.7
25.6
26.4
26.5
27.2
27.2
27.3
27.9
28.6
28.6
28.8
29.3
29.8
—
29.5
3O.6
31.5
31.5
32.2
32.5
34.9
34.9
36.4
42.7
43.2
45.1
—
19 to 30
25 to 34
18 to 3O
15to3O
15 to 32
15 to 32
12 to 34
22 to 34
23 to 32
Method
Detection
Limit (ng/L)
2.5
2.2
—
2.2
2.5
5.6
1.9
—
—
2.8
44
4.7
5.6
—
2.5
2.5
2.5
7.8
16.5
2.5
4.8
2.5
2.5
3.7
2.5
4.1
—
—
—
—
30
—
—
—
36
—
Characteristic ions
Electron Impact
Primary
149
353
237
202
79
246
202
81
237
235
184
235
272
67
149
149
228
228
252
149
252
252
252
276
278
276
42
373
159
224
19O
190
224
294
294
33O
Secondary
150
355
339
101
263
248
101
263
339
237
92
237
387
345
91
167
226
229
254
253
253
253
138
139
138
74
375
231
260
224
224
260
33O
330
362
104
351
341
WO
279
176
100
82
341
165
185
165
422
250
206
279
229
226
126
125
125
125
277
279
277
44
377
233
294
260
260
294
362
362
394
Chemical lonization
(Methane)
149
203
203
185
149
149
228
228
252
252
252
276
278
276
205
231
231
213
299
229
229
253
253
253
277
279
277
279
243
243
225
327
257
257
281
281
281
305
3O7
305
*See Section 1.2.
* * These compounds are mixtures of various isomers. (See Figures 2 to 12)
Gas Chromatographic conditions: Glass column 1.8m long x 2 mm ID packed with Supelcoport (100/12O mesh) coated with
3% SP-2250. Carrier gas: helium at a flow rate of 3O mL per min.
Temperature: Isothermal 'at 50 °C for 4 min., then 8 ° per min to 2 70 °C. Hold at 2 7O °C for 30 min.
Table 5. Chromatographic Conditions, Method Detection Limits and Characteristic Ions for Acid Extractables
Parameter
2-Chlorophenol
2-Nitrophenol
Phenol
2,4-Dimethylphenol
2,4-Dichlorophenol
2,4, 6-Trichlorophenol
4-Chloro-3-methylphenol
2,4-Dinitrophenol
2-Methyl-4, 6-dinitrophehol
Pentachlorophenol
4-Nitrophenol
1 IC7lC7/ft/l///
Time
(min.)
5.9
6.5
8.0
9.4
9.8
11.8
13.2
15.9
16.2
17.5
20.3
IVIGUIUU
Detection
Limit (\ng/L)
3.3
3.6
1.5
2.7
2.7
2.7
3.0
42
24
3.6
2.4
Electron Impact
Primary
128
139
94
122
162
196
142
184
198
266
65
Secondary
64
65
65
107
164
198
107
63
182
264
139
130
109
66
121
98
200
144
154
77
268
1O9
Chemical lonization
(Methane)
129
140
95
123
163
197
143
185
199
267
14O
131
168
123
151
165
199
171
213
227
265
168
157
122
135
163
167
201
183
225
239
269
122
Chromatographic conditions: 1.8m long x 2 mm ID glass column packedwith Supelcoport (WO/120 mesh) coatedwith 1%
SP-1240. Carrier gas: helium at a flow rate of 30 mL per min. Column temperature, isothermal at 7O°C for 2 min, then 8 ° per
min, to 2OO °. ',
625-11
July 1982
-------
Table 6. Accuracy and Precision for Base/Neutral Extractables
Reagent Water ~ Wastewater
Parameter
Acenaphthene
Acenaphthylene
Aldrin
Anthracene
Benzo(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(ghi)perylene
Benzofajpyrene
Benzidine
Butyl benzyl phthalate
P-BHC
6-BHC
B!s(2-chloroethoxy)methane
B!s(2-chloroethyl)ether
Bis(2-chlorolsopropyl)ether
Bis (2-ethylhexyl) phthalate
4-Bromophenyl phenyl ether
2-Chloronaphthalene
4-Chlorophenyl phenyl ether
Chrysene
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dibenzo(a,h)anthracene
Di-n-butyl phthalate
7, 2-Dichlorobenzene
1, 3-Dlchlorobenzene
1, 4-Dichlorobenzene
3, 3 '-Dichlorobenzidine
Diethylphthalate
Dimethyl phthalate
2,4-Dinitrotoluene
2, 6-Dinitrotoluene
Di-n-octylphthalate
Endosulfan sulfate
Fluoranthene
Fluorene
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexachlorobutadiene
Hexachlorocyclopentadiene
Hexachloroethane
Indeno (1,2,3-cd) pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-n-propylamine
N-N!trosodiphenylamine
PCB-1221
PCB-1254
Phenanthrene
Pyrene
1,2,4-Trichlorobenzene
Average
Percent
Recovery
77
78
72
84
83
96
96
8O
90
87
47
69
66
84
56
71
129
80
73
45
83
8O
69
63
82
70
59
55
61
184
42
25
83
79
97
79
89
77
69
82
79
46
27
46
65
75
6
72
68
84
77
8O
84
86
64
Standard
Deviation
(%)
23
22
6
14
19
68
68
45
22
61
32
25
18
33
36
33
50
17
24
11
19
9
20
15
39
25
27
28
31
174
28
33
32
18
37
29
19
16
6
7
2O
25
25
21
37
33
32
31
39
24
11
13
14
15
16
Average
Percent
Recovery
83
82
—
76
75
41
47
68
43
63
74 ;
—
—
82
72
71
82
75
79
—
75
—
—
— !
70 ;
S3
62
54
63
143
48
35
79
79
89
— :
80
80
— \
— •
71
48
12
52
81
77
75
82
76
86
— '•
i
76 •
80.
63 |
Standard
Deviation
,(%)
29
23
—
22
28
21
27
40
21
55
43
—
—
74
37
39
63
20
27
—
28
—
—
—
40
51
28
24
35
145
28
36
34
25
62
—
26
20
—
—
22
28
12
26
43
42
35
54
45
31
—
—
22
23
26
Spiked between 5 to 2400 \ig/L.
625-12
July 1982
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Table 7. Accuracy and Precision for Acid Extractables
Reagent Water
Parameter
4- Chloro-3-me th yl phenol
2-Chlorophenol \
2,4-Dichlorophenol
2,4-Dimethylphenol
2, 4-Dinitrophenol
2-Methyl-4, 6-dinitrophenol
4-Nitrophenol
2-Nitrophenol
Pentachlorophenol
Phenol '•
2,4,6- Trichlorophenol '
A verage
Percent
Recovery
79
70
74
64
78
83
41
75
86
36
77
Standard
Deviation
{%)
18
23
24
25 '
21
18
20
25
20
14
20
Wastewater
A verage
Percent
Recovery
75
71
80
58
JOS
90
43
75
66
36
81
Standard
Deviation
(%)
21
25
21
26
56
35
16
27
36
21
20
Spiked from 10 to 1500\ig/L
Table 8. Suggested Internal and Surrogate Standards
Base/Neutral Fraction
Acid Fraction
Aniline-d5 2-Fluorophenol '
Anthracene-d10 Pentafluorophenol
Benzofa)anthracene-d12 ', Phenol-ds
4,4'-Dibromobiphenyl ] 2-Perfluoromethyl phenol
4,4 '-Dibromooctafluorobiphen yl
Decafluorobiphenyl '•
2,2'-Difluorobiphenyl
4-Fluoroaniline
1-Fluoronaphthylene
2-Fluoronaphthylene
Naphthalene-d8 .
Nitrobenzene-ds
2,3,4,5,6-Pentafluorobiphenyl
Phenanthrene-d 1 o \
Pyridine-ds
Table 9. DFTPP Key Ions and Ion Abundance Criteria
Mass
51
68
70
127
197
198
199
275
365
441
442
443
Ion Abundance Criteria
3O-6O% of mass 198
less than 2% of mass 69
less than. 2 % of mass 69
4O-6O% of mass 198
less than 1% of mass 198
base peak, 10O% relative abundance
5-9% of mass 198
10-3O% of mass 198
greater than 1 % of mass 198
present but less than mass 443
greater than 4O% of mass 198
17-23% of mass 442
625-13
July 1982
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Column: 3% SP-2250 on Supelcoport
a; Program: 50°C, 4 min. 8° per min to 270°C
Detector: Mass spectrometer
I
£
2,4-DinitrotolueneJ N-Nitroso Diphenylamine
10
15
20 25 ; 30
Retention time, minutes
35
figure 1. Gas chroamatogram of base/neutral fraction.
40
45
Column: 1% SP-1240DA on Supelcoport
Program: 70°C-2 min, 8%/min to 200°C
Detector: Mass spectrometer
•5
CB
Column: 3% SP-2250 on Supelcoport
Program: 50°C-4 min. 8°/minute
to 270°C
Detector: Mass spectrometer
02 4 6 8 10 12 14 16 18 2O 22
Retention time, minutes
Figure 2, Gas chromatogram of acid fraction.
625-14
JO 15 20
Retention time, minutes
25
3O
Figure 3. Gas chromatogram of pesticide fraction.
July 1982
-------
Column: 3% SP-225O on Supelcoport
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
18 2O 22 24 26 28 30 32 34 36
Retention time, minutes
Figure 4. Gas chromatogram of chlordane.
m/z=233
22 24 26 28 30 32 34 36 38
Retention time, minutes
Figure 5. Gas chromatogram of toxaphene.
625-15
July 1982
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Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 6. Gas chromatogram of PCB-1016.
18 20 22 24 26 28 30
Retention time, minutes
Figure 7. Gas chromatogram of PCB-1221.
625-16
July 1982
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Column: 3% SP-2250 on Supelcopori
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
Column: 3% SP-225O on Supe/coport
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 8. Gas chromatogram of PCB-1232.
Figure 9.
20 22 24 26 28 30
Retention time, minutes
Gas chromatogram of PCB-1242.
32
625-17
July 1982
-------
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min., 8° per im. to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 10. Gas chromatogram of PCB-1248.
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min.,8° per min to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32 34 36 38
Retention time, minutes
Figure 11. Gas chromatogram of PCB-1254.
Column: 3% SP-2250 on Supelcoport
Program: 5O°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
625-18
18 20 22 24 26 28 30 32 34 36 38
Retention time, minutes
Figure 12. Gs chromatogram of PCB-1260.
July 1982
-------
BC
Tailing Factor = ——
Example calculation:
Peak Height = DE = 100mm
10% Peak Height = BD = JO mm
Peak Width at 10% Peak Height = AC = 23 mm
AB =11 mm
BC = 12 mm
Therefore: Tailing Factor = ~rr=1.1
/ > >
Figure 13. Tailing factor calculation.
625-19
July 1982
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Appendix A
Definition and Procedure for the Determination
of the Method Detection Limit
The method detection limit (MDL) is defined as the minimum concentration of a
substance that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero and determined from analysis of a
sample in a given matrix containing analyte.
Scope and Application
This procedure is designed for applicability to a wide variety of sample types
ranging from reagent (blank) water containing analyte to wastewater containing
analyte. The MDL for an analytical procedure may vary as a function of sample
type. The procedure requires a complete, specific and well defined analytical
method. It is essential that all sample processing steps of the analytical method be
included in the determination of the method detection limit.
t
The MDL obtained by this procedure is used to judge the significance of a single
measurement of a future sample.
The MDL procedure was designed for applicability to a broad variety of physical
and chemical methods. To accomplish this, the procedure was made device- or
instrument-independent.
Procedure
1. Make an estimate of the detection limit using one of the following:
(3) The concentration value that corresponds to an instrument signal/noise
ratio in th'e range of 2.5 to 5. If the criteria for qualitative identification of
the analyte is based upon pattern recognition techniques, the least
! abundant signal necessary to achieve identification must be considered in
making the estimate.
(b) The concentration value that corresponds to three times the standard
; deviation of replicate instrumental measurements for the analyte in
reagent water.
(c) The concentration value that corresponds to the region of the standard
- curve where there is a significant change in sensitivity at low analyte
I concentrations, i.e., a break in the slope of the standard curve.
(d) The concentration value that corresponds to known instrumental
limitations.
It is recognized that the experience of the analyst is important to this process.
However, the analyst musfinclude the above considerations in the estimate
of the detection limit.
2. Prepare reagent (blank) water that is as free of analyte as possible. Reagent or
interference free water is defined as a water sample in which analyte and
interferent concentrations are not detected at the method detection limit of
each analyte of interest. Interferences are defined as systematic errors in the
measured analytical signal of an established procedure caused by the
presence of interfering species (interferent). The interferent concentration is
presupposed to be normally distributed in representative samples of a given
matrix.
3. (a) If the MDL is to be determined in reagent water (blank), prepare a
• laboratory standard (analyte in reagent water) at a concentration which is
at least equal to or in the same concentration range as the estimated MDL.
(Recommend between 1 and 5 times the estimated MDL.) Proceed to Step
4.
A-1 July 1982
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(b) If the MDL is to be determined in Another sample matrix, analyze the
sample. If the measured level of the analyte is in the recommended range
of one to five times the estimated MDL, proceed to Step 4.
If the measured concentration of analyte is less than the estimated MDL,
add a known amount of analyte to: bring the concentration of analyte to
between one and five times the MDL. In the case where an interference is
coanalyzed with the analyte:
If the measured level of analyte is'greater than five times the estimated
MDL, there are two options:
(1) Obtain another sample of lower level of analyte in same matrix if
possible.
(2) The sample may be used as is for determining the MDL if the analyte
level does not exceed 10 times the MDL of the analyte in reagent
water. The variance of the analytical method changes as the analyte
concentration increases from the MDL, hence the MDL determined
under these circumstances ifiay not truly reflect method variance at
lower analyte concentrations.
4. (a) Take a minimum of seven aliquots of the sample to be used to calculate
the MDL and process each through the entire analytical method. Make all
computations according to the defined method with final results in the
method reporting units. If blank measurements are required to calculate
the measured level of analyte, obtain separate blank measurements for
each sample aliquot analyzed. The average blank measurement is
subtracted from the respective sample measurements.
(b) It may be economically and technically deirable to evaluate the estimated
MDL before proceeding with 4a. This will: (1) prevent repeating this entire
procedure when the costs of analyses are high and (2) insure that the
procedure is being conducted at the correct concentration. It is quite
possible that an incorrect MDL can be calculated from data obtained at
many times the real MDL even though the background concentration of
analyte is less than five times the calculated MDL. To insure that the
estimate of the MDL is a good estimate, it is necessary to determine that a
lower concentration of analyte will not result in a significantly lower MDL.
Take two aliquots of the sample to be used to calculate the MDL and
process each through the entire method, including blank measurements
as described above in 4a. Evaluate these data:
(1) If these measurements indicate the sample is in the desirable range for
determining the MDL, take five additional aliquots and proceed. Use
all seven measurements to cajculate the MDL
(2) If these measurements indicate the sample is not in the correct range,
reestimate the MDL, obtain new sample as in 3 and repeat either 4a or
4b.
5. Calculate the variance (S2) and standard deviation (S) of the replicate
measurements, as follows:
. 1 = 1
• = {S2)1/a I
)/"]
where: the xi, i = 1 to n are the analytical results in the final method reporting
units obtained from the n sample aliquots and ^- Xi2 refers to the sum of
the X values from i = 1 to n. i = 1
6. (a) Compute the MDL as follows:
= t(n-1,1-
-------
where:
MDL = the method detection
tin-i, i-« = .991 = the students' t value appropriate for a 99% confidence
level and a standard deviation estimate with n-1 degrees
of freedom. See Table.
S = standard deviation of the replicate analyses.
(b) The 95% confidence limits for the MDL derived in 6a are computed
; according to the following equations derived from percentiles of the chi
square over degrees of freedom distribution (XVdf) and calculated as
follows:
', MDLLCL = 0.69 MDL
i MDLucL = 1.92 MDL
where MDLixi. and MDLuct are the lower and upper 95% confidence limits
respectively based on seven aliquots.
7. Optional iterative procedure to verify the reasonableness of the estimated
MDL and calculated MDL of subsequent MDL determinations.
(a) If this is the initial attempt to compute MDL based on the estimated MDL
in Step 1, take the MDL as calculated in Step 6, spike in the matrix at the
calculated MDL and proceed through the procedure starting with Step 4.
(b) If the current MDL determination is an iteration of the MDL procedure for
which the spiking level does not permit qualitative identification, report the
MDL as that concentration between the current spike level and the
! previous spike level which allows qualitative identification.
(c) If the current MDL determination is an iteration of the MDL procedure and
the spiking level allows qualitative identification, use S2 from the current
MDL calculation and S2 from the previous MDL calculation to compute the
F ratio.
if |f< 3.05
; then compute the pooled standard deviation by the following equation:
o pooled I
+6S|T
2 J
12
if-Ta > 3.05, respike at the last calculated MDL and process the samples
OB
through the procedure starting with Step 4.
(c) Use the Spo0ied as calculated in 7b to compute the final MDL according to
the following equation:
MDL = 2.681 (Spoo,ed)
where 2.681 is equal to t(i2, i-a = .99).
(d) The 95% confidence limits for MDL derived in 7c are computed according
to the following equations derived from percentiles of the chi squared over
degrees of freedom distribution.
MDLLcL = 0.72 MDL
MDLucL = 1.65 MDL
where LCL and UCL are the lower and upper 95% confidence limits
respectively based on 14 aliquots.
Reporting
The analytical method used must be specifically identified by number or title and
the MDL for each analyte expressed in the appropriate method reporting units. If
the analytical method permits options which affect the method detection limit,
these conditions must be specified with the MDL value. The sample matrix used to
; A-3 July 1982
-------
determine the MDL must also be identified with the MDL value. Report the mean
analyte level with the MDL. If a laboratory'standard or a sample that contained a
known amount analyte was used for this' determination, report the mean recovery,
and indicate if the MDL determination was iterated.
i
If the level of the analyte in the sample matrix exceeds 10 times the MDL of the
analyte in reagent water, do not report a value for the MDL.
Reference
Glaser, J. A., Foerst, D. L., McKee, G. D., Quave, S. A., and Budde, W. L, "Trace
Analysis for Wastewaters," Environmental Science and Technology, 15, 1426
(1981). !
Table of Students' t Values at the 99 Percent Confidence Level
Number of Degrees of Freedom
Replicates (n-1) fm-i, i-«r = .9!
7 63.143
8 7 2.998
9 8 2.896
10 9 2.821
11 10 2.764
16 15, 2.602
21 20 2.528
26 25 2.485
31 30 2.457
61 60 2.390
°° °° 2.326
A-4 ' July 1982
ft U.S.GOVERNMENTPR1NTING OFFICE: 1982-559-092/0443
------- |